R3: Reviews, Responses & Reflections in Neuroscience

2020 Edition

R3

REVIEWS RESPONSES REFLECTIONS IN NEUROSCIENCE

Designed By: Andrea Pinto Cover Image from Pixabay

Copyright Š 2020 Human Biology Program, University of Toronto, Toronto HMB300H1S

2

Table of Contents Kynurenine Pathway and Gut Microbiota: Treatment Applications for Schizophrenia

5

Fecal microbiota transplantation as a defence against MPTP-induced Parkinson’s disease mice

13

Biophysical Modeling of Nonlinear Dendritic Computations

18

A Neuroligin-3 Mutation Results in Abnormal Behaviour in Mouse Model of Autism Spectrum Disorder

23

Behavioural and synaptic effect from EAAT3 overexpression on obsessive-compulsive disorder

28

An Express Train to the Diseased Brain: RVG-modified Exosomes for the Treatment of Alzheimer’s Disease

35

Injection of beta amyloid brain extract intravenously is shown to induce Alzheimer’s Like Disease in APPSwe/PS1dE9 41 mice –A Critical Review Effect of germ-free mice on monoamine neurotransmitter gene expression and anxiety-like behavior;

46

further evidence of the gut-brain connection.

The Effect of LSD On Brian Entropy and The Personality Trait ‘Openness’

55

Age-progressive β-amyloid depositions and altered hippocampal neurogenesis in Alzheimer’s Disease Tg2576 and APP Swedish PS1 dE9 mice

60

You Are What You Eat : Alzheimer’s and Diabetes Edition. A review of the effect of high fat and sugar diets on cognitive decline.

65

APOE genotype and chlorpyrifos: Examination of their roles in gut dysbiosis and influence on metabolites in the brain

69

Altered Feed-Forward Inhibition of Striosomes is Linked to Aberrant Value-Based Decision Making in Chronically Stressed Mice

74

Immune alteration using human mesenchymal stem cells in schizophrenia: a review

80

Human Amniotic Epithelial Stem Cells Aa A Therapy For Alzheimer’s Disease

85

The Role of Toll-Like Receptor 4 in Mediating Gut-Brain Axis Inflammation and Pathologies Seen in Parkinson’s Disease

91

Genome-edited Skin Transplants Offer a Safe and Enduring Gene Therapy Approach for Treating Drug Addiction

95

Behind Closed Doors: The Emerging Role of Focused Ultrasound in Alzheimer’s Disease

101

Go with your Gut: How inflammation can speed up Motor Dysfunction in Alpha‑Synuclein Mutant Mice

105

Positive Effects of Probiotic Treatment on Spatial Cognitive Performance and Synaptic Plasticity in a β-amyloid rat model of Alzheimer’s Disease

109

The Molecular Mechanisms Underlying Sleep Deprivation and Impaired Fetal Neurodevelopment

115

Aβ aggregation and Tau phosphorylation suggest PhIP correlation to Alzheimer’s disease

121

Apelin-36: An Overlooked Peptide with Promising Effects on Parkinson’s Disease

126

3

Restoration of the UPS through enhancement of UBA1 levels improves SMA models outcomes

132

Omega-3 Improves cognitive Dysfunction in Schizophrenia Via CREB S133 Phosphorylation

137

Hypersocialization, Social Blindness and Motor Deficits: the Role of PSD95 and PSD93

142

How a Hormone that Helps with Sleep can Reduce the Effects of Diabetes Towards the Hippocampus

150

The effects of oxytocin administration in the attenuation of autistic symptoms in ASD mice models

155

The Protein Pallidin and its Prognosis in Schizophrenia

161

Understanding the Link between Inflammation and Mental Health

167

Role of Lateral Hypothalamic Dopaminergic Mechanisms in Feeding Regulation: A Study

173

The Therapeutic Potential of the Novel Player Kcnn2 in Fetal Alcohol Syndrome Disorder Pathogenesis

179

Treating Parkinson’s Disease Symptoms Using Optogenic Deep Brain Stimulation in the Subthalamic Nucleus

186

Chronic Jet Lag and Its Long-Term Effects on Brain Function

190

Curcumin as a Potential Treatment in Parkinson’s Disease

195

Anterior Cingulate Cortex as Therapeutic Target for Autism Spectrum Disorder

201

4

Kynurenine Pathway and Gut Microbiota: Treatment Applications for Schizophrenia Ali Abdolizadeh

Previous studies reported increased pro-inflammatory markers in schizophrenia (SCZ) patients (MĂźller 2018). In addition, altered tryptophan (Trp) metabolism and dysregulation of its metabolites have been observed in inflammation-associated diseases (Dantzer 2017). The gut microbiota modulates different pathways of Trp metabolism and influences brain function (Gao et al. 2020). Significant contributions of dysregulated gut microbiota to neuropsychiatric disorders through the gut-brain axis have been highlighted in the past decade (Petra et al. 2015). However, it is still unclear whether microbiota alteration itself leads to neuropsychiatric diseases, or in turn, microbiota dysbiosis is a result of mental disorders. The original paper sought to further investigate the causation of this process (Zhu et al.

2019). They performed fecal microbiota transplantation (FMT) from antipsychotic-free individuals with schizophrenia (SZC) and healthy controls (CS) into specific pathogen-free (SPF) mice. Following FMT, behavioural tests revealed impaired spatial learning and memory, increased exploratory activity, and enhanced rearing behaviour in SPF mice treated with fecal-derived microbiota from SCZ individuals. Moreover, kynurenine (Kyn) and kynurenic acid (Kyna) levels of Trp metabolites were elevated in both peripheral tissues and the central nervous system of SCZ mice. Nonetheless, the exact mechanism by which the gut microbiota contributes to schizophrenia pathogenesis remains unclear. The current paper aims to review the role of the gut microbiota in brain functions, focusing on the Trp metabolism and Kyn pathway. This review further elucidates the link between Kyn metabolites and schizophrenia for potential therapeutic targets.

Key words: schizophrenia (SZC), tryptophan (Trp) metabolism, microbiota, brain-gut axis, impaired learning and memory, kynurenine (Kyn), Kynurenic acid (Kyna), fecal microbiota transplantation (FMT).

5

Introduction

(Lamas et al. 2016). Furthermore, the microbiota can mediate conversions of Trp into the downstream molecules, influencing The gut microbiota is the community of all microorganisms, the release of neurotransmitters, such as glutamate, serotonin, mainly bacteria, that live in our intestine (Turnbaugh et al. and dopamine (Agus, Planchais, and Sokol 2018).

2007). Accumulating evidence indicates that environmental factors, such as epigenetics, the mode of delivery, infant feeding, In the original paper, Zhu et al. (2019) sought to examdiet, antibiotics, may change the composition and diversity of ine whether fecal-derived microbiota of humans can affect the the gut microbiota (Wen and Duffy 2017). Studies in mice and normal brain function of the mice. Subsequently, they perhumans have revealed that the gut microbiota regulates signal- formed FMT from SCZ and healthy individuals into SPF mice. ling between the brain and the gut, contributing to the healthy Impaired spatial learning and memory and increased exploratory development of the brain (Dinan and Cryan 2017; Carlson et al. behaviour, known as SCZ-associated behavioural abnormalities 2018; Sharon et al. 2016). An earlier study showed that microbi- were observed in the mice treated with dysregulated fecalota has a significant role in mediating the stress response derived microbiota from SCZ patients. Further analysis revealed through the hypothalamic-pituitary-adrenal (HPA) axis (Sudo et that the Kyn pathway of Trp metabolism in SCZ mice is upregual. 2004). The primary microbiota metabolites, short-chain-fatty lated in both peripheral tissues and the central nervous system, acids (SCFA), are also essential for the normal function of the leading to neurotransmitter alterations. nervous system (Silva, Bernardi, and Frozza 2020). Moreover, gut microbiota plays an important role in the regulation of proteins involved in synaptic transmissions, such as synaptophysin Results (Heijtz et al. 2011). Further studies indicated that the gut microbiota is required for microglial cells’ function, and dysregulated microbiota may lead to neuroinflammation and mental disorders SCZ-like behaviors in SPF mice treated with FMT from SCZ pa(Erny et al. 2015). Vagus nerve is another pathway by which mi- tients crobiota influences the gut-brain crosstalk (Bravo et al. 2011).

Additionally, Trp metabolism, which is known to be important in inflammation and brain function, is regulated by the gut microbi- To examine the effects of the human gut microbiota on mice, ota (Gao et al. 2020). Thus, since our gut microbiota is involved behavioural tests, such as open field test (OPT) and Barnes maze in a broad spectrum of pathways to influence our brain function, (BM) task, were conducted following transplantation. OPT illusit can be plausibly targeted to help us better understand the trated that SCZ mice, compared to HC mice, exhibited hyperlocomotor activity since the number of lines crossed in the field pathogenesis of psychiatric disorders such as schizophrenia. and the total distance moved were all increased in the given Schizophrenia (SCZ) is known to be one of the most period (Fig.1 a and b). In addition, the number of rearing events complex mental disorders, and the symptoms may fall into three in SCZ mice was elevated, suggesting hyper-exploratory behavsubcategories; positive, negative, and cognitive (Kahn et al. iours. The Barnes Maze task was conducted to assess the learn2015). The most common characteristics of positive symptoms ing and memory of the mice (Fig. 1 c and d). In the BM task, esare hallucinations and delusions, while anhedonia and lack of cape latency measurements indicated that SCZ mice had promotivation are considered as negative symptoms. Individuals longed latency to find the escape box after trial training. The with schizophrenia may also experience cognitive symptoms results indicate spatial learning and memory impairments as such as cognitive dysfunctions (Kahn et al. 2015). A metaanother feature found in SCZ mice. In line with these findings, analysis of 12 studies illustrated the importance of genetic preZheng et al. (2019) showed that the behaviours of mice treated disposition and environmental factors for the development of with the microbiota of SCZ patients were associated with SCZ schizophrenia (Sullivan, Kendler, and Neale 2003). Therefore, it symptoms, such as increased hyperactivity. It is worth mentionis conceivable that the gut microbiota, which is highly regulated ing that locomotor activity and exploratory behaviours as posiby environments, to be involved in the SCZ-associated symptive symptoms and impaired memory as a negative symptom toms. In this context, recent studies used 16S rRNA sequencing have been previously reported as the behaviours associated to find a diagnostic biomarker in the gut microbiota of SCZ pawith the animal model of SCZ (Winship et al. 2019). tients. They were able to observe significant differences between the gut microbiota of SCZ patients and healthy controls (Shen et al. 2018; Nguyen et al. 2019). Miller et al. (2011) conducted a meta-analysis to investigate changes of proinflammatory cytokines in individuals with SCZ. They found elevated levels of TNF-alpha, IL-6, and IL-1B in SCZ patients, suggesting schizophrenia as a neuroinflammatory disorder.

Despite a slight discrepancy found between studies (Erhardt et al. 2001; Szymona et al. 2017), the impacts of inflammation on Trp metabolism and SCZ pathogenesis have been widely accepted (Pedraz-Petrozzi et al. 2020). Previous studies showed that microbiota-derived metabolites are involved in the regulation of inflammations through acting on Trp metabolism 6

Fig.1) OFT (a and b) and BM task (c and d) were performed to examine mice behaviours after FMT. HC mice and SCZ mice are shown in light pink and black, respectively. OFT showed increased locomotor activity and rearing events in SCZ mice (a) compared to HC (a). In BM task, SCZ mice exhibited impaired spatial learning and memory (d), in comparison to HC mice (c).

Fig.2 Expression of IDO and KAT-II in the Kyn pathway of Trp metabolism were measured immediately after and ten days after FMT. IDO mRNA significantly increased in the colon, liver, and spleen of SCZ mice compared to HC mice (a). mRNA levels of KAT-II were elevated only in the liver (b). This figure was presented in the original paper (Zhu et al. 2019).

Upregulation of the peripheral Kyn-Kyna pathway of Trp me- Increased Kyn-Kyna pathway of Trp metabolism in the brain of tabolism in SCZ mice SCZ mice Upon dissecting the mice’s peripheral and brain tissues, enzymelinked immunosorbent assay (ELISA), liquid chromatographymass spectrometry (LC/MS), and qPCR were used to measure downstream molecules concentrations and gene expressions of the enzymes present in Trp metabolism.

Zhu et al. targeted three brain regions of SCZ mice; prefrontal cortex (PFC), striatum, and hippocampus, to measure metabolites and enzymes involved in the Trp metabolism (Fig.3). The same tools and techniques previously mentioned were used to do the experiment. They similarly observed elevated Kyn-Kyna pathway of Trp metabolism in the brain of the SZC mice. In line with this finding, high brain Kyna in SCZ was also found in previous studies (Schwarcz et al. 2001). Moreover, 3-HK levels were decreased in PFC, striatum, and hippocampus of SCZ mice, while the mRNA levels of KMO remained almost the same, compared to HC. Even though the expression of IDO, which converts Trp to Kyn, did not increase, they found elevated Kyn levels in all three brain regions. Notably, increased IDO expressions were previously reported in the brain of the SCZ individuals (Miller et al.

2004). In addition, as the mRNA levels of KAT-II increased, they found the Kyna level was also elevated in the mentioned brain areas of SCZ mice. TPH-1 expression, which is responsible for Trp/5H-T conversion, increased in the hippocampus of SCZ mice, and subsequently, 5-HT was elevated. The increased 5-HT level was

It is noted that 5-hydroxytryptophan (5-HT) and Kynurenine (Kyn) are known as the two pathways of Trp metabolism (Höglund, Øverli, and Winberg 2019). Zhu et al. (2019) found that Trp levels in serum and peripheral tissues, such as intestine, spleen, and liver of the SCZ mice were decreased. Reduced 5-HT levels in serum and intestine of the SCZ mice were also observed. In addition, the expression of tryptophan hydroxylase (TPH-1), which controls the production of 5-HT from Trp, decreased in the peripheral tissues, suggesting that Trp metabolism in the 5-HT pathway is downregulated.

On the other hand, they reported increased Kyn levels in serum and peripheral tissues. Consistently, indoleamine 1,2dioxygenase 1 (IDO-1) enzyme that converts Trp to Kyn were overexpressed (Fig.2 a). Kyn can be further metabolized into two branches; Kynurenic acid (KA) and 3-hydroxykynurenine (3-HK) observed in the striatum as well. Furthermore, they reported (Höglund, Øverli, and Winberg 2019). They reported unchanged elevated dopamine levels in the striatum and PFC and increased 3-HK in peripheral tissues, whereas the level of Kyna was higher glutamate in PFC. in serum and peripheral tissues. In agreement with the increased Kyna, mRNA levels of kynurenine aminotransferase (KAT -II), which act as Kyn-Kyna conversion enzyme, were elevated in the liver of the SCZ mice (Fig.2 b). In consistent with the unchanged 3-HK, the Kynurenine-3- monooxygenase (KMO) enzyme that catalyzes Kyn to 3-HK did not change. These results indicated that the peripheral Kyn-Kyna pathway of Trp metabolism is activated in SCZ mice, compared to HC. A recent study also showed that elevated Kyna is associated with cognitive impairments in individuals with SCZ (Huang et al. 2020). Although many studies indicated that elevated immune response and inflammatory cytokines are associated with the pathogenesis of schizophrenia (Müller 2018), Zhu et al. (2019) could not find any significant differences in inflammatory markers between SCZ and HC mice. Thus, they postulated that direct or indirect effects of microbiota induced those changes in Trp metabolism.

7

Fig.3 Levels of Trp metabolites in PFC (a), striatum (b), and hippocampus (c) of SCZ and HC mice are illustrated above. Increased levels of Kyn and Kyna are noted in all three brain regions of SCZ mice, whereas 3-HK decreased, showing upregulation of central Kyn-Kyna pathway of Trp metabolism. Increased 5 -HT in striatum and hippocampus may explain positive symptoms in SCZ mice. This figure was presented in the original paper (Zhu et al. 2019).

regard, Kozak et al. (2014) showed that KAT-II inhibitors decreased brain Kyna level and improved cognitive functions in mice. It is worth mentioning that Olney and Farber (1995) had proposed that the dysfunction of glutamate receptors is linked with the pathogenesis of SCZ. Indeed, they showed that blockade of glutamate receptors resulted in the loss of inhibitory effects of GABAergic neurons, and further increased the release of glutamate in the brain. These findings may explain increased glutamate levels observed in the brain regions of SCZ mice, which are associated with positive symptoms. Studies have also Discussion demonstrated that high Kyna, as an NMDA receptor antagonist, can stimulate dopaminergic neurons in the midbrain of SCZ indiZhu et al. illustrated that altered gut microbiota from SCZ paviduals (Erhardt and Engberg 2002). In line with this observation, tients transplanted into SPC mice upregulated both peripheral Zhu et al. (2019) reported increased dopamine in striatum and and central Kyn-Kyna pathway of Trp metabolism. CorrespondPFC of SCZ mice. ingly, these changes were associated with SCZ-like symptoms.

Indeed, they provided new findings that may have implications Reviewing the original paper’s results and the relevance for future researches and therapeutic targets. The gut microbio- demonstrates that the Kyn pathway of Trp metabolism can be ta can regulate Trp metabolisms in three different pathways; considered an effective therapeutic target for SCZ patients. In indole, 5-HT, and Kyn (Agus, Planchais, and Sokol 2018) that the addition, once more, the importance of the gut microbiota in two later pathways will be discussed in this section. psychiatric disorders has been highlighted.

Interestingly, our gut microbiota has regulatory effects on the enzyme TPH (Yano et al. 2015; Reigstad et al. 2015), which converts Trp to 5-HT. It has been reported that microbiota metabolites, such as short-chain fatty acids, are involved in stimulating the expression of TPH (Yano et al. 2015). In line with this observation, a recent study showed 5-HT deficiency in germ-free mice (Reigstad et al. 2015). These findings may explain the original paper’s result indicating decreased peripheral 5-HT in SCZ mice with altered gut microbiota. In the Trp metabolism, IDO is the enzyme that catalyzes Trp to Kyn (Thomas and Stocker 1999). Recent studies have shown that IDO is stimulated by inflammatory cytokines, such as INF-gamma (O’Connor et al.

2009). Zhu et al. (2019) did not report increased inflammatory markers in SCZ mice, while the expression of IDO increased in the periphery. Therefore, they suggested that the influence of dysregulated microbiota led to such results. In parallel with this postulation, the effects of the gut microbiota on IDO activity were reported in Valladares et al. ‘s paper (2013).

Critical Analysis A recent study had indicated that germ-free mice exhibited depressive-like symptoms when received microbiota from individuals with major depressive disorder (Zheng et al. 2016). In this context, the original paper supported the result that microbiota can have casual effects in developing psychiatric disorders.

Zheng et al. (2019) very recently performed FMT from SCZ individuals to germ-free mice and observed SCZ-associated symptoms. In contrast to the present paper, Zheng et al. (2019) reported reduced anxiety behaviours and increased startle response in SCZ mice. As discussed in the original article, the authors suggested the use of germ-free vs. SPF mice in their experiments may account for these discrepancies.

Collecting fecal samples from a small number of individuals and using the same strain and gender of mice for FMT make the results difficult to be generalized. In addition, the authors did not address whether the SCZ individuals that donated their feces were at the early or late stage of illness. This may be important since the gut microbiota may differ throughout the disease and subsequently have different impacts on Trp metabolism, Kyn and Kyna levels. The authors should have divided SCZ mice into two groups and then induced inhibition to the Kyn pathway of Trp metabolism. This would allow them to compare their SCZ-like behaviours and ensure that the effects of altered gut microbiota on the Kyn pathway lead to such results. Furthermore, the authors did only focus on the positive and cognitive symptoms of SCZ mice, while negative symptoms were not examined.

It should be noted that Kyn can readily cross the bloodbrain barrier (BBB) using transporters (Fukui et al. 1991). This process is mediated by the microglia cells (Schwarcz and Pellicciari 2002), and interestingly maturation of these microglia is highly dependent on the gut microbiota (Erny et al. 2015). In addition, the ability of Kyn to cross the BBB may account for the increased brain Kyn, while IDO expression remained unchanged in the brain. Further, Kyn is metabolized in two distinct pathways, resulting in Kyna and 3-HK productions by the enzymes KAT-II and KMO, respectively (Guidetti et al. 2007; Kindler et al.

2019). Downstream, 3-HK leads to the formation of neurotoxic quinolinic acid (QA), which is known to be an N-methyl-daspartate (NMDA) receptor agonist. However, Kyna acts as an antagonist of the NMDA receptor (Schwarcz et al. 2012). Even though Kyna can reduce excitotoxicity effects of QA, increased Kyna level has been associated with impaired cognition in SCZ patients (Huang et al. 2020). Researchers suggested that inhibition of Kyna in the brain could be a potential target to restore cognition in SCZ patients (Wonodi and Schwarcz 2010). In this

It is worth emphasizing that increased Kyna levels are not observed in all cases of SCZ. For instance, in their clinical research, Szymona et al. (2017), reported significantly reduced Kyna in the periphery and elevated 3-HK in individuals with SCZ. Similarly, when Mynt et al. (2011) looked at the Kyn pathway of Trp metabolism in drug-free SCZ vs. medicated SCZ, they report8

ed that Kyna levels decreased, while 3-HK increased in the plas- the gut. It is also important to note that more studies are still ma of SCZ patients. Thus, it is plausible to consider the imbal- needed to replicate the previous results. anced Kyna/3-HK ratio, increased or decreased in either direction as an implication for SCZ development for future investigations.

Future Directions For the first time, the original paper postulated the casual effects of altered gut microbiota on the Kyn pathway of Trp metabolism and developing SCZ (Zhu et al. 2019). Therefore, future studies will be conceivable to delineate the exact mechanisms involved in this process, validate the results, and develop effective treatments for SCZ. Future investigations with larger sample sizes and balanced recruitment of both male and female mice are required for fecal microbiota transplantation. Phases of illness, acute vs. residual, or the disease progression must be taken into consideration when choosing SCZ subjects as donors in the experiment.

To further confirm that the casual links between altered gut microbiota and SCZ pathogenesis occur through the Kyn pathway, researchers should inject NDMA receptor agonists or KAT-II inhibitors to the mice immediately after treatments with microbiota from SCZ individuals. Inhibition of KAT-II would result in decreased brain Kyna and allow researchers to realize if it can improve mice's SCZ-like behaviours compared to HC. In other words, they would then be able to conclude better if altered gut microbiota disrupting the Kyn pathway is responsible for developing such behaviours. If decreased Kyna, as a result of KAT-II inhibition, did not improve SCZ-associated behaviours in mice, it would suggest that the gut microbiota can induce their effects through different pathways than Trp metabolism.

Moreover, the sucrose preference test should be obtained to evaluate the negative symptoms of the mice treated with SCZ microbiota. This would help researchers understand whether the upregulated Kyna induced by altered gut microbiota is only associated with psychotic episodes of SCZ or can also be related to negative symptoms.

Additionally, researchers should measure further downstream Kyn metabolites in SCZ mice, such as QA and Xanthurenic acid (XA), which are known to fluctuate in SCZ patients (Fazio et al. 2015). The results would allow them to understand if altered gut microbiota has an impact on SCZ pathogenies by acting on QA or XA metabolite and may introduce a new target for developing treatments for SCZ. Although it is difficult to determine the healthy composition of microbiota in humans, identification of the gut microbiota in SCZ individuals may have implications as a marker for diagnosis, prognosis, and even treatment response for SCZ. It is not apparent if a particular group of gut microbiota alone contributes to neuropsychiatric disorders, or the presence of certain bacteria with the effects of other microbes together leads to such results. Thus, it is plausible to investigate the patterns of gut microbiota and observe their relations with other bacteria in

9

REFRENCES 1.

Agus, Allison, Julien Planchais, and Harry Sokol. 2018. “Gut Microbiota Regulation of Tryptophan Metabolism in Health and Disease.” Cell Host & Microbe 23 (6): 716–24. https://doi.org/10.1016/j.chom.2018.05.003.

2.

Bravo, Javier A., Paul Forsythe, Marianne V. Chew, Emily Escaravage, Hélène M. Savignac, Timothy G. Dinan, John Bienenstock, and John F. Cryan. 2011. “Ingestion of Lactobacillus Strain Regulates Emotional Behavior and Central GABA Receptor Expression in a Mouse via the Vagus Nerve.” Proceedings of the National Academy of Sciences of the United States of America 108 (38): 16050–55. https:// doi.org/10.1073/pnas.1102999108.

3.

Carlson, Alexander L., Kai Xia, M. Andrea Azcarate-Peril, Barbara D. Goldman, Mihye Ahn, Martin A. Styner, Amanda L. Thompson, Xiujuan Geng, John H. Gilmore, and Rebecca C. Knickmeyer. 2018. “Infant Gut Microbiome Associated with Cognitive Development.” Biological Psychiatry 83 (2): 148–59. https://doi.org/10.1016/j.biopsych.2017.06.021.

4.

Dantzer, Robert. 2017. “Role of the Kynurenine Metabolism Pathway in Inflammation-Induced Depression – Preclinical Approaces.” Current Topics in Behavioral Neurosciences 31: 117–38. https://doi.org/10.1007/7854_2016_6.

5.

Dinan, Timothy G., and John F. Cryan. 2017. “Gut Instincts: Microbiota as a Key Regulator of Brain Development, Ageing and Neurodegeneration.” The Journal of Physiology 595 (2): 489–503. https://doi.org/10.1113/JP273106.

6.

Erhardt, S., K. Blennow, C. Nordin, E. Skogh, L. H. Lindström, and G. Engberg. 2001. “Kynurenic Acid Levels Are Elevated in the Cerebrospinal Fluid of Patients with Schizophrenia.” Neuroscience Letters 313 (1–2): 96–98. https://doi.org/10.1016/s0304-3940(01)02242-x.

7.

Erhardt, S., and G. Engberg. 2002. “Increased Phasic Activity of Dopaminergic Neurones in the Rat Ventral Tegmental Area Following Pharmacologically Elevated Levels of Endogenous Kynurenic Acid.” Acta Physiologica Scandinavica 175 (1): 45–53. https://doi.org/10.1046/ j.1365-201X.2002.00962.x.

8.

Erny, Daniel, Anna Lena Hrabě de Angelis, Diego Jaitin, Peter Wieghofer, Ori Staszewski, Eyal David, Hadas Keren-Shaul, et al. 2015. “Host Microbiota Constantly Control Maturation and Function of Microglia in the CNS.” Nature Neuroscience 18 (7): 965–77. https:// doi.org/10.1038/nn.4030.

9.

Fazio, Francesco, Luana Lionetto, Martina Curto, Luisa Iacovelli, Michele Cavallari, Cristina Zappulla, Martina Ulivieri, et al. 2015.

“Xanthurenic Acid Activates MGlu2/3 Metabotropic Glutamate Receptors and Is a Potential Trait Marker for Schizophrenia.” Scientific Reports 5 (December). https://doi.org/10.1038/srep177

10.

Fukui, Shinsuke, Robert Schwarcz, Stanley I. Rapoport, Yoshiaki Takada, and Quentin R. Smith. 1991. “Blood–Brain Barrier Transport of Kynurenines: Implications for Brain Synthesis and Metabolism.” Journal of Neurochemistry 56 (6): 2007–17. https://doi.org/10.1111/ j.1471-4159.1991.tb03460.x.

11.

Gao, Kan, Chun-long Mu, Aitak Farzi, and Wei-yun Zhu. 2020. “Tryptophan Metabolism: A Link Between the Gut Microbiota and Brain.” Advances in Nutrition 11 (3): 709–23. https://doi.org/10.1093/advances/nmz127.

12.

Guidetti, Paolo, Laura Amori, Michael T. Sapko, Etsuo Okuno, and Robert Schwarcz. 2007. “Mitochondrial Aspartate Aminotransferase: A Third Kynurenate-Producing Enzyme in the Mammalian Brain.” Journal of Neurochemistry 102 (1): 103–11. https://doi.org/10.1111/ j.1471-4159.2007.04556.x.

13.

Heijtz, Rochellys Diaz, Shugui Wang, Farhana Anuar, Yu Qian, Britta Björkholm, Annika Samuelsson, Martin L. Hibberd, Hans Forssberg, and Sven Pettersson. 2011. “Normal Gut Microbiota Modulates Brain Development and Behavior.” Proceedings of the National Academy of Sciences of the United States of America 108 (7): 3047–52. https://doi.org/10.1073/pnas.1010529108.

14.

Höglund, Erik, Øyvind Øverli, and Svante Winberg. 2019. “Tryptophan Metabolic Pathways and Brain Serotonergic Activity: A Comparative Review.” Frontiers in Endocrinology 10. https://doi.org/10.3389/fendo.2019.00158.

Huang, Xingbing, Wenhua Ding, Fengchun Wu, Sumiao Zhou, Shuhua Deng, and Yuping Ning. 2020. “Increased Plasma Kynurenic Acid Levels Are Associated with Impaired Attention/Vigilance and Social Cognition in Patients with Schizophrenia.” Neuropsychiatric Disease and Treatment 16 (January): 263–71. https://doi.org/10.2147/NDT.S239763.

15.

16.

17.

Kahn, René S., Iris E. Sommer, Robin M. Murray, Andreas Meyer-Lindenberg, Daniel R. Weinberger, Tyrone D. Cannon, Michael O’Donovan, et al. 2015. “Schizophrenia.” Nature Reviews Disease Primers 1 (1): 1–23. https://doi.org/10.1038/nrdp.2015.67.

Kindler, Jochen, Chai K. Lim, Cynthia Shannon Weickert, Danny Boerrigter, Cherrie Galletly, Dennis Liu, Kelly R. Jacobs, et al. 2019.

“Dysregulation of Kynurenine Metabolism Is Related to Proinflammatory Cytokines, Attention, and Prefrontal Cortex Volume in Schizophrenia.” Molecular Psychiatry, April, 1–13. https://doi.org/10.1038/s41380-019-0401-9.

10

16.

Kozak, Rouba, Brian M. Campbell, Christine A. Strick, Weldon Horner, William E. Hoffmann, Tamas Kiss, Douglas S. Chapin, et al. 2014.

“Reduction of Brain Kynurenic Acid Improves Cognitive Function.” The Journal of Neuroscience 34 (32): 10592–602. https:// doi.org/10.1523/JNEUROSCI.1107-14.2014.

17.

Lamas, Bruno, Mathias L. Richard, Valentin Leducq, Hang-Phuong Pham, Marie-Laure Michel, Gregory Da Costa, Chantal Bridonneau, et al. 2016. “CARD9 Impacts Colitis by Altering Gut Microbiota Metabolism of Tryptophan into Aryl Hydrocarbon Receptor Ligands.” Nature Medicine 22 (6): 598–605. https://doi.org/10.1038/nm.4102.

18.

Miller, Christine L, Ida C Llenos, Jeanette R Dulay, Meliza M Barillo, Robert H Yolken, and Serge Weis. 2004. “Expression of the Kynurenine Pathway Enzyme Tryptophan 2,3-Dioxygenase Is Increased in the Frontal Cortex of Individuals with Schizophrenia.” Neurobiology of Disease 15 (3): 618–29. https://doi.org/10.1016/j.nbd.2003.12.015.

19.

Miller, Brian J., Peter Buckley, Wesley Seabolt, Andrew Mellor, and Brian Kirkpatrick. 2011. “Meta-Analysis of Cytokine Alterations in Schizophrenia: Clinical Status and Antipsychotic Effects.” Biological Psychiatry 70 (7): 663–71. https://doi.org/10.1016/ j.biopsych.2011.04.013.

20.

Müller, Norbert. 2018. “Inflammation in Schizophrenia: Pathogenetic Aspects and Therapeutic Considerations.” Schizophrenia Bulletin 44 (5): 973–82. https://doi.org/10.1093/schbul/sby024.

21.

Myint, A. M., M. J. Schwarz, R. Verkerk, H. H. Mueller, J. Zach, S. Scharpé, H. W. M. Steinbusch, B. E. Leonard, and Y. K. Kim. 2011.

“Reversal of Imbalance between Kynurenic Acid and 3-Hydroxykynurenine by Antipsychotics in Medication-Naïve and Medication-Free Schizophrenic Patients.” Brain, Behavior, and Immunity 25 (8): 1576–81. https://doi.org/10.1016/j.bbi.2011.05.005.

Nguyen, Tanya T., Tomasz Kosciolek, Yadira Maldonado, Rebecca E. Daly, Averria Sirkin Martin, Daniel McDonald, Rob Knight, and Dilip V. Jeste. 2019. “Differences in Gut Microbiome Composition Between Persons with Chronic Schizophrenia and Healthy Comparison Subjects.” Schizophrenia Research 204 (February): 23–29. https://doi.org/10.1016/j.schres.2018.09.014.

22.

23.

O’Connor, Jason C., Caroline André, Yunxia Wang, Marcus A. Lawson, Sandra S. Szegedi, Jacques Lestage, Nathalie Castanon, Keith W. Kelley, and Robert Dantzer. 2009. “Interferon-γ and Tumor Necrosis Factor-α Mediate the Upregulation of Indoleamine 2,3-Dioxygenase and the Induction of Depressive-Like Behavior in Mice in Response to Bacillus Calmette-Guérin.” The Journal of Neuroscience 29 (13): 4200–4209. https://doi.org/10.1523/JNEUROSCI.5032-08.2009.

24.

Olney, J. W., and N. B. Farber. 1995. “Glutamate Receptor Dysfunction and Schizophrenia.” Archives of General Psychiatry 52 (12): 998– 1007. https://doi.org/10.1001/archpsyc.1995.03950240016004.

25.

Pedraz-Petrozzi, Bruno, Osama Elyamany, Christoph Rummel, and Christoph Mulert. 2020. “Effects of Inflammation on the Kynurenine Pathway in Schizophrenia — a Systematic Review.” Journal of Neuroinflammation 17 (1): 56. https://doi.org/10.1186/s12974-020-1721-z.

26.

Petra, Anastasia I., Smaro Panagiotidou, Erifili Hatziagelaki, Julia M. Stewart, Pio Conti, and Theoharis C. Theoharides. 2015. “GutMicrobiota-Brain Axis and Its Effect on Neuropsychiatric Disorders With Suspected Immune Dysregulation.” Clinical Therapeutics 37 (5): 984–95. https://doi.org/10.1016/j.clinthera.2015.04.002.

27.

Reigstad, Christopher S., Charles E. Salmonson, John F. Rainey Iii, Joseph H. Szurszewski, David R. Linden, Justin L. Sonnenburg, Gianrico Farrugia, and Purna C. Kashyap. 2015. “Gut Microbes Promote Colonic Serotonin Production through an Effect of Short-Chain Fatty Acids on Enterochromaffin Cells.” The FASEB Journal 29 (4): 1395–1403. https://doi.org/10.1096/fj.14-259598.

28.

Schwarcz, Robert, Arash Rassoulpour, Hui-Qiu Wu, Deborah Medoff, Carol A Tamminga, and Rosalinda C Roberts. 2001. “Increased Cortical Kynurenate Content in Schizophrenia.” Biological Psychiatry 50 (7): 521–30. https://doi.org/10.1016/S0006-3223(01)01078-2.

Schwarcz, Robert, and Roberto Pellicciari. 2002. “Manipulation of Brain Kynurenines: Glial Targets, Neuronal Effects, and Clinical Opportunities.” The Journal of Pharmacology and Experimental Therapeutics 303 (1): 110. https://doi.org/10.1124/jpet.102.034439.

Schwarcz, Robert, John P. Bruno, Paul J. Muchowski, and Hui-Qiu Wu. 2012. “Kynurenines in the Mammalian Brain: When Physiology Meets Pathology.” Nature Reviews Neuroscience 13 (7): 465–77. https://doi.org/10.1038/nrn3257.

29.

30.

31.

Sharon, Gil, Timothy R. Sampson, Daniel H. Geschwind, and Sarkis K. Mazmanian. 2016. “The Central Nervous System and the Gut Microbiome.” Cell 167 (4): 915–32. https://doi.org/10.1016/j.cell.2016.10.027.

32.

Shen, Yang, Jintian Xu, Zhiyong Li, Yichen Huang, Ye Yuan, Jixiang Wang, Meng Zhang, Songnian Hu, and Ying Liang. 2018. “Analysis of Gut Microbiota Diversity and Auxiliary Diagnosis as a Biomarker in Patients with Schizophrenia: A Cross-Sectional Study.” Schizophrenia Research 197 (July): 470–77. https://doi.org/10.1016/j.schres.2018.01.002.

11

33.

Silva, Ygor Parladore, Andressa Bernardi, and Rudimar Luiz Frozza. 2020. “The Role of Short-Chain Fatty Acids From Gut Microbiota in GutBrain Communication.” Frontiers in Endocrinology 11 (January). https://doi.org/10.3389/fendo.2020.00025.

34.

Sudo, Nobuyuki, Yoichi Chida, Yuji Aiba, Junko Sonoda, Naomi Oyama, Xiao-Nian Yu, Chiharu Kubo, and Yasuhiro Koga. 2004. “Postnatal Microbial Colonization Programs the Hypothalamic–Pituitary–Adrenal System for Stress Response in Mice.” The Journal of Physiology 558 (Pt 1): 263–75. https://doi.org/10.1113/jphysiol.2004.063388.

35.

Sullivan, Patrick F., Kenneth S. Kendler, and Michael C. Neale. 2003. “Schizophrenia as a Complex Trait: Evidence From a Meta-Analysis of Twin Studies.” Archives of General Psychiatry 60 (12): 1187. https://doi.org/10.1001/archpsyc.60.12.1187.

36.

Szymona, Kinga, Barbara Zdzisińska, Hanna Karakuła-Juchnowicz, Tomasz Kocki, Martyna Kandefer-Szerszeń, Marta Flis, Wojciech Rosa, and Ewa M. Urbańska. 2017. “Correlations of Kynurenic Acid, 3-Hydroxykynurenine, SIL-2R, IFN-α, and IL-4 with Clinical Symptoms During Acute Relapse of Schizophrenia.” Neurotoxicity Research 32 (1): 17–26. https://doi.org/10.1007/s12640-017-9714-0.

37.

Thomas, S. R., and R. Stocker. 1999. “Redox Reactions Related to Indoleamine 2,3-Dioxygenase and Tryptophan Metabolism along the Kynurenine Pathway.” Redox Report 4 (5): 199–220. https://doi.org/10.1179/135100099101534927.

38.

Turnbaugh, Peter J., Ruth E. Ley, Micah Hamady, Claire M. Fraser-Liggett, Rob Knight, and Jeffrey I. Gordon. 2007. “The Human Microbiome Project.” Nature 449 (7164): 804–10. https://doi.org/10.1038/nature06244.

39.

Valladares, Ricardo, Lora Bojilova, Anastasia H. Potts, Evan Cameron, Christopher Gardner, Graciela Lorca, and Claudio F. Gonzalez. 2013.

“Lactobacillus Johnsonii Inhibits Indoleamine 2,3-Dioxygenase and Alters Tryptophan Metabolite Levels in BioBreeding Rats.” The FASEB Journal 27 (4): 1711–20. https://doi.org/10.1096/fj.12-223339.

40.

Wen, Li, and Andrew Duffy. 2017. “Factors Influencing the Gut Microbiota, Inflammation, and Type 2 Diabetes.” The Journal of Nutrition 147 (7): 1468S-1475S. https://doi.org/10.3945/jn.116.2407

41.

Winship, Ian R., Serdar M. Dursun, Glen B. Baker, Priscila A. Balista, Ludmyla Kandratavicius, Joao Paulo Maia-de-Oliveira, Jaime Hallak, and John G. Howland. 2019. “An Overview of Animal Models Related to Schizophrenia.” Canadian Journal of Psychiatry. Revue Canadienne de Psychiatrie 64 (1): 5–17. https://doi.org/10.1177/0706743718773728.

42.

Wonodi, Ikwunga, and Robert Schwarcz. 2010. “Cortical Kynurenine Pathway Metabolism: A Novel Target for Cognitive Enhancement in Schizophrenia.” Schizophrenia Bulletin 36 (2): 211–18. https://doi.org/10.1093/schbul/sbq002.

43.

Yano, Jessica M., Kristie Yu, Gregory P. Donaldson, Gauri G. Shastri, Phoebe Ann, Liang Ma, Cathryn R. Nagler, Rustem F. Ismagilov, Sarkis K. Mazmanian, and Elaine Y. Hsiao. 2015. “Indigenous Bacteria from the Gut Microbiota Regulate Host Serotonin Biosynthesis.” Cell 161 (2): 264–76. https://doi.org/10.1016/j.cell.2015.02.047.

44.

Zheng, P., B. Zeng, C. Zhou, M. Liu, Z. Fang, X. Xu, L. Zeng, et al. 2016. “Gut Microbiome Remodeling Induces Depressive-like Behaviors through a Pathway Mediated by the Host’s Metabolism.” Molecular Psychiatry 21 (6): 786–96. https://doi.org/10.1038/mp.2016.44.

45.

Zheng, Peng, Benhua Zeng, Meiling Liu, Jianjun Chen, Junxi Pan, Yu Han, Yiyun Liu, et al. 2019. “The Gut Microbiome from Patients with Schizophrenia Modulates the Glutamate-Glutamine-GABA Cycle and Schizophrenia-Relevant Behaviors in Mice.” Science Advances 5 (2). https://doi.org/10.1126/sciadv.aau8317.

46.

Zhu, Feng, Ruijin Guo, Wei Wang, Yanmei Ju, Qi Wang, Qingyan Ma, Qiang Sun, et al. 2019. “Transplantation of Microbiota from DrugFree Patients with Schizophrenia Causes Schizophrenia-like Abnormal Behaviors and Dysregulated Kynurenine Metabolism in Mice.” Molecular Psychiatry, August, 1–14. https://doi.org/10.1038/s41380-019-0475-4

12

Fecal microbiota transplantation as a defence against MPTPinduced Parkinson’s disease mice Farzad Ahmadi

Parkinson’s disease (PD) patients show consistent dysbiosis of the gut microbiota providing evidence supporting a gut-brain axis. However, the mechanisms by which the gut microbiome influences the pathogenesis of PD is still unknown. Further, current therapies only treat symptoms, and none stop or slow the progression of PD due to a limited understanding of PD’s major molecular markers. The paper by Sun et. Al. (2018) investigates the involvement of the gut microbiota in PD progression and the neuroprotective effects of fecal microbiota transplantation (FMT) within PD mice. FMT is an effective method whereby the feces of one murine is infused into the GI tract of another, essentially causing a complete intervention of the microbial. FMT from α-Syn overexpressing mice into germ-free mice induced motor dysfunction and reduced neurotransmitter release within the germ-free mice showing the ability of the microbiome to induce PD. To test the neuroprotective effects of FMT, the gut microbiota from phosphate-buffered saline (PBS) treated mice were infused into MPTPinduced PD mice. FMT reduced microbiota dysbiosis, increased serotonin and dopamine release within the striatum, decreased fecal short-chain fatty acids (SCFAs) and rescued motor impairments within the PD mice. Microglia and astrocyte activation were also reduced within the substantia nigra showing a decrease in neuroinflammatory precursors. Since gut microbial dysbiosis shows consistent involvement in PD, future studies should aim at understanding the mechanism underlying this association and other neurological disorders, and targeting the microbiota as a possibly therapy for PD.

13

Background and Introduction

flammation of the intestinal epithelial cells are key factors in the pathogenesis of PD which led Dr. Sun’s group to investigate the Parkinson’s disease (PD) is a very common neurodegenerative neuroprotective effects of FMT. disorder resulting primarily from the loss of dopaminergic neurons in the substantia nigra (Dauer and Przedborski 2003). The risk of PD increases with age affecting approximately 1% of people over 60 (Jackson et al. 2019). The misfolding and aggregation Major Results of the protein, alpha-synuclein (α-Syn), into Lewy Bodies within A group of 8-week-old wild-type C57BL/6 mice were exposed to these dopaminergic neurons along with mitochondrial dysfunc- MPTP injections for 5 consecutive days to induce PD-like symption is theorized to cause apoptosis through a variety of mecha- toms while the other group served as a control. The MPTPnisms (Haikal, Chen, and Li 2019; Dauer and Przedborski 2003). induced PD mice were then introduced to FMT, from a healthy The characteristic motor symptoms such as bradykinesia and donor (wild-type mice), for 7 consecutive days via gavage or resting tremor are commonly preceded by dysfunction of the GI were given phosphate-buffered-saline (PBS) for 7 consecutive system such as constipation and weight loss, provoking re- days to act as the PD control for FMT. searchers to investigate the gut-microbiota-brain axis (Goldstein, Sewell, and Sharabi 2011). PD has been established FMT alleviates motor impairments in PD mice as a multifactorial disorder since studies show that the mono- Physical impairments such as bradykinesia, postural defects, and genic mutations account, only, for approximately 5-10% of PD resting tremor are hallmarks of PD. These impairments were cases leaving the remainder to be caused by complex combina- measured through a pole test, measuring the time it takes to tion between genetic and environmental factors (Lill 2016).The descent from the tip of the pole to the base, and a traction test, first autosomal dominant mutation to cause PD was discovered scoring the mice based on their ability to attach onto the to be in the SNCA gene responsible for the production and ex- platform with their hind limbs. The MPTP + PBS mice showed pression of α-Syn, however advances have been made and many significant motor dysfunction such as longer descent times and mutations have been identified (Lill 2016). an inability to use their hind limbs to attach resembling a lower Many studies have found evidence supporting the involvement traction score. Remarkably, the MPTP + FMT mice showed a of the gut microbiome in neuronal function and disease (Unger significant increase in motor function such as decreases in deet al. 2016; Jackson et al. 2019). Furthermore, a high-fiber Medi- scent time and higher traction scores. This data provides eviterranean diet has been shown to decrease PD risk as a result of dence supporting FMT as a viable method to rescue motor imchanges in the concertration of relative short-chain-fatty acid pairments in PD mice.

(SCFA) producing and lipopolysaccharide (LPS) containing bacteria as opposed to a high-fat, high-sugar Western diet (Jackson et al. 2019; Cani et al. 2008). This delicate balance within the gut is theorized to contribute to PD pathogenesis through the complex innervations between the vagus nerve, the enteric nervous system (ENS) and the GI tract (Braak et al. 2003). It has been proposed that a substance with prion-like properties, possibly αSyn, that moves from the GI tract to the CNS through these pathways and induce cell death (Vendrik et al. 2020). Recent studies have shown that chronic stress is a major contributor to the progression of PD through the dysfunction of the intestinal barrier leading to hyper-permeability and pro-inflammatory environment due to a decrease in anti-inflammatory bacteria (Lactobacillus) within the microbiota (Dodiya et al. 2020; Forsyth et al. 2011). Healthy intestinal epithelial cells produce low levels of TLR4; thereby, hyperpermeability will induce the production of pro-inflammatory substances such as TNF-α through increases in TLR4 which will not only cause peripheral immune activation leading to gut inflammation but can cross the blood brain barrier to cause neuroinflammation via microglia activation (Qin et al. 2007; Ransohoff 2016).

Fecal microbiota transplantation (FMT) is an effective technique used to transplant the fecal matter from a healthy donor to the GI tract of a patient. FMT is investigated as a treatment for a wide variety of disorders including neurological diseases such as PD, autism, multiple sclerosis and many others (Vendrik et al.

2020). These innovative discoveries show that the dysbiosis of the gut microbiome along with the hyper-permeability and in-

Figure 1. FMT improves motor function within PD mice. A) The

MPTP-induced PD mice which have undergone FMT resemble the normal controls and have significantly lower descent times.

B) The MPTP + FMT mice show significantly higher traction scores like those of the normal control group (Sun et al. 2018) Rescued dopamine and serotonin in PD mice In order to examine the neuroprotective effects of FMT within the brain, the concentration of neurotransmitters present along with their metabolites were examined within the striatum. Fluorescence detection was used after high performance liquid chromatography was performed. FMT rescued neurotransmitter release within the striatum as observed by their relative concentrations within figure 2. This shows that FMT not only alleviates the symptoms of PD but slows and influences the progression of PD by rescuing the neurotransmitter levels that are normally decreased within the PD brains.

14

Figure 3. The microbiome is responsible for the concentrations of SCFAs present and in turn these SCFAs can activate microglia and induce neuroinflammation and pathology.

Figure 4. The relative number of activated microglia and astrocytes within the substantia nigra (SN) are reduced significantly by FMT treatment as opposed to MPTP+PBS mice. (Sun et al.

2018)

Figure 2. FMT rescues the loss of striatal neurotransmitters mediated by MPTP. An overall increase in neurotransmitter and metabolite levels are measured within the MPTP+FMT group through fluorescence detection. (Sun et al. 2018)

FMT reduces microglial activation, neuroinflammation, and restore normal SCFAs MPTP induced PD causes dysbiosis of the microbiome resulting in alterations in the concentrations of SCFAs responsible for increased activation of microglia and inflammation. FMT is shown to decrease all SCFAs (acetic acid, propionic acid, butyric acid, and n-valeric acid) concentrations as opposed to MPTP+PBS group resembling the control group. Further, IF staining was used to examine the interactions between microglia, astrocytes and dopaminergic neurons. FMT significantly reduced the activation of astrocytes and microglia within the substantia nigra. This data suggests that through a restoration of normal SCFA concentrations, microglia and astrocyte activation were significantly reduced and thereby, so was the neuroinflammation

Discussions This paper investigates the neuroprotective effects of FMT on MPTP-induced PD mice. They have shown that FMT rescues striatal neurotransmitter levels and restores dopaminergic neurons within the substantia nigra. Furthermore, they have shown that FMT increases motor function and improves bradykinesia, muscle weakness, and postural instability. FMT reduces gut microbial dysbiosis and restores SCFA concentrations which contribute to reduced activation of microglia and astrocytes resulting in less neuroinflammation. The authors shown that the gut microbial dysbiosis within PD mice is consistent with dysbiosis observed in human PD patients suggesting that this PD model can be used in further research to investigate a possible therapy and provide improved understanding of PD pathology.

These results are relevant as they provide evidence for the neuroprotective effects of a healthy gut microbiome and FMT in PD mice. They build on evidence provided previously about the gut -brain axis and the influence of the gut microbiome on neurological health and disease (Sampson et al. 2016; Haikal, Chen, and Li 2019; Hill�Burns et al. 2017; Unger et al. 2016). It has been known that there is bidirectional communication between the CNS and the gut microbiome through the vagus nerve and ENS. Interestingly, this study shows that FMT can reverse, not only the symptoms of PD, but restore the neurotransmitter levels within the striatum. Furthermore, since GI symptoms precede other symptoms, it is suggested that the pathogen that eventually causes PD moves into the GI tract first then into the CNS (Dauer and Przedborski 2003). This pathogen alongside

15

v other possible factors such as chronic stress, promote a hyperpermeability of the intestinal epithelial barrier and promote a pro-inflammatory environment. The authors suggest this is due to the TLR4/TNF-α pathway as the damaged epithelial cells increase TLR4 activation which causes neuroinflammation through upregulation of other inflammatory factors such as TNF- α and cytokine secretion. They showed that FMT decreased the expression of this pathway and thereby, reduced gut and neuroinflammation. However, the authors acknowledge that this is only a possibility and there are many other pathways by which signalling can occur. Essentially, the gut microbiome is a very complex system that has many implications on the CNS and neurological disorders. The ability of FMT to rescue almost all PD symptoms and restore near normal brain status is remarkable and must be further studied.

Critical Analysis This paper provides a strong foundation for future studies examining the role of the gut microbiota in neurological diseases.

Since this study, along with many others, have shown that microbial dysbiosis leads to neuroinflammation and neurological disorders, future studies should investigate the methods by which the microbial balance becomes disturbed and the effects of diet and other environmental factors on this. The known genetic mutations only account for 10% of the PD cases therefore there must be a multitude of environmental factors that must account for the other 90% (Lill 2016). Therefore, future studies must investigate the effects of diet, stress, environmental conditions, viral effects as causes of microbial dysbiosis and PD pathology. Also, future studies must investigate more accurate molecular markers that can be used in tandem with microbial dysbiosis as an effective and accurate diagnostic tool for PD. The experiments and controls used in this study are well established. The neurotoxin MPTP is used to induce PD and they included controls to test for the possible effects of PBS. They found consistent evidence with other papers regarding the effects of the gut microbiome on PD (Dauer and Przedborski 2003; Sampson et al. 2016). There have been no contradictory studies published showing negative neurological effects following FMT in PD mice. Therefore, there is a well-established link between the gut microbiome and the brain and dysbiosis and neurological disorders. However, the mechanisms by which microbial dysbiosis influences PD pathology remains unclear and must be investigated further. More research must be conducted on the reasons why dysbiosis occurs (e.g. external pathogen, stress, diet, etc.) and how this dysbiosis contributes to αSyn aggregation within the CNS. The TLR4/TNF-α pathway proposed by the authors based on previous papers and this study, must be further investigated to examine if it has a direct role in PD pathogenesis. Interestingly, dysbiosis of the gut microbiome and alterations of SCFA concentrations are subjective as many papers provide a variety of findings, and this makes the search for possibly therapies and mechanisms more difficult.

Future Directions A future study should investigate the TLR4/TNF- α pathway as the primary pathway regulating neuroinflammation and microglial activation. This pathway consists of an overexpression of TLR4 which through a descending cascade of protein activation induces TNF- α activation which leads to cytokine release and inflammation via transcriptional activators. This study provided evidence that FMT reduces gut and neuroinflammation possibly through a reduction of this pathway, but the evidence is unclear. The TLR4 are commonly found in low concentrations on intestinal epithelial cells. Therefore, an artificial method of causing damage to the epithelial cells (e.g. MPTP) must be used in tandem with a TLR4-knock out (KO) mice strain to observe if the TLR4/TNF- α pathway is the main mechanism responsible for the neuroinflammation and PD pathogenesis following microbial dysbiosis.

The researcher must include two groups, one consisting of TLR4 -KO mice and the other must be wild-type C57BL/6 mice. Each of the mice strains must be divided into two groups and one will undergo i.p. injection of MPTP for 5 consecutive days in order to produce MPTP-induced PD mice. After the injection has occurred, behavioural tests such as the pole descent test and traction test must be done followed by tests measuring for neuroinflammatory molecular markers such as TNF- α, Iba-1 (microglial marker), and GFAP (astrocyte marker). If this pathway plays an important role in mediating neuroinflammation and PD pathogenesis, the following results should be expected.

The TLR4-KO + MPTP mice must show short descent times and high traction scores similar to the TLR4-KO and the wild-type C57BL/6 mice. The only mice strain showing PD-like bradykinesia and movement impairment should be the C57BL/6 mice.

Furthermore, once testing for neuroinflammatory markers, the levels within the TLR4-KO + MPTP mice must be similar to those of both controls as TLR4 is not present to initiate the TLR4/TNFα pathway and cause neuroinflammation. However, there should be gut inflammation present and hyper-permeability of the intestinal epithelial cells possibly through sigmoidoscopy.

Furthermore, if this pathway is not the main pathway responsible for the neuroinflammation following microbial dysbiosis, then the TLR4-KO + MPTP mice test results should mimic those of the C56BL/6 + MPTP mice as TLR4 would have no significant effects on PD pathogenesis. If this pathway is determined to be the major pathway contributing to neuroinflammation and PD pathogenesis, then further studies must be conducted investigating possible methods of suppressing the pathway and therapies targeting this pathway.

16

REFRENCES 1.

Braak, H., U. Rüb, W. P. Gai, and K. Del Tredici. 2003. “Idiopathic Parkinson’s Disease: Possible Routes by Which Vulnerable Neuronal Types May Be Subject to Neuroinvasion by an Unknown Pathogen.” Journal of Neural Transmission 110 (5): 517–36. https:// doi.org/10.1007/s00702-002-0808-2.

2.

Cani, Patrice D., Rodrigo Bibiloni, Claude Knauf, Aurélie Waget, Audrey M. Neyrinck, Nathalie M. Delzenne, and Rémy Burcelin. 2008.

“Changes in Gut Microbiota Control Metabolic Endotoxemia-Induced Inflammation in High-Fat Diet–Induced Obesity and Diabetes in Mice.” Diabetes 57 (6): 1470–81. https://doi.org/10.2337/db07-1403.

3.

Dauer, William, and Serge Przedborski. 2003. “Parkinson’s Disease: Mechanisms and Models.” Neuron 39 (6): 889–909. https:// doi.org/10.1016/S0896-6273(03)00568-3.

4.

Dodiya, Hemraj B., Christopher B. Forsyth, Robin M. Voigt, Phillip A. Engen, Jinal Patel, Maliha Shaikh, Stefan J. Green, et al. 2020. “Chronic Stress-Induced Gut Dysfunction Exacerbates Parkinson’s Disease Phenotype and Pathology in a Rotenone-Induced Mouse Model of Parkinson’s Disease.” Neurobiology of Disease 135 (Complete). https://doi.org/10.1016/j.nbd.2018.12.012.

5.

Forsyth, Christopher B., Kathleen M. Shannon, Jeffrey H. Kordower, Robin M. Voigt, Maliha Shaikh, Jean A. Jaglin, Jacob D. Estes, Hemraj B. Dodiya, and Ali Keshavarzian. 2011. “Increased Intestinal Permeability Correlates with Sigmoid Mucosa Alpha-Synuclein Staining and Endotoxin Exposure Markers in Early Parkinson’s Disease.” PLOS ONE 6 (12): e28032. https://doi.org/10.1371/journal.pone.0028032.

6.

Goldstein, David S., LaToya Sewell, and Yehonatan Sharabi. 2011. “Autonomic Dysfunction in PD: A Window to Early Detection?” Journal of the Neurological Sciences, Seventh Congress of Mental and other Non-motor Dysfunctions in Parkinson’s Disease, 310 (1): 118–22. https://doi.org/10.1016/j.jns.2011.04.011.

7.

Haikal, Caroline, Qian-Qian Chen, and Jia-Yi Li. 2019. “Microbiome Changes: An Indicator of Parkinson’s Disease?” Translational Neurodegeneration 8. https://doi.org/10.1186/s40035-019-0175-7.

8.

Hill‐Burns, Erin M., Justine W. Debelius, James T. Morton, William T. Wissemann, Matthew R. Lewis, Zachary D. Wallen, Shyamal D. Peddada, et al. 2017. “Parkinson’s Disease and Parkinson’s Disease Medications Have Distinct Signatures of the Gut Microbiome.” Movement Disorders 32 (5): 739–49. https://doi.org/10.1002/mds.26942.

9.

Jackson, Aeja, Christopher B. Forsyth, Maliha Shaikh, Robin M. Voigt, Phillip A. Engen, Vivian Ramirez, and Ali Keshavarzian. 2019. “Diet in Parkinson’s Disease: Critical Role for the Microbiome.” Frontiers in Neurology 10. https://doi.org/10.3389/fneur.2019.01245.

10.

Lill, Christina M. 2016. “Genetics of Parkinson’s Disease.” Molecular and Cellular Probes, Genetics of multifactorial diseases, 30 (6): 386– 96. https://doi.org/10.1016/j.mcp.2016.11.001.

11.

Qin, Liya, Xuefei Wu, Michelle L. Block, Yuxin Liu, George R. Breese, Jau-Shyong Hong, Darin J. Knapp, and Fulton T. Crews. 2007. “Systemic LPS Causes Chronic Neuroinflammation and Progressive Neurodegeneration.” Glia 55 (5): 453–62. https://doi.org/10.1002/glia.20467.

12.

Ransohoff, Richard M. 2016. “How Neuroinflammation Contributes to Neurodegeneration.” Science 353 (6301): 777–83. https:// doi.org/10.1126/science.aag2590.

13.

Sampson, Timothy R., Justine W. Debelius, Taren Thron, Stefan Janssen, Gauri G. Shastri, Zehra Esra Ilhan, Collin Challis, et al. 2016. “Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson’s Disease.” Cell 167 (6): 1469-1480.e12. https:// doi.org/10.1016/j.cell.2016.11.018.

14.

Sun, Meng-Fei, Ying-Li Zhu, Zhi-Lan Zhou, Xue-Bing Jia, Yi-Da Xu, Qin Yang, Chun Cui, and Yan-Qin Shen. 2018. “Neuroprotective Effects of Fecal Microbiota Transplantation on MPTP-Induced Parkinson’s Disease Mice: Gut Microbiota, Glial Reaction and TLR4/TNF-α Signaling Pathway.” Brain, Behavior, and Immunity 70 (May): 48–60. https://doi.org/10.1016/j.bbi.2018.02.005.

15.

Unger, Marcus M., Jörg Spiegel, Klaus-Ulrich Dillmann, David Grundmann, Hannah Philippeit, Jan Bürmann, Klaus Faßbender, Andreas Schwiertz, and Karl-Herbert Schäfer. 2016. “Short Chain Fatty Acids and Gut Microbiota Differ between Patients with Parkinson’s Disease and Age-Matched Controls.” Parkinsonism & Related Disorders 32 (November): 66–72. https://doi.org/10.1016/j.parkreldis.2016.08.019.

16.

Vendrik, Karuna E. W., Rogier E. Ooijevaar, Pieter R. C. de Jong, Jon D. Laman, Bob W. van Oosten, Jacobus J. van Hilten, Quinten R. Ducarmon, Josbert J. Keller, Eduard J. Kuijper, and Maria Fiorella Contarino. 2020. “Fecal Microbiota Transplantation in Neurological Disorders.”

Frontiers in Cellular and Infection Microbiology 10. https://doi.org/10.3389/fcimb.2020.00098.

17

Biophysical Modeling of Nonlinear Dendritic Computations Akshan Bansal

Nonlinear dendritic integration is crucial for the optimal processing of correlated inputs, may play a role in the location of dendritic clusters, and can be modeled biophysically. Linear integration is necessary for the processing of uncorrelated inputs where presynaptic neurons and postsynaptic dendrites are at a distance. The study make advancements in the field by expanding the study of dendritic computation to large scale networks using in vivo and in vitro statistics and produces high resolution results. Further studies need to involve optogenetic tools, and vary controls from spontaneous network activity to nonnaturalistic activity across single stimulus dimensions. Understanding of neural mechanisms related to processing can in the future be applied to neuromorphic hardware and machine learning for greater efficiency in processing.

18

Background and Introduction

tions, an optimal response dictated by dendritic nonlinearities can be arrived at. The optimal response was described as:

The computational ability of the human brain has previously been attributed to multi-layer neuronal networks capable of performing mathematical calculations individuals neurons could not, where neurons acted as simple point-like switches which merely summed up incoming potentials and upon exceeding a threshold, fired action potentials, (London and Hausser, 2020). Models that did view single neuron systems as a computational entity through the use of logic gates were highly restricted due to simplistic input measures, the lack of consideration given to the different internal structures of a neuron, and the lack of tools available to record activity along the various components of a dendrites, (Li, 2019). The finding that voltage signals decreased in magnitude whilst propagating along the axon pointed neuroscientists towards the idea that signals are compartmentalized and may be processed independently within the neuron, (Koch, 1982). This countered the long established view in computational neuroscience that single artificial neurons could not perform a nonlinear operation known as a XOR function, (Minsky and Papert, 1969). Although, developments in the field have led to plausible biophysical mechanisms modelling not only the ability of single neurons to act as a two-layer neural network but also the ability of dendrites to perform XOR and a host of other operations, it is unknown how dendritic nonlinearities contribute to computations at the level of the neural circuit, (Poirazi, 2003). Patterned stimulation of cortical pyramidal cells from the neocortex and hippocampus using two-photon glutamate uncaging and electrophysiology was executed to obtain in-vitro postsynaptic activity patterns consistent with a biophysical model that accounts for spatio-temporal variation and is built upon naturalistic in-vivo statistics. This depicted that the optimal response of a dendritic nonlinearity is dictated by presynaptic population statistics, that these nonlinearities are essential for the efficient integration of signals in neural circuits with analog computation, and the necessity of NMDA receptor activation for optimal dendritic integration. The associated novel biophysical model not only adds to the list of computational operations a neuron and its dendritic tree can perform but also draws light to the matter of single neuron systems and subsystems and their influence at the level of the neocortical circuit. This shift in the way we think about the processing power of the human brain will influence the representation of neuronal structures and the organization of information in machine learning, hebbian plasticity, and memory, (Richards, 2019).

Figure 1.0: Linear versus optimal response https://doi.org/10.7554/eLife.10056.010 Statistical models derived presynaptic statistics in line with in vitro and in vivo multielectrode readings of neuronal cortical population after which comparison between the linear response and the optimal response dictated by the aforementioned equation revealed that linear integration was optimal for uncorrelated inputs (A-C), and nonlinear integration was optimal for correlated inputs (D-F).

Figure 2.0: A simple model of nonlinear dendritic integration can approximate the optimal response https://doi.org/10.7554/eLife.10056.012

A simple, biophysically-motivated, canonical model of nonlinear dendritic integration was shown to closely approximate the optimal response when the majority of presynaptic neurons switch between a quiescent state and an active state. This model involves the linear integration of information within a branch, its conversion through a sigmoidal nonlinearity, and the Major Results Section ultimate formation of the local dendritic response. The sigmoidal nonlinearity model was found to be valid across supralinear Correlated information sources require nonlinear integration of and sublinear integration. presynaptic somatic membrane potentials due to the arisal of a bottleneck in circuit computations where the relationship between the presynaptic and postsynaptic neurons is dictated by analog variables as seen in cortical coding, (Cepelewicz, 2020; Clark and Hausser, 2006). By making this assumption about the arithmetic being performed by the postsynaptic cell, and restricting presynaptic values by in vivo data from cortical popula19

Figure 3.0: Nonlinear dendritic integration and presynaptic input statistics https://doi.org/10.7554/elife.10056.018 There’s an ideal fit between the optimal response and nonlinear integration in single cortical neurons, (Figure 3A, 3D). Response amplitudes (ms) were seen to depend on the interstimulus interval (ISI) and were well predicted by the optimal response, (Figures 3B, 3E). Correlations of the second order (cor2) or independent presynaptic firing (ind) resulted in poor fits with the optimal response in comparison to populations in the neocortex (NC) and the hippocampus (HP) across a varying range of conditions, (Figure 3C, 3F). Presynaptic cortical function was found to determine the location of the dendritic nonlinearity as seen by the inability of the optimal response to be a good fit when hippocampal rather than neocortical activities were matched for presynaptic statistics in cortical pyramidal neurons. Furthermore, NMDA receptor activation proved to be crucial for optimal dendritic integration.

Figure 4.0: Clustered connectivity and optimal response https://doi.org/10.7554/eLife.10056.015

across supralinearity and sublinearity, (Tzilivaki, 2019). It has been shown that linear integration with a single global dendritic nonlinearity can fit the response of neurons to naturalistic input patterns, (Ujfalussy, 2016). In addition, nonlinearities are essential for the efficient computing of information due to resources saved during encoding, (Tzilivaki, 2019). The effect of inter-stimulus intervals on response amplitudes and synaptic efficacies along with the presence of fast evolving, analog potentials and computation in cortical structures was noted, (Pfister, 2010). The majority of the results are in line with and build upon previous works. These findings can alter machine learning and neuromorphic hardware platforms to represent single neuron systems as 2 stage networks to increase processing ability, and storage, (Stockel, 2019).

Conclusions/Discussion The optimal response, the computation performed by a postsynaptic cell in relation to specific presynaptic statistics, was defined as:

The function successfully modelled in vitro measurements of the nonlinear integration of correlated inputs in single cortical neurons based on a given set of in vivo input statistics. The observed dependence of response amplitudes on interstimulus intervals points to the conclusion that not only is there nonlinear dendritic computation but also that dendritic nonlinearities are efficiently tuned to presynaptic activity patterns. Furthermore, it was also revealed that synaptic clustering of nonlinear neural assemblies exhibits an optimal integration of correlated inputs.

Novel interpretations come in the form of suggesting the essential role of NMDA receptors in dendritic nonlinearities, and the idea that short term neural plasticity plays a role in tuning the form of the optimal response to presynaptic statistics. In addition, the study pushes for further investigation of the relationship between the structure of correlations and morphological clustering. It was concluded that nonlinear dendrites are compulsory for the successful processing of varying spatio-temporal activity and that they hence play a vital role in higher level computation in neural circuits. These findings work towards establishing a causal link between single system and network level computations and naturalistic behaviors. This will aid in the better understanding of short term dendritic plasticity and processing ability in vertebrates and the application of neural structures to machine learning.

Figure 4.0 shows a comparison of a model with linear dendrites and a soma, a model in which only the soma is nonlinear, and two models with nonlinear dendrites with either random or clustered connectivity. Nonlinear dendrites with clustered connectivity between the presynaptic neuron and the dendritic branches proved to be at performance par with the optimal Critical Analysis response.

The authors have done an excellent job at extending controls The modeling of dendritic computational function by a simple for not only data sets extracted for in vivo neuronal population method of nonlinear integration has previously been seen in FS statistics from pre-existing works but also when examining the basket cells in both the hippocampus and prefrontal cortex link between nonlinear clustered dendrites and optimal re20

-sponse. Relationships were studied whilst varying various presynaptic statistics such as ISI, number of neurons, and hippocampal and neocortical input values. For results displayed in Figure 3.0, It was made sure that in vitro stimuli activated dendrites in a range comparable to in vivo conditions and hence integration was observed from a physiologically relevant context. Crucially, application of Bayesian Information Criterion ensured that the significant match between predicted and actual dendritic nonlinearities was not due to an overfitting error and the presence of an additional parameter.

distanced synapses. However, in the case that the null hypothesis is correct, it may be that neurons with overlapping receptive fields are creating excessive noise. An alternate theory would be that there exists only single neural systems initially, and then occurs rapid neural plasticity and reconfiguration to form local synaptic clusters upon encountering correlated activity.

This theory concurs with evidence that supports the existence of probationary or silent phases in newly formed synapses, “Poirazi and Mel, 2001). Summarily, this suggests that synapse clusters and their location in relation to presynaptic correlated inputs is dependent on the species and brain area in question There exists a wealth of resources supporting the fundamentals as well as the history a person may have in encountering tested used to build the framework of the study and the model of the stimulus. optimal response. Neuronal systems in the neocortex and hippocampal pyramidal cells display analog communication to perform basic arithmetic function economically and accurately, (Laughlin and Sejnowski, 2003). The study built upon this idea by suggesting that nonlinear integration would then be necessary for efficient processing of varying spatial-temporal patterns and that it could be modeled biophysically, (Poirazi and Mel, 2001; Grienberger et al., 2014). Although, the presence of nonlinear integration has previously been shown in the visual and somatosensory cortices, the authors expand the field by presenting proof in an unprecedented large scaled network model whilst not only varying input statistic spatially and temporally but also considering computations at the level of the dendrite, (Smith et al., 2013; Xu et al., 2012).

The authors should continue their pursuit of the accurate representation of neural assemblies by modifying in vitro stimulation parameters and structural details to match those in vivo as this will aid in the better understanding of short term dendritic plasticity and processing ability in vertebrates and the application of this neural structure to machine learning. Further experiments should test the ability of the model to predict the location and clustering of postsynaptic branches based on presynaptic correlations as there exists contradicting evidence based on varying controls like in vivo spontaneous network activity, and non-naturalistic activity across single stimulus dimensions, (Kirchner and Gjorgjieva, 2019).

Future Directions Future repetitions of the experiment should modify in vitro stimulation parameters and structural details to match those in vivo through the use of voltage dyes which allow for the detection of subthreshold synaptic potentials in addition to spiking activity, (Rost et al., 2017). The use of dendrite targeted optogenetic tools such as fluorescent sensors and optogenetic actuators to visualize signaling events and manipulate cellular activity should be used so that the authors may compare naturalistic data rather than predicted values for the biophysical model.

More importantly, these techniques can be used to further explore the disputed link between the presence and location of synaptic clusters and correlated inputs. I believe the results would be in line with the notion that correlated activity in presynaptic cells is tied to the nearby clustering of synapses whereas independent activity leads to linear processing in more 21

REFRENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

Ujfalussy, B. B., Makara, J. K., Branco, T., & Lengyel, M. (2015). Dendritic nonlinearities are tuned for efficient spike-based computations in cortical circuits. eLife, 4, e10056. https://doi.org/10.7554/eLife.10056 Grienberger, C., Chen, X., & Konnerth, A. (2014). NMDA Receptor-Dependent Multidendrite Ca2 Required for Hippocampal Burst firing in Viv. Neuron, 81(6), 1274-1281. Doi:10.1016/j.neuron.2014.01.014 Smith, S., Smith, I., Branco, T. et al. Dendritic spikes enhance stimulus selectivity in cortical neurons in vivo .

Nature 503, 115–120 (2013). https://doi.org/10.1038/nature12600 Xu, N., Harnett, M., Williams, S. et al. Nonlinear dendritic integration of sensory and motor input during an active sensing task. Nature 492, 247–251 (2012). https://doi.org/10.1038/nature11601 Poirazi, P., & Mel, B. W. (2001). Impact of Active Dendrites and Structural Plasticity on the Memory Capacity of Neural Tissue. Neuron, 29(3), 779-796. Doi:10.1016/s0896-6273(01)00252-5 Cepelewicz, J. (2020). Hidden Computational Power Found in the Arms of Neurons. Retrieved June 12, 2020, https:..www.quantamagazine.org/neural-dendrites-reveal-their-computational-power-20200114/ Poirazi, P., Brannon, T., & Mel, B. W. (2003). Pyramidal Neuron as Two-Layer Neural Network. Neuron, 37(6), 989-999. Doi: 10.1016/s0896-6273(03)00149-1 Rentinal ganglion cells: A functional interpretation of dendritic morphology. (1982). Philosophucal Transfactions of the Royal Society of London. B, Biological Sciences, 298(1090), 227-263. Doi:10.1098/rstb.1982.0084 Li, S., Liu, N., Zhang, X., Mclaughlin, D. W., Zhou, D., & Cai, D. (2019). Dendritic computation captured by an effective point neuron model. Proceedings of the National Academcy of Sciences, 116(30), 15244-15252.

Doi:10.1073/pnas.1904463116 Richards, B.A., Lillicrap, T.P., Beaudoin, P. et al. A deep learning framework for neuroscience. Nat Neurosci 22, 1761–1770 (2019). https://doi.org/10.1038/s41593-019-0520-2 Clark, B., & Hausser, M. (2006). Neural Coding: Hybrid Analog and Digital Signaling in Axons. Current Biology, 16 (15). Doi:10.1016/j.cub.2006.07.007 Tzilivaki, A., Kastellakis, G. & Poirazi, P. Challenging the point neuron dogma: FS basket cells as 2-stage nonlinear integrators. Nat Commun 10, 3664 (2019). https://doi.org/10.1038/s41467-019-11537-7 Ujfalussy, B. B., Makara, J. K., Lengyel, M., & Branco, T. (2018). Global and Multiplexed Dendritic Computations under in viv like Condition. Neuron, 100(3). Doi:10.1016/j.neuron.2018.08.032 Pfister, & Lengyel, Mate. (2010). Synapes with short term plasticity are optimal estimators of presynaptic membrane potentials. Nature Neuroscience. 13.1271-5. 10.1038/nn.2640 Sotckel, A., & Eliasmith, C. (2019). Passive nonlinear dendritic interactions as a general computational resource in functional spiking neural networks. 1-28.

Laughlin, S. B., & Sejnowski, T. J. (2003). Communication in neuronal networks. Science (New York, N.Y.), 301 (5641), 1870–1874. https://doi.org/10.1126/science.1089662

22

A Neuroligin-3 Mutation Results in Abnormal Behaviour in Mouse Model of Autism Spectrum Disorder Liang Chen

Repetitive, obsessive, and restricted behaviours are usually seen in individuals with Autism Spectrum Disorder (ASD), along with impaired social interaction. ASD is one of the common neurodevelopmental disorders that has been studied in many mouse models. The development of various regions of the brain is different in imaging tests of autistic individuals. Mutations in genes such as SHANK3, Neuroligin (NLGN), and Neurexin (NRXN) have been discovered and contribute to the cause of ASD. However, genetic mutations are not the only cause of ASD but also environmental factors, too. Individuals with autism experience a range of symptoms; therefore, it is difficult to pinpoint the exact cause of ASD and it still remains unknown. A study conducted by Burrows et al. (2017) examines how the Neuroligin-3 R451C mutation (NL3R451C ) in mouse model of ASD changes behaviours in social interaction and mating. The R451C mutation in Neuroligin-3 was originally found in a Swedish pair of siblings with ASD and Burrows et al. specifically investigated the mutation and wild type (WT) in male mice. Mice were pseudorandomly assigned to either social housing or isolation housing. The researchers identified that NL3R451C mice from both housings had increased interest in mating and were more aggressive toward female mice, especially NL3R451C mice from social housing. Thus, NL3R451C mice provide useful insights concerning atypical behaviour in ASD.

23

INTRODUCTION

mones. This study demonstrated that the genetic mutation of NL3R451C is involved in abnormal social behaviour that repreMany researchers continue to investigate autism spectrum dissents characteristics of ASD. order (ASD) despite its complex neurodevelopmental condition.

There has been a gradual increase in the prevalence of ASD and there are more males who are affected by the disorder than MAJOR RESULTS females (Lai et al., 2014). Autistic individuals typically display repetitive and restricted behaviour and they have difficulty communicating with people (Lai et al., 2014; Lewis et al., 2007).

Social interaction in WT and NL3R451C mice ASD is mainly caused by genetic factors with a relatively high R451C mice’s outcomes of different types of social heritability (Abrahams & Geschwind, 2008). Further evidence WT and NL3 supported by a study that illustrates the likelihood of inheriting interaction were recorded. The research was conducted over social impairments for ASD is about 61% and the concordance two stages (two exposure with three minute break in between) rates for identical twins and fraternal twins are 80% and 13.6%, for two weeks (once a week) (Burrows et al., 2017). There were R451C mice in social housing, and nine WT; nine respectively (Deng et al., 2015). Moreover, many genetic muta- ten WT; ten NL3 R451C mice in isolation housing. All mice contacted their fetions linked with ASD influence proteins that are essential for NL3 appropriate synaptic growth and function (Voineagu et al., male mouse in less than six seconds, and thus no differences in 2011). Specifically, genetic mutations that encode single-pass groups were observed in sniffing latency (Figure 1a). WT and R451C mice from isolation housing exhibited enhanced sociatransmembrane proteins such as neuroligin and neurexin have NL3 been discovered (Cao & Tabuchi, 2017; Jamain et al., 2003). bility toward the female mouse where they spent longer duraNeurexin and neuroligin interact with each other on both the tion of sniffing the female’s head and body (Figure 1b & 1c) as presynaptic and postsynaptic ends, which they are also called well as sniffing the female mouse’s genital region (Figure 1d). cell-adhesion molecules (Betancur et al., 2009; Cao & Tabuchi, The results are in agreement with a previous study that socially 2017; Jamain et al., 2003). Interruption to these cell-adhesion isolated mice are more eager to be involved in social interacmolecules may uncover novel understanding of the mecha- tion (Chabout et al., 2012). nisms of synaptic dysfunction in ASD (Betancur et al., 2009).

Besides genetic factors that are, for the most part, responsible for ASD, environmental factors are just as important to be taken into consideration since some could function as risk factors stimulating the development of ASD. Fortunately, environmental factors can be minimized and prevented from increasing the risk of giving birth to an autistic child. Pregnant mothers who have high intake of fatty acids in the first trimester lower the risk of giving birth to autistic newborns by 34% (Karimi et al., 2017). However, other environmental factors such as the MMR vaccine is found to be unassociated with ASD (Karimi et al., 2017; Taylor et al., 2002). ASD is indeed a complicated disorder that is caused not only by one factor but a mixture of numerous environmental and genetic risk factors.

Burrows et al. used WT and NL3R451C mice that were obtained from pairing NL3R451C male mice with heterozygous female mice. This produced even number of WT and NL3 R451C male progenies. Mice were pseudorandomly assigned to social housing (consisted of three to four mice in each housing) or isolation housing. All mice from both housing conditions subsequently experienced the Male-Female Social Interaction Test (MFSIT) at the age of 14 to 20 weeks where they interact with a new sexually full-grown female mouse individually. Total of two MFSIT were conducted once a week and each female mouse was paired with one male mouse pseudorandomly for each day. The Female Urine Sniffing Test (FUST) was also conducted. NL3R451C mice displayed atypical behaviour in terms of aggressiveness toward female mice and increased interest in mating. Socially isolated WT and NL3R451C mice had increased interest in interacting with female mice as well as NL3R451C mice from social housing showed a lack of interest in exploring female phero-

Figure 1. (a) No differences in groups were observed where all mice contacted female mouse within six seconds. (b)&(c) Longer duration of sniffing female mouse’s head and body in WT and NL3R451C mice from isolation housing. (d) NL3R451C mice from isolation housing were more interested in sniffing female mouse’s genital. The results in each graph is an average of four tests from the two stages. Figure derived from Burrows et al.

(2017) Mating behaviour in WT and NL3R451C mice There were ten WT; ten NL3R451C mice in social housing, and nine WT; nine NL3R451C mice in isolation housing (Burrows et al.,

24

2017). The research was conducted over two stages for two weeks. No differences were observed in mounting latency between all mice where they were all faster to mount during the first test after they had been separated for a period of time and were slower during the second test a week after (Burrows et al., 2017)(Figure 2a). The number of mounts is greater in NL3R451C mice than WT mice, especially NL3 R451C mice from social housing (Figure 2b). NL3R451C mice also spent longer duration of mounting the female mouse than WT mice (Figure 2c).

All mice spent longer time mounting the female mouse when the female was the same for both stages (Burrows et al., 2017).

Conversely, there was a decrease in mounting duration when a different female mouse was paired in the second stage Figure 3. (a) No effect of genotype on latency to stalk was ob(Burrows et al., 2017). served. (b) NL3R451C mice from social housing stalked female mouse for longer time, whereas WT and NL3 R451C mice from isolation housing had shorter stalk duration. (c) NL3 R451C mice had higher percentage of attacking female mice than WT mice, especially NL3R451C mice from isolation housing. The results in each graph is an average of four tests from the two stages. Figure derived from Burrows et al. (2017) NL3R451C mice from social housing showed a lack of interest in exploring and sniffing female mice’s urine based on FUST (Figure 4), whereas NL3R451C mice from isolation housing were Figure 2. (a) All mice were faster to mount during the first test highly interested in examining female mice’s urine where the after they had been separated for a period of time and were levels of duration is similar to WT mice (Figure 4). slower during the second test a week after, and thus no differences were observed. (b) NL3R451C mice mounted more times than WT mice, especially NL3R451C mice from social housing. (c) NL3R451C mice spent longer time mounting female mouse than WT mice. The results in each graph is an average of four tests from the two stages. Figure derived from Burrows et al. (2017) Unacceptable behaviour in NL3R451C mice and the Female Urine Sniffing Test (FUST) There were ten WT; ten NL3R451C mice in social housing, and nine WT; nine NL3R451C mice in isolation housing (Burrows et al., 2017). The research was conducted over two stages for two weeks. No effect of genotype on latency to stalk was observed (Figure 3a) and NL3R451C mice from social housing stalked the female mouse for longer time (Figure 3b). WT and NL3R451C mice from isolation housing had shorter stalk duration (Figure 3b). Furthermore, NL3R451C mice were hostile toward female mice and had higher chances of attacking female mice, especially NL3R451C mice from social housing (Figure 3c). These results are consistent with a recently study done on NL3 R451C mice that displayed atypical hostile behaviour (Hosie et al., 2018).

Figure 4. NL3R451C mice from social housing was uninterested in sniffing female mice’s urine, whereas NL3R451C mice from isolation housing were interested in sniffing female urine just as much as WT mice. The results in each graph is an average of four tests from the two stages. Figure derived from Burrows et al. (2017)

CONCLUSION/DISCUSSION Burrows et al. (2017) concluded that the mutation of NL3 R451C plays a role in altering social behaviour in mouse model of ASD where mice behave abnormally in terms of being hostile to25

ward female mice and wanting to mate more. Mice from isolation housing were more motivated to socialize with female mice no matter what the genotype is and NL3 R451C mice from social housing were uninterested in sniffing female mice’s urine.

are crucial to engage in social interaction and investigation (Hashikawa et al., 2016). Experiments on brain regions could potentially unveil additional insights into atypical behaviour caused by NL3R451C mutation. Further studies are required in assessing the concentration of testosterone in the blood to determine mating drive in NL3R451C mice. By comparing the baseline measure to the measure after being introduced to a Aggressive behaviour is common between male mice since it is female mouse, the level of mating drive can be established an intrinsic, social behaviour that is essential to gain social based on the concentration difference obtained. ranking and available resources from the surroundings (Miczek et al., 2001), but exhibiting hostile behaviour toward female mice is aberrant. Burrows et al. stated that NL3R451C mice’s ab- FUTURE DIRECTIONS normal aggression might not be territorial because they were Future studies should concentrate on examining specific areas being aggressive toward female mice as well. In addition, in the hypothalamus and olfactory systems that are responsible R451C NL3 mice exhibiting atypical hostile behaviour has been for different behavioral outcomes such as aggression in NL3 R451C reported that it is due to low levels of serotonin in the brain mice. The technique of optogenetics could be employed to inwhere they do not have sufficient tryptophan hydroxylase 2 in hibit or excite particular parts in these brain regions. If a spethe brain (Beis et al., 2015). cific group of neurons is thought to modulate aggressive behaviour in the hypothalamus, the researchers could excite or inhibit those particular neurons and observe the behavioural outMice sense pheromones to identify and interact with individucomes of the mouse. Similar procedure applies to olfactory als. NL3R451C mice that displayed a lack of interest in exploring systems. The expected outcomes of the hypothalamus would urine could be the cause to their hostility toward female mice. be if those specific neurons were being excited, the mouse The lack of interest could suggest that NL3R451C mice may have should display hostile behaviour because the area responsible olfactory dysfunction. However, a previous study showed that for aggression behaviour is activated. When those neurons are mice had no problem discriminating between different types of inhibited, the mouse should behave less hostile. The expected odors, including urine (Burrows et al., 2015). Moreover, outcomes of olfactory systems would be if those specific neuR451C NL3 mice from isolation housing explored pheromones in rons were inhibited, the mouse should become more aggresurine just as much as WT mice, implying that social housing sive since it could be difficult to identify individuals or could might be a factor in affecting the levels of interest in exploring misinterpret other mice as strangers. If no aggressive behaviour female mice’s urine. is observed in NL3R451C mice, then it can be concluded that the examined areas in the hypothalamus and olfactory systems are not responsible for eliciting hostile behaviour.

R451C NL3 mice exhibited high mating drive where they mounted female mice more times and for longer duration. This may be due to the concentration differences in blood testosterone beFuture studies should also concentrate on examining whether fore and after introducing to a female (Burrows et al., 2017). mating drive is influenced by testosterone. One possible experiment is that first, laboratory mice are castrated before conducting the study. Mice then are separated into two groups – Collectively, this neuroligin-3 R451C mutation does have an castrated mice that do not receive testosterone (control) and impact on modifying social behaviour in mice, which is also castrated mice that receive testosterone (experimental group). seen in individuals with ASD. Testosterone is given through injection. The researchers then pair each male mouse to a female mouse in a separate room and see whether there is a difference between the control CRITICAL ANALYSIS group and the experimental group. The expected outcomes The present study conducted by Burrows et al. (2017) obtained would be the experimental group has higher mating drive than several fascinating findings that are in line with other literature the control group. If no difference in mating drive is observed, in regard to the types of dysfunctional behaviour such as atypi- then it can be concluded that testosterone does not have any cal aggression that was detected in NL3R451C mice (Burrows et effect on mating drive and that other factors might be involved al., 2015; Miczek et al., 2001). Nevertheless, experiments re- in regulating mating drive instead. main to be performed are what brain regions are affected by NL3R451C that brings about abnormal behaviour in NL3R451C mice.

Experiments on brain regions such as hypothalamus and olfactory systems should be performed as well as the levels of neurotransmitters such as serotonin. Besides regulating homeostasis, the hypothalamus is known to release hormones that have an effect on sexual and social behaviours, and olfactory systems 26

REFRENCES 1.

Abrahams, B. S., & Geschwind, D. H. (2008). Advances in autism genetics: On the threshold of a new neurobiology. Nature Reviews Genetics, 9(5), 341–355. https://doi.org/10.1038/nrg2346

2.

Beis, D., Holzwarth, K., Flinders, M., Bader, M., Wöhr, M., & Alenina, N. (2015). Brain serotonin deficiency leads to social communication deficits in mice. Biology Letters, 11(3). https://doi.org/10.1098/rsbl.2015.0057

3.

Betancur, C., Sakurai, T., & Buxbaum, J. D. (2009). The emerging role of synaptic cell-adhesion pathways in the pathogenesis of autism spectrum disorders. Trends in Neurosciences, 32(7), 402–412. https://doi.org/10.1016/j.tins.2009.04.003

4.

Burrows, E. L., Eastwood, A. F., May, C., Kolbe, S. C., Hill, T., McLachlan, N. M., Churilov, L., & Hannan, A. J. (2017). Social Isolation Alters Social and Mating Behavior in the R451C Neuroligin Mouse Model of Autism. Neural Plasticity, 2017. https://doi.org/10.1155/2017/8361290

5.

Burrows, Emma L., Laskaris, L., Koyama, L., Churilov, L., Bornstein, J. C., Hill-Yardin, E. L., & Hannan, A. J. (2015). A neuroligin-3 mutation implicated in autism causes abnormal aggression and increases repetitive behavior in mice. Molecular Autism, 6(1), 62. https://doi.org/10.1186/s13229-015-0055-7

6.

Cao, X., & Tabuchi, K. (2017). Functions of synapse adhesion molecules neurexin/neuroligins and neurodevelopmental disorders. Neuroscience Research, 116, 3–9. https://doi.org/10.1016/j.neures.2016.09.005

7.

Chabout, J., Serreau, P., Ey, E., Bellier, L., Aubin, T., Bourgeron, T., & Granon, S. (2012). Adult Male Mice Emit ContextSpecific Ultrasonic Vocalizations That Are Modulated by Prior Isolation or Group Rearing Environment. PLoS ONE, 7(1). https://doi.org/10.1371/journal.pone.0029401

8.

Deng, W., Zou, X., Deng, H., Li, J., Tang, C., Wang, X., & Guo, X. (2015). The Relationship Among Genetic Heritability, Environmental Effects, and Autism Spectrum Disorders: 37 Pairs of Ascertained Twin Study. Journal of Child Neurology, 30(13), 1794–1799. https://doi.org/10.1177/0883073815580645

9.

Hashikawa, K., Hashikawa, Y., Falkner, A., & Lin, D. (2016). The neural circuits of mating and fighting in male mice. Current Opinion in Neurobiology, 38(Complete), 27–37. https://doi.org/10.1016/j.conb.2016.01.006

10.

Hosie, S., Malone, D. T., Liu, S., Glass, M., Adlard, P. A., Hannan, A. J., & Hill-Yardin, E. L. (2018). Altered Amygdala Excitation and CB1 Receptor Modulation of Aggressive Behavior in the Neuroligin-3R451C Mouse Model of Autism. Frontiers in Cellular Neuroscience, 12. https://doi.org/10.3389/fncel.2018.00234

11.

Jamain, S., Quach, H., Betancur, C., Råstam, M., Colineaux, C., Gillberg, I. C., Soderstrom, H., Giros, B., Leboyer, M., Gillberg, C., & Bourgeron, T. (2003). Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism. Nature Genetics, 34(1), 27–29. https://doi.org/10.1038/ng1136

12.

Karimi, P., Kamali, E., Mousavi, S. M., & Karahmadi, M. (2017). Environmental factors influencing the risk of autism. Journal of Research in Medical Sciences : The Official Journal of Isfahan University of Medical Sciences, 22. https:// doi.org/10.4103/1735-1995.200272

13.

Lai, M.-C., Lombardo, M. V., & Baron-Cohen, S. (2014). Autism. The Lancet, 383(9920), 896–910. https://doi.org/10.1016/ S0140-6736(13)61539-1

14.

Lewis, M. H., Tanimura, Y., Lee, L. W., & Bodfish, J. W. (2007). Animal models of restricted repetitive behavior in autism.

Behavioural Brain Research, 176(1), 66–74. https://doi.org/10.1016/j.bbr.2006.08.023

15.

Miczek, K. A., Maxson, S. C., Fish, E. W., & Faccidomo, S. (2001). Aggressive behavioral phenotypes in mice. Behavioural Brain Research, 125(1–2), 167–181. https://doi.org/10.1016/S0166-4328(01)00298-4

16.

Taylor, B., Miller, E., Lingam, R., Andrews, N., Simmons, A., & Stowe, J. (2002). Measles, mumps, and rubella vaccination and bowel problems or developmental regression in children with autism: Population study. BMJ : British Medical Journal, 324(7334), 393–396.

17.

Voineagu, I., Wang, X., Johnston, P., Lowe, J. K., Tian, Y., Horvath, S., Mill, J., Cantor, R. M., Blencowe, B. J., & Geschwind, D. H. (2011). Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature, 474(7351), 380– 384. https://doi.org/10.1038/nature10110

27

Behavioural and synaptic effect from EAAT3 overexpression on obsessive-compulsive disorder Yi -Chin Chen

Obsessive compulsive disorder (OCD) is a neurodevelopmental disorder that involves in repetitive thoughts and behaviours that are unwanted. Up to 3% of the general population are affected by this disorder and the symptoms can persist chronically if left untreated. Currently, antidepressant and cognitive behavioural therapy are the two main treatment that aids in alleviating OCD symptoms. A cure for this disorder has yet to be found. Delgado-Acevedo et al. (2019) explored the overexpression effect of EAAT3 on OCD and found an increase in anxious and repetitive behaviours along with greater spontaneous recovery of fear. They also found altered corticostriatal synapses from overexpression of EAAT3.

These findings provided evidences and connection between the involvement of EAAT3 expression on OCD-related behaviours. Therefore, EAAT3 could be a potential therapeutic target for OCD treatment.

Key words: obsessive-compulsive disorder (OCD), solute carrier family 1 member 1 (SLC1A1), excitatory amino acid transporter 3 (EAAT3), repetitive behaviours, N-methyl-D-aspartate receptor (NMDAR), GluN2B subunit, fear extinction, glutaminergic system, corticostrial synapse

28

INTRODUCTION

MAJOR RESULTS

Obsessive compulsive disorder (OCD) is a neurodevelopmental disorder that involves compulsive behaviours and persistent thoughts (Abramowitz et al., 2009). It affects 2-3% of adults in the general population and the symptoms can be chronic if left untreated (Mataix-Cols et al., 2002; S. E. Stewart et al., 2004).

Antidepressants, such as serotonin reuptake inhibitors, are often prescribed with or without conjunction of cognitive behavioral therapy to help alleviate OCD symptoms (Abramowitz et al., 2009). However, a cure for OCD has not yet been found.

Overexpression of EAAT3 increases anxious and repetitive behaviours

In the original study, Delgado-Acevedo et al. (2019) conducted four behaviour tests and found increases in anxious and repetitive behaviours in transgenic EAAT3 overexpressing mice, EAAT3glo/CMKII (Figure 1). In the open field test, EAAT3glo/CMKII mice spends less time in the box center than the control mice (Figure 1A). In the light-dark exploration test, EAAT3glo/CMKII mice spends more time in the dark compartment as oppose to control mice (Figure 1E). In the marble burying test, more marbles were buried by EAAT3glo/CMKII mice than the control mice (Figure 1F). In the grooming analysis, EAAT3 glo/CMKII mice spends more time grooming than the control mice (Figure 1G).

Studies have implicated that defective glutaminergic system are linked to OCD (Brennan et al., 2013; Chakrabarty et al., 2005).

Specifically, the glutaminergic neurons in the cortico-striatalthalamic-cortical (CSTC) circuitry is hyperactive in OCD (Hajcak & Simons, 2002; Johannes et al., 2001). The hyperactivity results from alternation of glutaminergic NMDR receptors that will ultimately promote synaptic plasticity (Pittenger, 2015). Hence, the Additionally, chronic treatment of antidepressants was found to reoccurrence of the intrusive thoughts leading to repetitive be- decrease anxious and repetitive behaviours in EAAT3glo/CMKII mice. Administration of fluoxetine or clomipramine were given haviours can be seen in OCD. to EAAT3glo/CMKII mice for one day or 21 days (Figure 1). This result supports literature evidences on antidepressant as a treatAlthough there are environmental contributors to this disorder, ment to alleviate anxious and repetitive behaviours in OCD studies on twins suggested genetic factors can held up to 65% (Abramowitz et al., 2009). liability for the observed OCD symptoms (Grootheest et al., 2005). In particular, solute carrier family 1 member 1 (SLC1A1) has been identified as a candidate gene for OCD that expresses excitatory amino acid transporter 3 (EAAT3), a glutamate transporter (Pittenger, 2015; Ting & Feng, 2008). EAAT3 can regulate synaptic level of glutamate and N-methyl-D-aspartate receptor (NMDAR) function (Bjørn-Yoshimoto & Underhill, 2016; Escobar et al., 2019). Previous studies has shown that EAAT3 knockout mice shows unaltered baseline anxious and repetitive behaviours (Gonzålez et al., 2017; Peghini et al., 1997; Quinlan et al., 1999; Zike et al., 2017). Several SLC1A1 variants were found to promote transcription of SLC1A1 in OCD brain (Liang et al., 2008; Shugart et al., 2009; S. Evelyn Stewart et al., 2007). Therefore, EAAT3 overexpression may increase susceptibility to OCD.

The original paper by Delgado-Acevedo et al. (2019) investigated the effect of EAAT3 overexpression on behaviour and synapses of OCD. Transgenic mice were used to overexpress EAAT3. The mice then underwent a series of behavioral tests and fear conditioning and extinction tasks. Subsequently, oral administration of antidepressants was given to the mice acutely or chronically.

Corticostraital synapses were examined using electrophysiological and molecular analyses. An increase in anxious and compulsive behaviours as well as greater spontaneous recovery of fear were evident in EAAT3 overexpressed mice. Results from the analyses showed changes in NMDAR subunits in EAAT3 overexpressed mice. Overall, this study provides evidence on involvement of glutamatergic system at play in OCD and managing levels of EAAT3 as a possible new therapeutic target.

Figure 1. The results of the four behaviour tests and the effect of antidepressants on overexpressing EAAT3 mice (EAAT3glo/CMKII). EAAT3glo is the control mice. Panel A shows the results from the open field test. Panel E shows the results from the light-dark exploration test. Panel F shows the result from the marble burying test. Panel G shows the results from the grooming analysis. Clomipramine and fluoxetine are the administered antidepressants.

Figure adapted from Delgado-Acevedo et al. (2019).

Overexpression of EAAT3 is associated with greater spontane-

29

In the study by Delgado-Acevedo et al. (2019), they also examined the performance of EAAT3glo/CMKII mice in the fear conditioning and extinction tasks (Figure 2). One day after fear learning, EAAT3glo/CMKII mice showed higher tendency to freeze from extinction retrieval than the control mice (Figure 2C).

EAAT3glo/CMKII mice also showed higher tendency to freeze from spontaneous recovering of fear than the control mice two weeks after fear learning (Figure 2D). This result supports welldocumented OCD characteristics of having elevated fear and trouble in extinction of fear (Fullana et al., 2014; Milad et al., 2013).

Figure 2. The results on extinction retrieval and spontaneous recovery of fear on overexpressing EAAT3 mice (EAAT3 glo/CMKII).

EAAT3glo is the control mice. Panel C shows amount of freezing during retrieval given the number of conditioned tone. Panel D shows the amount of freezing during spontaneous recovery of fear given the number of conditioned tone presented. Figure adapted from Delgado-Acevedo et al. (2019).

Overexpression of EAAT3 is associated with corticostriatal alternations When Delgado-Acevedo et al. (2019) examined the corticostriatal synapses of EAAT3glo/CMKII mice using western blot, it was found NMDAR that contains GluN2B subunit are more prevalent than GluN2A-containing NMDAR. High-frequency afferent stimulation that was carried out at the synapse failed to induce NMDA -dependent long-term depression in EAAT3glo/CMKII mice. These results reinforces previous evidences on EAAT3 ability to regulate the function of NMDAR to promote hyperactivity and synaptic plasticity in CSTC that render the observed restless behaviours and thoughts in OCD (Bjørn-Yoshimoto & Underhill, 2016; Escobar et al., 2019). The results ultimately support evidences on involvement of the glutaminergic system in OCD through alteration in NMDAR composition (Brennan et al., 2013; Chakrabarty et al., 2005).

DISCUSSIONS/ CONCLUSIONS The study by Delgado-Acevedo et al. (2019) was able to demonstrate overexpression of EAAT3 increases repetitive behaviours, promote changes in corticostriatal synapses and decrease susceptibility to fear extinction. These findings are evidences in supporting connection between EAAT3 and OCD pathogenesis. Ultimately, the results implicated that EAAT3 can be a potential therapeutic target for OCD due to its ability to alter OCD-related behaviours from level of expression.

Several SNP in SLC1A1 gene has been identified in OCD studies that correlate with increase SLC1A1 expression in the OCD brain (Liang et al., 2008; Shugart et al., 2009; S. Evelyn Stewart et al., 2007). Delgado-Acevedo et al. (2019) thereby hypothesized that expression of EAAT3 is positively correlated with OCD susceptibility. By using transgenic EAAT3 overexpressing mice (EAAT3 glo/ CMKII), Delagto et al. 2019 was able to induce a series of OCDrelated behaviours. The results showed support to the proposed hypothesis. Further indirect evidences that reinforces the hypothesis was found in previous studies where baseline OCDrelated behaviours in EAAT3 knockout mice remained unaffected (GonzĂĄlez et al., 2017; Peghini et al., 1997; Quinlan et al., 1999; Zike et al., 2017).

Grooming analysis was one of the behavioural tests carried out to examine presence of repetitive behaviours. Increased groom time was found in EAAT3glo/CMKII mice (Figure 1G). However, the extent of grooming in EAAT3glo/CMKII mice did not reach the point of injury. Conversely, knockout model of other OCD candidate genes, SAPAP3 and SLITRK5, showed excess grooming to the point of skin bleeding (Shmelkov et al., 2010; Welch et al., 2007). Expression level may differ among all the candidate genes that account for the spectrum differences observed in OCD phenotype. Additionally, the administration of antidepressants, clomipramine and fluoxetine, was used in the study to relieve anxious and repetitive behaviours like those of excess grooming observed in EAAT3glo/CMKII mice. Antidepressants are mainly used as a measure of validity for studies on OCD due to its prevalence as treatment for OCD (Chakrabarty et al., 2005; Coric et al., 2005).

EAAT3glo/CMKII mice was also shown to have a greater spontaneous recovery of fear, an indicator of defect in retaining fear extinction that can be seen in OCD individuals and from previous OCD-related studies (Figure 2D)(Milad et al., 2013; Reimer et al., 2018). Ventromedial prefrontal cortex (vmPFC) and amygdala are areas of the brain involved in fear extinction (Burgos-Robles et al., 2007; Do-Monte et al., 2010; Milad et al., 2007, 2013).

Previous studies on OCD has linked reduced activation in both vmPFC and amygdala to decreased ability to retain fear extinction (Milad et al., 2013; Phelps et al., 2004). Therefore, overexpression EAAT3 mice may have a low activation of vmPFC and amygdala that give rise to the observed fear extinction deficits.

Many studies have implicated that functions of NMDAR can differ depending on the subunit composition of the receptor (Das et al., 1998; Paoletti et al., 2013). Specifically, previous studies have found involvement of GluN2B-containing NMDAR in generating long term depression and subsequently represses conditioned fear response in fear extinction whereas GluN2Acontining NMDAR in generating long term potentiation (Dalton et al., 2012; Park et al., 2012). In one study, highly anxious rat was found to express more GluN2B-containing NMDAR in the prefrontal cortex and amygdala (Lehner et al., 2011). This is line with the results obtained by Delgado-Acevedo et al. (2019)

30

where elevated level of GluN2B-containing NMDAR in corticostriatal synapse of EAAT3glo/CMKII mice which has been shown to exhibit anxious behaviours. The evidences above supported the role of EAAT3 on altering NMDAR function.

CRITICAL ANALYSIS Little to no significant discrepancy was found in the literature that opposes the findings by Delgado-Acevedo et al. (2019). In line with the results from Delgado-Acevedo et al. (2019), similar results has been found examining other OCD candidate genes, SAPAP3 and SLITRK5 (Shmelkov et al., 2010; Welch et al., 2007).

SAPAP3 and SLITRK5 along with EAAT3 are all expressed in the corticostriatal synapse (Delgado-Acevedo et al., 2019; Shmelkov et al., 2010; Welch et al., 2007). Grooming behaviours was explored in SAPAP3 knockout (SAPAP3 KO) mice, SLITRK5 knockout (SLITRK5 KO) mice, and EAAT3 overexpressing (EAAT3glo/CMKII) mice (Delgado-Acevedo et al., 2019; Shmelkov et al., 2010; Welch et al., 2007). All three mice model showed prolonged grooming behaviour with SAPAP3 KO and SLITRK5 KO to the point of self-injury (Delgado-Acevedo et al., 2019; Shmelkov et al., 2010; Welch et al., 2007). Open field test in all three mice model also showed little time spend in the center of the box, indicating presence of anxiety in all the mice (DelgadoAcevedo et al., 2019; Shmelkov et al., 2010; Welch et al., 2007).

Similar to EAAT3glo/CMKII mice, marble burying test was explored in SLITRK KO mice where increase marble burying was also found (Delgado-Acevedo et al., 2019; Shmelkov et al., 2010). Light-dark exploration test was explored in both EAAT3glo/CMKII mice and SAPAP3 KO mice and both showed longer time spent in the dark (Delgado-Acevedo et al., 2019; Welch et al., 2007). Overall, behaviour tests conducted among the three mice model all showed OCD-related behaviours. Upon chronic administration of antidepressant, all three model showed alleviation on OCD-related behaviours. Additionally, corticostriatal electrophysiology was also carried out on SAPAP3 mice (Welch et al., 2007). Similar to the electrophysiological results from EAAT3glo/CMKII mice, activity of NMDAR activities were upregulated (Welch et al., 2007).

FUTURE DIRECTION Future studies can incorporate positron emission tomography (PET) imaging techniques to visually observe areas of the EAAT3glo/CMKII mice brain activity upon fear learning and extinction paradigm. Overexpression EAAT3 mice will first be transgenically generated. A synthetic radiotracers can injected into the mice. A signal will be generated by the radiotracers that can be measured by specialized equipment. Success of the experiment will show decreased brain activity in vmPFC and amygdala in extinction retrieval. Failure of the experiment will show decreased activity in other brain regions.

31

REFRENCES

1.

Abramowitz, J. S., Taylor, S., & McKay, D. (2009). Obsessive-compulsive disorder. The Lancet, 374(9688), 491–499. https:// doi.org/10.1016/S0140-6736(09)60240-3

2.

Bjørn-Yoshimoto, W., & Underhill, S. M. (2016). The Importance of the Excitatory Amino Acid Transporter 3 (EAAT3). Neurochemistry International, 98, 4–18. https://doi.org/10.1016/j.neuint.2016.05.007

3.

Brennan, B. P., Rauch, S. L., Jensen, J. E., & Pope, H. G. (2013). A critical review of magnetic resonance spectroscopy studies of obsessive-compulsive disorder. Biological Psychiatry, 73(1), 24–31. https://doi.org/10.1016/j.biopsych.2012.06.023

4.

Burgos-Robles, A., Vidal-Gonzalez, I., Santini, E., & Quirk, G. J. (2007). Consolidation of Fear Extinction Requires NMDA Receptor-Dependent Bursting in the Ventromedial Prefrontal Cortex. Neuron, 53(6), 871–880. https://doi.org/10.1016/ j.neuron.2007.02.021

5.

Chakrabarty, K., Bhattacharyya, S., Christopher, R., & Khanna, S. (2005). Glutamatergic Dysfunction in OCD. Neuropsychopharmacology, 30(9), 1735–1740. https://doi.org/10.1038/sj.npp.1300733

6.

Coric, V., Taskiran, S., Pittenger, C., Wasylink, S., Mathalon, D. H., Valentine, G., Saksa, J., Wu, Y.-T., Gueorguieva, R., Sanacora, G., Malison, R. T., & Krystal, J. H. (2005). Riluzole augmentation in treatment-resistant obsessive-compulsive disorder: An open-label trial. Biological Psychiatry, 58(5), 424–428. https://doi.org/10.1016/j.biopsych.2005.04.043

7.

Dalton, G. L., Wu, D. C., Wang, Y. T., Floresco, S. B., & Phillips, A. G. (2012). NMDA GluN2A and GluN2B receptors play separate roles in the induction of LTP and LTD in the amygdala and in the acquisition and extinction of conditioned fear. Neuropharmacology, 62(2), 797–806. https://doi.org/10.1016/j.neuropharm.2011.09.001

8.

Das, S., Sasaki, Y. F., Rothe, T., Premkumar, L. S., Takasu, M., Crandall, J. E., Dikkes, P., Conner, D. A., Rayudu, P. V., Cheung, W., Chen, H.-S. V., Lipton, S. A., & Nakanishi, N. (1998). Increased NMDA current and spine density in mice lacking the NMDA receptor subunit NR3A. Nature, 393(6683), 377–381. https://doi.org/10.1038/30748

9.

Delgado-Acevedo, C., Estay, S. F., Radke, A. K., Sengupta, A., Escobar, A. P., Henríquez-Belmar, F., Reyes, C. A., Haro-Acuña, V., Utreras, E., Sotomayor-Zárate, R., Cho, A., Wendland, J. R., Kulkarni, A. B., Holmes, A., Murphy, D. L., Chávez, A. E., & Moya, P. R. (2019). Behavioral and synaptic alterations relevant to obsessive-compulsive disorder in mice with increased EAAT3 expression. Neuropsychopharmacology, 44(6), 1163–1173. https://doi.org/10.1038/s41386-018-0302-7

10.

Do-Monte, F. H. M., Kincheski, G. C., Pavesi, E., Sordi, R., Assreuy, J., & Carobrez, A. P. (2010). Role of beta-adrenergic receptors in the ventromedial prefrontal cortex during contextual fear extinction in rats. Neurobiology of Learning and Memory, 94(3), 318–328. https://doi.org/10.1016/j.nlm.2010.07.004

11.

Escobar, A. P., Wendland, J. R., Chávez, A. E., & Moya, P. R. (2019). The Neuronal Glutamate Transporter EAAT3 in Obsessive-Compulsive Disorder. Frontiers in Pharmacology, 10. https://doi.org/10.3389/fphar.2019.01362

12.

Fullana, M. A., Cardoner, N., Alonso, P., Subirà, M., López-Solà, C., Pujol, J., Segalàs, C., Real, E., Bossa, M., Zacur, E., Martínez-Zalacaín, I., Bulbena, A., Menchón, J. M., Olmos, S., & Soriano-Mas, C. (2014). Brain regions related to fear extinction in obsessive-compulsive disorder and its relation to exposure therapy outcome: A morphometric study. Psychological Medicine, 44(4), 845–856. https://doi.org/10.1017/S0033291713001128

13.

González, L. F., Henríquez-Belmar, F., Delgado-Acevedo, C., Cisternas-Olmedo, M., Arriagada, G., Sotomayor-Zárate, R., Murphy, D. L., & Moya, P. R. (2017). Neurochemical and behavioral characterization of neuronal glutamate transporter EAAT3 heterozygous mice. Biological Research, 50(1), 29. https://doi.org/10.1186/s40659-017-0138-3

14.

Grootheest, D. S. van, Cath, D. C., Beekman, A. T., & Boomsma, D. I. (2005). Twin Studies on Obsessive–Compulsive Disorder: A Review. Twin Research and Human Genetics, 8(5), 450–458. https://doi.org/10.1375/twin.8.5.450

15.

Hajcak, G., & Simons, R. F. (2002). Error-related brain activity in obsessive-compulsive undergraduates. Psychiatry Research, 110(1), 63–72. https://doi.org/10.1016/s0165-1781(02)00034-3

16.

Johannes, S., Wieringa, B. M., Nager, W., Rada, D., Dengler, R., Emrich, H. M., Münte, T. F., & Dietrich, D. E. (2001). Discrepant target detection and action monitoring in obsessive–compulsive disorder. Psychiatry Research: Neuroimaging, 108(2), 101–110. https://doi.org/10.1016/S0925-4927(01)00117-2

32

17.

Lehner, M., Wislowska-Stanek, A., Skorzewska, A., Maciejak, P., Szyndler, J., Turzynska, D., Sobolewska, A., Krzascik, P., & Plaznik, A. (2011). Expression of N-methyl-D-aspartate (R)(GluN2B)—Subunits in the brain structures of rats selected for low and high anxiety. Journal of Physiology and Pharmacology: An Official Journal of the Polish Physiological Society, 62(4), 473–482.

18.

Liang, K.-Y., Wang, Y., Shugart, Y. Y., Grados, M., Fyer, A. J., Rauch, S., Murphy, D., McCracken, J., Rasmussen, S., Cullen, B., Hoehn-Saric, R., Greenberg, B., Pinto, A., Knowles, J., Piacentini, J., Pauls, D., Bienvenu, O., Riddle, M., Samuels, J., & Nestadt, G. (2008). Evidence for potential relationship between SLC1A1 and a putative genetic linkage region on chromosome 14q to obsessive-compulsive disorder with compulsive hoarding. American Journal of Medical Genetics. Part B, Neuropsychiatric Genetics: The Official Publication of the International Society of Psychiatric Genetics, 147B(6), 1000–1002. https:// doi.org/10.1002/ajmg.b.30713

19.

Mataix-Cols, D., Rauch, S. L., Baer, L., Eisen, J. L., Shera, D. M., Goodman, W. K., Rasmussen, S. A., & Jenike, M. A. (2002).

Symptom Stability in Adult Obsessive-Compulsive Disorder: Data From a Naturalistic Two-Year Follow-Up Study. American Journal of Psychiatry, 159(2), 263–268. https://doi.org/10.1176/appi.ajp.159.2.263

20.

Milad, M. R., Furtak, S. C., Greenberg, J. L., Keshaviah, A., Im, J. J., Falkenstein, M. J., Jenike, M., Rauch, S. L., & Wilhelm, S. (2013). Deficits in Conditioned Fear Extinction in Obsessive-Compulsive Disorder and Neurobiological Changes in the Fear Circuit. JAMA Psychiatry, 70(6), 608–618. https://doi.org/10.1001/jamapsychiatry.2013.914

21.

Milad, M. R., Wright, C. I., Orr, S. P., Pitman, R. K., Quirk, G. J., & Rauch, S. L. (2007). Recall of Fear Extinction in Humans Activates the Ventromedial Prefrontal Cortex and Hippocampus in Concert. Biological Psychiatry, 62(5), 446–454. https:// doi.org/10.1016/j.biopsych.2006.10.011

22.

Paoletti, P., Bellone, C., & Zhou, Q. (2013). NMDA receptor subunit diversity: Impact on receptor properties, synaptic plasticity and disease. Nature Reviews Neuroscience, 14(6), 383–400. https://doi.org/10.1038/nrn3504

23.

Park, S., Lee, S., Kim, J., & Choi, S. (2012). Ex vivo depotentiation of conditioning-induced potentiation at thalamic input synapses onto the lateral amygdala requires GluN2B-containing NMDA receptors. Neuroscience Letters, 530(2), 121–126. https://doi.org/10.1016/j.neulet.2012.10.011

24.

Peghini, P., Janzen, J., & Stoffel, W. (1997). Glutamate transporter EAAC-1-deficient mice develop dicarboxylic aminoaciduria and behavioral abnormalities but no neurodegeneration. The EMBO Journal, 16(13), 3822–3832. https:// doi.org/10.1093/emboj/16.13.3822

25.

Phelps, E. A., Delgado, M. R., Nearing, K. I., & LeDoux, J. E. (2004). Extinction learning in humans: Role of the amygdala and vmPFC. Neuron, 43(6), 897–905. https://doi.org/10.1016/j.neuron.2004.08.042

26.

Pittenger, C. (2015). Glutamatergic agents for OCD and related disorders. Current Treatment Options in Psychiatry, 2(3), 271–283. https://doi.org/10.1007/s40501-015-0051-8

27.

Quinlan, E. M., Olstein, D. H., & Bear, M. F. (1999). Bidirectional, experience-dependent regulation of N-methyl-d-aspartate receptor subunit composition in the rat visual cortex during postnatal development. Proceedings of the National Academy of Sciences, 96(22), 12876–12880. https://doi.org/10.1073/pnas.96.22.12876

28.

Reimer, A. E., de Oliveira, A. R., Diniz, J. B., Hoexter, M. Q., Miguel, E. C., Milad, M. R., & Brandão, M. L. (2018). Fear extinction in an obsessive-compulsive disorder animal model: Influence of sex and estrous cycle. Neuropharmacology, 131, 104– 115. https://doi.org/10.1016/j.neuropharm.2017.12.015

29.

Shmelkov, S. V., Hormigo, A., Jing, D., Proenca, C. C., Bath, K. G., Milde, T., Shmelkov, E., Kushner, J. S., Baljevic, M., Dincheva, I., Murphy, A. J., Valenzuela, D. M., Gale, N. W., Yancopoulos, G. D., Ninan, I., Lee, F. S., & Rafii, S. (2010). Slitrk5 deficiency impairs corticostriatal circuitry and leads to obsessive-compulsive-like behaviors in mice. Nature Medicine, 16(5), 598–602, 1p following 602. https://doi.org/10.1038/nm.2125

30.

Shugart, Y. Y., Wang, Y., Samuels, J. F., Grados, M. A., Greenberg, B. D., Knowles, J. A., McCracken, J. T., Rauch, S. L., Murphy, D. L., Rasmussen, S. A., Cullen, B., Hoehn-Saric, R., Pinto, A., Fyer, A. J., Piacentini, J., Pauls, D. L., Bienvenu, O. J., Riddle, M. A., Liang, K. Y., & Nestadt, G. (2009). A family-based association study of the glutamate transporter gene SLC1A1 in obsessive-compulsive disorder in 378 families. American Journal of Medical Genetics. Part B, Neuropsychiatric Genetics: The Official Publication of the International Society of Psychiatric Genetics, 150B(6), 886–892. https://doi.org/10.1002/ ajmg.b.30914

31.

Stewart, S. E., Geller, D. A., Jenike, M., Pauls, D., Shaw, D., Mullin, B., & Faraone, S. V. (2004). Long-term outcome of pediatric obsessive–compulsive disorder: A meta-analysis and qualitative review of the literature. Acta Psychiatrica Scandinavica, 110(1), 4–13. https://doi.org/10.1111/j.1600-0447.2004.00302.x

33

32.

Stewart, S. Evelyn, Fagerness, J. A., Platko, J., Smoller, J. W., Scharf, J. M., Illmann, C., Jenike, E., Chabane, N., Leboyer, M., Delorme, R., Jenike, M. A., & Pauls, D. L. (2007). Association of the SLC1A1 glutamate transporter gene and obsessivecompulsive disorder. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics, 144B(8), 1027–1033. https://doi.org/10.1002/ajmg.b.30533

33.

Ting, J. T., & Feng, G. (2008). Glutamatergic Synaptic Dysfunction and Obsessive-Compulsive Disorder. Current Chemical Genomics, 2, 62–75. https://doi.org/10.2174/1875397300802010062

34.

Welch, J. M., Lu, J., Rodriguiz, R. M., Trotta, N. C., Peca, J., Ding, J.-D., Feliciano, C., Chen, M., Adams, J. P., Luo, J., Dudek, S. M., Weinberg, R. J., Calakos, N., Wetsel, W. C., & Feng, G. (2007). Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3 -mutant mice. Nature, 448(7156), 894–900. https://doi.org/10.1038/nature06104

35.

Zike, I. D., Chohan, M. O., Kopelman, J. M., Krasnow, E. N., Flicker, D., Nautiyal, K. M., Bubser, M., Kellendonk, C., Jones, C. K., Stanwood, G., Tanaka, K. F., Moore, H., Ahmari, S. E., & Veenstra-VanderWeele, J. (2017). OCD candidate gene SLC1A1/ EAAT3 impacts basal ganglia-mediated activity and stereotypic behavior. Proceedings of the National Academy of Sciences, 114(22), 5719–5724. https://doi.org/10.1073/pnas.1701736114

34

An Express Train to the Diseased Brain: RVG-modified Exosomes for the Treatment of Alzheimer’s Disease Yoobin Cho

Rabies Virus Glycoprotein (RVG) modification has been used successfully to target exosome delivery to the brain in various studies on neurodegenerative diseases. This technique has not yet been implemented with mesenchymal stem cell (MSCs) transplantation, a method known to be effective in Alzheimer’s disease (AD) symptoms. In a study by Cui et al., MSC-derived exosomes conjugated with RVG-peptide are delivered intravenously to APP/PS1 mice. A significant improvement in cognitive function is seen with this treatment compared to standard MSC-exosome injections and saline injections, as well as the decreasing of neuroinflammatory factors, Aß accumulation, and astrocytic activation. This novel technique provides guidance for future directions for stem cell derived exosome research for Alzheimer’s Disease.

Key words: Alzheimer’s Disease, rabies viral glycoprotein (RVG), mesenchymal stem cells, neuroinflammation, cognitive impairment, modified exosomes

35

Background and Introduction

modified MSC-derived exosomes (Haney et al. 2015), CNStargeted engraftment of exosomes significantly increases with Alzheimer’s disease (AD) remains the most common the protein modification. cause of dementia despite the extensive research in progress to b) find treatments for its irreversible impacts on cognitive func- a) tion. Although difficult to diagnose at early stages, the most important features of the disease are widely accepted to be βamyloid plaque deposition, and neuroinflammation (Weller and Budson 2018).

Many questions remain unanswered in AD research and a recent interest involves the use of stem cell derived exosomes as a potential treatment method. Although the ability of brain-derived exosomes to cross the blood-brain-barrier have led for them to be regarded as an important biomarker and cause of neurodegeneration in AD (Jiang et al. 2019; Cai et al.

2018; Counil and Krantic 2020), the transplantation of MSCderived exosomes from bone marrow or adipose tissue have been found to improve some Alzheimer’s symptoms in mouse models (Cui et al. 2019; Lee et al. 2012). Although the mechanisms behind this phenomenon are still under investigation, it is known that MSC-derived exosomes are crucial players in intercellular communication and that they transfer factors that promote neural stem cell proliferation as well as Aβ-degrading enzymes like neprilysin (Reza-Zaldivar et al. 2018). In addition, MSC-derived exosomes have shown to decrease neuroinflammation by balancing inflammatory factors and by modulating microglial activation (Ding et al. 2018).

Previous experiments have expressed difficulties when delivering MSC-exosomes to the brain and have noted accumulation in the spleen and liver, rather than in the CNS. Fortunately, neuron-targeted delivery of exosomes and other cellular components has been enhanced successfully in the past few decades by conjugating them with the CNS-specific RGV peptide (Alvarez-Erviti et al. 2011; J.-Y. Kim et al. 2013). A novel method of brain-targeted delivery of MSC-derived exosomes using the RVG peptide is presented by Cui et al. (2019). After peptid modification via DOPE-NHS linker, double-transgenic APP/PS1 (B5C3-Tg 85Dbo/J) mice in three experimental groups (MSC-RGV-Exo, MSC-Exo, saline) were injected monthly with their respective treatments for 4 consecutive months. Significant improvements in exosome engraftment to the CNS were seen with the MSC-RGV-Exo injections compared to the standard MSC-exosome treatment. The Alzheimer’s pathological outcome was also significantly reduced in the MSC-RGV-Exo group compared to the other groups, resulting in improved balancing of inflammatory factors, decreased ß-amyloid plaque deposition, decreased astrocyte activation, as well as improved cognitive function. Modification of exosomes with the RVG peptide may be a promising field of study in Alzheimer’s disease for future drug research.

Fig 1. Molecular changes in the brains of APP/PS1 mice after respective group treatment. a) Engraftment of MSC-derived exosomes in the brain is enhanced with RVG-modification in the hippocampus and the cortex. b) ß-Amyloid plaque deposition and accumulation is most reduced with MSC-RVG-Exo treatment.

Cui, Guo-Hong, Hai-Dong Guo, Han Li, Yu Zhai, Zhang-Bin Gong, Jing Wu, Jian-Sheng Liu, You-Rong Dong, Shuang-Xing Hou, and Jian-Ren Liu. 2019. “RVG-Modified Exosomes Derived from Mesenchymal Stem Cells Rescue Memory Deficits

by Regulating Inflammatory Responses in a Mouse Model of Alzheimer’s Disease.” Immunity & Ageing: I & A 16: 10. https://doi.org/10.1186/s12979-019-0150-2.

As seen in Fig. 1a), the mean fluorescence intensity 5 hours post-injection of the DiI-labeled MSC-exosomes is higher in the MSC-RVG-Exo group compared to the MSC-Exo group.

Fig. 1b shows the Thioflavin-S staining and ELISA analysis which reveals significantly decreased plaque deposition in the cortex as well as decreased soluble Aß40 and Aß42 in both MSC-Exo and MSC-RVG-Exo groups, compared to the saline group. However, the MSC-RVG-Exo group has generally the lowest ß-amyloid accumulation in comparison to both groups.

RVG modification to the delivered exosomes significantly alleviates plaque deposition and ß-amyloid accumulation.

Reduced Astrocyte Activation and Neuroinflammation

Major Results Enhanced Engraftment of Exosomes, Decreased Plaque Deposition and ß-Amyloid Accumulation As expected based on various past studies with RVG36

Fig 2. Decreased GFAP expression with MSC-RVG-Exo injection compared to MSC-Exo or control.

As expected, based on prior cognitive tests post-exosome treatment on APP/PS1 mice, there is an increased number of platform location crosses and time spent in the target quadrant, shown in Fig. 3a). MSC-RVG-Exo injections lead to an even greater improvement in performance in the same task. Fig. 3b) notes that swimming speed is similar in all groups, thus the observed results can be attributed to cognitive functioning rather than differing behavioral and motor skills.

Cui, Guo-Hong, Hai-Dong Guo, Han Li, Yu Zhai, Zhang-Bin Gong, Jing Wu, Jian-Sheng Liu, You-Rong Dong, Shuang-Xing Hou, and Jian-Ren Liu. 2019. “RVG-Modified Exosomes Derived from Mesenchymal Stem Cells Rescue Memory Deficits

by Regulating Inflammatory Responses in a Mouse Model of The immunofluorescence images in Fig. 2 shows the expression of GFAP, an astrocytic marker in the brain. GFAP expression is low in the MSC-Exo group but lowest in the MSCConclusions/Discussions RVG-Exo group, and highest in the saline-injected group. The decreased astrocytic marker in the RVG-modified treatment The results suggest that RVG modification is an effecsuggests that the peptide contributes to alleviating overactiva- tive method for improving MSC-derived exosome delivery to tion of astrocytes, a common pathological outcome in AD. the CNS, enhancing the decrease of AD-like symptoms in mice.

The brain-targeted exosomes improve learning and memory, Both MSC-RVG-Exo and MSC-Exo groups support rereduce Aß accumulation and the activation of glial cells, and cent findings which suggest the alleviation of neuroinflammabalance neuroinflammatory factors, each at significantly greattion by the injection of MSC-derived exosomes (Ding et al. er levels than an unmodified MSC-exosome treatment. Alt2018). qRT-PCR shows that pro-inflammatory mediators such as hough various peptides, ligands, and receptor-specific antibodTNFalpha, IL-B, and IL6 are reduced in both MSC-RVG-Exo and ies have previously been used successfully to target the CNS via MSC-RVG groups compared to saline group, but INFalpha and conjugation with the RVG peptide (Kim et al. 2013; AlvarezIL1ß are reduced in the MSC-RVG-Exo group only. The MSCErviti et al. 2011), MSC-derived exosomes have not yet been RVG-Exo injections also significantly decrease IL10, IL4, and IL13 tested with this method. Cui et al. successfully implements the compared to both other groups. Even in comparison to the altechnique to improve AD-like symptoms in APP/PS1 mice. ready effective MSC-Exo treatment, the injections of RVGmodified exosomes lead to a significantly enhanced balance of The authors express the importance of researching AD inflammatory factors, with a comparatively greater decrease in therapeutics using MSC-derived exosomes specifically, as they pro-inflammatory factors and greater increase in anti- have not only been known to produce exosomes in a larger inflammatory factors. scale but they also contain active NEP, an enzyme whose presence suggests the mechanism for how MSC-exosomes can decrease ß-amyloid levels in AD-affected individuals (Katsuda et Improved Cognitive Function al. 2013).

Spatial learning and memory, assessed via Morris WaThe results presented by Cui et al. contribute to the ter Maze tests, is dramatically impaired in saline-injected mice existing literature on stem-cell derived exosome delivery and compared to the other groups. its growing potential in AD treatment. The findings replicate the current understanding of the advantages of MSC-derived exoa) some injections, such as improved cognitive function and regub) lation of inflammatory factors (Cui et al. 2017; de Godoy et al.

2018; Reza-Zaldivar et al. 2018). The effectiveness of this known treatment is made clear by the inclusion of the nonconjugated MSC-Exo group to compare with the saline control group. The accumulation of MSC-exosomes in the liver and spleen has been a constant concern in this field of research (Harrell et al. 2019) and the conjugation of the RVG peptide to Fig 3. Improved Morris Water Maze Test results with RVG- the exosomes is a novel and successful method for CNSmodification of delivered exosomes. a) Increased time spent in targeted exosome delivery. The treatment is proven effective target quadrant with MSC-RVG-Exo treatment. b) Improved by comparing the conjugated MSC-RVG-Exo group with the learning and memory is attributed to improved cognitive pro- unconjugated MSC-Exo group. cesses as swimming speed and behavior remains constant across treatment groups.

Critical Analysis Cui, Guo-Hong, Hai-Dong Guo, Han Li, Yu Zhai, Zhang-Bin Gong, Like most preceding studies, the authors cannot comJing Wu, Jian-Sheng Liu, You-Rong Dong, Shuang-Xing Hou, and Jian-Ren Liu. 2019. “RVG-Modified Exosomes Derived from Mes- ment on whether or not their findings could be adapted for enchymal Stem Cells Rescue Memory Deficits by Regulating In- human use. The study provides direction for future experiflammatory Responses in a Mouse Model of Alzheimer’s Dis- ments involving RVG-peptide modifications and MSC-derived ease.” Immunity & Ageing: I & A 16: 10. https:// exosomes, but it is unable to provide information on whether or not it is dosage-dependent, its potential side-effects, or if it doi.org/10.1186/s12979-019-0150-2.

37

is potentially applicable to human individuals with Alzheimer’s disease. The use of a singular transgenic mouse type (APP/PS1) is a limitation as this model is known to not show neuronal loss (Arendash and King 2002). To yield results generally conclusive for AD, pathological characteristics as important as neuronal loss should not be overlooked. It is also unjustified to accept claims based on cognitive improvement observed via one cognitive test. As many other aspects of cognition such as working memory and memory recall are impaired in AD (Kelley and Petersen 2007), the inclusion of other assessments to test them may have helped solidify the conclusions.

mouse models with other AD pathologies may help alleviate the limitations that using only one model may hold. For example, since the APP/PS1 model does not express neuronal loss as seen in normal Alzheimer’s pathology, the rTg(tauP301L) 4510 model can be observed to compare the effects of MSC-RVG-Exo on its neuronal loss from the previously observed pathologies.

Similar results would confirm that the RVG-modified exosomes have an effect on multiple AD pathological outcomes, not just the ones testable by the APP/PS1 model (Spires et al. 2006). If engraftment or AD outcomes in the rTg 4510 model differ significantly from the APP/PS1 model, the results by Cui et al.

(2019) may not propose that RVG-Exo is generally effective on mouse AD models, but instead only specifically on the pathology of the APP/PS1 model. It may raise important questions inquiring the true effectiveness of MSC-derived exosomes and RVG-modification as a treatment for AD. Future experiments using different mouse models should also include additional methods of testing for improvements in AD pathology after exosome treatment. For example, object recognition tests for working memory, changes in neurotransmission, and changes in neuronal loss (Sasaguri et al. 2017) are all assessments that may yield more compelling evidence.

The techniques used to assess the effects of RVGexosomes such as immunofluorescence and ELISA were effective in showing that modified exosome delivery yielded the most favorable results in decreasing plaque deposition, astrocyte activation, and neuroinflammation. However, other important molecular markers in AD were not included in the experiment; the dissemination of tau and late endosome markers, increased activation of microglia and regulatory RNA, and many other biomarkers have previously been shown to be affected by MSC-derived exosome treatments (Reza-Zaldivar et al. 2018). Such details are necessary in an exploration of a novel technique which could potentially be used in future treatments Cui et al. make important advances in generating a for neurodegenerative diseases. treatment for the currently incurable Alzheimer’s disease, with the conjugation of RVG-peptide to MSC-derived exosomes. The Nonetheless, the study presents an original method to results drawn from the study provide suggestions for future use target potential AD treatments directly to the CNS in transgenic of exosome modification techniques for targeted exosome deAPP/PS1 mice. It provides a pathway for future research to be livery to brains affected by neurodegenerative diseases. conducted on protein modification of MSC-derived exosomes.

Future Directions Although the results provide robust evidence of the enhancement of exosome targeting by the CNS-specific RVG peptide, further improvements to the potential treatment should be made as steps towards the development of future implications of their findings on humans.

To provide further support leading up to clinical trials, an additional experiment to be conducted by Cui et al.’s group may be to separate the MSC-RVG-Exo treatment group into dosage groups to analyze the optimal dosage of the injections on AD pathology and exosome engraftment. The dosage may vary in frequency (eg. bi-weekly injections, rather than monthly injections) or in concentration. For example, the groups in the experiment may include mice injected with a varying and specific dosage of MSC-RVG-Exo, while another group receives a non-conjugated MSC-Exo injection. A saline-injected control group should also be included for comparison. It is expected, based on previous findings by Venugopal et al. (2017), that a lower dosage of MSC-RVG-Exo would yield a greater effect on AD recovery, such as improved cognitive function, while higher dosages would be detrimental. Differing results would suggest that improved AD pathology by the delivery of MSC-RVG exosomes is either not dosage dependent or that higher dosages may be beneficial with this particular treatment.

Similar experiments conducted on other transgenic 38

REFRENCES 1.

Alvarez-Erviti, Lydia, Yiqi Seow, Haifang Yin, Corinne Betts, Samira Lakhal, and Matthew J. A. Wood. 2011. “Delivery of SiRNA to the Mouse Brain by Systemic Injection of Targeted Exosomes.” Nature Biotechnology 29 (4): 341–45. https:// doi.org/10.1038/nbt.1807.

2.

Arendash, Gary W., and David L. King. 2002. “Intra- and Intertask Relationships in a Behavioral Test Battery given to Tg2576 Transgenic Mice and Controls.” Physiology & Behavior 75 (5): 643–52. https://doi.org/10.1016/s0031-9384(02) 00640-6.

3.

Cai, Zhi-You, Ming Xiao, Sohel H. Quazi, and Zun-Yu Ke. 2018. “Exosomes: A Novel Therapeutic Target for Alzheimer’s Disease?” Neural Regeneration Research 13 (5): 930–35. https://doi.org/10.4103/1673-5374.232490.

4.

Counil, Hermine, and Slavica Krantic. 2020. “Synaptic Activity and (Neuro)Inflammation in Alzheimer’s Disease: Could Exosomes Be an Additional Link?” Journal of Alzheimer’s Disease 74 (4): 1029–43. https://doi.org/10.3233/JAD-191237.

5.

Cui, Guo-Hong, Hai-Dong Guo, Han Li, Yu Zhai, Zhang-Bin Gong, Jing Wu, Jian-Sheng Liu, You-Rong Dong, Shuang-Xing Hou, and Jian-Ren Liu. 2019. “RVG-Modified Exosomes Derived from Mesenchymal Stem Cells Rescue Memory Deficits by Regulating Inflammatory Responses in a Mouse Model of Alzheimer’s Disease.” Immunity & Ageing: I & A 16: 10. https:// doi.org/10.1186/s12979-019-0150-2.

6.

Cui, Guo-Hong, Jing Wu, Fang-Fang Mou, Wei-Hua Xie, Fu-Bo Wang, Qiang-Li Wang, Jie Fang, et al. 2017. “Exosomes Derived from Hypoxia-Preconditioned Mesenchymal Stromal Cells Ameliorate Cognitive Decline by Rescuing Synaptic Dysfunction and Regulating Inflammatory Responses in APP/PS1 Mice.” The FASEB Journal 32 (2): 654–68. https:// doi.org/10.1096/fj.201700600R.

7.

Ding, Mao, Yang Shen, Ping Wang, Zhaohong Xie, Shunliang Xu, ZhengYu Zhu, Yun Wang, et al. 2018. “Exosomes Isolated From Human Umbilical Cord Mesenchymal Stem Cells Alleviate Neuroinflammation and Reduce Amyloid-Beta Deposition

by Modulating Microglial Activation in Alzheimer’s Disease.” Neurochemical Research 43 (11): 2165–77. https:// doi.org/10.1007/s11064-018-2641-5.

8.

Godoy, Mariana A. de, Leonardo M. Saraiva, Luiza R. P. de Carvalho, Andreia Vasconcelos-dos-Santos, Hellen J. V. Beiral, Alane Bernardo Ramos, Livian R. de Paula Silva, et al. 2018. “Mesenchymal Stem Cells and Cell-Derived Extracellular Vesicles Protect Hippocampal Neurons from Oxidative Stress and Synapse Damage Induced by Amyloid-β Oligomers.” Journal of Biological Chemistry 293 (6): 1957–75. https://doi.org/10.1074/jbc.M117.807180.

9.

Haney, Matthew J., Natalia L. Klyachko, Yuling Zhao, Richa Gupta, Evgeniya G. Plotnikova, Zhijian He, Tejash Patel, et al.

2015. “Exosomes as Drug Delivery Vehicles for Parkinson’s Disease Therapy.” Journal of Controlled Release: Official Journal of the Controlled Release Society 207 (June): 18–30. https://doi.org/10.1016/j.jconrel.2015.03.033.

10.

Harrell, Carl Randall, Nemanja Jovicic, Valentin Djonov, Nebojsa Arsenijevic, and Vladislav Volarevic. 2019. “Mesenchymal Stem Cell-Derived Exosomes and Other Extracellular Vesicles as New Remedies in the Therapy of Inflammatory Diseases.”

Cells 8 (12). https://doi.org/10.3390/cells8121605.

11.

Jiang, Liqun, Huijie Dong, Hua Cao, Xiaofei Ji, Siyu Luan, and Jing Liu. 2019. “Exosomes in Pathogenesis, Diagnosis, and Treatment of Alzheimer’s Disease.” Medical Science Monitor: International Medical Journal of Experimental and Clinical Research 25 (May): 3329–35. https://doi.org/10.12659/MSM.914027.

12.

Katsuda, Takeshi, Reiko Tsuchiya, Nobuyoshi Kosaka, Yusuke Yoshioka, Kentaro Takagaki, Katsuyuki Oki, Fumitaka Takeshita, Yasuyuki Sakai, Masahiko Kuroda, and Takahiro Ochiya. 2013. “Human Adipose Tissue-Derived Mesenchymal Stem Cells Secrete Functional Neprilysin-Bound Exosomes.” Scientific Reports 3 (1): 1197. https://doi.org/10.1038/ srep01197.

13.

Kelley, Brendan J., and Ronald C. Petersen. 2007. “Alzheimer’s Disease and Mild Cognitive Impairment.” Neurologic Clinics 25 (3): 577–v. https://doi.org/10.1016/j.ncl.2007.03.008.

14.

Kim, Ja-Young, Won Il Choi, Young Ha Kim, and Giyoong Tae. 2013. “Brain-Targeted Delivery of Protein Using Chitosanand RVG Peptide-Conjugated, Pluronic-Based Nano-Carrier.” Biomaterials 34 (4): 1170–78. https://doi.org/10.1016/ j.biomaterials.2012.09.047.

15.

Lee, Hyun Ju, Jong Kil Lee, Hyun Lee, Janet E. Carter, Jong Wook Chang, Wonil Oh, Yoon Sun Yang, et al. 2012. “Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Improve Neuropathology and Cognitive Impairment in an Alzheimer’s Disease Mouse Model through Modulation of Neuroinflammation.” Neurobiology of Aging 33 (3): 588–602. https:// doi.org/10.1016/j.neurobiolaging.2010.03.024.

39

16.

Reza-Zaldivar, Edwin E., Mercedes A. Hernández-Sapiéns, Benito Minjarez, Yanet K. Gutiérrez-Mercado, Ana L. MárquezAguirre, and Alejandro A. Canales-Aguirre. 2018. “Potential Effects of MSC-Derived Exosomes in Neuroplasticity in Alzheimer’s Disease.” Frontiers in Cellular Neuroscience 12. https://doi.org/10.3389/fncel.2018.00317.

17.

Sasaguri, Hiroki, Per Nilsson, Shoko Hashimoto, Kenichi Nagata, Takashi Saito, Bart De Strooper, John Hardy, Robert Vassar, Bengt Winblad, and Takaomi C Saido. 2017. “APP Mouse Models for Alzheimer’s Disease Preclinical Studies.” The EMBO Journal 36 (17): 2473–87. https://doi.org/10.15252/embj.201797397.

18.

Spires, Tara L., Jennifer D. Orne, Karen SantaCruz, Rose Pitstick, George A. Carlson, Karen H. Ashe, and Bradley T. Hyman.

2006. “Region-Specific Dissociation of Neuronal Loss and Neurofibrillary Pathology in a Mouse Model of Tauopathy.” The American Journal of Pathology 168 (5): 1598–1607. https://doi.org/10.2353/ajpath.2006.050840.

19.

Venugopal, Chaitra, Christopher Shamir, Sivapriya Senthilkumar, Janitri Venkatachala Babu, Peedikayil Kurien Sonu, Kusum Jain Nishtha, Kiranmai S. Rai, Shobha K, and Anandh Dhanushkodi. 2017. “Dosage and Passage Dependent Neuroprotective Effects of Exosomes Derived from Rat Bone Marrow Mesenchymal Stem Cells: An In Vitro Analysis.” Current Gene Therapy 17 (5): 379–90. https://doi.org/10.2174/1566523218666180125091952.

20.

Weller, Jason, and Andrew Budson. 2018. “Current Understanding of Alzheimer’s Disease Diagnosis and Treatment.”

F1000Research 7 (July). https://doi.org/10.12688/f1000research.14506.1.

40

Injection of beta amyloid brain extract intravenously is shown to induce Alzheimer’s Like Disease in APPSwe/PS1dE9 mice –A Critical Review Nikol Digtyar

Alzheimer’s Disease is one of the most common neurodegenerative diseases. There has been much significant research in the field to determine whether Alzheimer’s Disease could be a transmissible disease. The spread of Alzheimer’s disease has been linked to seeding of β-amyloid protein plaques in the brain. However, the studies which were completed injected Human Alzheimer’s disease brain extracts intracerebrally into the hypothalamus and other corresponding brain regions. The following study was completed on transgenic APPSwe/PS1dE9 mice. There were three groups in the experiment.

A control group was injected with brain extracts from a healthy male adult (HTC), the experimental groups were injected with brain extracts from two male human Alzheimer’s Disease patients (AD1, AD2). Both experimental groups were additionally subdivided. One subdivision was injected with the brain extract intracerebrally and the other was injected intravenously. Mice were then sacrificed 180, 270, and 360 days post-injection. The brain was then extracted and placed in formaldehyde and embedded in wax. Several histological analyses were completed on the tissue samples. β -amyloid was detected using anti-Abeta 4G8 antibody. The results of this study demonstrate that 180 days post intravenous injection of AD1 brain extract, mice began to develop β-amyloid plaques in the vasculature of the thalamus. This suggests that the β-amyloid brain extract was able to bypass the blood-brain barrier.

The intracerebrally injected mice also showed similar results 360 days post injection.

41

with C57BL/6 mice to produce heterozygous mice. APPswe/ PS1dE9 mice overexpress APP and have a mutated PS1 gene which contributes to Alzheimer’s like disease development (Malm T et. al. 2011). C57BL/6 are wild-type normal mice commonly used in a laboratory setting. These mice were injected with AD brain extracts both intracerebrally and intravenously.

Their results showed that the mice injected intravenously had similar results to the mice injected with the intracerebral method. Furthermore, they showed that even at 180 days post intravenously injected the mice had β -amyloid plaques in the brain (Burwinkel M et. al. 2018). This is a new outlook on Alzheimer’s disease and the first paper to show that β -amyloid injected intravenously could enter the blood-brain barrier and include Figure 1:A visual representation of the methods and major findAlzheimer’s like disease in mice. ings of the research study.

Introduction:

Major Results:

Alzheimer’s Disease (AD) is a lethal neurodegenerative disorder which is characterized by severe dementia. Despite the vast scientific leaps in Alzheimer's research, there are still many unknowns and no definitive treatment nor cure. There have been precursor genes identified such as the amyloid precursor protein APP and others which increase the chances of acquiring AD (Lane, C., Hardy et. al. 2017). Other factors can also contribute to the development of Alzheimer’s Disease; however, they are not well understood or definitive. Alzheimer’s is a disease in which β-amyloid plaques and Lewy bodies accumulate within the areas of the brain responsible for memory such as the hippocampus (Lane, C., Hardy et. al. 2017).

The disease slowly spreads throughout the brain causing atrophy, neuronal death, and is fatal. There is a seeding phenomenon that occurs in AD. Seeding is the deposition or “infection” of surrounding brain areas with the β -amyloid protein (Lane, C., Hardy et. al. 2017). There is still some debate in the scientific community concerning the notion of Alzheimer’s Disease transmission and the cascade of events which occur once amyloid plaques begin to accumulate. There has been research done in the past which consisted of injecting mice with brain extracts directly acquired from AD patients. The results of these numerous studies were varied based on their methods and criteria.

The mice used for these experiments are generally transgenic amyloid precursor protein mice APP23 or tg2576. These mice overexpress the β -amyloid protein gene and with age acquire an increasing amount of beta-amyloid plaques in the brain. A study conducted by Michael D. Kane et. al. (2000) showed that intracerebral injection of brain extracts from AD patients did cause an accumulation of β -amyloid plaques (Kane MD et. al.

2000). Which does support the notion that seeding could significantly increase the chances of developing AD. Other studies have also been done on these mice with oral, intranasal, and intraocular administration of β –amyloid extracts. In these studies, only intracranial administration of AD brain extracts induced the development of β- amyloid plaques (Eisele YS et. al.

2009). A more recent study completed by Michael Burwinkel et. al. 2018 used a different approach to test the hypothesis that intravenous administration of AD brain extracts would be sufficient to induce Alzheimer’s like disease in mice (Burwinkel M. et. al. 2018). This study uses APPswe/PS1dE9 mice crossbred

The histological study results for the following study showed that for both the experimental and the control group, mice developed β-amyloid plaques 360 days post-injection of the extract in the intracerebrally injected mice (Burwinkel M et. al. 2018). These findings are consistent with the transgenic mouse line which is predisposed to develop Alzheimer’s Disease with age.

However, the AD1, AD2 mice showed the deposition of β-amyloid plaques in the vasculature of the thalamus (Figure 2).

This was not observed in HTC mice which were injected with brain extract from a non-AD patient. The methodology of injecting the extract directly into the brain has been tested in other studies. In a 2000 study conducted by Michael D. Kane researchers injected βAPP transgenic mice with brain extracts from human patients with AD, unilaterally into the neocortex and hippocampus ( Kane MD et. al. 2000). Their results revealed that 5 months post-injection, mice developed β–amyloid plaques in the injected areas as well as some of the vasculature. Therefore, the findings from the study done by Michael Burwinkel et. al. 2018 are consistent with the results of the previous research.

A key finding of the 2018 study however is the result of injecting the mice intravenously using the tail vein. The results for the intravenous injection of brain extracts of both AD1 and AD2 showed a substantially higher amount of plaques located in the vasculature of the thalamus when compared to the control at 180 days post-injection (Figure 3). They also found increased pathology in the cortices in the intravenous group.

These results show that there is a significantly higher amount of β-amyloid plaques in mice even 180 days post-injection when compared to the intracerebral group at 360 days. However, both the hippocampal and cortical regions both at 180 and 270 days post-injection did not show significant amyloid angiopathy.

42

component of Alzheimer’s disease. With further understanding and research, the information uncovered in this study could contribute to preventing the transmission of β-amyloid plaques in human patients.

Critical Analysis:

Figure 2:A)Intracerebral Injection of HTC (non-AD extract) hippocampus and thalamus 360 days after injection. B) Intracerebral Injection of AD1 (AD + extract after injection). C)&D) Increased magnification showing the deposition in the vasculature of the thalamus (Burwinkel M. et. al 2018).

The study uncovered some new concepts concerning the transmission of β-amyloid in mice models. In order to fully understand these findings, further research needs to be completed. The intravenous injection did result in β -amyloid plaques in the vasculature of the brain however it is poorly understood how and why this occurred. A question that the authors should investigate is how and why the human β -amyloid brain extract was able to pass through the blood-brain barrier, which has not been observed before. A research paper conducted by Eisele YS et. al. 2009 showed that oral, intravenous, intraocular, and intranasal administration of human AD brain extract did not show angiopathy or plaques within the brain.

This is completely controversial to the study completed in 2018. However, the 2009 study used APP23 transgenic mice while the 2018 study used APPSwe/PS1dE9, C57BL/6 crossbred mice.

There are various animal models of human neurodegenerative diseases and the differences in the altered genes of the mice can lead to different results in studies.

In the APP23 mouse model, the APP gene is the amyloid precursor protein which is composed of β-amyloid and an amino acid peptide (Van Dan D et. al. 2005). This is an autosomal dominant mutation that is identified in some Alzheimer's disease families with early-onset pathologies (Van Dan D et. al.

2005). This mouse model is neuron-specific and expresses a promoter which leads to overexpression of a human β-amyloid precursor (Van Dan D et. al. 2005). These mice are usually bred Conclusion/ Discussion: with C7 wild-type mice to achieve a heterozygous transgenic The major findings of the study supported previous mice model. As the mice age they do develop β-amyloid studies on intracerebral administration of AD brain extracts. plaques in the hippocampus and neocortex (Van Dan D et. al.

The researchers found increased amyloid angiopathy in the 2005). injected areas of the brain and the surrounding vasculature.

On the contrary, the APPSwe/PS1dE9 mouse model The findings exclusive to this study showed that intravenous includes the APP mutation as well as a PS-1 mutation. PS1 is a injection also induced amyloid angiopathy in the thalamus and protease catalyst and when mutated or inhibited can increase vasculature of the brain. This was a key finding of the study the development of β-amyloid plaques (Malm T et. al. 2011). In which has not been observed before. A study was conducted in addition, this mouse model is not neuron-specific and can 2009 by Yvonne S Eisele et. al. in which researchers adminisaffect the CNS and allow for β-amyloid accumulation in other tered β -amyloid extracts orally, intravenously, intraocularly, areas of the brain such as the parenchyma of the brain and the and intranasally (Eisele YS et. al. 2009). Their results showed no vasculature (Malm T et. al. 2011). Research has also shown that β-amyloidosis in the mice. However, this study used the APP23 this mice model does not spontaneously develop β-amyloid transgenic mice in comparison to the crossbred APPSwe/ plaques within the brain even at 30 months of age (Prado, M. PS1dE9, C57BL/6 mice which did yield results in the 2018 study.

A., Baron, G. 2012). Intracerebral injection of β -amyloid in The results of the study are especially significant because bethese mice showed the acceleration of amyloidosis in the brain fore this time other studies were unable to demonstrate the in other studies (Rosen, R. F. et. al. 2012). effects of intravenous injection. The study shows that βamyloid extracts can pass through the blood-brain barrier and Therefore, this may be the reason for the lack of results and transmit to the vasculature of the CNS in the brain. These find- evidence of intravenous injections affecting the formation of β ings could aid researchers in understanding the transmissible amyloid plaques in the brain in past studies.

Figure 3:A) Intravenous Injection of HTC (non-AD extract) control, hippocampus and thalamus at 180 days after injection. B) Intravenous Injection of AD (AD + extract) at 180 days after injection. C) Thalamus after AD injection with higher magnification D) Thalamus after AD injection higher magnification.

(Burwinkel M. et. al 2018).

43

Future Directions: A possible future direction and experiment which could be explored by future researchers is to determine the cascade of events that occurs after a venous injection of the AD brain extract is administered. Viral GFP tagging of the β amyloid could be used to determine which pathways the transmission of β -amyloid extract is taking to reach the CNS and the vasculature of the brain. Cultures of the AD brain extracts can be transfected and combined with fluorescent GFP in culture and then be administered intravenously to the test animals.

Imaging techniques and other histological studies could also be used to track the progression and the path that the extract takes, from entering the vein of the tail to crossing the bloodbrain barrier. Instead of sacrificing animals 180 days postinjection, histological studies can be done earlier on nerves and vasculature of the mice near the injection site to determine that path the β-amyloid takes prior to reaching the brain. In particular, it is important to understand how β-amyloid is able to pass the blood-brain barrier and reach the brain vasculature only 180 days post-injection into the tail. It is difficult to hypothesize what results such experiments would lead to. This is due to the notion that this discovery is fairly recent and would be particularly difficult to track, and infer by which path this particular prion-like molecule would take to reach the CNS and the brain. Future research should focus on determining this pathway and how it is able to go through the blood-brain barrier, as this knowledge would provide great insight into understanding the underlying aspects of β -amyloid plaques and their destruction of brain areas. With this knowledge, possible treatment for prevention and isolation of transmission from one area of the brain to the surrounding regions could be obtained.

With newer technology being invented finding a treatment or cure could be possible in the future.

44

REFRENCES 1.

Burwinkel M, Lutzenberger M, Heppner FL, Schulz-Schaeffer W, Baier M. Intravenous injection of beta-amyloid seeds promotes cerebral amyloid angiopathy (CAA). Acta Neuropathol Commun. 2018;6(1):23. Published 2018 Mar 5. doi:10.1186/s40478-018 0511-7

2.

Eisele YS, Bolmont T, Heikenwalder M, et al. Induction of cerebral beta-amyloidosis: intracerebral versus systemic Abeta inoculation. Proc Natl Acad Sci U S A. 2009;106(31):12926-12931. doi:10.1073/pnas.0903200106

3.

Kane MD, Lipinski WJ, Callahan MJ, et al. Evidence for seeding of beta -amyloid by intracerebral infusion of Alzheimer brain extracts in beta -amyloid precursor protein transgenic mice. J Neurosci. 2000;20(10):3606-3611. doi:10.1523/ JNEUROSCI.20-10 03606.2000

4.

Lane, C., Hardy, J., & Schott, J. (2017, October 19). Alzheimer's disease. Retrieved June 15, 2020, from https:// onlinelibrary.wiley.com/doi/full/10.1111/ene.13439

5.

Malm, T., Koistinaho, J., & Kanninen, K. (2011). Utilization of APPswe/PS1dE9 Transgenic Mice in Research of Alzheimer's Disease: Focus on Gene Therapy and Cell-Based Therapy Applications. International journal of Alzheimer's disease, 2011, 517160. https://doi.org/10.4061/2011/517160

6.

Van Dam, D., Vloeberghs, E., Abramowski, D., Staufenbiel, M., & De Deyn, P. P. (2005).

APP23 mice as a model of Alzheimer's disease: an example of a transgenic approach to modeling a CNS disorder. CNS spectrums, 10(3), 207– 222.https://doi.org/10.1017/s1092852900010051

7.

Prado, M. A., & Baron, G. (2012). Seeding plaques in Alzheimer's disease. Journal of neurochemistry, https://doi.org/10.1111/j.1471-4159.2011.07574.x

8.

Rosen, R. F., Fritz, J. J., Dooyema, J., Cintron, A. F., Hamaguchi, T., Lah, J. J., LeVine, H., 3rd, Jucker, M., & Walker, L. C. (2012). Exogenous seeding of cerebral β-amyloid deposition in βAPP-transgenic rats. Journal of neurochemistry, 120(5), 660–666.https://doi.org/10.1111/j.1471-4159.2011.07551.x

9.

Zhou, J., & Liu, B. (2013). Alzheimer's disease and prion protein. Intractable & rare diseases https://doi.org/10.5582/irdr.2013.v2.2.35

45

120(5),

641–643.

research, 2(2), 35–44.

Effect of germ-free mice on monoamine neurotransmitter gene expression and anxiety-like behavior; further evidence of the gut-brain connection.

Alexander Geiger

Studies have demonstrated gut microbiota are important for cognitive health, as revealed when gut microbiome balance is disrupted. Neurological, behavioral, and mental health issues can result. Questions arise as to what specific effects an abnormal microbiome have on the brain and, importantly, whether those effects can be reversed. The current study, represented by the visual abstract below, examined hippocampal monoamine neurotransmitter gene expression in mice with absent gut microbiota, and observed behavioral aspects, compared to mice with more normal gut microbiota (Pan et al.

2019). The researchers chose monoamine neurotransmitters as a promising biological indicator due to several previous studies linking this system to neuropsychiatric disorders. They compared Germ Free (GF) mice to Specific-Pathogen-Free (SPF) mice and found that 19 monoamine genes were expressed at different levels in GF mice. Additionally, GF mice portrayed much less anxiety than SPF mice, as measured using the open field test (OFT) and the novelty suppressed feeding test (NSF).

The GF mice displayed higher locomotor activity and reduced latency for feeding compared to SPF mice. Finally, the researchers attempted to re-colonize gut microbiota in a set of GF mice (CGF) to reverse the effects of differential gene expression. However, CGF mice continued to show less anxiety compared with SPF mice, and only 50% of the monoamine neurotransmitter genes were successfully expressed to the same levels as SPF mice.

Keywords: gut microbiota, gut-brain axis, monoamine neurotransmitters, GF/SPF mice, hippocampus

46

Major Results

Monoamine Gene Expression

Background and Introduction Studies on gut microbiota and their connection to the brain include such areas as mental illness (Misiak et al. 2020), obesity and inflammation (Soto et al. 2018), and neural-related behavior (Huo et al. 2017). Understanding the neurological and behavioral impact of biological processes is a key goal, making these studies truly relevant to neuroscience. The gut microbiome can affect the brain commensally, but also may be the culprit behind neurological disorders. Misiak et al. (2020) have accumulated a body of evidence from studies that link dysregulation of the hypothalamic-pituitary-adrenal axis (HPA), stemming from abnormal gut microbiota, to mood disorders and more severe mental illness. Additionally, the gut microbiota and HPA were linked to anxiety and stress disorders (Frankiensztajn, 2020). The reciprocal path has also been studied related to how neuronal systems affect the gut. In one study, it was shown that altered serotonin levels affected gut microbes which in turn can affect homeostasis, leading to many symptoms including depression (Reigstad, 2015). As complex connections are discovered, there remain questions about the impact gut microbes have on specific areas within the brain what accounts for differences in their behavioral and cognitive health effects. An additional important question is whether the effects can be reversed.

In the current study, Pan et al. (2019) were able to expand upon the gut-brain link and directly correlate hippocampal monoamine neurotransmitter gene expression levels with a lack of a microbiota. They included a neuro-behavioral link by measuring anxiety-like behavior as well. Briefly, the method and materials involved three types of mice split into three groups each. All were tested at around 9 weeks, which were considered to be adults. GF mice were generated and kept in strictly controlled, sterile environments with sterilized water and food. SPF mice were purchased and kept in the same feeding conditions. CGF mice were created from adolescent male GF mice. The recolonization procedure was accomplished by placing them with SPF bedding and fecal matter.

Each of the groups were randomly assigned to one of three separate tests. One group underwent behavioral activity testing in the OFT and NSF tests in a quiet environment, another group was used for western blotting and the third underwent PCR array analysis. This was done in separate groups so that the behavioral testing itself would not affect gene expression testing.

Hippocampal slices were analyzed, and bioinformatics software was used to research genes. Overall, 19 genes in the GF mice were found to be up- or down-regulated differently when compared to the SPF group’s monoamine levels. Upregulated genes with significant differences included 15 genes as seen in Figure 1. The genes that were downregulated included: Fos, Grk6, and Nr4a1. Bioinformatics analysis revealed the most probable function and pathways involving the genes and were categorized into four types as seen in Figure 1. These included dopamine and serotonin receptor, signal transduction, transporters and metabolism and downstream signaling genes. The most effected genes were related to dopaminergic and serotonergic synapses.

Figure 1. Gene expression results of Pan et al. study (2019).

Represents the levels of hippocampal monoamine neurotransmitter genes across the three groups of mice. Graph (A) represents dopamine and serotonin receptor genes. Recolonization was successful only in Htr7 in this category, (B) is transporters & metabolism genes, where no CGF genes successfully recolonized; Graph (C) represents the monoamine neurotransmitters related to signal transduction. Several genes were expressed to the same level as SPF. Graph (D) represents the downstream signaling gene targets.

Behavioral Test Results Locomotor activity and latency in feeding are two measures of anxiety in mice studies. Groups of mice; GF (n=22), and SPF (n=25) were given standard anxiety-index tests in quiet, low stress environment. OFT measures the movement and provides the total distance traveled, as well as time spent in the center cage. For the NSF test, mice were unfed for 24 hours to induce hunger and placed in an open-field novel arena with a single pellet of food. The time it took to leave the edges and eat the pellet was measured. It was found that the GF mice were less anxious, as indicated by more locomotor activity and reduced latency in feeding compared to the SPF mice. These results coincide with another study that compared GF and SPF motor activity under non-stressed and stressed conditions; the

47

non-stressed results of that study also showed less anxiety in campus. They found similar anxiety-like behavioral effects in GF GF mice compared to SPF (Huo et al. 2017). mice, no significant difference in the hippocampus turnover rate of noradrenaline, dopamine, or serotonin, but did find differential gene expression in the hippocampus consistent CGF Results with the current study. The current study expanded upon the previous one by looking at the reversibility of these differences.

In experiments designed to examine reversibility of GF In another study that looked at the bidirectional impact on ineffects, adolescent GF mice were recolonized (CGF mice) by testinal microbes, hormonal levels and behavior in stressed and placing them with SPF fecal matter. Ten of the 19 genes were un-stressed GF and SPF mice, they found similar results in beexpressed at the same level as SPF mice. The OFT and NSF tests havioral tests (Huo et al. 2017). Pan et al. (2019) expanded were administered, and results showed that the CGF mice reupon this study by looking at the hippocampal connection. tained lower anxiety-like behaviors compared to SPF mice, similar to levels of GF mice, as can be seen in Figure 2. These results seem to conflict with a study by Soto et al. (2017), where Critical Analysis effects associated with changes in BDNF and GABA related to high fat diet mice were successfully transferred to GF mice, The methodologies utilized by Pan et al. (2019) alhowever, they used fecal transplant as a method to transfer gut lowed the researchers to target and analyze specific gene activmicrobiota. ity in the hippocampus. Results of varied monoamine gene expressions were able to be visualized and correlated with behavioral effects. This is a complex connection and important area to study given all the potential detrimental effects on mental and general health. Additionally, by looking at behavioral impact and utilizing bioinformatics, a great deal of information was gathered that can be used in future studies.

Though in the current study they found that the recolonization Figure 2: This figure represents results from the behavioral procedure did not result in fully reversing the effects of GF, tests for anxiety. Graph (A) and (B) show the results of OFT, attempting to understand additional aspects helps to advance distance, and time spent in the center, respectively. Graph C research in this area. represents the latency to feed time during the NSF test. CGF mice did not achieve SPF anxiety-like results.

Improvement areas lie first in the behavioral analysis.

Results showed that anxiety-like behaviors were reduced in both CGF and GF mice, but in CGF mice, the NSF test showed Conclusion/Discussions Section increased latency in feeding over GF. The authors should perform an additional behavioral test such as dark-light or elevatPan et al. (2019) concluded with the two main findings. ed zero-maze testing that could be used to further validate that First, a lack of gut microbiota did distinctly affect the dopamindifferences between mice groups are related to anxiety. Addiergic and serotoninergic pathways in the hippocampi of mice as tionally, in this experiment, behavioral tests were conducted in evidenced by differential gene expression. Additionally, the a quiet, stress free environment. The authors concluded that absence of a microbiome resulted in behavioral changes, as the GF mice were less anxious, but this was found to be not were indicated by less anxiety-like activities of GF mice. The significant when GF and SPF mice were put in stress-induced researchers were not able to reverse these effects in adult mice environments (Geng et al. 2020). Stress situations activate the but pointed out that at least 50% of genes were brought to HPA axis which may negate differences in GF and SPF behaviornormalized levels using their method of recolonization. al results. The authors could add the additional condition of The study performed is important because it adds ex- controlled stress to further determine if GF mice are less anxpanded evidence that the gut microbiota effect the brain and ious. provided new insight on specific hippocampal impact of altered There are some additional experiments that the augut microbiome. The authors combined the behavioral aspect thors discuss should be completed. The bioinformatics analysis by including some standard anxiety tests that allowed them to revealed expected pathways and effects. Metabolomic studies show an association between the affected hippocampal mononeed to be completed to validate those pathways are correct. amine neurotransmitter system and behavior. They concluded One other factor the authors could review, as mentioned, is the that their results suggest the absence of early life microbiota method used to recolonize mice, as it might be insufficient to can disrupt the entire monoamine neurotransmitter system, conclude that the effects of GF are not reversible in adult mice. which can cause neuropsychiatric effects.

Fecal transplant is an example alternate method.

The results of this study supported earlier experiFuture Directions Section ments, one of which was by Diaz Heijtz et al. (2011). These researchers looked at neurochemistry changes in the hippoThis study still leaves some unanswered questions, such campus, frontal cortex, and striatum in GF and SPF mice as well as how the absence of gut microbiota changes the brain and as gene expression in five brain regions including the hippo- whether a specific developmental stage of life most adversely 48

effects the ability to colonize gut microbiomes. Pan et al. (2019) studied a single age-group, adult mice, but it has been shown that in humans, microbiota composition changes in stages from birth until a shift to a more adult-like colonization at about 3-5 years (Koenig, 2010). There may be a more susceptible period to recolonization (Rodriguez, 2015). The future experiment should expand to multiple ages and stages of development to determine whether recolonization would be successful at alternate ages and, if so, whether there is an optimal time to reverse the effects of altered microbiota. The study would include GF, CGF, and SPF mice, each with a neonatal group, breast-fed group, and weaning stage group. PCR array analysis and western blotting could be used for analyzing hippocampal gene expression levels along with metabolomic studies as suggested by the authors to validate pathways predicted based on bioinformatics programs.

Additionally, different behavioral measures would need to be administered to better suit the age of the mice, such as in vocalization and movement comparisons. Expected results should show that the effects are reversible at all the earlier stages. Therefore, recolonizing at such a young age should result in normalized behaviors and gene expression levels. The importance of knowing developmental stage-related impact would be beneficial if eventually would lead to testing, intervention, and prevention of neuropsychological effects of abnormal gut microbiomes.

49

REFRENCES

1.

Diaz Heijtz, Rochellys, Shugui Wang, Farhana Anuar, Yu Qian, Britta Björkholm, Annika

2.

Samuelsson, Martin L. Hibberd, Hans Forssberg, and Sven Pettersson. 2011. “Normal Gut Microbiota Modulates Brain Development and Behavior.” Proceedings of the National Academy of Sciences of the United States of America 108 (7): 3047– 52. https://doi.org/10.1073/pnas.1010529108.

3.

Frankiensztajn, Linoy Mia, Evan Elliott, and Omry Koren. “The Microbiota and the

4.

Hypothalamus-Pituitary-Adrenocortical (HPA) Axis, Implications for Anxiety and Stress Disorders.” Current Opinion in Neurobiology 62 (June 2020): 76–82. https://doi.org/10.1016/j.conb.2019.12.003.

5.

Geng, Shaohui, Liping Yang, Feng Cheng, Zhumou Zhang, Jiangbo Li, Wenbo Liu, Yujie Li, et

6.

al. 2020. “Gut Microbiota Are Associated With Psychological Stress-Induced Defections in Intestinal and Blood–Brain Barriers.” Frontiers in Microbiology 10 (January): 3067. https://doi.org/10.3389/fmicb.2019.03067.

7.

Huo, Ran, Benhua Zeng, Li Zeng, Ke Cheng, Bo Li, Yuanyuan Luo, Haiyang Wang, et al. 2017.

8.

“Microbiota Modulate Anxiety-Like Behavior and Endocrine Abnormalities in Hypothalamic-Pituitary-Adrenal Axis.” Frontiers in Cellular and Infection Microbiology 7 (November): 489. https://doi.org/10.3389/fcimb.2017.00489.

9.

Koenig, Jeremy E., Aymé Spor, Nicholas Scalfone, Ashwana D. Fricker, Jesse Stombaugh, Rob

10.

Knight, Largus T. Angenent, and Ruth E. Ley. “Succession of Microbial Consortia in the Developing Infant Gut Microbiome.”

Proceedings of the National Academy of Sciences of the United States of America 108 Suppl 1 (March 15, 2011): 4578–85. https://doi.org/10.1073/pnas.1000081107

11.

Misiak, Błażej, Igor Łoniewski, Wojciech Marlicz, Dorota Frydecka, Agata Szulc, Leszek

12.

Rudzki, and Jerzy Samochowiec. “The HPA Axis Dysregulation in Severe Mental Illness: Can We Shift the Blame to Gut Microbiota?” Progress in Neuro-Psychopharmacology and Biological Psychiatry 102 (August 2020): 109951. https:// doi.org/10.1016/j.pnpbp.2020.109951.

13.

Pan, Jun-Xi, Feng-Li Deng, Ben-Hua Zeng, Peng Zheng, Wei-Wei Liang, Bang-Min Yin, Jing

14.

Wu, et al. “Absence of Gut Microbiota during Early Life Affects Anxiolytic Behaviors and Monoamine Neurotransmitters System in the Hippocampal of Mice.” Journal of the Neurological Sciences 400 (May 2019): 160–68. https:// doi.org/10.1016/j.jns.2019.03.027.

15.

Reigstad, Christopher S., Charles E. Salmonson, John F. Rainey Iii, Joseph H. Szurszewski,

16.

David R. Linden, Justin L. Sonnenburg, Gianrico Farrugia, and Purna C. Kashyap. “Gut Microbes Promote Colonic Serotonin Production through an Effect of Short‐chain Fatty Acids on Enterochromaffin Cells.” The FASEB Journal 29, no. 4 (April 2015): 1395–1403. https://doi.org/10.1096/fj.14-259598.

17.

Rodríguez, Juan Miguel, Kiera Murphy, Catherine Stanton, R. Paul Ross, Olivia I. Kober,

18.

Nathalie Juge, Ekaterina Avershina, et al. “The Composition of the Gut Microbiota throughout Life, with an Emphasis on Early Life.” Microbial Ecology in Health and Disease 26 (2015): 26050. https://doi.org/10.3402/mehd.v26.26050.

19.

Soto, M., Herzog, C., Pacheco, J.A. et al. Gut microbiota modulate neurobehavior through changes in brain insulin sensitivity and metabolism. Mol Psychiatry 23, 2287–2301 (2018). https://doi.org/10.1038/s41380-018-0086-5

50

Arianna Gholami

For many years, the public has been aware of the detrimental effects of smoking, but until recently, knowledge about how the main active component of cigarettes, nicotine, affects the brain has remained somewhat elusive. This literature review seeks to explore the results of a paper studying the effects of perinatal nicotine exposure on the genetic profile of VTA dopaminergic neurons and whether it can disrupt various molecular pathways. The authors conducted this study by treating isolated ventral tegmental areas (VTA) of rat pups with either nicotine or saline during the perinatal period of development. They then conducted experiments such as FACS, mRNA & miRNA expression microarrays, and other analysis techniques to gain more information as to the effect that nicotine can have specifically on the dopaminergic neurons of the VTA. It is important to dedicate some time to this topic because it is important to gain new information as to how smoking affects the fetal brain and what effects these are. This will allow the public to become better educated, hopefully leading to a decline in the incidence of smoking, especially in expecting mothers.

51

Once the authors isolated the VTA of both the nicotine treated and control rat pups, they then sorted the samples of VTA neurons into those that expressed both NeuN and TH. This was done using FACS, to measure the differences in expression of these neurons. When analyzing the results, no statistically significant difference was found compared to control (p>0.5).

The dopaminergic neurons were then collected, and a microarray miRNA and mRNA expression analysis was conducted to determine the differential expression profiles of the dopaminergic neurons due to perinatal nicotine exposure. MiRNAs (microRNAs) are non-coding, short RNA sequences with the ability to regulate genes in a post-transcriptional manner by targeting the 3’-UTR of other mRNA sequences. In order to elucidate the points of regulation as well as enriched pathways caused by nicotine exposure, the authors used the transcriptome and miRNome of the VTA dopaminergic neurons. When searching for changes in mRNA exposure following perinatal nicotine exposure, it was found that there was a statistically significant difference in differential expression when compared to control, with 862 upregulated and 1,774 down regulated differentially expressed genes (DEGs). These results support the theory that perinatal nicotine exposure is able to cause differential gene expression, and furthermore, that the majority of genes affected are downregulated. In addition to this finding, the authors then conducted an miRNA expression analysis and found that of the 74 differentially expressed miRNAs, 58 were upregulated and 16 were downregulated in response to perinatal nicotine exposure. This is consistent with the data found for mRNA differential expression, due to the fact that the majority of mRNAs were downregulated while the majority of miRNAs were upregulated, and miRNAs are known to regulate mRNA expression. This was validated when using MultimiR, showing that the majority of the DEmiRNAs (differentially expressed miRNAs) that were upregulated were paired with an mRNA DEG that was downregulated.

After gaining information as the differential expression of both mRNAs and miRNAs, the authors investigated the different pathways that were enriched in response to perinatal nicotine exposure. It was found that the downregulation of the DEG mRNAs caused enrichment in pathways such as neuroactive ligand-receptor interactions, calcium signaling, cAMP signaling, long term potentiation. These pathways are all known to be involved in drug addiction and are known as the Kyoto Encyclopedia Genes and Genomes (KEGG) pathways. In addition to the enrichment of the KEGG pathways, it was also found that additional signaling and synapse pathways were also affected in the same manner. This enrichment was found to be particularly significant in the dopaminergic synapse pathway, a key interest to the authors studying the effects of perinatal nicotine exposure. However, the glutamatergic and serotonergic synapse pathways were less affected than the dopaminergic pathway. Four genes in particular were found to be significantly downregulated in response to perinatal nicotine exposure. The first two genes, Gabrd and Gabrg2, are subunits of the GABA(a) receptor, and have been shown to be involved in the response of dopaminergic neurons and dopaminergic

neurotransmission in response to drugs. The second two genes are subunits of the glutamate receptor, Grin2d and Gria3, and are known to be involved in the nucleus accumbens, leading to an increase in dopamine release. These are significant findings because these four genes are involved in modulating the function of the dopaminergic system, leading to a possible vulnerability to drug addiction in individuals exposed to nicotine at a young age.

In addition to the above findings, the authors were able to identify specific miRNAs as well as mRNAs that resulted in a significant alteration within the dopaminergic system. For example, the authors found that there was a reduction in nAChR subunits such as the Beta4 mRNA subunit, Chrnb4, found to be expressed in dopaminergic neurons. This receptor subunit functions in the neuroactive ligand-receptor interaction, and the cholinergic synapse pathways; pathways that were shown to be enriched due to DEG downregulation. Additionally, the authors validated the results of the miRNA and mRNA microarray conducted previously, using RT-qPCR. This validation was conducted using the miRNAs and mRNAs that were deemed significant; Cck and Gabrg2 for the mRNA microarray validation, and Scn1a, Ntrk2, and Ablim3 for the miRNA microarray validation. It was found that the microarray was consistent with previous results from the microarray. This allowed for the researchers to implicate nicotine in drug addiction by showing a correlation between addiction and altered activity of miRNA located in the dopaminergic system, known to be involved in addiction. In terms of nicotine addiction specifically, the authors were able to show that in response to perinatal nicotine exposure, miR-140-3p and miR-140-5p were both differentially expressed. This was a significant finding due to the fact that these two miRNAs have been shown to be directly involved in nicotine addiction by increasing the reinforcing effects of substance abuse within the mesocorticolimbic dopaminergic pathway. This result was validated by a previous study conducted by Bosch et. al., using methamphetamine self-administration rats.

In this study, miR-125a-5p in the VTA was shown to play a significant role in the reinforcement of the addictive behaviors displayed by the rats.

As the authors predicted, perinatal nicotine exposure was able to change the differential expression of many miRNAs as well as mRNAs. Additionally, they concluded that this exposure not only led to genetic alterations within these neurons, but also to post-transcriptional gene regulation via genemiRNA target interactions. Given their results, the authors were able to further confirm that these genetic alterations can lead to the reinforcement of addictive behaviors in the mesocorticolimbic pathway of the dopaminergic system. The authors did however recommend further studies into how gender may play a role in how dopaminergic neurons in the VTA are affected by perinatal nicotine exposure, and urged further investigation to help elucidate the effect of miRNAs on other biological pathways. This study is important to acknowledge because it shows a clear link between the effects of nicotine and the brain. Many

52

people may not take into consideration the lasting consequences that nicotine can have on their brain, and instead only focus on issues such as cancer. However, given this study and studies that have been conducted previously, it has now become clear that more data must be collected to determine how else nicotine may affect our brains.

53

REFRENCES

1.

Keller, Renee F., et al. “Investigating the Genetic Profile of Dopaminergic Neurons in the VTA in Response to Perinatal Nicotine Exposure Using MRNA-MiRNA Analyses.” Scientific Reports, vol. 8, no. 1, 2018, doi:10.1038/ s41598-018-31882-9.

2.

Bosch, P J et al. “mRNA and microRNA analysis reveals modulation of biochemical pathways related to addiction in the ventral tegmental area of methamphetamine self-administering rats.” BMC neuroscience vol. 16 43.

19 Jul. 2015, doi:10.1186/s12868-015-0186-y

54

The Effect of LSD On Brian Entropy and The Personality Trait ‘Openness’ Nathaniel Green

In the paper “LSD-Induced Entropic Brain Activity Predicts Subsequent Personality Change,” the relationship between brain entropy while taking Lysergic acid diethylamide (LSD), and the personality trait of openness two weeks later was examined (Lebedev et al. 2016). Classic psychedelics such as psilocybin have been known to change aspects of adult personality, despite being relatively fixed past the age of 30 (MacLean, Johnson, and Griffiths 2011). Nineteen healthy individuals, all of whom had prior experience with LSD, were given a dose of 75µg of LSD. These individuals then underwent three 7.5 minute fMRI scans to observe change in brain entropy. The first and third scans were in silence while the second was while listening to music. After each scan, subjects were questioned on the extent of ego dissolution experienced. Two weeks later the levels of ‘openness’ were assessed compared to a baseline previously taken. The results showed that LSD significantly increased openness, as well as global brain entropy, affecting all hierarchal levels of the brain, but most importantly the upper levels.

Increases in entropy accurately predicted the increase of openness two weeks later, especially during and after the music listening session, and when ego dissolution was achieved. These findings illuminate the medium-term positive effects of psychedelics and correlates these with acute brain circuit changes. These findings support emerging arguments that LSD and other psychedelics could have a role in treatments for illnesses including treatment-resistant depression and PTSD.

55

Background and introduction The human use of psychedelics for ritual and recreation dates back thousands of years (“The Medical History of Psychedelic Drugs”, 2007). These early psychedelics included peyote, which contained mescaline, and psilocybin. It wasn’t until 1938 that LSD, a psychedelic originally synthesized from the poisonous fungus ergot, was synthesized by Albert Hofmann (Williams, 1999). Early research included by the Central Intelligence Agency into the potential use of LSD as a truth serum, alongside other therapeutic uses. At the same time, the counterculture movement started welcoming and encouraging its (and other psychedelics) use (Carhart-Harris and Goodwin 2017). As the use of psychedelics in counterculture progressed into the 1960s, in part lead by Harvard professor Tim Leary, fear of the negative cultural side effects grew until the US government severely regulated its use and halted research (Williams, 1999). After 40 years, research into the therapeutic use of psychedelics began again (Carhart-Harris and Goodwin, 2017).

Since emerging from the psychedelic dark age, research has made great strides in the classification of LSD and other psychedelics’ effects and mechanisms. LSD, psilocybin, and mescaline act as 5-HT2A (serotonin receptor) agonists, secondarily increasing glutamate release and effecting subjective perception and sense of self (Kraehenmann et al., 2017; Carhart-Harris and Friston, 2019). These changes are understood as being related to the brain’s functional hierarchy in a new theory called ‘Relaxed Beliefs Under Psychedelics’ (REBUS) (Carhart-Harris and Friston, 2019). In normal brain function, information is gathered from the environment and filtered through the progressive hierarchy to multimodal associative areas. As it enters through sensory areas of the brain and moves through the brain’s hierarchy, it is compared to predictions using an approach employing Bayesian statistics: predictive coding (Friston, 2010). Predictions are formed from priors encoded in the upper echelons – higher cortical structures - of the brain's hierarchy (Carhart-Harris and Friston, 2019). New sensations and data from the World are weighed against these predictions and are either confirmed true (confidence) or false (surprise). False predictions may reshape priors if those priors are malleable enough, and the surprise significant (Carhart-Harris and Friston, 2019). Psychedelics act to decrease the activity of high levels of the brain, disrupting the influence of these prior on lower input, therefore increasing malleability and the surprise factor. This theory has been supported by high concentrations of 5-HT2A receptors in high level regions, such as the default mode network (DMN) (CarhartHarris and Friston, 2019).

py is correlated with the intensity of the psychedelic experience (Carhart-Harris and Friston, 2019).

This phenomenon is studied in the paper “LSD-Induced Entropic Brain Activity Predicts Subsequent Personality Change,” as it shows a correlation between increase in both brain entropy and the personality trait of openness. This paper connects the two concepts of the entropic brain hypothesis and the free energy principle in a real-world example, as increased levels of brain entropy show less input from priors. Brian entropy was measured three times using fMRI, once with music playing. These scans were done under LSD and placebo conditions. After each scan, it was determined whether ego dissolution had occurred.

Ego dissolution is the loss of understanding of ‘self’ and is correlated with the global connection of brain regions such as the DMN (Tagliazucchi et al., 2016). Openness was measured two weeks after taking LSD using the NEO-PI-R test which measures five main categories of personality (Carhart-Harris and Friston, 2019; “NEO Personality Inventory-Revised | NEO PI-R” n.d.).

Major results The results of the target paper demonstrate that LSD had a significant effect on 11 of 17 functional systems of the brain (figure 1), showing there was increased global entropy, as predicted by the REBUS model. Another significant result that was supported by past research (Carhart-Harris and Friston, 2019) was a significant increase in the personality trait of openness, despite the fixation of personality once adulthood is reached. This effect is logical concerning the free energy principle, as priors are relaxed during psychedelic use. A significant correlation was found between increased brain entropy and increased personality change, demonstrating LSD-induced increase in global brain entropy is a predictor of personality change two weeks later. Interestingly, the correlation was strongest during and after listening to music (figure 2). Also, those with high ego dissolution showed greater personality change. These two results provide important information that could allow for more future success in psychedelic therapy (Kaelen et al., 2018; Carhart-Harris, 2019).

Conclusion and discussion

Administration of LSD caused a significant increase in global brain entropy, correlating with increased openness, indicating that brain entropy may predict personality change. This effect was especially strong during and after music listening, as Another aspect of the REBUS theory is the property of well as when ego dissolution had occurred, offering evidence increased brain entropy under psychedelics, known as the en- that music and ego dissolution may play an important role in the tropic brain hypothesis (Carhart-Harris and Friston, 2019). Brian therapeutic use of LSD. entropy is a measure of the activity of brain networks differing The findings of the target paper that state that LSD infrom its natural order (Carhart-Harris et al., 2014). For example, creased brain entropy is significant to the newly resurrected the networks in the brain that contribute to the supposed defield of psychedelic therapy and research. LSD acts on the fault mode network, which is responsible for a stable sense of brain’s higher levels, decreasing the influence of priors on inself and day-dreaming states, shows reduced co-activity, recoming sensory data streams. This allows for more information sulting in increased connections between brain regions that entering the brain to be interpreted without the constraining would otherwise be relatively muted (Carhart-Harris et al., 2014; lens of prior assumptive understanding, potentially aided by Carhart-Harris and Friston, 2019). Interestingly, increased entro56

increased entropy in high-level brain networks such as the DMN (Carhart-Harris and Friston, 2019). Considering this, the result that individuals showed increased openness begins to make sense. While this effect is not well understood, the paper theorizes that through mystical experience or ego dissolution, prior understanding of the world may begin to unravel, allowing the individual to question their assumptions of the World and accept what they may have denied without psychedelic aid. Increased openness is a well-recognized effect of psychedelics, and was seen even one year after use (MacLean, Johnson, and Griffiths, 2011). Another aspect of the psychedelic experience that may play a role in personality change is the registered value of the experience. While personality is mostly fixed in adulthood, significant life experiences may have a transformative effect (MacLean, Johnson, and Griffiths, 2011; Lebedev et al., 2016). In one study, a majority of psychedelic naïve volunteers who took psilocybin reported that their psychedelic experience was deeply profound and spiritual, over 60% rating it in their top 5 most meaningful encounters. (Griffiths et al., 2006). The importance of the experience may cause a greater change in personality.

These findings are supported by the biological processes of stress modulated serotonin release to the 5-HT2A receptors, causing a change in perspective and neuroplasticity (Murnane, 2019).

Music and ego dissolution were shown to affect openness by increasing change in personality and predictive power of entropy change. Music may trigger the change of personality as these effects were only found during and after listening to music. One explanation the paper proposes is that music may guide changes in brain entropy that affect personality. Both music and ego dissolution have been discussed in prior research. Music has been linked with increased mystical experiences, and positive therapeutic results (Kaelen et al., 2018) while ego dissolution has been regarded as an effect of increased brain entropy and dysregulation of the DMN and has also been linked to therapeutic benefit and increased openness (Carhart-Harris and Friston, 2019).

Critical analysis REBUS is largely theoretical and somewhat speculative.

While experiments such as the one conducted in the target paper support the REBUS model, much is still disputed and unknown. One issue posed by the target paper was that brain entropy correlated strongly with increased personality change during or after listening to music. However, in the experiment, music listening was always done 7.5 minutes after the first silent fMRI, and before a third silent fMRI. This was not controlled for, as the order of listening to music was not changed. This means that the predictive power increase is possibly a result of the timing rather than the music. Another notable problem with this study is that all participants had taken LSD in the past, though not within two weeks of the experiment. While it is unlikely that this discredits the results of the study, it is important, as individuals were aware of the effect of the drug, and most likely felt a level of comfort and expectation. LSD may have different effects on LSD naïve people than in people with experience.

Future direction Because of the novelty of these experiments and theories, there are many directions research into this field could be taken. The results of this paper offer a reason to explore the possibility of LSD and other psychedelics as therapeutic aids.

Such research has already begun, and promising results have been documented for diseases such as treatment-resistant depression, PTSD, addiction and even eating disorders (CarhartHarris and Friston, 2019).

Psychedelic therapy has the potential to treat disorders that have evaded members of the medical community for years.

The methods continue to be refined through research and critique, improving the safety and utility of psychedelics. To strengthen the results of the target paper, the experimenters could change the order of music listening sessions or exclude music altogether. This experiment would still demonstrate that music had an important effect, as other sources have shown this phenomenon to some extent (Kaelen et al. 2018). In the case that music did not show a meaningful effect, it would discredit the theory that music may aid in how the brain increases entropy. It may also support evidence that indicated that the benefit of music is variable and may not affect everyone equally (Kaelen et al., 2018). The possibility that music may “guide” LSD users in a particular way proposes an important aspect of psychedelic treatment: the subjective experience. It is already known that the environment and mental attitude of the individual plays a role in the unique experience that each LSD experience offers (Carhart-Harris and Friston, 2019). However, the mechanism of this is still not well understood. This information is essential in standardizing LSD research, as different environments may affect each experimenter's results significantly (Unger, 1963). It is also important to gauge environments that could improve therapeutic results.

In an attempt to verify that LSD-naïve and experienced people would have the same outcome from this, both groups should be studied. It is important to note that government regulation of LSD and other classic psychedelics is extremely strict, and it is difficult to ethically enter naïve individuals into a psychedelic study. Had this study been completed, however, I predict that the results would be the same. In the case results were different, I predict a possibly stronger effect for LSD naïve individuals, as psychedelics can have lessening effects with repeated use, and because personality change may decrease from repeat use. This information would be important when exploring the possibility of using psychedelics therapeutically, as it would allow therapists to weight the benefit of undergoing this novel therapeutic method.

The results of this study provide evidence of the possible benefit of psychedelics in a therapeutic way, as well as offering ways to predict the medium-term effects of LSD use. With increased research and testing, it is highly plausible that psychedelics will soon play a role in the treatment of many psychological diseases.

57

Figure 1 Boxed numbers depict brain networks with increased entropy after LSD exposure 1. Secondary Visual; 2. Primary Visual; 3. Superior Sensorimotor; 4. Inferior Sensorimotor; 5. Superior Parietal; 6. Posterior Sensorimotor; 7. Posterior Salience; 8. Anterior Salience; 9. Anterior MTL; 10. Orbitofrontal; 11. Precuneus; 12. Inferior Frontoparietal; 13. Superior Frontoparietal; 14. Auditory; 15. Hippocampal; 16. Default Mode Network; 17. Frontotemporal (Lebedev et al. 2016).

Figure 2 red = first fMRI; green = second fMRI; blue = third fMRI. These data demonstrate the effect of music on the predictive power of entropy and openness. Green took place while listening to music, and blue after music. These results demonstrate the increase of predictive power during and after listening to music (Lebedev et al. 2016).

58

REFRENCES

1.

Carhart-Harris, R. L., and K. J. Friston. 2019. “REBUS and the Anarchic Brain: Toward a Unified Model of the Brain Action of Psychedelics.” Edited by Eric L. Barker. Pharmacological Reviews 71 (3): 316–44. https://doi.org/10.1124/pr.118.017160.

2.

Carhart-Harris, Robin L. 2019. “How Do Psychedelics Work?” Current Opinion in Psychiatry 32 (1): 16–21. https:// doi.org/10.1097/YCO.0000000000000467.

3.

Carhart-Harris, Robin L, and Guy M Goodwin. 2017. “The Therapeutic Potential of Psychedelic Drugs: Past, Present, and Future.” Neuropsychopharmacology 42 (11): 2105–13. https://doi.org/10.1038/npp.2017.84.

4.

Carhart-Harris, Robin L., Robert Leech, Peter J. Hellyer, Murray Shanahan, Amanda Feilding, Enzo Tagliazucchi, Dante R. Chialvo, and David Nutt. 2014. “The Entropic Brain: A Theory of Conscious States Informed by Neuroimaging Research with Psychedelic Drugs.” Frontiers in Human Neuroscience 8. https://doi.org/10.3389/fnhum.2014.00020.

5.

Friston, Karl. 2010. “The Free-Energy Principle: A Unified Brain Theory?” Nature Reviews Neuroscience 11 (2): 127–38. https://doi.org/10.1038/nrn2787.

6.

Kaelen, Mendel, Bruna Giribaldi, Jordan Raine, Lisa Evans, Christopher Timmerman, Natalie Rodriguez, Leor Roseman, Amanda Feilding, David Nutt, and Robin Carhart-Harris. 2018. “The Hidden Therapist: Evidence for a Central Role of Music in Psychedelic Therapy.” Psychopharmacology 235 (2): 505–19. https://doi.org/10.1007/s00213-017-4820-5.

7.

Kraehenmann, Rainer, Dan Pokorny, Helena Aicher, Katrin H. Preller, Thomas Pokorny, Oliver G. Bosch, Erich Seifritz, and Franz X. Vollenweider. 2017. “LSD Increases Primary Process Thinking via Serotonin 2A Receptor Activation.” Frontiers in Pharmacology 8 (November). https://doi.org/10.3389/fphar.2017.00814.

8.

Lebedev, A. V., M. Kaelen, M. Lövdén, J. Nilsson, A. Feilding, D. J. Nutt, and R. L. Carhart‐Harris. 2016. “LSD-Induced Entropic Brain Activity Predicts Subsequent Personality Change.” Human Brain Mapping 37 (9): 3203–13. https://doi.org/10.1002/ hbm.23234.

9.

MacLean, Katherine A., Matthew W. Johnson, and Roland R. Griffiths. 2011. “Mystical Experiences Occasioned by the Hallucinogen Psilocybin Lead to Increases in the Personality Domain of Openness.” Journal of Psychopharmacology (Oxford, England) 25 (11): 1453–61. https://doi.org/10.1177/0269881111420188.

10.

Murnane, Kevin Sean. 2019. “Serotonin 2A Receptors Are a Stress Response System: Implications for Post-Traumatic Stress Disorder.” Behavioural Pharmacology 30 (2-): 151–62. https://doi.org/10.1097/FBP.0000000000000459.

11.

“NEO Personality Inventory-Revised | NEO PI-R.” n.d. Accessed June 12, 2020. https://www.parinc.com/Products/Pkey/276.

12.

Tagliazucchi, Enzo, Leor Roseman, Mendel Kaelen, Csaba Orban, Suresh Muthukumaraswamy, Kevin Murphy, Helmut Laufs, et al. 2016. “Increased Global Functional Connectivity Correlates with LSD-Induced Ego Dissolution.” Current Biology 26 (April). https://doi.org/10.1016/j.cub.2016.02.010.

13.

“The Medical History of Psychedelic Drugs.” 2007, 52.

14.

Unger, Sanford ML. 1963. “Mescaline, LSD, Psilocybin, and Personality Change a Review †.” Psychiatry 26 (2): 111–25. https://doi.org/10.1080/00332747.1963.11023344.

15.

Williams, Luke. 1999. “Human Psychedelic Research: A Historical And Sociological Analysis.” MAPS. MAPS. 1999. https:// maps.org/index.php.

59

Age-progressive β-amyloid depositions and altered hippocampal neurogenesis in Alzheimer’s Disease Tg2576 and APP Swedish PS1 dE9 mice Zahin Hafiz

Neurogenesis refers to the birth of new neurons within the brain. Numerous studies have been done on hippocampal neurogenesis as these neurons regulate brain functions which are closely associated to learning and memory. In this review, the study conducted by Unger and colleagues (2016) was thoroughly examined through the lens of the most common form of dementia, Alzheimer’s Disease.

The major findings of this paper revealed that β-amyloid plaque formation is age-progressive in APP Swedish PS1 dE9 mice, the hyperproliferation of proliferating cells and neuroblasts occur prior to plaque formation in both Tg2576 and APP Swedish mice, and the survival and differentiation of proliferating cells and numbers of newly formed neurons significantly reduce before plaque formation in APP Swedish mice. In addition, the role of hyperproliferating DCX positive cells remains to be elucidated.

Altogether, these findings suggest that there is a link between β-amyloid plaque depositions and impaired health of hippocampal cells.

Keywords: Neurogenesis, hippocampus, Alzheimer’s Disease, β-amyloid plaques, proliferation, APP Swedish mice, Tg2576 mice

60

INTRODUCTION

Fig.1: Age-progressive β-amyloid deposition in APP-PS1 mice and dendate gyrus granule cell layer (GCL) perforation. 3-month old mice showed no signs of β-amyloid plaques. 4-month old mice showed initial signs of β-amyloid deposits (indicated by white arrows). 10 and 13-month old mice showed a significant increase in β-amyloid deposition and plaque size. The structure was perforated by β-amyloid deposition and showed a significant reduction in volume at 13 months compared to the control. Figure adapted from Unger et al. (2016). Early Changes in Hippocampal Neurogenesis Transgenic Mouse Models for Alzheimer’s Disease.

Mol Neurobiol, 53(8), 5796-5806.

Neurogenesis, the formation of new neurons, is prevalent throughout life in mammalian brains. Neurogenesis mainly occurs within two distinct zones in the mammalian brain, the subventricular zone (SVZ) of the lateral ventricles and the dendate gyrus of the hippocampus (Braun & Jessberger, 2014). Moreover, the hippocampus lies within the temporal lobe and plays a major role in learning and memory (Anand and Dhikav, 2012). This review will explore age-progressive β-amyloid plaque deposition in Alzheimer’s Disease mouse models, APP Swedish PS1 dE9 (APPPS1) and Tg2576, and how its toxicity can drastically affect hippocampal neurogenesis, which involves cell survival, differentiation, and proliferation (Unger et al., 2016). As a general background, Alzheimer’s Disease is the most frequent form of dementia in elderly individuals worldwide. It has characteristic pathological hallmarks, such as the formation of β-amyloid plaques and neurofibrillary tangles (Korolev, 2014, Unger et al., 2016, Yamazaki et al., 2019). However, this review would mainly focus on the β-amyloid aspect of Alzheimer’s Disease in transHyperproliferation of PCNA+ and PCNA+/DCX+ in APP-PS1 and genic mice models.

Tg2576 Unger et al. selected female APP-PS1 and Tg2576 mice, of equal Immunohistochemistry analysis showed hyperproliferation of numbers, in order to conduct their study. APP-PS1 mice conproliferating cells (PCNA+) and proliferating neuroblasts that cotained the chimeric mouse/human mutant amyloid precursor expressed DCX (PCNA+/DCX+) in 3-month old APP-PS1 and protein (APP) and the human mutant presenilin 1 (PS1). MoreoTg2576 mice before the formation of β-amyloid plaques. Hyver, the Tg2576 contained the overexpressing APP (APP OE) perproliferation of most of the proliferating neuroblasts (PCNA +/ (Unger et al., 2016). Prior to brain extraction, both APP-PS1 and DCX+) took place within the sub-granular cell layer (SGCL) of the Tg2576 mice were anesthetised and transcardially perfused. APP dendate gyrus. This hyperproliferation of proliferating cells and -PS1 mice were intraperitoneally administered with BrdU neuroblasts was not found in 10 and 13-month old APP-PS1 mice (bromodeoxyuridine) for the analysis of cell survival and differand 5-month old Tg2576 mice. However, both mice models entiation. Brains were analyzed at different timepoints for both showed no hyperproliferation of DCX positive neurons (Unger et mice models and immunohistochemical analysis was performed al., 2016). Similar to this finding, hyperproliferation of proliferon tissues in order to examine β-amyloid plaque deposits and ating cells within the hippocampus was noticed in 3-month old the conditions of the hippocampal cell niche. Furthermore, UnTg2576 mice (Krezymon et al., 2013). ger et al. showed that there was an age-progressive β-amyloid deposition in APP-PS1 mice, hyperproliferation of proliferating Decreased Survival and Differentiation of Cells and Diminishing + + cells and proliferating neuroblasts in both Tg2576 and APP-PS1 Number of Newly Formed Neurons (BrdU /NeuN ) in APP-PS1 mice, and reduced numbers of proliferating cells and newly After 30 days of BrdU (bromodeoxyuridine) intraperitoneal adformed neurons in APP-PS1 mice (Unger et al., 2016). Overall, ministration in 3-month old APP-PS1, immunohistochemistry these findings show that there is a link between the deposition analysis revealed that the survival and differentiation of proliferof β-amyloid plaques and impaired health of hippocampal cells ating cells (BrdU+) significantly reduced in APP-PS1 compared to in Alzheimer’s Disease mice models.

WT. BrdU+ co-stained with markers for newly formed neurons (BrdU+/NeuN+) showed a number reduction in APP-PS1 comMAJOR RESULTS pared to WT. BrdU+ co-stained with markers for oligodendroAge-Progressive β-Amyloid Plaque Deposition and Dendate cytes (BrdU+/Olig2+) and astrocytes (BrdU+/GFAP+) showed no Gyrus GCL Perforation in APP PS1 dE9 (APP-PS1) differences between APP-PS1 and WT. BrdU+ co-stained with + + 3, 4, 10, and 13-month APP-PS1 mice experienced an age- markers for neuronal progenitors (BrdU /DCX ) did not vary sigprogressive deposition of β-amyloid plaques within the hippo- nificantly between APP-PS1 and WT [Fig. 2] (Unger et al., 2016). campus and cortex. Thioflavin S staining was used to locate β- Similar to this finding, a reduced number of newly formed neuamyloid plaques within the hippocampus and cortex. DAP1 stain- rons was observed in 3-month old Tg2576 mice (Krezymon et al., ing was used to stain nuclei of neurons located within the den- 2013). date gyrus GCL. Same-aged controls showed no signs of βamyloid deposition within the hippocampus and cortex. The dendate gyrus granule cell layer (GCL) was perforated by the deposition of these β-amyloid plaques [Fig.1] (Unger et al., 2016). Similar to this finding, age-progressive β-plaque deposition within the hippocampus and reduction of dendate gyrus GCL neurogenesis were noticed in 3xTg-AD mice (Rodriguez et al., 2008).

61

Fig.2: Analysis of the survival and differentiation of cells from neuronal and glial lineages in APP-PS1 and WT. Survival and differentiation of proliferating cells (BrdU+) significantly decreased in APP-PS1. Newly formed neurons (BrdU+/NeuN+) were significantly reduced in APP-PS1. Astrocyte (BrdU+/ GFAP+), oligodendrocyte (BrdU+/Olig2+), and neuronal progenitor (BrdU+/DCX+) numbers did not vary between APP-PS1 and WT. Figure adapted from Unger et al. (2016). Early Changes in Hippocampal Neurogenesis Transgenic Mouse Models for Alzheimer’s Disease. Mol Neurobiol, 53(8), 5796-5806.

CONCLUSIONS/DISCUSSION According to Unger and colleagues, APP-PS1 mice showed ageprogressive β-amyloid deposition within the hippocampus and cortex starting from 4 months of age, a greater prominence of plaque size and deposition at 10 and 13 months of age, and a significant reduction in the survival and differentiation of newly formed neurons at 3 months of age. Both Tg2576 and APP-PS1 mice models showed hyperproliferation of proliferating cells and proliferating neuroblasts at 3 months of age, which diminished at 5 months for Tg2576 and 10 and 13 months for APPPS1. The authors have concluded that these early alterations in hippocampal neurogenesis in Tg2576 and APP-PS1 mice can affect cell proliferation, survival, and differentiation (Unger et al., 2016), which in turn, can drastically deteriorate learning and memory.

Numerous studies have supported the fact that β-amyloid deposition is age-progressive, and that it disrupts hippocampal neurogenesis in developing mice (Rodriguez et al., 2008, Taniuchi et al., 2007). Rodriguez et al. found an age-progressive deposition of plaques in 3x-Tg AD mice and Taniuchi et al. showed an age-progressive deposition of plaques in APP-PS1 mice. Unger et al. found that there was a decreased survival and differentiation of hippocampal proliferating cells and newly formed neurons in APP-PS1 mice (Unger et al., 2016). Moreover, they also mentioned the hyperproliferation of proliferating cells and proliferating neuroblasts co-expressing DCX in both Tg2576 and APP-PS1 mice (Unger et al., 2016). On the other hand, Krezymon et al. showed hyperproliferation of proliferating cells and a significant number reduction of newly formed neurons in Tg2576 mice (Krezymon et al., 2013). In comparison to Krezymon et al.’s findings, Unger et al.’s finding of proliferating neuroblasts co-expressing DCX is relatively new because other studies in hippocampal neurogenesis did not report this finding. Moreover, most of the past literature utilized the BrdU labelling method in order to detect cell proliferation (Valero et al., 2005) but Unger et al. used PCNA labelling to detect cell proliferation. Overall, the findings show that Alzheimer’s Disease worsens with age and the toxicity of β-amyloid plaques harms neurogenesis, mainly within the hippocampus.

of 3, 4, 10, and 13-month old female APP-PS1 mice were extracted and analyzed by using immunohistochemical methods.

A similar study was performed on both female and male APPPS1 mice, which were of 3, 5, 7, and 9 months of age. Immunohistochemical analysis revealed that these mice also experienced an age-progressive β-amyloid deposition within the hippocampus (Taniuchi et al., 2007). The authors of this study did not take male APP-PS1 mice into consideration (Unger et al., 2016) and hence, have decreased the external validity of this study. Moreover, the authors might have only considered female APP-PS1 mice because a previous study mentioned that elderly women have a higher chance of developing Alzheimer’s Disease compared to their male counterparts (Fratiglioni et al., 1997). This is also true for female Alzheimer’s Disease mice models, as a previous study mentioned that they experienced an age-progressive deposition of β-amyloid plaques, which were much higher compared to male Alzheimer’s Disease mice models (Taniuchi et al., 2007, Hardy & Selkoe, 2002). In addition, the authors of the study chose four different timepoints, with unequal intervals, to analyze the brains of APP-PS1 mice (Unger et al., 2016). Taniuchi et al. however, chose timepoints with equal intervals (after every one month) to analyze APP-PS1 mice brains, which was a well-designed timeframe to track ageprogressive β-amyloid plaque formation within the hippocampus (Taniuchi et al., 2007). Hence, the implementation of this timeframe would be helpful to effectively track age-progressive deposition of β-amyloid plaques.

In addition, the authors observed reduced survival and differentiation of proliferating cells (BrdU+) in 3-month old APP-PS1 mice compared to the wildtype (WT). The BrdU (bromodeoxyuridine) labelling method is commonly used to detect proliferating cells at the S phase of the cell cycle (Valero et al., 2005). As an efficient alternative, the authors could use the EdU (5-ethynyl-2’-deoxyuridine) labelling method to detect proliferating cells. EdU labelling was initially used in neurogenesis research over a decade ago (Zeng et al., 2010). Unlike BrdU labelling, EdU labelling does not involve the DNA denaturation process and utilizes “click” chemistry to detect proliferating cells within a tissue of interest (Chehrehasa et al., 2009).

Furthermore, one of the key hallmarks of Alzheimer’s Disease (AD) is β-amyloid plaque formation (Unger et al., 2016). Even though there are several environmental and genetic factors that contribute to the development of AD, studies have mentioned that the inhibition of the Wnt/β-catenin signalling pathway leads to the formation of β-amyloid plaques (Tapia-Rojas & Inestrosa, 2018, Wan et al., 2014). In the normal brain, the Wnt/β-catenin signalling pathway serves as a neuroprotective mechanism to alleviate the effects of β-amyloid toxicity (TapiaRojas & Inestrosa, 2018). Hence, several researchers have suggested therapeutic strategies to activate the Wnt/β-catenin pathway in AD mice models (Jin et al., 2017, De Ferrari et al., CRITICAL ANALYSES 2003). Jin et al. found that sodium selenate enhanced the activAs mentioned previously, the authors noticed an age- ity of protein phosphatase 2 enzyme (PP2A) and raised βprogressive deposition of β-amyloid plaques within the hippo- catenin levels, which further inhibited the glycogen synthase campus and cortex of APP-PS1 mice (Unger et al., 2016). Brains kinase 3 beta (GSK-3β) enzyme and promoted gene transcription in the Wnt/β-catenin pathway (Jin et al., 2017). Further62

more, De Ferrari et al. showed that lithium inhibited the GSK-3β enzyme and activated the Wnt/β-catenin pathway (De Ferrari et al., 2003). Similar to these studies, the authors could also look into the therapeutic side of AD prevention, by focusing on how the activation of the Wnt/β-catenin pathway might aid in hippocampal neurogenesis.

FUTURE DIRECTIONS According to the authors, the role of hyperproliferating DCX positive cells in APP-PS1 and Tg2576 mice is yet to be determined (Unger et al., 2016). As a future suggestion, the authors could examine the Wnt/β-catenin signalling pathway, as it activates multiple genes which are associated with cell proliferation and differentiation (Varella-Nallar & Inestrosa, 2013).

ti-step experiment by performing a pharmacogenomic test on all mice, before the administration of the sodium-selenate solution. Pharmacogenomic testing should allow authors to understand if the mice have different responses to the chemical due to their genetic composition. In this manner, the authors should be able to administer personalized doses of the chemical to each mouse, which should be therapeutically effective (Mroziewicz & Tyndale, 2010).

Moreover, to make this study more applicable to therapeutics and behavioural neuroscience, the authors should perform a multi-step experiment. For all steps, the authors could choose any one of the two mice models, APP-PS1 or Tg2576, but must consider collecting equal numbers of male and female mice from that species (4 male and 4 female mice/group). For the first step, the authors should create three groups – the APPPS1/Tg2576 experimental group, the APP-PS1/Tg2576 positive control, and the non-transgenic APP-PS1/Tg2576 negative control. Similar to Jin et al.’s study, the authors should orally administer sodium selenate-containing water to the experimental group and pure water to both positive and negative controls (Jin et al., 2017). All mice should be 2 months of age and authors should administer their appointed solutions on a daily basis, for a total of 10 months (Jin et al., 2017). For the second step, all 12-month old mice should undergo a Morris Water Maze Test. The experimental group and negative control should show a lower latency period to notice the tagged platform underwater compared to the positive control, which should have a higher latency period due to their impaired spatial learning and memory (Wolf et al., 2016). For the third step, the authors should analyze the brains of all mice groups. In order to detect proliferating cells within the hippocampus, the authors should use EdU labelling instead of BrdU labelling. This should allow them to skip the denaturation process which is associated with BrdU staining (Chehrehasa et al., 2009) and should save them the time and cost that is required to perform DNA denaturation. The experimental group and negative control should show the presence of proliferating cells within the hippocampus. The experimental group should show this result because the sodium-selenate treatment should activate their Wnt/β-catenin signalling pathway, which should reduce β-plaque deposition within their hippocampus and allow cell proliferation to occur, similar to the negative control (Jin et al., 2017, Varella-Nallar & Inestrosa, 2013). The positive control should show no presence of proliferating cells (Unger et al., 2016). This is because their Wnt/β-catenin signalling pathway should be inactivated, due to the high deposition of β-amyloid plaques. As this pathway is responsible for regulating cell proliferation (Varella-Nallar & Inestrosa, 2013), chances are that its inactivation should also hinder its ability to cause cell proliferation. The results for the positive control seem to be consistent with past literature.

However, if the experiments do not work for the experimental group and negative control, the authors should repeat the mul63

REFRENCES

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

Braun, S. M. G., & Jessberger, S. (2014). Adult neurogenesis: mechanisms and functional significance. Development, 141 (10), 1983–1986. doi: 10.1242/dev.104596 Chehrehasa, F., Meedeniya, A. C., Dwyer, P., Abrahamsen, G., & Mackay-Sim, A. (2009). EdU, a new thymidine analogue for labelling proliferating cells in the nervous system. Journal of Neuroscience Methods, 177(1), 122–130. doi: 10.1016/ j.jneumeth.2008.10.006 Dhikav, V., & Anand, K. (2012). Hippocampus in health and disease: An overview. Annals of Indian Academy of Neurology, 15(4), 239–246. doi: 10.4103/0972-2327.104323 Ferrari, G. V., Chacón, M. A., Barría, M. I., Garrido, J. L., Godoy, J. A., Olivares, G., . . . Inestrosa, N. C. (2003). Activation of Wnt signaling rescues neurodegeneration and behavioral impairments induced by β-amyloid fibrils. Molecular Psychiatry, 8(2), 195-208. doi:10.1038/sj.mp.4001208 Fratiglioni, L., Viitanen, M., Strauss, E. V., Tontodonati, V., Herlitz, A., & Winblad, B. (1997). Very Old Women at Highest Risk of Dementia and Alzheimers Disease: Incidence Data from the Kungsholmen Project, Stockholm. Neurology, 48(1), 132-138. doi:10.1212/wnl.48.1.132 Hardy, J., & Selkoe, D. J. (2002). The Amyloid Hypothesis of Alzheimer's Disease: Progress and Problems on the Road to Therapeutics. American Association for the Advancement of Science, 297(5580), 353-356. Retrieved from http:// www.jstor.com/stable/3077168 Jin, N., Zhu, H., Liang, X., Huang, W., Xie, Q., Xiao, P., . . . Liu, Q. (2017). Sodium selenate activated Wnt/β-catenin signaling and repressed amyloid-β formation in a triple transgenic mouse model of Alzheimers disease. Experimental Neurology, 297, 36-49. doi:10.1016/j.expneurol.2017.07.006 Korolev, I. O. (2014). Alzheimer’s Disease: A Clinical and Basic Science Review. Medical Student Research Journal, 4(Fall), 24–33. doi: doi:10.3402/msrj.v3i0.201333 Krezymon, A., Richetin, K., Halley, H., Roybon, L., Lassalle, J.-M., Francès, B., … Rampon, C. (2013). Modifications of Hippocampal Circuits and Early Disruption of Adult Neurogenesis in the Tg2576 Mouse Model of Alzheimer’s Disease. PLoS ONE, 8(9), 1–14. doi: 10.1371/journal.pone.0076497 Mroziewicz, M., & Tyndale, R. F. (2010). Pharmacogenetics: A Tool for Identifying Genetic Factors in Drug Dependence and Response to Treatment. Addict Sci Clin Pract., 5(2), 17–29.

Rodríguez, J. J., Jones, V. C., Tabuchi, M., Allan, S. M., Knight, E. M., Laferla, F. M., … Verkhratsky, A. (2008). Impaired Adult Neurogenesis in the Dentate Gyrus of a Triple Transgenic Mouse Model of Alzheimers Disease. PLoS ONE, 3(8), 1–8. doi: 10.1371/journal.pone.0002935 Taniuchi, N., Niidome, T., Goto, Y., Akaike, A., Kihara, T., & Sugimoto, H. (2007). Decreased proliferation of hippocampal progenitor cells in APPswe/PS1dE9 transgenic mice. NeuroReport, 18(17), 1801–1805. doi: 10.1097/ wnr.0b013e3282f1c9e9 Tapia-Rojas, C. & Inestrosa, N. (2018). Loss of canonical Wnt signaling is involved in the pathogenesis of Alzheimers disease. Neural Regeneration Research, 13(10), 1705-1710. doi:10.4103/1673-5374.238606 Unger, M. S., Marschallinger, J., Kaindl, J., Höfling, C., Rossner, S., Heneka, M. T., … Aigner, L. (2016). Early Changes in Hippocampal Neurogenesis in Transgenic Mouse Models for Alzheimer’s Disease. Molecular Neurobiology, 53(8), 5796–5806. doi: 10.1007/s12035-016-0018-9 Valero, J., Weruaga, E., Murias, A. R., Recio, J. S., & Alonso, J. R. (2005). Proliferation markers in the adult rodent brain: Bromodeoxyuridine and proliferating cell nuclear antigen. Brain Research Protocols, 15(3), 127-134. doi:10.1016/ j.brainresprot.2005.06.001 Varela-Nallar, L., & Inestrosa, N. C. (2013). Wnt signaling in the regulation of adult hippocampal neurogenesis. Frontiers in Cellular Neuroscience, 7, 1-11. doi:10.3389/fncel.2013.00100 Wan, W., Xia, S., Kalionis, B., Liu, L., & Li, Y. (2014). The Role of Wnt Signaling in the Development of Alzheimer’s Disease: A Potential Therapeutic Target? BioMed Research International, 2014, 1-9. doi:10.1155/2014/301575 Wolf, A., Bauer, B., Abner, E. L., Ashkenazy-Frolinger, T., & Hartz, A. M. (2016). A Comprehensive Behavioral Test Battery to Assess Learning and Memory in 129S6/Tg2576 Mice. PLoS ONE, 11(1). doi:10.1371/journal.pone.0147733 Yamazaki, Y., Zhao, N., Caulfield, T. R., Liu, C.-C., & Bu, G. (2019). Apolipoprotein E and Alzheimer disease: pathobiology and targeting strategies. Nature Reviews Neurology, 15(9), 501–518. doi: 10.1038/s41582-019-0228-7 Zeng, C., Pan, F., Jones, L. A., Lim, M. M., Griffin, E. A., Sheline, Y. I., … Mach, R. H. (2010). Evaluation of 5-ethynyl-2′deoxyuridine staining as a sensitive and reliable method for studying cell proliferation in the adult nervous system. Brain Research, 1319, 21–32. doi: 10.1016/j.brainres.2009.12.092 64

You Are What You Eat : Alzheimer’s and Diabetes Edition A review of the effect of high fat and sugar diets on cognitive decline.

Michelle Ha

Is there a relationship between a high fat diet, insulin resistance within the brain, and cognitive impairment? The aim of a 2017 study sought to determine whether a fatty diet, able to induce insulin sensitivity, has the ability to alter brain signalling pathways impacting cognitive functions. High fat diets have been observed to induce obesity, and obesity is often associated with insulin resistance and dementia.

It is likely obesity and insulin resistance have some sort of association since approximately 85% of patients with type 2 diabetes mellitus are also obese (Kang et al., 2016). Furthermore, prior studies have observed that early stage Alzheimer’s patients tend to also show an impairment in insulin signalling (Ferreira et al., 2018).

One of the most prevalent forms of dementia is Alzheimer’s disease, and is characterized by the presence of amyloid plaques and neurofibrillary tangles in the brain. Kang’s study analyzed the effect of insulin induced changes in protein expression relating to cognitive impairment, and whether a high fat diet played a role. It was ultimately concluded that the mice given the high fat and sugar diet showed a statistically significant increase in obesity, lower insulin insulin sensitivity and glucose tolerance, and higher fasting insulin levels compared to the control group. Interestingly, impaired glucose uptake and insulin resistance was also seen in the brain. The researchers concluded that high fat diets cause inflammatory and stress responses which lead to the dysregulation of amyloid-β production and clearance rates, the formation of neurofibrillary tangles, and decreased synaptic plasticity within the brain.

Furthermore, insulin resistance is a major pathological feature in patients with Type 2 Diabetes Mellitus, and could also be involved in Alzheimer’s since 80% of patients with Alzheimer’s are also diagnosed with type 2 diabetes, or at the very least, an impaired resting level of glucose (Kothari et al., 2017).

Key words: western diet, obesity, insulin resistance, Alzheimer’s disease.

65

Background & Introduction: The global burden of Alzheimer’s is projected to affect over 74.4 million people by 2030, 131.1 million people by 2050, and double every 20 years (Kothari et al., 2017). As such, it’s imperative to fully understand the mechanisms in order to find a cure, or at the very least, decrease the risk of the disease.

Evidence suggests that Alzheimer’s is closely linked to metabolic disorders since patients with diabetes are much more likely to develop Alzheimer’s. This risk goes both ways as Alzheimer’s patients also have an increased risk for developing diabetes, and seems to be due to shared mechanisms between metabolic disorders and Alzheimer’s disease (Ferreira et al., 2014). Furthermore, in subjects with and without pre-existing insulin resistance, a diet rich in saturated fats was able to induce systemic insulin resistance (Koska et al., 2016).

It has been speculated that Western diets which tend to be higher in saturated fats, may play a role in reducing cognitive function, especially within the hippocampal region (Hansen et al., 2018). Furthermore, animal models chronically exposed to diets high in fats and sugars perform worse in memory tests, suggesting a link to worsening hippocampal memory function. It is likely that systemic changes in the phosphotyrosine signalling are involved in the relationship between fattier diets and cognitive decline. Through tyrosine phosphorylation, changes to enzymes involved in the metabolic pathways are made resulting in the development of insulin resistance, and potentially diabetes (Dittmann et al., 2019). It has also been observed that people who frequently consume high fat or high cholesterol diets often have lipoprotein profiles similar to Alzheimer’s patients. Animal models fed diets high in fat and sugars also show changes in protein markers associated with Alzheimer’s disease (Hansen et al., 2018).

administered at twelve weeks of age also revealed that the mice on a high fat diet also showed a higher fasting insulin level, impaired glucose tolerance, and decreased insulin sensitivity (Figure B and Figure C). Following the glucose tolerance test, not only did the high fat diet mice show a higher concentration of blood glucose, they also required a longer period of time to bring these glucose levels back down. The decrease in glucose tolerance suggests a development in insulin resistance. These changes have previously been observed in similar studies and generally falls in line with the results of other publications.

These metabolic changes are seen systemically, and the decrease in insulin sensitivity in the brain, as noted via HOMA-IR testing, (figure D) may be due to the increased degradation of the insulin receptor substrate-1 (IRS-1) protein.

Additionally, the western blots of mice fed the fattier diet had a statistically significant decrease in the expression of glucose transporters GLUT1 and GLUT3 within the brain (figure E). These transporters are responsible for glucose uptake, and their decrease has also been noted in both diabetes and Alzheimer’s patients. Researchers also found an increase in the expression of β-secretase which is responsible for cleaving amyloid precursor protein (APP) into β-amyloid. On the other hand, expression of insulin degrading enzyme (IDE) responsible for moderating β-amyloid clearance, was found to have decreased.

As a result of this dysregulation, β-amyloid plaque formation is observed. Furthermore, the researchers noted a visible increase in Tau phosphorylation. In its phosphorylated form, Tau encourages the growth of neurofibrillary tangles; this is also a major indicator of Alzheimer’s pathology.

Kothari’s team (2017) randomly assigned six week old male C57BL/6NHsd mice into experimental groups. The control group was fed a standard chow diet with 12% of energy coming from lipids. In comparison, the high fat and sugar group was fed a diet where 40% of energy came from lipids. Additionally, while the control group were given pure water to drink, the experimental group had a water source supplemented with 42g of sugar/L. At twelve weeks of age, the mice underwent a glucose tolerance test where blood glucose measured following an intraperitoneal glucose injection. Western blots were used to visualize the expression of proteins in the whole brain lysate.

Figure A: A comparison of the body weights between mice givUltimately it was concluded that the high fat and sugar diet en the high fat and sugar diet versus the control group over a could successfully induce systemic insulin resistance, impair span of 13 weeks. (Kothari et al., 2017) glucose uptake, and cause chronic neuroinflammation. Additionally, insulin resistance was observed in the brain resulting in biochemical changes to the rate of β-amyloid deposition, the formation of neurofibrillary tangles, and decrease in synaptic plasticity often seen in Alzheimer’s pathology.

Major Results: The mice given a diet high in fat and sugar all showed a statistically significant increase in total body mass in comparison to the control group (Figure A). The glucose tolerance test 66

Figure B: Fasting insulin levels taken at 12 weeks (left), and ages yet another pathological hallmark of Alzheimer’s known as changes to blood glucose levels following an intraperitoneal neurofibrillary tangles. All together, the combination of changes injection of glucose (right). (Kothari et al., 2017). stimulated by a fattier and sweeter diet are similar to the biological changes in Alzheimer’s patients. Therefore, diet itself could play a vital role in the process of cognitive decline. The data taken from this study shows that a high fat and sugar diet is able to induce insulin resistance within the brain, alter insulin signaling pathways within the brain, affect β-amyloid plaque formation, encourage neurofibrillary tangle formation, and decrease synaptic plasticity.

Critical Analysis Section The results of this study were fascinating and covered a Figure C: Mice following a diet high in sugar and fats (white wide variety of bases. Going forward, it may be beneficial to run boxes) have impaired glucose tolerance due to decreased insua similar study with varying levels of fat and sugar within the lin sensitivity. This is shown by the higher levels of blood gludiet. Rather than confirming whether diet induced insulin recose (Kothari et al., 2017). sistance has the ability to cause changes, researchers should now focus on the level at which fats and sugars begin to impact insulin signaling and cognitive dysfunction. To do so, the levels of fat and sugar in the feed should be changed linearly with a control group given regular chow, a low fat high sugar control group, and a high fat low sugar control group. Since this study analyzed the effect of a fatty diet supplemented with sugar water, it may very well be that a combination of these two factors are responsible for the observed changes. By varying the composition of the mice’s feed, it would be possible to note whether a fatty diet, or sugary diet alone is able to induce these Figure D: HFS mice show higher insulin resistance in the HOMA- changes. Furthermore, Hanses (2018) proposed that obesity IR test; measured in (µU/mL) x glucose (mg/dL)/405 (Kothari et caused by high fat diets may not be the mechanism responsible for the difference in neuronal signaling, but rather dyslipidemia. al., 2017).

An abnormal amount of lipid in the blood may be behind the changes to neuronal signaling and is thought to reduce cognitive function. Rather than using a higher fat diet to induce obesity, perhaps increasing serum lipid concentration would be sufficient in generating a response.

Figure E: Western blot and boxplot of corresponding expression of glucose transporters GLUT1 and GLUT3, which were significantly decreased in the mice given the high fat and sugar diet.

Samples were taken from whole brain lysate after 13 weeks.

(Kothari et al., 2017)

Furthermore, it may be beneficial to have a regulated exercise regime for all mice rather than allow them exercise ad libitum. It was noted in the original experiment that the mice on the higher fat diet had an overall lower level of physical activity in comparison to the control group (Kothari et al., 2017). With mounting evidence that physical activity can ameliorate cognitive decline and the stimulation of glucose uptake via exercise (Brasure et al., 2018) and (Cockroft et al. 2019), it would be worthwhile to moderate their level of activity in order to remove physical activity levels as a confounding variable.

Discussion & Conclusion: High fat diets potentiate systemic insulin resistance, thus resulting in altered protein expression of key transporters and proteins within the brain. Decreased glucose uptake in addition to the downregulation of insulin degrading enzymes are responsible for changing metabolic pathways. This will tip the balance between β-amyloid production and degradation and encourage the formation of β-amyloid plaques seen in Alzheimer pathology. Furthermore, the increase in Tau phosphorylation encour67

REFRENCES

1.

Brasure, Michelle, Priyanka Desai, Heather Davila, Victoria A. Nelson, Collin Calvert, Eric Jutkowitz, Mary Butler, et al.

“Physical Activity Interventions in Preventing Cognitive Decline and Alzheimer-Type Dementia.” Annals of Internal Medicine 168, no. 1 (December 19, 2017): 30–38. https://doi.org/10.7326/M17-1528.

2.

Cockcroft, Emma J., Bert Bond, Craig A. Williams, Sam Harris, Sarah R. Jackman, Neil Armstrong, and Alan R. Barker. “The Effects of Two Weeks High-Intensity Interval Training on Fasting Glucose, Glucose Tolerance and Insulin Resistance in Adolescent Boys: A Pilot Study.” BMC Sports Science, Medicine and Rehabilitation 11, no. 1 (December 2019): 29. https:// doi.org/10.1186/s13102-019-0141-9.

3.

Dittmann, Antje, Norman J Kennedy, Nina L Soltero, Nader Morshed, Miyeko D Mana, Ömer H Yilmaz, Roger J Davis, and Forest M White. “High-Fat Diet in a Mouse Insulin-Resistant Model Induces Widespread Rewiring of the Phosphotyrosine Signaling Network.” Molecular Systems Biology 15, no. 8 (August 1, 2019): e8849. https://doi.org/10.15252/ msb.20198849.

4.

Ferreira, Laís S. S., Caroline S. Fernandes, Marcelo N. N. Vieira, and Fernanda G. De Felice. “Insulin Resistance in Alzheimer’s Disease.” Frontiers in Neuroscience 12 (2018). https://doi.org/10.3389/fnins.2018.00830.

5.

Ferreira, Sergio T., Julia R. Clarke, Theresa R. Bomfim, and Fernanda G. De Felice. “Inflammation, Defective Insulin Signaling, and Neuronal Dysfunction in Alzheimer’s Disease.” Alzheimer’s & Dementia 10, no. 1S (February 1, 2014): S76–83. https://doi.org/10.1016/j.jalz.2013.12.010.

6.

Grice, Brian A., Kelly J. Barton, Jacob D. Covert, Alec M. Kreilach, Lixuan Tackett, Joseph T. Brozinick, and Jeffrey S. Elmendorf. “Excess Membrane Cholesterol Is an Early Contributing Reversible Aspect of Skeletal Muscle Insulin Resistance in C57BL/6NJ Mice Fed a Western-Style High-Fat Diet.” American Journal of Physiology-Endocrinology and Metabolism 317, no. 2 (June 25, 2019): E362–73. https://doi.org/10.1152/ajpendo.00396.2018.

7.

Hansen, Stine Normann, David Højland Ipsen, Anne Marie Schou-Pedersen, Jens Lykkesfeldt, and Pernille Tveden-Nyborg.

“Long Term Westernized Diet Leads to Region-Specific Changes in Brain Signaling Mechanisms.” Neuroscience Letters 676 (May 29, 2018): 85–91. https://doi.org/10.1016/j.neulet.2018.04.014.

8.

Kang, Young-Ho, Mi-Hyang Cho, Ji-Young Kim, Min-Seo Kwon, Jong-Jin Peak, Sang-Wook Kang, Seung-Yong Yoon, and Youngsup Song. “Impaired Macrophage Autophagy Induces Systemic Insulin Resistance in Obesity.” Oncotarget 7, no. 24 (May 25, 2016): 35577–91. https://doi.org/10.18632/oncotarget.9590.

9.

Kasper, J. M., A. J. Milton, A. E. Smith, F. Laezza, G. Taglialatela, J. D. Hommel, and N. Abate. “Cognitive Deficits Associated with a High-Fat Diet and Insulin Resistance Are Potentiated by Overexpression of Ecto-Nucleotide Pyrophosphatase Phosphodiesterase-1.” International Journal of Developmental Neuroscience, Special Issue: Developmental Perspectives on Obesity and Energy Balance, 64 (February 1, 2018): 48–53. https://doi.org/10.1016/j.ijdevneu.2017.03.011.

10.

Koska, Juraj, Marlies K. Ozias, James Deer, Julie Kurtz, Arline D. Salbe, S. Mitchell Harman, and Peter D. Reaven. “A Human Model of Dietary Saturated Fatty Acid Induced Insulin Resistance.” Metabolism 65, no. 11 (November 1, 2016): 1621–28. https://doi.org/10.1016/j.metabol.2016.07.015.

11.

Kothari, Vishal, Yuwen Luo, Talia Tornabene, Ann Marie O’Neill, Michael W Greene, Thangiah Geetha, and Jeganathan Ramesh Babu. “High Fat Diet Induces Brain Insulin Resistance and Cognitive Impairment in Mice.” Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1863, no. 2 (February 1, 2017): 499–508. https://doi.org/10.1016/ j.bbadis.2016.10.006.

68

APOE genotype and chlorpyrifos: Examination of their roles in gut dysbiosis and influence on metabolites in the brain Charlene Hoang

During birth to adulthood the gut microbiome can undergo a series of changes (Backhed et al., 2015), and dysbiosis of the gut microbiota is a result of diet changes, lifestyle changes, genetic factors and environmental factors and is a target of study for neurodevelopmental disorders like Alzheimer’s Disease. Guardia-Escote et al. (2020) explores the Apolipoprotein E (APOE) gene’s isoforms apoE3 and apoE4 – apoE4 is a genetic risk factor for Alzheimer’s Disease (Hersi et al., 2017) – and their suspected roles in modulating gut microbiota composition. They further explored dysbiosis and metabolite level changes in response to exposure to chlorpyrifos (CPF), a commonly used pesticide found at low levels in our diet, which was administered postnatally between days 10-15. Short-chain fatty acid (SCFA) levels and genomic differences of gut microbiota were examined across three transgenic mouse types – C57BL/6, apoE3-TR, and apoE4-TR at postnatal day 15. ApoE4-TR mice were found to have significantly reduced levels of A. muciniphilla, luteolibacter, and rubritaleo in response to CPF exposure and apoE3-TR mice were found to have increased levels of SCFAs including isovaleric acid and 4-methylvaleric acid. The results suggest that the gut microbiota can be altered substantially at early stages of development by both common genetic and environmental factors, and that alterations of metabolites like SCFA in the brain can lead to neurodevelopmental disorders later in life.

Keywords: Apolipoprotein E (APOE), chlorpyrifos (CPF), dysbiosis, short-chain fatty acid (SCFA), gut microbiota, Alzheimer’s Disease

69

Background and Introduction

effects when done during development. The gut composition is associated with the production of SCFAs which are key signaling factors found in the brain. Acetate and butyrate play key roles as energy sources for the brain and can also provide neuroprotection for the brain however, levels of various SCFAs like isovaleric acid are to be monitored as overexpression can cause neurodevelopmental defects (Zhao et al., 2016).. The effects CPF have on gut microbiome composition and SCFA levels are therefore both indirectly and directly influencing human health.

The symbiotic relationship between the gut microbiome, its composition, and humans are a major determinant of human health. Metabolism, immune system function, digestion, and even mental health are all affected by the gut microbiota, and when dysregulation or dysbiosis occurs human health can be influenced. Dysbiosis has been known to cause neurodegenerative diseases (Marizzoni et al., 2017) including Alzheimer’s Disease, dementia, and Parkinson’s Disease. Alterations in the gut microbiome can occur from diet changes (Makki There is still a lack of direct connection between how et al., 2018), lifestyle changes, genetic factors and environmen- alterations to the gut microbiome changes and affects brain tal factors like pesticide exposure (Yuan et al., 2019). development as well as neural behavior. However through this paper, the authors have tried to examine a link between vulThe APOE gene exists in isoforms in the human body, nerabilities to environmental factors in developing gut microbiof which the isoform apoE4 has been linked as a prevalent risk omes and brains of mice with varying genetic backgrounds and factor for Alzheimer’s Disease (Hersi et al., 2017). Normally, it how such interactions can implicate future studies of neuroexists as a gene important for lipid efflux and lipid transportadegenerative and neuropsychiatric diseases. The results of this tion (Rebeck G. W., 2017) serving as a low-density lipoprotein paper suggests that there is more to be learned of about how receptor (LDLR) (Zhao et al., 2019) however, apoE4 has been genetic and environmental factors work together to modulate linked to hallmarks of Alzheimer’s Disease such as accumulaboth the gut and the brain’s activities, and how predispositions tion of amyloid beta plaques (Munoz et al., 2018) and is a risk and external factors can be therapeutically targeted in both factor for the development of alpha synuclein derived Lewy treatment and prevention of neurodevelopmental and neuroBodies (DLB) – a pathological hallmark of dementia and Parkindegenerative diseases. son’s Disease (Fyfe I., 2020). A quarter of the US population carries the apoE4 allele (Rebeck G.W., 2017) and of the patients suffering from AD, 50% are linked to this allele (Teter B., 2004), rendering almost 13% of the population susceptible to develop- Major Results ing late onset AD and dementia. The genomic composition of Baseline difference in relative abundance of microbes between the microbiota of individuals are also influenced by APOE genodifferent genetic backgrounds type (Tran et al., 2019) and so the relative abundance of miThe phylum Verrucomicrobia was found at a generally crobes can therefore determine differing metabolite production as well as interactions with CPF. Thus it is important to higher level in apoE4 genotypes but levels had dropped to examine the role of APOE genetic backgrounds on gut microbi- numbers relatively similar to that of apoE3-TR and C57BL/6 ome susceptibility to CPF and its further implications on human mice after CPF exposure (Fig 1). health.

Chlorpyrifos are a commonly used pesticide that causes inhibition of acetylcholinesterase (AchE) (Burke et al., 2017) – an enzyme responsible for the catalyzation of acetylcholine which is a neurotransmitter involved in neuronal signaling. The gut microbiota is the most vulnerable to the toxicity of CPF and can cause dysbiosis (Liang et al., 2019) as CPF exposure is elicited through low doses during consumption of foods and is therefore in direct contact with the microbiome The authors administered CPF to postnatal mice at 10-15 days old orally to Fig. 1. The phylum Verrcomicrobia had significant examine the effects it had on gut microbiome composition in mice of differing genetic backgrounds on day 15. Modulation of baseline differences of relative abundance in apoE4 control the microbiome depends on the genetic composition of the pre mice compared to apoE3 and C57BL/6 mice -existing gut, but changes from CPF can cause long lasting 70

Akkermansia, Luteolibacter and Rubritalea modulation is uniquely found in apoE4 genotype compared to apoE3 in response to CPF exposure (Fig. 2). ApoE4-TR control mice showed significantly higher levels of Verrucomicrobia, A.muciniphilla, luteolibacter, and rubritalea compared to apoE3 -TR and C57BL/6 mice indicating a baseline difference of relative abundance of microbes between different genetic backgrounds. This indicates that genetic profiles influence the susceptibility of individuals to external factors as in this case where apoE4 carriers were more susceptible to effects of CPF on gut microbiota composition.

Fig. 3. Short-chain fatty acid level between control and CPF treated mice of (a) acetic acid, (b) propionic acid, and (c) butyric acid. Changes in all SCFAs were increased in CPF treated apoE3 mice.

Fig. 2. ApoE3, apoE4, and C57BL/6 control mice and CPF treated mice’s relative abundance of (A) akkermansia,(G) luteolibacter, and (H)rubritalea Akkermansia muciniphilla’s heightened susceptibility to CPF Fig. 4. Isovaleric acid and 4-methylvaleric acid are SCFAs that exposure in apoE4 genotypes are less abundant in control mice, but are increased after expoA.muciniphilla levels in apoE4 were most abundant sure to CPF indicating CPF treatment could favor the release of amongst the three groups, with levels being almost 24 times less abundant SCFAs. that of apoE3 and C57BL/6 control mice. CPF treated apoE4 mice exhibited significant loss of A. muciniphilla as levels conDiscussion siderably diminished (Fig. 2A) suggesting that apoE4 genotype was more susceptible to effects of CPF during early developApoE4-TR mice exhibited naturally greater amounts of ment A.muciniphilla and greater loss after exposure to CPF as well.

SCFA levels in apoE3 genotype are increased after CPF exposure Important SCFAs including butyric acid and acetic acid were found at higher relative abundance after treatment with CPF in apoE3 mice (Fig 3A and 3B). Levels of SCFAs in apoE3 CPF treated mice were higher compared to control mice for butyric acid, acetic acid, isovaleric acid and 4-methylvaleric acid indicating that exposure to CPF increased the production of these SCFAs (Fig 4). These SCFAs are involved in brain signaling therefore alterations to levels by CPF can change brain function and behavior.

This microbe is a mucin degrading bacterium that is positively correlated with healthier individuals during early development as it promotes stronger immune defense and function. Individuals lacking this species are negatively correlated with obesity and diabetes, therefore the presence of this species is important for implication in early human development and later health. After exposure to CPF, A. muciniphilla experienced great loss indicating this species has a higher susceptibility to CPF and therefore apoE4 individuals are at greater risk of losing this species and its immune protective properties during development. These results indicate that there is a genetic and environmental factor that determines gut microbiota composition.

71

ApoE3-TR mice exhibited naturally higher levels of butyrate which acts as an inhibitor of histone deacetylases as well as having higher levels of acetate which acts as an energy source for the brain, therefore conferring higher neuroprotective properties compared to apoE4 genotype. After exposure to CPF, levels of these SCFAs increased inferring even higher neuroprotective properties in those with the apoE3 genetic profile after exposure to CPF. However, isovaleric acid and 4methylvaleric acid levels also increased compared to control in apoE3-TR mice. Higher levels of these less abundant SCFAs have been linked to individuals suffering from depression, another neurobehavioral disorder. This result further suggests that there is a link between genetic profiles, external factors, their affects on the gut microbiome, and its potential link to neurodegenerative diseases like Alzheimer’s disease.

The results of this paper further support the idea that neurodegenerative diseases are multifactorial and that many factors act together to further potentiate the onset of certain developmental diseases. They suggest that early development and alterations to gut microbiome during this time can have major affects on later health, and that studying these effects can help formulate ideas of new therapeutic targets for major diseases like dementia and Alzheimer’s Disease.

Critical Analysis Neurodegenerative diseases like AD are influenced by many factors ranging from genetic to environmental and can be caused by gut microbiota composition. These neurodegenerative diseases, although occurring later in life, can be of higher risk to certain individuals because of early development of gut microbiota composition. As seen in the results apoE4 CPF treated mice, susceptibility to such toxins are greater in mice carrying said genotype indicating that genetic factors and environmental risk factors work together to alter gut microbiota compositions and therefore human health later on in life. The decrease in A. muciniphilla negatively correlates with health complications like diabetes and obesity later on in life and well as diminishing immune protective properties, and with apo4 being a prevalent predisposition for 50% of AD patients. This leads us to believe that early and recurring exposure to pesticides commonly found in food can cause an even higher risk of AD development in those carrying the E4 genotype. In apoE3 individuals, CPF exposure induces higher neuroprotective properties, however increases in less abundant SCFAs have also been correlated with depression suggesting again that early gut microbiome

alterations from external factors paired with genetic profiles lead individuals to higher susceptibility of neurodevelopmental and health issues. The authors should further investigate the neurodegenerative properties that are a result of changes in the gut microbiota and SCFA production from CPF exposure in genetically predisposed individuals, namely to Alzheimer’s Disease.

Future Directions As stated above, further investigation will allow for future therapeutic directions in terms of treatment and prevention of neural developmental diseases like AD. Experiments should be done using genetically predisposed people like carriers of the apoE4 genotype following their lives and diets that are pesticide free or with pesticides. Along the way, they should monitor microbial composition as well as SCFAs levels in the plasma, and record behavior and mental health. If the results of these experiments show that common pesticide use increases the risk even more in almost 13% of the US population for neurodevelopmental diseases like AD, then this could further implicate the use of pesticide in wide spread farming practices. However if results fail to show an increased risk of disease, then further research should be done with other known genetic predispositions, or known environmental factors. This experiment can be replicated with not only neurodegenerative and neurobehavioral disease, but other disease such as diabetes, developmental, immunosuppressive diseases and obesity.

72

REFRENCES

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

Guardia-Escote, L. et al. (2020). “APOE genotype and postnatal chlorpyrifos exposure modulate gut microbiota and cerebral short-chain fatty acids in preweaning mice”. Food and Chemical Tosicology. 135: 110872. doi: 10.1016/ j.fct.2019.110872 Rebeck, G. W. (2017). “The role of APOE on lipid homeostasis and inflammation in normal brains”. J lipid Res.58(8): 14931499. doi: 10.1194/jlr.R075408 Munoz, S. S. (2019). “Understanding the Role of ApoE Fragments in Alzheimer’s Disease”. Neurochemical Research. 44: 1297-1305. https://doi-org.myaccess.library.utoronto.ca/10.1007/s11064-018-2629-1 Zhao, N. et al. (2019). “Apolipoprotein E, Receptors and Modulation of Alzheimer’s Disease”. Biol Psychiatry. 83(4): 347357. doi: 10.1016/j.biopsych.2017.03.003 Fyfe, I. (2020). “APOE*E4 promotes synucleinopathy”. Nature Reviews Neurology. 16: 185. https://doiorg.myaccess.library.utoronto.ca/10.1038/s41582-020-0335-5 Teter, B. (2004). “ApoE-dependent Plasticity in Alzheimer’s Disease”. J Mol Neurosci.23(3): 167-9. doi: 10.1385/ JMN:23:3:167.

Carter, D. B. (2005). “The Interaction of amyloid-beta with ApoE”. Subcell Biochem. 38: 255-72. doi: 10.1007/0-387-232265_13.

Tran, T. et al. (2019). “APOE genotype influences the gut microbiome structure and function in humans and mice: relevance for Alzheimer’s disease pathophysiology”. FASEB J. 33(7): 8221-8231. doi: 10.1096/fj.201900071R Liang, Y. et al. (2019). “Organophosphorus pesticide chlorpyrifos intake promotes obesity and insulin resistance through impacting gut and gut microbiota” Microbiome. 7: 19. doi: 10.1186/s40168-019-0635-4 Yuan, X. et al. (2019). “Gut microbiota: An underestimate and unintended recipient for pesticide-induced toxicity”. Chemosphere. 227: 425-434. https://doi.org/10.1016/j.chemosphere.2019.04.088 Burke, R. D. et al. (2017) “Developmental neurotoxicity of the organophosphorus insecticide chlorpyrifos: from clinical findings to preclinical models and potential mechanisms”. J Neurochem. 142(Suppl 2): 162-177. doi: 10.1111/jnc.14077 Lee, C. J. et al. (2019). “Gut microbiome and its role in obesity and insulin resistance”. The New York Academy of Sciences.

1461(1). https://doi-org.myaccess.library.utoronto.ca/10.1111/nyas.14107 Makki, K. et al. (2018). “The Impact of Dietary Fiber on Gut Microbiota in Host Health and Disease”. Cell Host & Microbe.

23(6): 705-715. https://doi.org/10.1016/j.chom.2018.05.012 Zhao, Y. et al. (2016). “Effects of chlorpyrifos on the gut microbiome and urine metabolome in mouse (Mus musculus)”.

Chemosphere. 153: 287-293.

Marizzoni, M. et al. (2017). “Microbiota and Neurodegenerative Diseases”. Curr Opin Neurol. 30(6):630-638. doi: 10.1097/ WCO.0000000000000496.

73

Altered Feed-Forward Inhibition of Striosomes is Linked to Aberrant Value-Based Decision Making in Chronically Jessica Jenkins

Individuals suffering from disorders such as anxiety and depression often exhibit difficulty with rational decision making. Since these disorders are frequently a consequence of chronic stressors, Friedman et al. (2017) hypothesized that stress may also be responsible for maladaptive changes in the neural circuitry involved in evaluative thinking and decision making. Specifically, their region of interest was the prefrontal corticostriatal circuit since previous research identified this area to be important for evaluation. A T-maze task was used to identify differences in value-based decision-making behavior (decisions based on rewards and costs) between chronically stressed rats and control. Compared to control, stressed rats demonstrated flawed cost-benefit evaluation, since they chose absolute highrisk high-reward options more frequently than alternatives with a maximum benefit-cost difference (maximum value). This discrepancy in behavior correlated with a decrease in the spike activity in the prefrontal-prelimbic cortex neurons (PFC-PL), which in return caused a decrease in the firing of synapsing fast-spiking interneurons (FSI) and an increase in the activity of projecting neurons in the striosome (SPN). Optogenetic excitation of the PFC-PL neurons during the T-maze task successfully rescued decision making in chronically stressed mice, increasing confidence that the regulation of striosome activity plays an important role in healthy executive function. Freidman and colleagues (2017) referred to previous literature that identified striosomal projections that synapse directly with subsets of dopaminergic neurons in the substantia nigra. These neurons respond to rewarding and aversive stimuli, therefore it is possible that stress-induced overactivity in the striosome may be causing the erratic responses to costs and rewards in the T-maze task. Future studies are encouraged to implement some changes in the design of the T-maze task to overcome confounding effect, and to further explore the effect of chronic stress on the connection between the striosome and its downstream projections to the substantia nigra.

Key words: chronic stress, cost-benefit evaluation, striosome, value-based decision making, prefrontalprelimbic cortex, striosomal projecting neurons, fast spiking interneurons, T-maze task, prefrontal corticostriatal circuit, feed-forward inhibition, foot shock, optogenetics

74

Introduction

Chronic stress is one of the leading factors that cause anxiety and depressive disorders (Khan & Khan, 2017). According to the American Psychological Association, reported levels of stress are on the incline, with an average 6% annual increase in stress levels between 2012 and 2017 in the United States (Bethune & Lewan, 2017). Furthermore, a 2017 report by the APA put forth the alarming statistic that at a third of individuals experience symptoms of nervousness, headaches and feelings of depression due to the stressors in their lives (Bethune & Lewan, 2017).

Impairment in cognitive functions such as attention, memory and evaluative thinking are common symptoms of both anxiety and depression according to the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (2013). Experimental evidence for this was provided through a case-control study, whereby individuals with Major Depressive Disorder performed significantly worse in value-based decision tasks that involve punishment and reward learning. (Mukherjee et al., 2020). Previous repeated trans-magnetic stimulation studies on humans and lesion studies with macaque monkeys have collected evidence for the importance in the connectivity within the prefrontal, orbitofrontal and prelimbic systems during reward-guided learning and decision making (Amemori & Graybiel, 2012). In 2015, Friedman and colleagues’ discovery of prefrontal corticostriatal circuit lead to their suggestion that this circuitry likely contributes to the executive control of value-based decision making, since it facilitates the recruitment of the reward system during evaluation (Friedman et al., 2015). Given the compilation of the research listed, it is possible that the stress-inflicted impairment of value-based decision making is brought by changes in the connectivity of the prefrontal corticostriatal circuit.

logical measurements during a T-maze cost-benefit conflict decision task. The prefrontal corticostriatal circuit was implicated by previous research to be important for cost, effort and reward evaluation (Amemori & Graybiel, 2012). This was done to compare neuronal activity levels in chronically stressed rats versus control during value-based decisionmaking. In doing so, it was confirmed that there were differences in brain activity between the two groups of rats, and these differences correlated with stress-induced impairment of cost-benefit evaluation in stressed rats. The reduction in the feed-forward inhibition of striosome projecting neurons was of particular interest to the authors, since these projections synapse with neurons in the substantia nigra that regulate motivated behaviors guided by reward and aversion (Sousa & Almeida, 2012). Confidence in these results was increased when optogenetic intervention successfully rescued stress-induced impairments in decision making.

MAJOR RESULTS Experimental rats underwent 14 days of foot shock as chronic stress, and then were tested in a costbenefit T-maze task. The cost-benefit T-maze task is an assay commonly used in psychology and neuroscience research to study reward-based decision making (Cousins et al., 1996; Schweimer & Hauber, 2006). Friedman et al. (2017) used fluorescent light as an aversive stimulus and chocolate milk as a reward. Different combinations of light intensity and chocolate milk concentration were presented to the rat at the left and right arm of the T-maze, to vary the size of the costs and rewards at each arm across trials. This was done to prompt the rats to evaluate which option was worth choosing, given the size of the combined reward and cost at each arm. The experiment was repeated after the rats were fitted with electrodes to measure spike activity at the PFCPL, FSI and SPN. After stress-induced spike activity changes were identified, optogenetic manipulation was introduced at the PFC-PL of stressed rats to counteract the effect of chronic stress on behavior.

Nonetheless, until Friedman and colleagues’ 2017 study, there was no concrete evidence to suggest that these changes could be brought by stress, and nor was there a clear understanding of how these changes bring upon maladaptive decision-making. To 14 days of chronic stress causes changes in responaddress these questions, Friedman et al. (2017) siveness to costs and rewards in a T-Maze chose three components of interest within prefroncost-benefit conflict task. tal corticostriatal circuit, to carry out electrophysio75

1A

HIGH

Figure 2: Changes in the prefrontal corticostriatal circuit activity following 14 days of chronic stress: spike activity in the PFCPL, FSI and SNP.

LOW

Spike activity recorded during the T-maze test revealed that chronically stressed rats had lower PFC-PL and FSI neural activity measures compared to control, p < 0.01 (KolmogorovSmirnov test and t test). In contrast, neural activity in the SPN was greater in chronically stressed mice, compared to control p < 0.01 (Kolmogorov-Smirnov test and t test). These differences were speculated to cause the stress-induced behavioral changes in value-based decision-making.

1B

Optogenetic rescue of value-based decision-making behavior in chronically stressed rats.

3A

3B

1C

Figure1: T-maze experiment investigates cost-benefit evaluation behavior: A) Schematic depicting the T-maze task with a cost-benefit conflict. Authors varied size of rewards on each arm of the T-maze. B) Ratsâ&#x20AC;&#x2122; preference for milk chocolate varying with concentration. C) Time spent under light exposure at low (7lux) versus high (2000 lux) intensity.

Compared to control, chronically stressed rats exhibited higher sensitivity to increases in chocolate milk concentration, and lower responsiveness to increased light intensity. As a result, chronically stressed rats were consistently attracted to the high concentrated milk (high reward) even when it was coupled with high intensity light (high cost), p<0.001 (one-way ANOVA with Bonferroni correction). Control rats were less inclined to choose high reward options if they were coupled with high costs, and therefore settled for options with lower concentrations of chocolate so long as they were coupled with low intensity light.

Figure 3: Optogenetic excitement of the PFC-PL projections rescue decision-making behavior in chronically stressed rats.

A) Schematic of transfection of viral load onto PFC-PL neurons, and optogenetic control of synapses between PFC-PL and striosomal SPNs. B) Optogenetic excitation of PFC-PL during the Tmaze task.

Optogenetic excitation of the PFC-PL neurons in chronically stressed rats lead them to make decisions that account for maximum value over absolute high risk-high reward. This means that, like control, the chronically stressed optogenetic mice settled for options with low intensity light over options with high intensity light coupled with high reward, p < 0.001 (one-way ANOVA with Bonferroni correction. This is considered to be adaptive behavior and indicative of successful costbenefit evaluation.

Evidence for changes in the prefrontal cortiostriatal circuit acDISCUSSIONS AND CONCLUSIONS tivity following 14 days of chronic stress.

Prior to this study, it was known that chronic stress is a major contributor to the development of depression and anxiety, and that the impairment of value-based decision making is commonly experienced by individuals with depression or anxiety.

Friedman and colleagues (2017) used behavioral and physiological measures to shed light on the direct link between chronic stress and decision making, and they concluded with compelling evidence to support their hypothesis. Firstly, it was found 76

that chronically stressed rats were unable to conduct correct cost-benefit evaluation, which lead to poor value-based decision-making. The stress-induced behavioral change also correlated with alterations in activity of the prefrontal corticostriatal circuitry known to be involved in decision-making.

Following chronic stress, electrophysiological recording revealed reduced activity of this feed-forward inhibitory circuit, which lead to the increased excitation of striosomal projecting neurons. Uncoincidentally, these projections synapse with neurons of the substantia nigra that respond to motivating stimuli (rewarding and aversive) (Sousa & Almeida, 2012). The authors speculated that uncontrolled and excessive activity of the substantia nigra may be the mechanism through which chronic stress causes impairment in appropriate cost-benefit evaluation. This prediction was tested and supported when optogenetic excitation of the PFC-PL neurons rescued the decisionmaking behavior of chronically stressed mice.

Overall, Friedman and colleagues’ (2017) study contributed greatly to our understanding of the intersection between environmental stressors, brain physiology and behavioral output.

Until recently, the relationship between stress and decision making was mainly considered in the context of psychiatric disorders (Goschke, 2014; Stetz et al., 2007). This is because difficulties with decision making was often attributed to abnormal fluctuations in norepinephrine neurotransmission brought by stress-induced depression or anxiety (Goddard et al., 2010; Schildkraut, 1965). Although impairment in decision-making is often comorbid with such disorders, Friedman and colleagues’ (2017) study shows that the specialized circuit for value-based decision-making may be influenced by stress separate from other brain regions that control mood and anxiety, such as the anterior cingulate cortex, insula and amygdala (Pandya et al., 2012). This could explain why some individuals still suffer from stress induced decision-making impairment without necessarily experiencing depression or anxiety (Porcelli & Delgado, 2017). Likewise, Friedman et al.’s findings may have important clinical implications, since it supports the notion that stress is linked to a cluster of symptoms associated but not exclusive to psychiatric disorders.

CRITICAL ANALYSIS Although the cost-benefit conflict T-maze behavioral assay and methodology was described in Friedman et al. (2017)’s paper, the results from this assay was not depicted clearly by the authors. A brief description in the supplemental materials mentions that different combinations of concentration of chocolate milk were coupled with either high (2000lux) or low (7lux) light intensity, however, the authors did not present the ‘frequency of selection’ data for each combination of chocolate concentration and light intensity. Recording and presenting this data could give insight to trends in decision making, and reveal the threshold levels of costs and rewards where rats experience the most conflict.

could be criticized. Bright fluorescent light is aversive for rats, and it is considered appropriate to apply as punishment in place of foot shocks (Barker et al., 2010). However, punishment is not synonymous with cost; for cost-benefit paradigms, cost is generally defined by the amount effort that is exerted to obtain a reward (Braun & Hauber, 2011; Walton et al., 2002). In contrast, punishment elicits negative affect and often causes distress, which could lead to confounding effects that influence judgement (Molm, 1994). Therefore, in the case of Friedman et al. (2017)’s experiment, chronically stressed rats may have behaved differently from control, not because of impaired costbenefit evaluation, but because of increased or decreased sensitivity to distressing punishment.

Lastly, the authors suggested that ineffective feedforward inhibition in the prefrontal corticostriatal circuit is the cause for excessive activity of the substantia nigra neurons that control responses to rewarding and aversive stimuli. Although they quoted previous research to explain this line of logic, Friedman et al. (2017) did not carry out any physiological measures or optogenetic manipulations at the substantia nigra to confirm this. Hyperactivity in the striosomal projecting neurons may be influencing other brain regions besides the substantia nigra, and therefore decision-making impairment in this study cannot be confidently attributed to the substantia nigra, without evidence.

FUTURE DIRECTIONS To avoid confounding effects from distressing punishment with bright light, future studies continuing Freidman et al.’s (2017) work could implement a cost-benefit conflict T-maze task that uses barriers as cost instead of light. This setup was used in the past by Braun and Hauber (2011) and Walton et al. (2002), both of whom were interested in rodent brain function during decision-making. In both of these studies, the barriers were triangular shaped wooden blocks, and the rats had to climb up the vertical wall and climb down the steep slope to reach the reward on the other side. The cost was modified by increasing the height of the vertical wall of the barrier, from 15cm to 30cm in Walton et al.’s (2002) study, and from 20cm to 30cm in Braun and Hauber’s (2011) study. Braun and Hauber (2011) also manipulated reward by varying food reward size between full to half size. Considering experimental designs from previous literature, Friedman et al.’s study could implement block barriers of heights 15cm, 20cm, and 30cm, to provide 3 magnitudes of cost, and use 30%, 60% and 90% chocolate concentrations to provide 3 magnitudes of reward. This will make it simpler to match different sizes of costs and rewards, and it will ensure that the obtained results will not be influenced by confounding effects resulting from punishment.

Furthermore, authors Friedman et al. could improve the transparency of their work by presenting their results clearly. For each trial, the cost-benefit options at the two arms of the TMaze should be reported, as well as the option that the rat chose. This will allow readers to follow the authors’ line of logic, Likewise, the authors’ choice of fluorescent light as a ‘cost’ and to derive more extensive interpretations from the data.

77

Similarly, the missing link between the substantia nigra and decision impairment must be addressed. Repeating the present experimental design and adding electrophysiological measures at the substantia nigra during the cost-benefit conflict T-maze task will be the first indicator of whether the prefrontal corticostriatal feed-forward inhibition increases activity in the subtantia nigra, as proposed by the authors.

Friedman and colleagues’ (2017) study could be extended to expand our understanding of the relationship between chronic stress and addictive behaviors. Friedman et al. (2017)’s study found that chronically stressed rats gravitate to high-risk highreward alternatives, which is also the maladaptive behavior exhibited by individuals with gambling addictions (Fujimoto et al., 2017). Fujimoto and colleagues put forth evidence for reduced risk-attitude flexibility in participants with gambling disorders associated with reduced activity in the dlPFC. With this in mind, future studies could identify the influence of chronic stress on brain activity in the prefrontal corticostriatal circuit and the dlPFC while testing with the rodent model of the Iowa Gambling Task (IGT) (Van Den Bos et al., 2006). IGT is an experiment that tests behavioral responses to outcome uncertainty and payoffs between immediate and long-term gains. Applying this assay will increase the sophistication of Friedman et al.’s cost-benefit conflict paradigm by adding the dimension of future planning into decision making. According to Friedman et al. (2017), chronically stressed rats cannot evaluate costs and benefits as well as control, therefore stressed rats’ behavior is expected to reflect this as an increase impulsion in the IGT test, as well as a significant shift to short-term risky decisions that forgo low-risk/high-gain long-term options. Overall, implementing the IGT test as an extension to Friedman et al. (2017)’s study will not only expose the gravity of influence that chronic stress has on time-dependent value-decision making, but it will also have important implications for the interaction between chronic stress and addictive behaviors.

78

REFRENCES 1.

Amemori, K. I., & Graybiel, A. M. (2012). Localized microstimulation of primate pregenual cingulate cortex induces negative decision-making. Nature Neuroscience, 15(5), 776–785. https://doi.org/10.1038/nn.3088

2.

American Psychiatric Association. (2013). Diagnostic and statistical manual of mental disorders (5th ed.). https:// doi.org/10.1176/appi.books.9780890425596

3.

Barker, D. J., Sanabria, F., Lasswell, A., Thrailkill, E. A., Pawlak, A. P., & Killeen, P. R. (2010). Brief light as a practical aversive stimulus for the albino rat. Behavioural Brain Research, 214(2), 402–408. https://doi.org/10.1016/j.bbr.2010.06.020

4.

Braun, S., & Hauber, W. (2011). The dorsomedial striatum mediates flexible choice behavior in spatial tasks. Behavioural Brain Research, 220(2), 288–293. https://doi.org/10.1016/j.bbr.2011.02.008

5.

Cousins, M. S., Atherton, A., Turner, L., & Salamone, J. D. (1996). Nucleus accumbens dopamine depletions alter relative response allocation in a T-maze cost/benefit task. Behavioural Brain Research, 74(1–2), 189–197. https://doi.org/10.1016/0166-4328(95) 00151-4

6.

Friedman, A., Homma, D., Gibb, L. G., Amemori, K. I., Rubin, S. J., Hood, A. S., Riad, M. H., & Graybiel, A. M. (2015). A corticostriatal path targeting striosomes controls decision-making under conflict. Cell, 161(6), 1320–1333. https://doi.org/10.1016/ j.cell.2015.04.049

7.

Fujimoto, A., Tsurumi, K., Kawada, R., Murao, T., Takeuchi, H., Murai, T., & Takahashi, H. (2017). Deficit of state-dependent risk attitude modulation in gambling disorder. Translational Psychiatry, 7(4). https://doi.org/10.1038/tp.2017.55

8.

Goddard, A. W., Ball, S. G., Martinez, J., Robinson, M. J., Yang, C. R., Russell, J. M., & Shekhar, A. (2010). Current perspectives of the roles of the central norepinephrine system in anxiety and depression. In Depression and Anxiety (Vol. 27, Issue 4, pp. 339– 350). John Wiley & Sons, Ltd. https://doi.org/10.1002/da.20642

9.

Goschke, T. (2014). Dysfunctions of decision-making and cognitive control as transdiagnostic mechanisms of mental disorders: Advances, gaps, and needs in current research. International Journal of Methods in Psychiatric Research, 23(S1), 41–57. https:// doi.org/10.1002/mpr.1410

10.

Khan, S., & Khan, R. A. (2017). Chronic Stress Leads to Anxiety and Depression.

11.

Many Americans Stressed about Future of Our Nation, New APA Stress in America TM Survey Reveals. (n.d.). Retrieved June 9, 2020, from https://www.apa.org/news/press/releases/2017/02/stressed-nation

12.

Molm, L. D. (1994). Is Punishment Effective? Coercive Strategies in Social Exchange. Social Psychology Quarterly, 57(2), 75. https://doi.org/10.2307/2786703

13.

Mukherjee, D., Lee, S., Kazinka, R., D Satterthwaite, T., & Kable, J. W. (2020). Multiple Facets of Value-Based Decision Making in Major Depressive Disorder. Scientific Reports, 10(1), 3415. https://doi.org/10.1038/s41598-020-60230-z

14.

Pandya, M., Altinay, M., Malone, D. A., & Anand, A. (2012). Where in the brain is depression? In Current Psychiatry Reports (Vol. 14, Issue 6, pp. 634–642). NIH Public Access. https://doi.org/10.1007/s11920-012-0322-7

15.

Porcelli, A. J., & Delgado, M. R. (2017). Stress and decision making: effects on valuation, learning, and risk-taking. In Current Opinion in Behavioral Sciences (Vol. 14, pp. 33–39). Elsevier Ltd. https://doi.org/10.1016/j.cobeha.2016.11.015

16.

Schildkraut, J. J. (1965). The catecholamine hypothesis of affective disorders: a review of supporting evidence. In The American journal of psychiatry (Vol. 122, Issue 5, pp. 509–522). American Psychiatric Publishing. https://doi.org/10.1176/ajp.122.5.509

17.

Schweimer, J., & Hauber, W. (2006). Dopamine D1 receptors in the anterior cingulate cortex regulate effort-based decision making. Learning and Memory, 13(6), 777–782. https://doi.org/10.1101/lm.409306

18.

Sousa, N., & Almeida, O. F. X. (2012). Disconnection and reconnection: The morphological basis of (mal)adaptation to stress. In Trends in Neurosciences (Vol. 35, Issue 12, pp. 742–751). Elsevier Current Trends. https://doi.org/10.1016/j.tins.2012.08.006

19.

Stetz, M. C., Thomas, M. L., Russo, M. B., Stetz, T. A., Wildzunas, R. M., McDonald, J. J., Wiederhold, B. K., & Romano, J. A. (n.d.).

Stress, Mental Health, and Cognition: A Brief Review of Relationships and Countermeasures.

20.

Van Den Bos, R., Lasthuis, W., Den Heijer, E., Van Der Harst, J., & Spruijt, B. (2006). Toward a rodent model of the Iowa gambling task. Behavior Research Methods, 38(3), 470–478. https://doi.org/10.3758/BF03192801

21.

Walton, M. E., Bannerman, D. M., & Rushworth, M. F. S. (2002). The role of rat medial frontal cortex in effort-based decision making. Journal of Neuroscience, 22(24), 10996–11003. https://doi.org/10.1523/JNEUROSCI.22-24-10996.2002

79

Immune alteration using human mesenchymal stem cells in schizophrenia: a review Xiaoke Jiang

Schizophrenia is a serious mental disorder characterized by positive, negative and cognitive symptoms, the word “Schizophrenia” is proposed almost a century ago, yet we do not know the pathology behind it. There are many lines of evidence pointing out the correlation between microglial activation and neuroinflammation in schizophrenia proposing the control of microglial activation potentially could be a therapeutic direction in schizophrenia treatment. In this review, we assess the potential use of human mesenchymal stem cells with its immunosuppression and immunomodulatory property to decrease schizophrenia-relevant behavioral abnormality targeting microglial activation. This review answers two questions, “Can cytokine level be a diagnostics marker in schizophrenia” and “Can human mesenchymal stem cells be a therapeutic drug for schizophrenia” Using the answers to these two questions, critical analysis and future prospect of clinical interpretation is proposed.

80

Background and Introduction

of macrophage-derived cytokines TNF-α, IL-6and IL-1β in periphery and the brain of schizophrenic patients, which is normalized after APDs treatment. The study considered APDs and clinical Schizophrenia is a complex, serious mental disorder affecting status to eliminate potential confounding and the overall concluapproximately 1% of the population, it is characterized by posision is consistent with the immune-cytokine hypotheses of schiztive symptoms such as delusions, hallucinations and paranoia, ophrenia negative symptoms such as social withdrawal and emotional withdrawal, and cognitive symptoms such as learning and attention disorders. Abnormalities in dopaminergic receptors (e.g., D2 Figure 1 Immunohistochemical staining of microglial cell （A） receptor) have established the current understanding of pathoImmunohistochemical staining from physiology of schizophrenia, although antipsychotic (APDs) medfrontal cortex of control (C) Immunoications (both first- and second-generation) aiming dopamine histochemical staining from frontal cortex of schizophrenia patients transmission by blocking D2 receptor could alleviate positive symptoms of schizophrenia, frequent occurring negative and Adapted fromBayer, Thomas A, Rolf Buslei, Laszlo Havas, and Peter Falkai. cognitive symptoms remain untackled by current APDs. Moreo“Evidence for Activation of Microglia in ver, these medications have also been associated with side Patients with Psychiatric Illnesses.” effects including rigidity, tremor, weight gain, metabolic dysregNeuroscience Letters 271, no. 2 (August 20, 1999): 126–28. https:// ulation and sedation, suggesting an increasing need for novel doi.org/10.1016/S0304-3940(99)00545-5. therapeutic approaches and targets for schizophrenia.

The abnormality of cytokine level have been proposed to be associated with schizophrenia in many studies, although that level of cytokine level often correlate with the severity of clinical symptoms, elevation of puerperal level of IL-1β, IL-6, and TNF-α are concise in schizophrenia patients contrast to control suggesting immune-inflammatory mechanism related to these proinflammatory cytokine maybe involved in schizophrenia pathology.

However, although all studies above are informative, they are also innately limited, as the pathology behind schizophrenia remains unknown. This review focuses on linking neuroinflammation with microglial activation in schizophrenia. We provide a review on experiments done by You et al. on stem cell therapy on schizophrenia suggested the potential use of immunomodulatory medication/therapy targeting microglial activation as future treatment for schizophrenia, followed by critical analysis and future prospect.

The microglia hypothesis of schizophrenia proposing inhibition of free radical cytokines including TNF-α, IL-6 and IL-1β to treat developing/onset/relapse of schizophrenia was also proposed in a review article (Fig.2), the article showed extensive knowledge in schizophrenia related neuroinflammation focus on the role on microglial activation and suggested controlling anti-inflammatory cytokine as well as immunomodulatory drug as future treatment for schizophrenia .

Results Microglial activation and cytokine More recent studies have suggested activated microglial cells associated with neuroinflammation maybe involved in neuropsychiatric disorders like schizophrenia. The idea of microglia activation in brains of patients with severe psychiatric illnesses like schizophrenia was first proposed two decades ago by Thomas A Bayer, who brought the first insight into pathophysiological process for schizophrenia, post-mortem study was conducted using immunohistochemical staining and HLA-DR antigen as an indicator for microglial reactivity. Out of 14 patients with schizophrenia, three exhibited activated microglial cells and all positive cases were late onset schizophrenia, suggesting microglial activation as a key factor in evidence of pathologic changes in the brains of severe psychiatric illnesses patients including some subgroups of schizophrenia patients (Fig.1). A meta-analysis of cytokine alteration found significant increase in circulating level

Fig.2 The microglial hypothesis of schizophrenia Adapted from Monji et al. “Neuroinflammation in Schizophrenia Especially Focused on the Role of Microglia.” Progress in NeuroPsychopharmacology and Biological Psychiatry, Special Issue: Inflammatory Pathways as new drug targets in Schizophrenia, 42 (April 5, 2013): 115–

Human mesenchymal stem cells The use of human mesenchymal stem cells (MSCs) were reported known for possessing numbers of therapeutic effect with its immunomodulatory properties. MSCs are multipotent stem cells that can derived into varieties of cell types such as neuronal cells, with its immunosuppression and immunomodulatory properties, MSCs can regulate regulatory T cells, supress T cell proliferation, cytokine secretion, cytotoxicity and promotes cellmediated immune response (details shown in Fig.3) . MSCs have

81

been examined to have the ability to repair for CNS injury, tis- Conclusion sue reconstructions as In conclusion, although the current understanding of the pathology of schizophrenia still remains unknown and to our current knowledge, there is no single-target drug, however neuroinflammation and microglial activation might be a key pathologic marker for diagnostics in schizophrenia, experiments performed above suggested stem cell therapy using MSCs maybe effective for schizophrenia patients with elevated TNF-α, controlling inflammatory cytokine signaling maybe a new therapeutic approach for schizophrenia.

Fig.3(Previous page) MSCs immunomodulatory effect on immune cell.

Critical Analysis

Adapted from Gao, F., Chiu, S., Motan, D. et al. Mesenchymal stem cells and immunomodulation: current status and future prospects. Cell Death Dis 7, e2062 (2016). https://doi.org/10.1038/cddis.2015.327

Animal models

cell-therapy, etc. The article we reviewed proposed a potent therapeutic alteration using human umbilical

Due to the multi-factorial property of schizophrenia, there is no “perfect” animal model as the genetic differences often lead cord derived MSCs for treating neuroinflammation in schizo- to limited interpretation from animals to humans, the author used AMP mice to induce schizophrenia-relevant behaviour, phrenia. however heterogeneous schizophrenic disorder group cannot MSCs has been reported to possess immunomodulatory propbe represented. Several studies have shown elevated periphererties in schizophrenia in a meta-analysis for its efficiency and al IL-6 circulation in schizophrenia patients (Table.1), safety, results were collected double-blinded, internationally to eliminate potential bias. Based on this conclusion, the first experiments using MSCs for reducing clinical symptoms of schizophrenia using mice model was performed and the results were interpreted.

The immunomodulatory effect of MSCs was associated by the introduction of regulatory T cells with the production of antiinflammatory cytokine IL-10 and prohibit the sustained neuroinflammation cause by the elevation of mRNA expression of TNF-α, IL-1β, and KMO in amphetamine-sensitized mice (AMP mice) with schizophrenia-relevant behaviour (Fig.4). Vitro study concluded that the neuroinflammation and microglial activation maybe correlated with elevation is circulating TNF-α instead of by the direct effect of amphetamine which was inhibited by MSCs. Behavioural test performed after MSCs injection in AMP mice showed some degree of decrease in behavioural abnormality proposing a possible therapeutic option in schizophrenic subgroup with elevated TNF-α.

Fig.4 Underlying mechanism of MSCs in AMP mice Adapted from You et al. “Human Umbilical Cord-Derived Mesenchymal Stem Cells Alleviate Schizophrenia-Relevant Behaviors in AmphetamineSensitized Mice by Inhibiting Neuroinflammation.” Translational Psychiatry 10, no. 1 (December 2020): 123. https://doi.org/10.1038/s41398020-0802-1.

Table.1 Summary of cytokine level in schizophrenia patients based on metastudy.

Adapted from Momtazmanesh, Sara, Ameneh Zare-Shahabadi, and Nima Rezaei. “Cytokine Alterations in Schizophrenia: An Updated Review.” Frontiers

which is contradicting to the non-significant result of IL-6 in the experiment You et al. conducted (Fig.5).

Moreover, the contention of using prepulse inhibition (PPI) in detecting schizophrenia is well

established and PPI deficits are identified to be one of the most distinguished characteristics in schizophrenia patients. In contrast to this founding, You et al.’s finding reported to show no PPI deficits in AMP mice compare to control. From the review above, it is apparent that genetically modified rodent model or neurodevelopmental rodent model would be more appropriate for studying schizophrenia. The prenatal methylazoxymethanol acetate (MAM) rodent model would be more relevant since it epitomizes developmental disruption bringing out histological, neurophysiological and behavioral deficits more closely resem82

bles to schizophrenia, and more comprehensive mechanism behind the pathology of schizophrenia and effect of MSCs on microglial activation can be well studied.

IL-10 Anti-inflammatory cytokine IL-10 have been explored to have an association with schizophrenia. A meta-study has shown single nucleotide polymorphisms (SNPs) of IL-10 significantly correlate with the risk factor for schizophrenia, and level of IL10 is seen to be negatively associated with severity of clinical symptoms of schizophrenia particular in first episode and drug -naive (FEDN) psychosis and first episode psychosis (FEP).

Moreover, in a different study performed by Lee et al. on the effect of MSCs on Niemann-Pick type C (NP-C) disease, the effect of MSCs is also reported to be associated with increase of IL-10 circulation to modulate inflammation response. All pointing out increase in IL-10 circulation may have an effect on microglial activation. In vitro study performed by You et al. who hypothesized IL-10 to exert similar effect of MSCs but found no effect of IL-10 infusion rescue schizophrenia-relevant behavior. The author should perform further study to block the effect of IL-10 using anti-IL-10R monoclonal antibody then test the effect of MSCs and to gain deeper understanding behind the mechanisms of MSCs.

Future studies To resolve these gaps, proper mice model (suggest MAM mice model) for schizophrenia must be used in future studies and to test for the role of IL-10 in modulating neuroinflammation in schizophrenia, MAM mice must be inject with MSCs first followed by IL-10 antibody injection to test for behavioural and vitro studies. Furthermore, the experiment should have longer experimental duration to see whether MSCs could potentially have a long-term effect. If the results show no degree of behavioural rescue and persistent neuroinflammation, it can be interpreted that MSCs without inducing IL-10 itself may not have the direct effect on microglial activation modulation.

Vitro study using cultured microglial cell lines can be performed, thus the role of MSCs and IL-10 must be concisely studied. Moreover, it may be beneficial to repurpose other immunomodulatory drug/therapy previously proposed for autoimmune diseases in severe psychosis like schizophrenia.

Taken together, additional studies are required before MSCs can be considered an efficient therapeutic strategy for schizophrenia.

83

REFRENCES 1.

Bayer, Thomas A, Rolf Buslei, Laszlo Havas, and Peter Falkai. “Evidence for Activation of Microglia in Patients with Psychiatric Illnesses.” Neuroscience Letters 271, no. 2 (August 20, 1999): 126–28. https://doi.org/10.1016/S0304-3940(99) 00545-5.

2.

Chen, Faye H., and Rocky S. Tuan. “Mesenchymal Stem Cells in Arthritic Diseases.” Arthritis Research & Therapy 10, no. 5 (October 10, 2008): 223. https://doi.org/10.1186/ar2514.

3.

Donegan, Jennifer J., and Daniel J. Lodge. “Cell-Based Therapies for the Treatment of Schizophrenia.” Brain Research 1655 (January 15, 2017): 262–69. https://doi.org/10.1016/j.brainres.2016.08.010.

4.

Gao, F., S. M. Chiu, D. a. L. Motan, Z. Zhang, L. Chen, H.-L. Ji, H.-F. Tse, Q.-L. Fu, and Q. Lian. “Mesenchymal Stem Cells and Immunomodulation: Current Status and Future Prospects.” Cell Death & Disease 7, no. 1 (January 2016): e2062– e2062. https://doi.org/10.1038/cddis.2015.327.

5.

Gao, Lei, Zhao Li, Suhua Chang, and Jing Wang. “Association of Interleukin-10 Polymorphisms with Schizophrenia: A Meta -Analysis.” PLOS ONE 9, no. 3 (March 6, 2014): e90407. https://doi.org/10.1371/journal.pone.0090407.

6.

Kállai, Veronika, Attila Tóth, Rita Gálosi, László Péczely, Tamás Ollmann, Zoltán Petykó, Kristóf László, et al. “The MAME17 Schizophrenia Rat Model: Comprehensive Behavioral Analysis of Pre-Pubertal, Pubertal and Adult Rats.” Behavioural Brain Research 332 (August 14, 2017): 75–83. https://doi.org/10.1016/j.bbr.2017.05.065.

7.

Lee, Hyun, Jae-Sung Bae, and Hee Kyung Jin. “Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Improve Neurological Abnormalities of Niemann-Pick Type C Mouse by Modulation of Neuroinflammatory Condition.” Journal of Veterinary Medical Science 72, no. 6 (2010): 709–17. https://doi.org/10.1292/jvms.09-0495.

8.

Lodge, Daniel J. “The MAM Rodent Model of Schizophrenia.” Current Protocols in Neuroscience / Editorial Board, Jacqueline N. Crawley ... [et Al.] 0 9 (April 2013): Unit9.43. https://doi.org/10.1002/0471142301.ns0943s63.

9.

Miller, Brian J., Peter Buckley, Wesley Seabolt, Andrew Mellor, and Brian Kirkpatrick. “Meta-Analysis of Cytokine Alterations in Schizophrenia: Clinical Status and Antipsychotic Effects.” Biological Psychiatry 70, no. 7 (October 1, 2011): 663– 71. https://doi.org/10.1016/j.biopsych.2011.04.013.

10. Momtazmanesh, Sara, Ameneh Zare-Shahabadi, and Nima Rezaei. “Cytokine Alterations in Schizophrenia: An Updated Review.” Frontiers in Psychiatry 10 (2019). https://doi.org/10.3389/fpsyt.2019.00892.

11. Monji, Akira, Takahiro A. Kato, Yoshito Mizoguchi, Hideki Horikawa, Yoshihiro Seki, Mina Kasai, Yusuke Yamauchi, Shigeto Yamada, and Shigenobu Kanba. “Neuroinflammation in Schizophrenia Especially Focused on the Role of Microglia.” Progress in Neuro-Psychopharmacology and Biological Psychiatry, Special

84

Human Amniotic Epithelial Stem Cells Aa A Therapy For Alzheimer’s Disease Reetu Khan

Alzheimer’s disease (AD) is an irreversible neurodegenerative condition associated with cognitive decline in older populations1. Pathological hallmarks of this disorder include the accumulation of amyloid plaques, cerebral angiopathy and the loss of neuronal and synaptic function2. Despite being the most common cause of dementia, there is currently no cure for AD2. Modern therapies aim to cease progression of the disease. One novel treatment that has recently been brought to light is stem cellbased therapy. Kim et al. (2020) study the therapeutic benefits of human amniotic epithelial cells (hAESCs) on a transgenic mouse model of AD3. Upon receiving intracerebral injections of either hAESC or vehicle, Tg2576 transgenic mice and wildtype mice performed a set of behavioral tests to assess spatial and working memory. In the Morris water maze test, Tg2576-hAESC mice had shorter escape latencies than Tg2576-vehicle mice. In the Y-Maze test, Tg2576-hAESC mice displayed higher rates of spontaneous alternation compared to Tg-vehicle treated mice. Injection of hAESCs reduce Beta-secretase (BACE) activity, the protein responsible for development of amyloid plaque generation4, and improve cognitive function. These findings illustrate the potential hAESCs carry as a future therapeutic agent.

Key words: Alzheimer’s disease (AD), human amniotic stem cells (hAESCs), BACE, amyloid plaques, neurofibrillary tangles, transgenic Tg2576 mouse model .

85

Background and Introduction

hAESC (WT-hAESC) or vehicle (WT-vehicle). Tg2576 transgenic mice were used model AD. These mice overexpress mutant amyloid precursor protein (APP) to mimic elevated levels of Ab and plaque central to AD15. The mice performed two sets of behavioral tests to assess spatial and working memory. TghAESC mice exhibited decreased escape latencies and higher rates of spontaneous alternation, signifying better spatial and working memory than Tg-vehicle mice. These findings highlight the important neurological mechanisms by which hAESC transplantation may influence amyloid burden and BACE activity to improve cognitive decline in AD. Understanding and exploring these mechanisms may give rise to a novel treatment or cure.

Alzheimer’s disease (AD), a progressive neurodegenerative disorder, is the prevalent cause of dementia following senescence3,5. It is characterized by memory loss, language impairment, attention-deficits and cognitive decline1. Histological markers of AD include the combined presence of amyloid-b (Ab) plaques and neurofibrillary tangles containing hyperphosphorylated and misfolded tau protein2. Amyloid precursor protein (APP) is sequentially cleaved by b- and g-secretases, resulting in the accumulation of beta amyloid aggregates. These manifestations lead to microtubule collapse, thus impairing structural support and axonal transport and leading to neuronal death6. The buildup of amyloid plaque occurs before the onset of cognitive dysfunction2, thus making early diagnosis a chalMajor Results lenge. Despite its prevalence in individuals over the age of 651, At 11 months of age, mice received intracerebral injections of a cure for AD is yet to be found. hAESC or vehicle in the dentate gyri of the bilateral hippocamCurrent pharmacological treatments, such as the cholinesteraspus. Stereotactic injections were administered relative to the es donepezil and rivastigmine, are prescribed for mildbregma at the coordinates: AP = −0.15 mm, ML = ±0.13 mm, DV moderate AD to slow its progression7. While the mechanisms = −0.19 mm. At 14 months, all four groups of mice performed are still unknown, researchers believe these medications pretwo sets of behavioral tests to assess cognitive function: the vent the breakdown of acetylcholine, a molecule important for Morris water maze test and the Y-maze test. memory and learning7. N-methyl-D-aspartate (NMDA) antagonists are another form of treatment that regulate glutamatergic activity and block excessive levels from eliciting cell death 7.

--- hAESCs Improve Spatial Learning: Morris Water Maze --These drugs, however, have limited efficacy and simply alleviThe Morris water maze is the gold standard test used to assess ate symptoms without providing long-lasting relief8. spatial learning and memory 3,16. Rodents navigate around the Stem cell-based therapy has been a focus of interest in the onperimeter of an open pool, using distal cues and memory, in going search for neurodegenerative cures. Several studies have order to locate a hidden escape platform beneath the outlined their therapeutic effects on spinal cord injury repair 9, water16,17. The time required to correctly locate the platform is amyotrophic lateral sclerosis10 and Huntington’s disease11. Novmeasured as the rodent’s escape latency17. el methods aim to target degenerating neuronal networks observed in AD to induce regeneration and repopulation 3. In or- Overall, Tg mice displayed significantly longer escape latencies der to develop a practical stem-cell based therapy, cells must compared to the WT mice. Tg-hAESC mice, however, showed be extracted from the most appropriate source. These may decreased escape latencies than Tg-vehicle mice, indicating include pluripotent embryonic stem cells involved in the devel- improved spatial memory. Both WT groups showed no signifiopment of germ layers, mesenchymal stem cells capable of cant differences in escape latency time (Figure 1A). forming mesenchymal tissue, neural stem cells responsible for growth of neural cell types or human amniotic epithelial stem cells (hAESCs)6. These stem cells are derived from the inner- To determine if the groups had memorized the location of the most layer of the placenta. They bear the ability to differentiate platform in zone 4, a probe trial was performed 48 hours after like embryonic stem cells while also retaining immunomodula- the final trial. In this trial, the mice were able to swim freely 17 tory properties of adult stem cells12. Furthermore, studies claim without the platform . Tg-hAESC mice showed significantly they are able to synthesize neurotrophic and growth factors recovered escape latencies than the Tg-vehicle mice; in fact, essential for neuronal regeneration and survival13. In a study their latency times closely matched the latency times of the WT conducted by Xue et al., transplantation of hAESCs into double groups (Figure 1B). Both WT groups and the Tg-hAESC group transgenic mice expressing APP increased hippocampal acetyl- spent more time in zone 4 than zones 1-3. choline levels and enhanced spatial memory14. This effect is quite similar to those exerted by pharmacological cholinesterase treatments, suggesting that hAESCs may function through a similar mechanism and serve as a promising candidate for the treatment of AD. In the original paper, Kim et al. explored the potential therapeutic benefits of hAESCs in a mouse model of AD. In their study, four groups of mice were experimented on: Tg2576 transgenic mice treated with either hAESC (Tg-hAESC) or vehicle (Tg-vehicle) and age-matched wildtype mice treated with 86

Figure 1. A) The Morris water maze test was performed for six tected in Tg-hAESC mice 60 minutes after treatment compared consecutive days, three months after mice received intracere- to Tg-vehicle mice (Figure 3B). bral injections. Escape latency times were recorded. B) 48 hours after their final trial, a probe test was conducted. Escape latency times were recorded.

Figure origin: Kim, Ka Young, Yoo-Hun Suh, and Keun-A Chang.

Therapeutic Effects of Human Amniotic Epithelial Stem Cells in a Transgenic Mouse Model of Alzheimer’s Disease. April 10, 2020. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178120/pdf/ ijms-21-02658.

--- hAESCs Improve Working Memory: Y-Maze Test --The Y-Maze is a learning and memory test that assesses the tendency of a rodent to explore a new environment 18. The test measures how often the rodent enters a different arm of the maze instead of returning to the one it previously visited; this is known as spontaneous alternation11.

Figure 3. A) The number of plaques per a slice tissue were recorded in Tg-hAESC and Tg-vehicle mice. B) BACE activity was recorded in Tg-vehicle and Tg-hAESC mice.

Figure origin: Kim, Ka Young, Yoo-Hun Suh, and Keun-A Chang.

Therapeutic Effects of Human Amniotic Epithelial Stem Cells in a Transgenic Mouse Model of Alzheimer’s Disease. April 10, 2020.

The total number of arm entries remained the same between https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178120/pdf/ groups (Figure 2A), however, Tg-hAESC mice exhibited signifiijms-21-02658. cantly higher rates of spontaneous alternation than Tg-vehicle mice (Figure 2B).

Conclusions/Discussions As researchers advance in their knowledge on the etiology of AD, a well-established long-term cure remains to be discovered. As of now, current therapies include cholinesterases and NMDA antagonists, which only halt the progression of the disease7. However, stem cell-based therapy appears to be revolutionizing the field. hAESCs, stem cells derived from the placenta, can differentiate into cells of the germ layer, such as the endoderm, mesoderm and ectoderm3. Previous studies have examined the role of these cells and discovered that intravenous injection of hAESCs into APPswe mice correlated with fewer amyloid plaques and improved spatial learning19. Furthermore, hAESCs have reduced amyloid deposition in C57BL/6JFigure 2. A) The Y-maze test was performed, three months APP by attenuating oxidative stress and stimulating antioxidaafter mice received intracerebral injections. The total number tive enzymes20. These observations are in line with the results of arm entries was recorded. B) The rate of spontaneous alterobtained in the current study, further elucidating the therapeunation after performing the Y-maze test was recorded. tic effects of hAESCs. Figure origin: Kim, Ka Young, Yoo-Hun Suh, and Keun-A Chang.

Therapeutic Effects of Human Amniotic Epithelial Stem Cells in a Transgenic Mouse Model of Alzheimer’s Disease. April 10, 2020. In this study, transplantation of hAESCs into the bilateral hippohttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178120/pdf/ campus of Tg2576 transgenic mice reduced BACE activity and amyloid plaque burden, thus improving cognitive impairment. ijms-21-02658.

When compared to Tg mice treated with vehicle, Tg-hAESC mice exhibited exceptionally better spatial memory and working memory. The authors concluded that transplantation of --- hAESCs Reduce BACE Activity --hAESCs successfully alleviated cognitive decline in transgenic To examine the abundance of amyloid present in Tg and WT mouse models of AD, suggesting hAESCs could be a novel thermice, their cortices and hippocampi were stained with Congo apeutic treatment for AD memory impairment. red. Tg-hAESC mice displayed significant reductions of amyloid plaques compared to the Tg-vehicle group overall (Figure 3A).

Beta-secretase (BACE) activity was analyzed as well to gain an Additionally, the authors interpret that reduced BACE activity understanding of the amyloid plaque reduction observed upon could be the mechanism by which hAESCs exert their effects to hAESC transplantation. BACE activity increased over time in all reduce amyloid plaque burden in Tg2576 mice3. BACE is an enfour Tg and WT groups, however, decreased activity was de87

enzyme responsible for the proteolysis of APP, leading to production of the N-terminus of Ab4. It has been the prime drug target for AD. Studies using BACE1 knockout (BACE1 -/-) mice demonstrated that when these mice were bred with APP transgenic mice, Ab generation, amyloid burden and cognitive deficits were prevented21. Moreover, inhibition of BACE has been shown to stop the generation of Ab3, thus making it a potential therapeutic approach to treating AD. Not only do these important revelations shed light the unknown processes of hAESC transplantation and their effects on BACE activity, they lead scientists closer to finding a potential cure for AD.

Critical Analysis Kim et al. effectively examine the potential short-term and long -term benefits of stem-cell transplantation, however, they fail to acknowledge the possibility of graft/transplantation rejection. Non-autologous hAESCs incur the risk of immune rejection22, thus rendering them ineffective for some individuals.

While hAESCs are less susceptible to rejection than other types of stem cells, due to their low immunogenicity 23, a study conducted by Chiavegato et al. observed the rejection of amniotic stem cells in an immunodeficient/immunosuppressed rat model24. They concluded that the xenotransplantation of amniotic stem cells in cell therapy is hindered by their immunogenic factors and phenotypic unpredictability 24. One possible solution may be to use immunosuppressive drugs, however, these may induce unwanted side effects25 or introduce variability to the study.

The potential of hAESCs in the treatment of AD becomes evident in transgenic mouse models, however, it is questionable whether these model organisms are representative of the human body. While most cases of AD occur sporadically in a population of genetically heterogenous individuals 6,26, transgenic mouse models are based on familial mutations in genetically homogenous populations14. Transgenic 2576 mice used in this study do not exhibit the neuronal loss characteristic of human AD6,26 and therefore do not fully replicate the human disease.

Future AD studies may need to experiment with higher-order animals that accurately embody the clinical features of the disease.

Furthermore, the authors primarily studied the prefrontal cortex, entorhinal cortex and hippocampus, the dominant areas affected by the development of amyloid plaques and neurofibrillary tangles. However, neurofibrillary tangles can further accumulate in the amygdala and thalamus, eventually spreading to associative isocortical regions2. These are possible brain structures to study in upcoming experiments and reviews to fully understand the neurological etiology of AD.

used in place of transgenic Tg2576 mice. Octodon degus mice naturally display AD-like pathologies and share a close bA sequence homology with humans27, making them an ideal candidate to physiologically model sporadic AD. Compared to Tg2576 mice that only display intraneuronal Ab and parenchymal Ab plaques, Octodon Degus mice exhibit a full range of neuropathological features: intraneuronal Ab, parenchymal Ab plaques, hyperphosphorylated tau, neurofibrillary tangles, and synaptic dysfunction27. It is unclear if neuronal loss is present in these animals27.

Octodon Degus mice and wildtype mice will either be injected with hAESCs or vehicle in the bilateral hippocampus. Three months later, their cognitive function will be assessed through a set of behavioral tests. Upon completing the tests, different stains will be used to analyze their hippocampi, prefrontal and entorhinal cortices. One stain will identify amyloid plaques, another stain will identify neurofibrillary tangles, and so forth â&#x20AC;&#x201C; they will examine whether levels have been upregulated, downregulated or remained the same (baseline measurements will be taken prior to injection of hAESCs or vehicle).

If amyloid burden, neurofibrillary tangles and hyperphosphorylated tau levels decrease in Octodon Degus-hAESC mice, we should expect to see them exhibit improved spatial and working memory compared to Octodon Degus-vehicle mice. This may suggest that there is some underlying mechanism by which hAESCs are reducing amyloid and neurofibrillary tangle burden, and a consequence of this mechanism is the alleviation of cognitive decline. One possible mechanism could be that hAESCs are secreting neural growth factors that are reconstructing pathways in memory-related areas of the brain. Alternatively, they could be secreting factors that inhibit the formation of amyloid plaques and neurofibrillary tangles or inhibit kinases responsible for the hyperphosphorylation of tau 3.

If amyloid burden, neurofibrillary tangles and hyperphosphorylated tau levels increase or remain the same upon transplantation, we would expect to see Octodon Degus-hAESC mice performing just as poorly as Octodon Degus-vehicle mice. Their spatial and working memory will remain impaired. While this is an unlikely outcome, based on the reported beneficial effects of hAESCs, it is plausible and might indicate that hAESCs have no therapeutic influence on AD.

In similar experiments, perhaps other areas of the brain can be investigated, such as the amygdala, thalamus, and associative isocortex. When staining for amyloid plaques, hyperphosphorylated tau and neurofibrillary tangles, we should expect to see levels decrease in these particular areas, too. Overall, these studies should demonstrate how hAESCs can alleviate cognitive decline in AD and direct us toward a human cure.

Future Directions In future experiments, perhaps Octodon Degus mice can be 88

REFRENCES 1. “What Is Alzheimer’s Disease?” n.d. National Institute on Aging. Accessed June 18, 2020. https://www.nia.nih.gov/health/whatalzheimers-disease.

2. Serrano-Pozo, Alberto, Matthew P. Frosch, Eliezer Masliah, and Bradley T. Hyman. 2011. “Neuropathological Alterations in Alzheimer Disease.” Cold Spring Harbor Perspectives in Medicine: 1 (1). https://doi.org/10.1101/cshperspect.a006189.

3. Kim, Ka Young, Yoo-Hun Suh, and Keun-A Chang. 2020. “Therapeutic Effects of Human Amniotic Epithelial Stem Cells in a Transgenic Mouse Model of Alzheimer’s Disease.” International Journal of Molecular Sciences 21 (7). https://doi.org/10.3390/ ijms21072658.

4. Vassar, Robert, Dora M. Kovacs, Riqiang Yan, and Philip C. Wong. 2009. “The β-Secretase Enzyme BACE in Health and Alzheimer’s Disease: Regulation, Cell Biology, Function, and Therapeutic Potential.” The Journal of Neuroscience 29 (41): 12787–94. https://doi.org/10.1523/JNEUROSCI.3657-09.2009.

5. Scheltens, Philip, Kaj Blennow, Monique M. B. Breteler, Bart de Strooper, Giovanni B. Frisoni, Stephen Salloway, and Wiesje Maria Van der Flier. 2016. “Alzheimer’s Disease.” Lancet (London, England) 388 (10043): 505–17. https://doi.org/10.1016/S01406736(15)01124-1.

6. Duncan, Thomas, and Michael Valenzuela. 2017. “Alzheimer’s Disease, Dementia, and Stem Cell Therapy.” Stem Cell Research & Therapy 8 (1): 111. https://doi.org/10.1186/s13287-017-0567-5.

7. “How Is Alzheimer’s Disease Treated?” n.d. National Institute on Aging. Accessed June 18, 2020. https://www.nia.nih.gov/ health/how-alzheimers-disease-treated 8. Liu, Alan King Lun. 2013. “Stem Cell Therapy for Alzheimer’s Disease: Hype or Hope?” Bioscience Horizons: The International Journal of Student Research 6 (January). https://doi.org/10.1093/biohorizons/hzt011.

9. Sankar, V, and R Muthusamy. 2003. “Role of Human Amniotic Epithelial Cell Transplantation in Spinal Cord Injury Repair Research.” Neuroscience 118 (1): 11–17. https://doi.org/10.1016/S0306-4522(02)00929-6.

10. Lunn, J. Simon, Stacey A. Sakowski, and Eva L. Feldman. 2014. “Stem Cell Therapies for Amyotrophic Lateral Sclerosis: Recent Advances and Prospects for the Future.” Stem Cells (Dayton, Ohio) 32 (5): 1099–1109. https://doi.org/10.1002/stem.1628.

11. Maucksch, Christof, Elena M. Vazey, Renee J. Gordon, and Bronwen Connor. 2013. “Stem Cell-Based Therapy for Huntington’s Disease.” Journal of Cellular Biochemistry 114 (4): 754–63. https://doi.org/10.1002/jcb.24432.

12. Miki, Toshio. 2018. “Stem Cell Characteristics and the Therapeutic Potential of Amniotic Epithelial Cells.” American Journal of Reproductive Immunology (New York, N.Y.: 1989) 80 (4): e13003. https://doi.org/10.1111/aji.13003.

13. Xu, Huiming, Jiaofei Zhang, Kam Sze Tsang, Hao Yang, and Wei-Qiang Gao. 2019. “Therapeutic Potential of Human Amniotic Epithelial Cells on Injuries and Disorders in the Central Nervous System.” Review Article. Stem Cells International. Hindawi. November 20, 2019. https://doi.org/10.1155/2019/5432301.

14. Xue, Shouru, Chongfang Chen, Wanli Dong, Guozhen Hui, Tianjun Liu, and Lihe Guo. 2012. “Therapeutic Effects of Human Amniotic Epithelial Cell Transplantation on Double-Transgenic Mice Co-Expressing APPswe and PS1ΔE9-Deleted Genes.” Science China. Life Sciences 55 (February): 132–40. https://doi.org/10.1007/s11427-012-4283-1.

15. “Tg2576 | ALZFORUM.” n.d. Accessed June 18, 2020. https://www.alzforum.org/research-models/tg2576.

16. Vorhees, Charles V, and Michael T Williams. 2006. “Morris Water Maze: Procedures for Assessing Spatial and Related Forms of Learning and Memory.” Nature Protocols 1 (2): 848–58. https://doi.org/10.1038/nprot.2006.116.

17. Morris, Richard G. M. 2008. “Morris Water Maze.” Scholarpedia 3 (8): 6315. https://doi.org/10.4249/scholarpedia.6315.

18. “Y Maze Spontaneous Alternation Test | Behavioral and Functional Neuroscience Laboratory | Stanford Medicine.” n.d. Accessed June 18, 2020. https://med.stanford.edu/sbfnl/services/bm/lm/y-maze.html.

19. Kim, Kyung-Sul, Hyun Sook Kim, Ji-Min Park, Han Wool Kim, Mi-Kyung Park, Hyun-Seob Lee, Dae Seog Lim, Tae Hee Lee, Michael Chopp, and Jisook Moon. 2013. “Long-Term Immunomodulatory Effect of Amniotic Stem Cells in an Alzheimer’s Disease Model.” Neurobiology of Aging 34 (10): 2408–20. https://doi.org/10.1016/j.neurobiolaging.2013.03.029.

20. Jiao, Hongliang, Ke Shi, Weijie Zhang, Liang Yang, Lu Yang, Fangxia Guan, and Bo Yang. 2016. “Therapeutic Potential of Human Amniotic Membrane-Derived Mesenchymal Stem Cells in APP Transgenic Mice.” Oncology Letters 12 (3): 1877–83. https:// doi.org/10.3892/ol.2016.4857.

89

21. Vassar, Robert, and Patty C. Kandalepas. 2011. “The β-Secretase Enzyme BACE1 as a Therapeutic Target for Alzheimer’s Disease.” Alzheimer’s Research & Therapy 3 (3): 20. https://doi.org/10.1186/alzrt82.

22. Hayashi, Yoshihito, Huan-Ting Lin, Cheng-Che Lee, and Kuen-Jer Tsai. 2020. “Effects of Neural Stem Cell Transplantation in Alzheimer’s Disease Models.” Journal of Biomedical Science 27 (1): 29. https://doi.org/10.1186/s12929-020-0622-x.

23. Elias, Maya, Jaclyn Hoover, Hung Nguyen, Stephanny Reyes, Christopher Lawton, and Cesar V Borlongan. 2015. “Stroke Therapy: The Potential of Amniotic Fluid-Derived Stem Cells.” Future Neurology 10 (4): 321–26. https://doi.org/10.2217/FNL.15.19.

24. Chiavegato, Angela, Sveva Bollini, Michela Pozzobon, Andrea Callegari, Lisa Gasparotto, Jenny Taiani, Martina Piccoli, et al.

2007. “Human Amniotic Fluid-Derived Stem Cells Are Rejected after Transplantation in the Myocardium of Normal, Ischemic, Immuno-Suppressed or Immuno-Deficient Rat.” Journal of Molecular and Cellular Cardiology 42 (4): 746–59. https:// doi.org/10.1016/j.yjmcc.2006.12.008.

25. Davulcu, Eren Arslan, and Filiz Vural. 2018. “Immunosuppressive Agents in Hematopoietic Stem Cell Transplantation.” Trends in Transplantation 11 (1). https://doi.org/10.15761/TiT.1000240.

26. Elder, Gregory A., Miguel A. Gama Sosa, and Rita De Gasperi. 2010. “Transgenic Mouse Models of Alzheimer’s Disease.” The Mount Sinai Journal of Medicine, New York 77 (1): 69–81. https://doi.org/10.1002/msj.20159.

27. Castro-Fuentes, Rafael, and Rosario Socas-Pérez. 2013. “Octodon Degus: A Strong Attractor for Alzheimer Research.” Basic and Clinical Neuroscience 4 (1): 91–96.

90

The Role of Toll-Like Receptor 4 in Mediating Gut-Brain Axis Inflammation and Pathologies Seen in Parkinsonâ&#x20AC;&#x2122;s Disease Kung Min Kim

The gut-brain axis is suggested as the important contributor to the neurodegenerative diseases such as Parkinsonâ&#x20AC;&#x2122;s disease (PD). It has been proposed that perturbation of intestinal environment activates enteric glial cells, whose inflammatory response propagates the pathophysiology from periphery to central nervous system (CNS) via the vagus nerve (Sampson et al. 2016, 1475). However, despite neuroinflammation being the common hallmark of neurodegenerative diseases, there is much to learn about the precise mechanism in which the inflammatory cascade is triggered. Perez-Pardo et al. studied the colon biopsy samples of PD patients to examine the molecular profiles and abnormalities.

Compared to healthy controls, PD patients were found to have dysfunctional and inflamed intestinal wall. More importantly, it was noted that molecular markers pertaining to the immune activation of Toll-like receptor 4 (TLR4) by bacterial endotoxin were elevated. These findings prompted the authors to hypothesize that a leaky gut resulting from dysbiosis allows the invasion of gram-negative bacteria into the mucosa. Consequently, the pattern recognition receptor TLR4 is activated and proinflammatory cascade is initiated. To test, TLR4 knock-out (KO) mice and wildtype (WT) were treated with daily dose of rotenone to induce PD-like symptoms. Whereas the WT mice exhibited the similar pathophysiology and molecular profiles as the human PD patients, TLR4-KO mice were partially protected from the pathogenesis. They showed less epithelial disruption, low level of inflammation, superior motor functions, and healthier neurons. This translational study is highly relevant to the future direction of developing early diagnostic tools and treatment options for PD and other neurodegenerative conditions associated with TLR4 and the gut-brain axis.

91

Background

The second part of the study was designed as a translational study to replicate the findings in mice. The goal was to test whether TLR4 activation plays a significant role in triggering the inflammatory cascade in the gut-brain axis. Rotenone, a mitochondrial Complex 1 inhibitor, is a neurotoxin known to induce PD-like symptoms by causing the death of dopaminergic neurons. Adult TLR4-KO mice and the WT were fed rotenone daily for 4 weeks and their gut and brain tissue samples were collected. Like the human counterpart, the WT had altered gut microbiota, hyperpermeable epithelium, and higher degree of enteric glial cell-mediated inflammation. On the other hand, the TLR4-KO mice were found to be partially protected from the rotenone-induced effects in terms of intestinal disruption, immune activation, deterioration of motor functions, and neuropathy. This novel knowledge that silencing TLR4 pathway serves as a preventative and protective measure against PD is highly relevant to the current field of research, as it may be investigated as a potential therapeutic target.

It has been more than 200 years since James Parkinson first described the â&#x20AC;&#x2DC;shaking palsyâ&#x20AC;&#x2122; patients with slowness of movement and resting tremor (Parkinson 2002, 224). However, much about the disease including its etiology, genetics, preventive measures, and cures are not clearly known. Given that itâ&#x20AC;&#x2122;s the second most common neurogenerative disease that affects 572 per 100,000 individuals aged over 45 in North America (Marras et al. 2018, 2), the lack of definitive understanding of this disease is frustrating to the patients and to the researchers alike. Although many benefits from the dopamine replacement therapy, long term complication such as dyskinesia is common, and we currently do not have an effective way to fundamentally treat PD by stopping and reversing the neuropathy (Tambasco, Romoli, and Calabresi 2017, 1241). The misfolded alpha-synuclein protein aggregates known as the Lewy bodies are one of the pathological hallmarks and thought to be contributing to neuronal death. Yet, except for the few rare autosomal mutations that account for less than 10% of the cases, sporadic PD is believed to have multifactorial causes and there are numerous risks associated with the toxic oligomerization (Rocha, Miranda, and Sanders 2018, 250). A breakthrough came when Braak et al. (2002) published the finding that symptoms arising from dysbiosis such as constipation precedes the motor dysfunction by nearly a decade. More studies followed, solidifying the association between gut microbiota, intestinal health, and PD. And a new hypothesis was proposed: the altered gut microbiota activates neuroglial cells to launch immune responses through short chain fatty acid (SCFA) signaling (Sampson et al. 2016, 1472) and the oxidative stress from neuroinflammation and associated cytokines contribute to the on- Major Results set of PD (Garcia-Esparcia et al. 2014, 584) in the genetically Human colon biopsy studies susceptible individuals.

With works of the forerunners as the foundation, the authors further examined the nature of the immune responses that initiate neuroinflammation. First, they examined the colon biopsies of PD patients and noted that the integrity of gut barrier was significantly disrupted as measured by the degree of urinary excretion of ingested sugar. Specifically, the tight junctions which create an impermeability barrier were compromised. Immunocytochemistry analysis showed the elevated level of molecular markers for TLR4 and activated CD3+ T cells, indicative of active innate and adaptive immune responses against bacterial pathogens. Colonic gene expression analysis further supported the findings: the mRNA level of inflammatory cytokines and LPS binding protein (LBP) were indeed higher in the PD patients. LBP is an acute-phase protein that plays an integral role in TLR4 activation and its level is directly correlated.

Compared to healthy subjects, PD patients showed disruption in their gut barrier even in the absence of clinical gastrointestinal dysfunction and the expression of tight junction protein ZO1 was found to be significantly reduced. When stained with antibodies, it was evident that PD subjects have greater TLR4 and CD3+ T cells immunoreactivity, which can be translated as the result of frequent invasions by gram-negative bacteria. Indeed, the mRNA expression level of LBP and associated proinflammatory cytokines were also elevated in the lamina propria (LP). These results precisely replicate the observations made by Forsyth et al. (2011) who had additionally singled out E. coli as the major contributor, as its presence in LP was 4 times higher in PD cases. Defining the intestinal characteristics of PD patients is highly important, as they can serve as biological markers for early diagnosis.

TLR4-KO mice study Both TLR4-KO and WT mice experienced a change in their microbiota composition in response to daily dose of rotenone.

Specifically, there was a significant decrease in Bifidobacteria which is believed to be beneficial, while endotoxin-producing bacteria increased in abundance. Again, this data fits what has been routinely observed in the human PD patients as reported 92

by Gerhardt and Mohajeri (2018). However, TLR4-KO mice were partially protected from the detrimental effects of dysbiosis and PD-like pathologies induced by rotenone. Not only was the integrity of gut epithelium preserved with functional tight junctions, less signs and molecular markers of inflammation and TLR4 activation were observed. It was also demonstrated for the first time that the absence of TLR4 pro-inflammatory signaling is associated with a significantly lower level of enteric alphasynuclein expression and microglial activation. More importantly, same trend was observed in the CNS. This suppressive effect is highly relevant, for it can be further studied as a potential treatment to slow down the progression of PD at an early stage.

not explored in this experiment despite it being extremely important for the risk of developing the disease. A future study in which alpha-synuclein overexpressing (ASO) transgenic animal model is incorporated would be a great addition to gather more applicable results. In addition, nowhere in the paper did the authors explore the deleterious effects of silencing TLR4 receptor which is an integral part of our innate immune defense. Mutations to TLR4 are highly associated with atherosclerosis, asthma, and Crohnâ&#x20AC;&#x2122;s disease (Lin, Verma, and Hodgkinson 2012, 635) not to mention the greatly increased susceptibility to infection that can be life-threatening. At least mention this aspect would have added more scientific objectivity and credibility.

Future Directions

Discussion In this paper, Perez-Pardo et al. demonstrated that TLR4mediated immune activation in the gut is a crucial contributor to neuroinflammation characterized in PD. By blocking the TLR4 signalling pathway, the development of PD and associated symptoms were largely mitigated in the animal model treated with rotenone. As such, the authors concluded by supporting the gut-brain axis model of pathogenesis in PD. Although there are number of other pattern recognition receptors such as TLR2, 5, and NLRP3 known to be associated with gut dysbiosis (Jin, Henao-Mejia, and Flavell 2013, 879), this study is first to clearly show the causal link between TLR4 activation and PD pathologies through an experimental manipulation.

The next suggested step involves repeating the experiment with ASO mice to test the effects of TLR4 activation in neuroinflammation and alpha-synuclein pathology. Two groups of ASO mice and a control WT will receive fecal transplant from a PD patient and only one ASO group will also receive a pharmacological intervention to inhibit TLR4. After few weeks, the gut and brain tissues will be analyzed for epithelial integrity, proinflammatory markers, microglial activation, and presence of alpha-synuclein pathology. It is expected that ASO mice will develop PD-like pathology as demonstrated by Sampson et al.

(2016) following a dysbiotic microbiota transplant. However, the ASO group treated with the TLR4 inhibitor is expected to be partially protected as in the case for the TLR4-KO mice. The control WT may also suffer from dysbiosis, inflamed gut, and increased microglial activation, yet they are not expected to develop PD-related symptoms due to lack of genetic predisposition.

The result may not match the expectation, that is, the ASO group treated the TLR4 inhibitor may still develop similar degree of neuroinflammation and alpha-synuclein pathology. If so, the ASO mice may have a lower threshold for initiating oligomerization compared to the TLR4-KO mice. Other modes of activating microglia, such as through SCFAs and TLR2 (Caputi and Giron 2018, 1689) may be suffice for those with strong genetic disposition to develop PD even in the absence of TLR4 signaling.

Critical Analysis The authors used rotenone as the method of quickly inducing PD in mice. While highly effective, it is not the most relevant model of PD pathogenesis; exposure to the environmental neurotoxins is much more subtle and chronic in the most sporadic PD cases. In addition, the element of genetic predisposition was 93

REFRENCES 1.

Caputi, Valentina, and Maria Giron. “Microbiome-Gut-Brain Axis and Toll-Like Receptors in Parkinson’s Disease.” International Journal of Molecular Sciences 19, no. 6 (2018): 1689. https://doi.org/10.3390/ijms19061689.

2.

Forsyth, Christopher B., Kathleen M. Shannon, Jeffrey H. Kordower, Robin M. Voigt, Maliha Shaikh, Jean A. Jaglin, Jacob D. Estes, Hemraj B. Dodiya, and Ali Keshavarzian. “Increased Intestinal Permeability Correlates with Sigmoid Mucosa AlphaSynuclein Staining and Endotoxin Exposure Markers in Early Parkinson's Disease.” PLoS ONE 6, no. 12 (2011). https:// doi.org/10.1371/journal.pone.0028032.

3.

Garcia-Esparcia, Paula, Franc Llorens, Margarita Carmona, and Isidre Ferrer. “Complex Deregulation and Expression of Cytokines and Mediators of the Immune Response in Parkinson's Disease Brain Is Region Dependent.” Brain Pathology 24, no. 6 (2014): 584–98. https://doi.org/10.1111/bpa.12137.

4.

Gerhardt, Sara, and M. Mohajeri. “Changes of Colonic Bacterial Composition in Parkinson’s Disease and Other Neurodegenerative Diseases.” Nutrients 10, no. 6 (2018): 708. https://doi.org/10.3390/nu10060708.

5.

Jin, Chengcheng, Jorge Henao-Mejia, and Richard A. Flavell. “Innate Immune Receptors: Key Regulators of Metabolic Disease Progression.” Cell Metabolism 17, no. 6 (2013): 873–82. https://doi.org/10.1016/j.cmet.2013.05.011.

6.

Lin, Yi-Tzu, Amanda Verma, and Conrad P. Hodgkinson. “Toll-Like Receptors and Human Disease: Lessons from Single Nucleotide Polymorphisms.” Current Genomics 13, no. 8 (2012): 633–45. https://doi.org/10.2174/138920212803759712.

7.

Marras, C., J. C. Beck, J. H. Bower, E. Roberts, B Ritz, G. W. Ross, R. D. Abbott, et al. “Prevalence of Parkinson’s Disease across North America.” npj Parkinson's Disease 4, no. 1 (2018). https://doi.org/10.1038/s41531-018-0058-0.

8.

Parkinson, James. “An Essay on the Shaking Palsy.” The Journal of Neuropsychiatry and Clinical Neurosciences 14, no. 2 (2002): 223–36. https://doi.org/10.1176/jnp.14.2.223.

9.

Perez-Pardo, Paula, Hemraj B Dodiya, Phillip A Engen, Christopher B Forsyth, Andrea M Huschens, Maliha Shaikh, Robin M Voigt, et al. “Role of TLR4 in the Gut-Brain Axis in Parkinson’s Disease: a Translational Study from Men to Mice.” Gut 68, no. 5 (2018): 829–43. https://doi.org/10.1136/gutjnl-2018-316844.

10.

Rocha, Emily M., Briana De Miranda, and Laurie H. Sanders. “Alpha-Synuclein: Pathology, Mitochondrial Dysfunction and Neuroinflammation in Parkinson’s Disease.” Neurobiology of Disease 109 (2018): 249–57. https://doi.org/10.1016/ j.nbd.2017.04.004.

11.

Sampson, Timothy R., Justine W. Debelius, Taren Thron, Stefan Janssen, Gauri G. Shastri, Zehra Esra Ilhan, Collin Challis, et al. “Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson’s Disease.” Cell 167, no. 6 (2016): 1469-1480. https://doi.org/10.1016/j.cell.2016.11.018.

12.

Tambasco, Nicola, Michele Romoli, and Paolo Calabresi. “Levodopa in Parkinson’s Disease: Current Status and Future Developments.”

Current Neuropharmacology 16, no.

8 (2018): 1239–52. https:// doi.org/10.2174/1570159x15666170510143821.

94

Genome-edited Skin Transplants Offer a Safe and Enduring Gene Therapy Approach for Treating Drug Addiction John Luu

Canonical treatments of cocaine dependence are primarily behavioural therapy-based as there are no approved drugs or gene-based therapies for treatment (Fischer et al. 2015). Human butyrylcholinesterase (hBChE) has been engineered for extremely high specificity and catalytic potential for hydrolysis of cocaine (Zheng et al. 2014), but prior delivery strategies using fusion proteins and viral vectors have produced insubstantial results or are prone to high expenses and risk for therapeutic application (Gilgun-Sherki et al. 2016; Naldini 2015). Li and colleagues (2019) demonstrate the viability of editing epidermal stem cells for expression of engineered hBChE and grafting edited tissues into mice for protection against cocaine-induced toxicity and behaviour. Mice with hBChE-releasing skin grafts exhibited protection against cocaine overdose as well as significant reductions in cocaine seeking, hyperactivity, and relapse. The results support a powerful gene therapy approach for expression of therapeutic proteins that circumvents the primary obstacle of the hBChE enzymeâ&#x20AC;&#x2122;s short half-life (Brimijoin 2011) by enabling continued expression long-term. However, the authors did not address the impact on cocaine relapse induced by environmental cuesâ&#x20AC;&#x201D;powerful conditioned stimuli primarily responsible for chronic relapse behaviour (Lee, Milton, and Everitt 2006); the authors may employ a conflict model of cue-induced relapse to test this (Katzir et al. 2007).

95

BACKGROUND

MAJOR RESULTS

Cocaine addiction initiates from overstimulation of reward pathways of the brain—specifically via ventral tegmental area (VTA) dopaminergic signaling to the nucleus accumbens and prefrontal cortex (Penberthy et al. 2010). Ensuing drug dependence is characterized by cue-induced cravings and hedonic homeostatic dysregulation which underlie compulsive drug seeking, abuse, and relapse behaviours despite socioeconomic, psychological, and physical burdens (Dackis and O’Brien 2001).

Typical treatment approaches involve behavioural interventions which include cognitive behavioural therapy (CBT)—an individualized method teaching patients new mental and behavioural tools to cope with drug-related stress—and contingency management (CM)—providing reward incentives based on the principle of operant conditioning (Penberthy et al.

2010). There currently exists no approved pharmaceutical interventions and the collective literature on pharmacological and immune-based therapies remains unconvincing (Fischer et al. 2015).

CRISPR-mediated expression of hBChE in mice epidermal stem cells

One potential therapeutic protein for treating cocaine abuse is human butyrylcholinesterase (BChE), an enzyme that endogenously functions to detoxify environmental toxicants— including hydrolysis of cocaine into inactive products—and in hydrolysis of acetylcholine (ACh) neurotransmitter (Lockridge 2015). Natural plasma BChE is limited in its therapeutic application by very low efficiency for hydrolysing cocaine, thus modified versions of human BChE (hBChE) have been engineered for drastically greater catalytic efficiency and greater specificity for cocaine (Zheng et al. 2014). A short half-life compared to wildtype hBChE and potentially inefficient parenteral delivery are the primary obstacles to therapeutic application of engineered hBChE (Brimijoin 2011). Past attempts at delivering recombinant hBChE via albumin-hBChE fusion proteins delivered intramuscularly failed to attenuate cocaine dependence in human participants (Gilgun-Sherki et al. 2016). Viral-mediated delivery methods are also plagued by challenges of precision cell targeting, efficient gene transduction, and avoidance of host immunity (Naldini 2015).

Li and colleagues (2019) performed genome editing via clustered regularly interspaced short palindromic repeats (CRISPR) using vectors encoding Cas9D10A nickase—functioning in pairs for double-stranded DNA cleavage (Ran et al. 2013). Additional vectors encoded two guide RNAs targeting the Rosa26 locus for ubiquitous expression (Chu et al. 2016). The targeting vector itself contained the expression cassette for homology directed repair (HDR)-mediated insertion of hBChE—encoding the enzyme hydrolyzing cocaine—and Puro—encoding puromycin resistance for selection (Fig. 1). Constitutive expression of cassette genes is driven by UbiC ubiquitin promoter. CRISPR elements were transformed into epidermal basal cells of newborn mice via electrophoresis.

Figure 1 Targeting vector containing expression cassette for CRISPR-mediated expression of engineered hBChE for cocaine hydrolysis. Figure adapted from: Li et al. (2019)

Quantitative validation of hBChE secretion from genome-edited and wild-type epidermal progenitor cells via enzyme-linked immunosorbent assay (ELISA) revealed strong, detectable expression in genome-edited cells only (Fig. 2A). Additionally, the cocaine hydrolysis activity of secretions from cell cultures expressing hBChE and similarly engineered mouse BChE (mBChE) in comparison with wild-type control cultures was determined via clearance assay; only hBChE cultures showed significant clearance of cocaine (Fig. 2B). Thus, the engineered epidermal progenitors were successfully edited for cocaine degrading acin vivo.

In their 2019 publication, Li and colleagues demonstrate a de- tivity livery approach based on a 2017 study by Yue et. al utilizing (A) epidermal stem cells edited ex vivo to express engineered (B) hBChE which were grafted into a host for long-term protein delivery. A gene therapy approach based on skin benefits from great accessible of the organ enabling ease of extraction, monitoring, and removal. Furthermore, well-established protocols for growing skin implants in vitro for treating burns exist (O’Connor et al. 1981). Epidermal keratinocytes are also excellent candidates for transplantation due to low immunogenicity offered by the absence of MHC class II expression (Haniffa, Gunawan, and Jardine 2015). Previous experiments have prov- Figure 2 (A) Detection of serum hBChE levels via ELISA in conen successful in engineering skin cells for secretion of large trol and hBChE-expressing mice. Detectable levels are only preproteins into circulation for therapeutic effect (Fakharzadeh et sent in hBChE-expressing mice. (B) Cocaine clearance assay for al. 2000). The authors successfully demonstrated cocaine clearance in mice grafted with engineered skin implants, in addition control, mBChE-expressing mice, and hBChE-expressing mice. to attenuation of cocaine seeking, relapse, and acute systemic Significant clearance only seen for hBChE-expressing mice. Figure adapted from: Li et al. (2019). toxicity (Li et al. 2019).

96

Transplantation of hBChE-expressing skin organoid diminishes Figure 4 (A) Conditioned place preference (CPP) paradigm cocaine-induced hyperactivity and overdose in mice for cocaine conditioning in GWT and GhBChE mice. Cocaine associated CPP acquisition only seen in GWT mice. (B) CPP Wild-type mice were grafted with skin organoids generated paradigm for cocaine preference reinstatement following from in vivo differentiation and stratification of epidermal stem extinction in GWT and GhBChE mice. Cocaine associated cells engineered to express hBChE (GhBChE mice) and compared to controls grafted with unedited skin organoids (GWT CPP reinstatement only seen in GWT mice; identical CPP mice) (Li et al. 2019). ELISA-mediated detection of blood hBChE acquisition between mice groups pre-surgery for skin graftlevels over a 10-week period revealed significant levels in ing. Figure adapted from: Li et al. (2019).

GhBChE mice only. Extracellular cocaine and associated dopamine levels within the nucleus accumbensâ&#x20AC;&#x201D;involved in the CRISPR-mediated hBChE expression in human epidermal stem reward pathway of the brainâ&#x20AC;&#x201D;from microdialysis collections cells revealed significant reductions in both cocaine and dopamine levels after cocaine injection in GhBChE mice when compared The authors used a similar CRISPR approach as shown in Figure to GWT mice. Elevated locomotion induced by cocaine injection 1a for genome-editing of human epidermal keratinocytes from was attenuated in hBChE-producing mice compared to controls newborn foreskin for hBChE expression (Li et al. 2019). Signifi(Fig. 3A). GhBChE mice were also protected from cocaine- cant hBChE secretions in the blood of nude mice grafted with induced acute toxicity at cocaine levels that proved lethal in hBChE-expressing human skin organoid was validated via ELISA GWT mice (Fig. 3B). in comparison to control mice grafted with unedited cells. Thus, CRISPR-edited human skin cells are a viable medium for deliver(A) ing hBChE for treatment of cocaine addiction.

(B)

DISCUSSION

Li et al. (2019) successfully used CRISPR to edit mouse epidermal stem cells for expression of engineered hBChE for cocaine hydrolysis. Transplantation of hBChE-expressing skin organoids, originating from mice or humans, into mice demonstrated cocaine clearance after drug administration as well as protection from cocaine-induced acute systemic toxicity, or overdose.

Figure 3 (A) GhBChE mice show reduced locomotion at all Most notably hBChE-expression in mice attenuated cocainecocaine dosages compared to GWT mice. (B) GhBChE mice induced behaviours including hyperactivity, cocaine seeking, and relapse. Thus, the experimental results further support the rescued from cocaine-induced lethality at dosages lethal to viability of cutaneous gene therapy for therapeutic protein deGWT mice. Figure adapted from: Li et al. (2019). livery first presented by Yue et al. in 2017. This is also the first application of the approach for treating the effects of addictive Transplantation of hBChE-expressing skin organoid diminishes stimulants affecting behaviour. cocaine-seeking and relapse in mice GhBChE mice maintained stable hBChE levels in serum for more than 10 weeks, with the oldest mice living up to 6 months withA conditioned place preference (CPP) paradigm was used to out signs of immune rejection (Li et al. 2019). The authors also evaluate cocaine-seeking and relapse behaviour (Li et al. 2019). reference the historical use of autologous skin grafts for the CPP acquisition following cocaine conditioning was only evident treatment of severe burns and skin diseases, including those in GWT mice and not GhBChE mice (Fig. 4A). CPP re-acquisition generated from genome-edited stem cell cultures, as evidence following skin grafting and a CPP extinction period revealed for safe and long-lasting skin-based gene therapy. The therareinstatement of CPP in GWT mice only, despite identical pre- peutic advantages of their cutaneous gene therapy approach grafting CPP acquisition and post-grafting CPP extinction are highlighted in the key cocaine-related toxicity and behavpatterns between mice groups (Fig. 4B). Ethanol conditioning ioural issues protected against by hBChE-producing skin grafts was used as a control, with no differences in CPP acquisition or in mice. Therapeutic application is also made easier by exreinstatement between mice groups. tremely high specificity and catalytic activity for cocaine alleviating the need for individualization based on cocaine dosage.

(A) The cutaneous genome-edited transplantation strategy can be applied for treating abuse beyond cocaine as demonstrated by Yue et al. (2017) when they CRISPR-engineered mouse and human epidermal stem cells for secretion of glucagon-like peptide 1 (GLP1) for protection against obesity and diabetes induced by high-fat consumption.

CRITICAL ANALYSIS 97

A major challenge to overcoming cocaine and general drug dependence is recurrent relapse, despite long periods of abstinence, primarily mediated by drug-associated cues (Gawin and Kleber 1986). These cues function like conditioned environmental stimuli in a Pavlovian classical conditioning paradigm and thus drug-seeking and relapse responses are at least, in part, driven by involuntary psychological drives explaining drug abuse despite negative outcomes (Di Ciano and Everitt 2004).

As such, it is worth investigating the effectiveness of hBChEexpressing skin implants for attenuation of cue-induced relapse behaviour.

It is worth addressing if there is a cocaine dosage threshold for which the protective qualities of hBChE secretions are diminished. Cocaine esterase (COcE)â&#x20AC;&#x201D;another cocaine hydrolysing enzyme derived from bacteriaâ&#x20AC;&#x201D;failed to attenuate cocainereinforced responses at sufficiently high doses (Collins et al.

2009). However, this may be a non-issue as experimental mice have been shown to tolerate BChE dosages at 1500-fold greater than endogenous levels (Murthy et al. 2014), and thus it may be reasonable to just increase hBChE dosages to levels sufficient to combat high cocaine administration.

FUTURE DIRECTIONS To evaluate the capacity of mice grafted with hBChE-expressing skin transplants to resist cue-triggered cocaine relapse, the authors should adapt the conflict model of cue-induced relapse outlined by Katzir and colleagues (2007). This paradigm better reflects the human experience of self-conflict between the impulsions to use a drug and the desire to avoid drug-associated negative outcomes. For this procedure, mice are conditioned to press a lever paired with a light-cue to receive cocaine after which an electrified barrier on the floor grows in intensity until animals avoid the lever for a few days straight. The following testing period involves random light-cue presentations with the active electrified barrier and lever presses are recorded. When testing mice grafted with unedited skin (GWT mice) and mice grafted with hBChE-expressing skin (GhBChE mice), it expected that more GhBChE mice will relapse in response to the lone light-cue compared to GWT mice by merit of hBChE-mediated clearance of cocaine drastically reducing hedonistic response to the drug in the first place. However, performing grafting surgery after cue-conditioning may show no differences between mice groups because the brain systems underlying contextual learning and cued learning are not identical. For instance, the hippocampus is necessary for contextual learning, which was studied when the authors used a CPP paradigm, but it is not necessary for associative learning of cues (Curzon, Rustay, and Browman 2009); the hippocampus should be unaffected by hBChE-expression post-cue conditioning.

98

REFRENCES 1.

Brimijoin, Stephen. 2011. “Interception of Cocaine by Enzyme or Antibody Delivered with Viral Gene Transfer: A Novel Strategy for Preventing Relapse in Recovering Drug Users.” CNS & Neurological Disorders Drug Targets 10 (8): 880–91.

2.

Chu, Van Trung, Timm Weber, Robin Graf, Thomas Sommermann, Kerstin Petsch, Ulrike Sack, Pavel Volchkov, Klaus Rajewsky, and Ralf Kühn. 2016. “Efficient Generation of Rosa26 Knock-in Mice Using CRISPR/Cas9 in C57BL/6 Zygotes.” BMC Biotechnology 16 (1): 4. https://doi.org/10.1186/s12896-016-0234-4.

3.

Collins, Gregory T., Remy L. Brim, Diwahar Narasimhan, Mei-Chuan Ko, Roger K. Sunahara, Chang-Guo Zhan, and James H. Woods. 2009. “Cocaine Esterase Prevents Cocaine-Induced Toxicity and the Ongoing Intravenous Self-Administration of Cocaine in Rats.” The Journal of Pharmacology and Experimental Therapeutics 331 (2): 445–55. https://doi.org/10.1124/ jpet.108.150029.

4.

Curzon, Peter, Nathan R. Rustay, and Kaitlin E. Browman. 2009. “Cued and Contextual Fear Conditioning for Rodents.” In Methods of Behavior Analysis in Neuroscience, edited by Jerry J. Buccafusco, 2nd ed. Frontiers in Neuroscience. Boca Raton (FL): CRC Press/Taylor & Francis. http://www.ncbi.nlm.nih.gov/books/NBK5223/.

5.

Dackis, Charles A., and Charles P. O’Brien. 2001. “Cocaine Dependence: A Disease of the Brain’s Reward Centers.” Journal of Substance Abuse Treatment 21 (3): 111–17. https://doi.org/10.1016/S0740-5472(01)00192-1.

6.

Di Ciano, Patricia, and Barry J. Everitt. 2004. “Conditioned Reinforcing Properties of Stimuli Paired with Self-Administered Cocaine, Heroin or Sucrose: Implications for the Persistence of Addictive Behaviour.” Neuropharmacology 47 Suppl 1: 202– 13. https://doi.org/10.1016/j.neuropharm.2004.06.005.

7.

Fakharzadeh, S. S., Y. Zhang, R. Sarkar, and H. H. Kazazian. 2000. “Correction of the Coagulation Defect in Hemophilia A Mice through Factor VIII Expression in Skin.” Blood 95 (9): 2799–2805.

8.

Fischer, Benedikt, Peter Blanken, Dartiu Da Silveira, Andrea Gallassi, Elliot M. Goldner, Jürgen Rehm, Mark Tyndall, and Evan Wood. 2015. “Effectiveness of Secondary Prevention and Treatment Interventions for Crack-Cocaine Abuse: A Comprehensive Narrative Overview of English-Language Studies.” The International Journal on Drug Policy 26 (4): 352–63. https:// doi.org/10.1016/j.drugpo.2015.01.002.

9.

Gawin, F. H., and H. D. Kleber. 1986. “Abstinence Symptomatology and Psychiatric Diagnosis in Cocaine Abusers. Clinical Observations.” Archives of General Psychiatry 43 (2): 107–13. https://doi.org/10.1001/archpsyc.1986.01800020013003.

10.

Gilgun-Sherki, Yossi, Rom E. Eliaz, David J. McCann, Pippa S. Loupe, Eli Eyal, Kathleen Blatt, Orit Cohen-Barak, Hussein Hallak, Nora Chiang, and Shwe Gyaw. 2016. “Placebo-Controlled Evaluation of a Bioengineered, Cocaine-Metabolizing Fusion Protein, TV-1380 (AlbuBChE), in the Treatment of Cocaine Dependence.” Drug and Alcohol Dependence 166 (September): 13– 20. https://doi.org/10.1016/j.drugalcdep.2016.05.019.

11.

Haniffa, Muzlifah, Merry Gunawan, and Laura Jardine. 2015. “Human Skin Dendritic Cells in Health and Disease.” Journal of Dermatological Science 77 (2): 85–92. https://doi.org/10.1016/j.jdermsci.2014.08.012.

12.

Katzir, Ayelet, Noam Barnea-Ygael, Dino Levy, Yavin Shaham, and Abraham Zangen. 2007. “A Conflict Rat Model of CueInduced Relapse to Cocaine Seeking.” Psychopharmacology 194 (1): 117–25. https://doi.org/10.1007/s00213-007-0827-7.

13.

Lee, Jonathan L. C., Amy L. Milton, and Barry J. Everitt. 2006. “Cue-Induced Cocaine Seeking and Relapse Are Reduced by Disruption of Drug Memory Reconsolidation.” The Journal of Neuroscience 26 (22): 5881–87. https://doi.org/10.1523/ JNEUROSCI.0323-06.2006.

14.

Li, Yuanyuan, Qingyao Kong, Jiping Yue, Xuewen Gou, Ming Xu, and Xiaoyang Wu. 2019. “Genome-Edited Skin Epidermal Stem Cells Protect Mice from Cocaine-Seeking Behaviour and Cocaine Overdose.” Nature Biomedical Engineering 3 (2): 105– 13. https://doi.org/10.1038/s41551-018-0293-z.

99

15.

Lockridge, Oksana. 2015. “Review of Human Butyrylcholinesterase Structure, Function, Genetic Variants, History of Use in the Clinic, and Potential Therapeutic Uses.” Pharmacology & Therapeutics 148 (April): 34–46. https://doi.org/10.1016/ j.pharmthera.2014.11.011.

16.

Murthy, Vishakantha, Yang Gao, Liyi Geng, Nathan K. LeBrasseur, Thomas A. White, Robin J. Parks, and Stephen Brimijoin.

2014. “Physiologic and Metabolic Safety of Butyrylcholinesterase Gene Therapy in Mice.” Vaccine 32 (33): 4155–62. https:// doi.org/10.1016/j.vaccine.2014.05.067.

17.

Naldini, Luigi. 2015. “Gene Therapy Returns to Centre Stage.” Nature 526 (7573): 351–60. https://doi.org/10.1038/ nature15818.

18.

O’Connor, NicholasE., JohnB. Mulliken, Susan Banks-Schlegel, Olaniyi Kehinde, and Howard Green. 1981. “GRAFTING OF BURNS WITH CULTURED EPITHELIUM PREPARED FROM AUTOLOGOUS EPIDERMAL CELLS.” The Lancet, Originally published as Volume 1, Issue 8211, 317 (8211): 75–78. https://doi.org/10.1016/S0140-6736(81)90006-4.

19.

Penberthy, Jennifer K., Nassima Ait-Daoud, Michelle Vaughan, and Tasmin Fanning. 2010. “Review of Treatment for Cocaine Dependence.” Current Drug Abuse Reviews 3 (1): 49–62. https://doi.org/10.2174/1874473711003010049.

20.

Ran, F. Ann, Patrick D. Hsu, Chie-Yu Lin, Jonathan S. Gootenberg, Silvana Konermann, Alexandro Trevino, David A. Scott, et al. 2013. “Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity.” Cell 154 (6): 1380–89. https://doi.org/10.1016/j.cell.2013.08.021.

21.

Yue, Jiping, Xuewen Gou, Cynthia Li, Barton Wicksteed, and Xiaoyang Wu. 2017. “Engineered Epidermal Progenitor Cells Can Correct Diet-Induced Obesity and Diabetes.” Cell Stem Cell 21 (2): 256-263.e4. https://doi.org/10.1016/j.stem.2017.06.016.

22.

Zheng, Fang, Liu Xue, Shurong Hou, Junjun Liu, Max Zhan, Wenchao Yang, and Chang-Guo Zhan. 2014. “A Highly Efficient Cocaine-Detoxifying Enzyme Obtained by Computational Design.” Nature Communications 5 (1): 3457. https:// doi.org/10.1038/ncomms4457.

100

Behind Closed Doors: The Emerging Role of Focused Ultrasound in Alzheimerâ&#x20AC;&#x2122;s Disease Justin Mendoza

Alzheimerâ&#x20AC;&#x2122;s Disease (AD) has become recognized as a neurodegenerative condition in which declination in cognitive function progressively worsens over time. Hallmarks of AD include basal forebrain cholinergic neuron (BFCN) dysfunction, leading to the loss of post-synaptic connections associated with memory functioning. Another known defect that has been correlated with the early onset of this particular condition is the aberrant decline in the levels of nerve growth factor (NGF), a selective neurotrophin that propagates survival of mature neurons, once bound to its cognate receptor, tropomyosin-related kinase A (TrkA). The NGF-mediated TrkA pathway has been elucidated to support survival mechanisms, as well as the mediation of apoptotic signaling, depending on the bound ligand. With the available information of the pathophysiology and intracellular mechanisms of AD, current research aims to mitigate the severity of early hallmarks; however, a major limitation that prevents the delivery and efficacy of therapeutic regimens is the tightly regulated barrier that is the blood-brain barrier (BBB). As a result, current research has adapted to this limitation, proposing the use of non-invasive methods, such as magnetic resonance imaging-guided focused ultrasound (MRIgFUS) to increase the permeability of the BBB, as a means to deliver therapeutic compounds to elicit the TrkA cascade pathway. Through the work of Xhima et al., the TrkA agonist known as D3, had rescued cholinergic functioning within AD murine models, as opposed to monotherapy involving NGF alone, using MRIgFUS to deliver the agonist. As a result, the use of protective neurotrophins, along with the use of MRIgFUS to circumvent the BBB holds promising results, which can be used to further clinical trials in AD patients. Further research is warranted to elucidate the mechanisms of NGF-mediated therapy, as NGF alone is unable to stimulate TrkA-dependent signaling in an AD murine model.

Key words: Alzheimerâ&#x20AC;&#x2122;s disease, nerve growth factor, blood-brain barrier, magnetic resonance imagingguided focused ultrasound, TrkA, D3

101

Introduction

of cholinergic neuron functioning. The results of this study provide evidence of a potential therapeutic regimen to reverse the The neurodegenerative disease that is Alzheimerâ&#x20AC;&#x2122;s Disease (AD), neurodegenerative effect that AD entails. is commonly associated with a decline in cognitive function, being a major factor that plays a role in the progression of dementia [1]. With many pathways leading to neuronal deficits in AD, Results: such as amyloid-b (Ab) plaques, with subsequent tau aggregation, a susceptible brain region is the basal forebrain cholinergic TgCRND8 Murine Models as an acceptable model of human nuclei (BFCN), which has been associated for learning and Alzheimerâ&#x20AC;&#x2122;s memory [2]. The reason as to why the BFCN exhibit a decline in First, a phenotypic analysis of the TgCRND8 murine model was activity in AD is due to its dependence on the neurotrophin conducted, to determine its eligibility as a model for human AD. nerve growth factor (NGF), a biomolecule associated with neuThis was elucidated through the measurement of NGF, TrkA and ronal survival [3]. Dysfunction of this particular neurotrophinp75NTR mRNA and protein levels through Western blot. By studyreceptor axis is a common hallmark of AD, and begins to appear ing comparisons of age-matched TgCRND8 murine models to during the prodromal period of AD. During the preclinical stages their wild type counterparts, a noticeable decline in NGF and of AD, occurring as early as 20 years prior to onset of clinical TrkA mRNA levels within the Tg mice, keeping consistent with symptoms, there is a decrease in neuroprotective capacity, as the human phenotype of AD [5]. Thus, this result verified the cholinergic neurons consist of altered NGF levels, accompanied possibility of representing the human phenotype of AD with with defects in the processing pathways [4].

TgCRND8 murine models.

The role of NGF has been elucidated to induce neuronal survival and synaptic plasticity, when bound to its cognate receptors, p75 neurotrophin receptor (p75NTR) and tropomyosin- D3 stimulates TrkA signaling cascade, as opposed to NGF alone related kinase A (TrkA). In response to the formation of the NGF/ Another finding centralized on the roles of D3 and NGF. Within TrkA axis at the BFCN terminals, NGF signals elicit a survival rethis particular experiment, an in vivo analysis of these neurotrosponse through downstream molecules, such as the phosphophins was conducted in Tg mice by examining the signaling casinositide 3 kinase (PI3K)/Akt pathway. As a result, BFCN have cades elicited upon binding of the cognate TrkA receptor. Upon enhanced cholinergic activity, which occurs through increased intraparenchymal injection into the basal forebrain, D3, as optranscription of an enzyme known as choline acetyltransferase posed to the neurotrophin NGF, had stimulated the TrkA(ChAT), essential for the biosynthesis of acetylcholine. However, dependent signaling cascade, which was measured through levin AD, where a declination of both NGF and TrkA is observed, the els of phosphorylated TrkA (pTrkA), pMAPK, and pCREB. This other cognate receptor p75NTR has a higher affinity for the Ab particular result had provided significant evidence of a potential plaques, and the precursor of NGF, known as proNGF. Binding of therapeutic regimen designed to reverse cholinergic neuron this receptor signals a neurodegenerative pathway, and is the degradation in AD, thus furthering the existing evidence of resreason for selective degradation of BFCN in AD [5]. cuing cholinergic function through a selective TrkA agonist [8].

With the available information on the intracellular mechanisms of AD, current research aims to develop a therapeutic regimen centralizing on NGF-mediated therapy. However, a limi- Delivering D3 to the Basal Forebrain through MRIgFUS tation that is commonly addressed is the seemingly impermeaWith evidence of a therapeutic regimen that may prove to be ble blood-brain barrier (BBB). Moreover, NGF is unable to cross optimal in treating cholinergic neuron degradation in AD, the the BBB. To address this issue, current research aims to increase only obstacle yet to be overcome was the tightly regulated BBB. the permeability of the BBB, through non-invasive techniques The non-invasive method of MRIgFUS was employed to ensure such as focused ultrasound. Writing in Science Advances, Xhima an efficient delivery of the selective TrkA agonist to the basal et al. [6] proposes the use of magnetic resonance imagingforebrain. To measure the efficacy of this delivery method, highguided focused ultrasound (MRIgFUS) to deliver a selective TrkA performance liquid chromatography was used to measure D3 agonist into the BFCN, as a means of rescuing cholinergic funcconcentrations in both the Tg mice and the non-Tg control mice tioning within a TgCRND8 murine model that encapsulates the upon intravenous administration of D3. The hypothesis of phenotype of human AD. MRIgFUS as an efficient non-invasive method to deliver neuroWith the known mechanism of action of MRIgFUS, protective agents was confirmed by an increase in D3 concentrawhich centralizes on increasing the permeability of the blood- tion within Tg mice, as opposed to non-Tg mice. This result was brain barrier [7], the authors aimed to deliver D3 to the basal consistent with other findings using focused ultrasound to delivforebrain, a site of interest that is associated with cognitive de- er therapeutic agents in patients with AD [9,10]. cline in AD. Through an appropriate murine model of AD, combined with the non-invasive mechanism of MRIgFUS, the researchers were able to efficiently deliver D3, a TrkA agonist that MRIgFUS D3 delivery rescues cholinergic functioning through elicits a survival mechanism, similar to that of NGF, when bound TrkA-dependent signaling to TrkA. MRIgFUS facilitated D3 delivery to the basal forebrain of Examining the functional effects of MRIgFUS-mediated delivery a TgCRND8 murine model. D3 activity, not NGF, led to rescuing 102

of the TrkA agonist was done through Western blot, in which pTrkA was measured. There was an observed increase in pTrkA expression within the D3-FUS mice, as opposed to the D3 nonFUS mice. Furthermore, this indirectly led to the increased activity of cholinergic neurons within the Tg model. Normally, the enzymatic activity of ChAT was reduced in Tg mice, ultimately leading to decreased biosynthesis of ACh. 90 days after sonication, there was an observed increase of ChAT activity within D3-FUS treatment. Overall, this study provides evidence of a possible therapeutic regimen with an efficient delivery method, which may be implemented into future clinical trials as a means of dealing with the neurodegenerative effects of AD.

can be run. However, rather than the sole administration of D3, a combination therapy can be run, in which anti-Ab antibodies and NGF administration occurs. Through the use of noninvasive MRIgFUS, the elimination of Ab pathology is now possible. Moreover, while TrkA expression is compromised within AD, a quantitative analysis through radiolabeling prior to therapy can provide an accurate measurement of the concentration of TrkA receptors, which can determine the optimal concentration of NGF needed to engage TrkA receptors. Moreover, through MRIgFUS, larger antibodies targeting Ab plaques can cross through the BBB, targeting Ab plaques, which have been associated with dysregulation of the metabolic processing of NGF. To measure the efficacy of NGF-based therapy, an immunoprecipitation and Western blot of the downstream mediaDiscussion/Critical Appraisal (Talk about dismissing Ab, NGF tors relevant in TrkA signaling can be used. If there is increased selectivity, and impaired memory) levels of pCREB, pAKT, or pMAPK, this provides evidence of an efficient NGF-based therapy.

In this study, significant evidence was observed to propose a possible treatment to AD, through the use of MRIgFUS and the TrkA agonist, D3. Moreover, it further validated the use of a TgCRND8 murine model as a potential model for human AD. The importance of these new findings centralizes on the ability to recapitulate human circumstances of AD, and develop possible mechanisms to increase the efficacy of the delivery of therapeutic agents. Generally, the common route of treatment for AD is the use of pharmaceutical compounds, such as acetylcholinesterase inhibitors (AChEIs), which prevent the breakdown of acetylcholine within the cholinergic neurons, maintaining some cholinergic function. However, the authors conclude that through MRIgFUS and a selective TrkA antagonist would maintain the morphology of the BFCN, as it would lead to a more profound effect through increased enzymatic activity of ChAT, which would compensate for the loss of ACh, whilst increasing neuronal survival, which cannot be achieved through AChEI alone.

While the work of Xhima et al. provides evidence of a potential therapeutic regimen to reverse the neurodegenerative effects of AD, there are some unanswered questions that need to be addressed. For instance, the study finds a mechanism of rescuing cholinergic neurons by enhancing the survival pathway (ie. D3 activating TrkA, leading to the Akt pathway), but does not address a method for eliminating the Ab pathology, which is known to associate with the p75NTR receptor, in an apoptotic signaling cascade. Within the study, a major finding centralized on the efficacy of D3 administration, accompanied with the inefficacy of native NGF administration. The authors had hypothesized that NGF-mediated therapies within previous trials were limited to the Ab and tau pathologies, ultimately leading to dysregulation of NGF metabolism. Moreover, due to the invasive method of surgery to administer NGF-based agents, the authors conclude that the risks of NGF-based therapies outweigh the benefits.

Future Directions Proceeding with the findings of Xhima et al., to elucidate the efficacy that NGF-based therapy entails, a similar experiment 103

REFRENCES 1.

Tiwari, S., Atluri, V., Kaushik, A., Yndart, A., & Nair, M. (2019). Alzheimer's disease: pathogenesis, diagnostics, and therapeutics. International journal of nanomedicine, 14, 5541–5554.

2.

Baxter, M. G., & Chiba, A. A. (1999). Cognitive functions of the basal forebrain. Current opinion in neurobiology, 9(2), 178– 183.

3.

Fahnestock, M., & Shekari, A. (2019). ProNGF and Neurodegeneration in Alzheimer's Disease. Frontiers in neuroscience, 13, 129

4.

Mufson, E. J., Counts, S. E., Ginsberg, S. D., Mahady, L., Perez, S. E., Massa, S. M., Longo, F. M., & Ikonomovic, M. D. (2019).

Nerve Growth Factor Pathobiology During the Progression of Alzheimer's Disease. Frontiers in neuroscience, 13, 533.

5.

Canu, N., Amadoro, G., Triaca, V., Latina, V., Sposato, V., Corsetti, V., Severini, C., Ciotti, M. T., & Calissano, P. (2017). The Intersection of NGF/TrkA Signaling and Amyloid Precursor Protein Processing in Alzheimer's Disease Neuropathology. International journal of molecular sciences, 18(6), 1319.

6.

Xhima K, Markham-Coultes K, Nedev H, et al. Focused ultrasound delivery of a selective TrkA agonist rescues cholinergic function in a mouse model of Alzheimer's disease. Sci Adv. 2020;6(4):eaax6646

7.

Lee, E. J., Fomenko, A., & Lozano, A. M. (2019). Magnetic Resonance-Guided Focused Ultrasound : Current Status and Future Perspectives in Thermal Ablation and Blood-Brain Barrier Opening. Journal of Korean Neurosurgical Society, 62(1), 10–26.

8.

Josephy-Hernandez S, Pirvulescu I, Maira M, et al. Pharmacological interrogation of TrkA-mediated mechanisms in hippocampal-dependent memory consolidation. PLoS One. 2019;14(6):e0218036.

9.

Jordão, J. F., Ayala-Grosso, C. A., Markham, K., Huang, Y., Chopra, R., McLaurin, J., Hynynen, K., & Aubert, I. (2010). Antibodies targeted to the brain with image-guided focused ultrasound reduces amyloid-beta plaque load in the TgCRND8 mouse model of Alzheimer's disease. PloS one, 5(5), e10549.

10.

Raymond, S. B., Treat, L. H., Dewey, J. D., McDannold, N. J., Hynynen, K., & Bacskai, B. J. (2008). Ultrasound enhanced delivery of molecular imaging and therapeutic agents in Alzheimer's disease mouse models. PloS one, 3(5), e2175.

104

Go with your Gut: How inflammation can speed up Motor Dysfunction in Alpha‑Synuclein Mutant Mice Sonita Mohammadi

Parkinson's Disease (PD) is a neurodegenerative disorder that causes motor symptoms bradykinesia, rigidity, and tremor (de Lau & Breteler, 2006) PD prevalence increases with age and affects 1% of the population over the age of 60 (de Lau & Breteler, 2006). No cure exists and pharmacological therapies are available to diminish the symptoms of the disease. Recent studies suggest gut microbiota can lead to the formation of alpha-synuclein in the enteric nervous system (ENS) and can travel via the vagus nerve to the central nervous system (CNS) (Fitzgerald et al., 2019). Alpha-synuclein is an important factor of Lewy body’s production that comes from in the loss of dopaminergic neurons in the substantia nigra as a cause of PD (Pickrell et al., 2015). Studies are trying to prevent Lewy body development by focusing on alpha-synuclein aggregates (Pickrell et al., 2015). The initial paper by Kishimoto et al., (2019) explored the role of chronic mild gut inflammation and how it plays a role in hastening the onset of motor dysfunction in PD mice (Kishimoto et al., 2019). In their animal model, Parkinson's disease (PD) mice treated with dextran sodium sulfate (DSS) in their water for 12 weeks and observed that the onset of motor disorder sped up (Kishimoto et al., 2019).

The PD mutant DSS- treated mice exhibited motor dysfunction considerably earlier than their control group (Kishimoto et al., 2019). This study concluded that a chronic mild increase in gut inflammation speeds up the onset of motor dysfunction in PD. Key words: Parkinson’s disease, inflammation, alpha-synuclein, Enteric neurons, Neuroinflammation

105

INTRODUCTION

mildly ease the motor symptoms. This review will seek to shed light chronic mild gut inflammation and Parkinsonâ&#x20AC;&#x2122;s disease Parkinson's Disease (PD) is a neurodegenerative movement dis(Kishimoto et al., 2019). order that is a multi-etiological condition with uncertain etiopathogenesis (de Lau & Breteler, 2006). Genes and environ- Visual Abstract mental hazards both play a part in the pathology of PD (Chen et al., 2019). PD is an age-related disorder affecting individuals over the age of 6o years of age (Antony et al., 2013). The hallmark of PD is the destruction of dopaminergic neurons in substantia nigra and alpha-synuclein presence in Lewy-bodies (Weil, R. S., Lashley). Losing these neurons results in manifestation of motor symptoms such as bradykinesia, resting tremor, and other non- Figure 1. Visual abstract to represent the experiment carried out motor symptoms like depression, constipation, (Chen et al., by Kishimoto et al. regarding the role of chronic mild gut inflam2019). The modern mechanism of PD is that mitochondrial im- mation and onset of motor dysfunction in PD (Kishimoto et al., pairment can lead to oxidative damage and that aggregation of 2019). In this experiment, the authors reported that chronic mild alpha-synuclein. These aggregates play a key role in degradation gut inflammation is suffices to speed motor disorder symptoms of dopaminergic neurons (Hu & Wang, 2016). In addition, micro- in PD mice (Kishimoto et al., 2019). glial cells produce proinflammatory cytokines with neuronal degradation in PD (Kishimoto et al., 2019). Kishimoto et al., (2019), animal models suggest that alpha-synuclein aggregation may Major Results and Methods first emerge from the peripheral neurons (Kishimoto et al., 2019). Kishimoto et al., (2019) propose that alpha-synuclein The authors used female transgenic mice, over-expressing alphatravels retrogradely from the gut to different regions of the synuclein (PD mice) and age-matched wild type (WT) mice in brain (Fitzgerald et al., 2019). They base this idea on the under- their experiments. They randomly assigned PD and WT mice to standing that patients with PD often have a history of chronic different treatments, either treated DSS in their drinking water constipation and suffer from vagal cholinergic tone deficit or just water for 12 weeks and mice euthanized. To determine (Kishimoto et al., 2019). Earlier studies found that alpha- motor performance, Kimshimo and colleagues used 3 unique synuclein pathology is present in gut 20 years before they diag- motor test tasks, a rotarod apparatus, rotating rod and gate and nosed individuals with PD (Kishimoto et al., 2019). Therefore, grip analysis (Kishimoto et al., 2019). The authors observed that studies shifted their attention to the processes by which the gut mutant PD mice developed discernable motor symptoms and inflammation contribute to the production of alpha-synuclein in become progressively worse at performing motor test tasks performance until the mice no longer could ambulate (Kishimoto et the enteric nervous system (Fitzgerald, Emily, et al.). al., 2019). DSS-treated mice showed a significant decline in The original paper examines whether chronic mild gut inflamma- rotarod performance Figure 1 (a), grip strength (b) and stride tion in PD mouse model can speed up motor dysfunction symp- length (c) (Kishimoto et al., 2019). These data are significant as toms (Kishimoto et al., 2019). Kishimoto et al. 2010, hypothe- they showed that there was a considerable difference in disease sized that chronic mild gut inflammation can speed up the onset progression in PD mice induced with compared to the Wilde of PD and increased neuroinflammation in the brain (Kishimoto type mice (Kishimoto et al., 2019). Kimshimo et al. results are et al., 2019). They induced inflammation in the gut via dextran consistent with previous findings in literature. Figure 2 results sodium sulfate (DSS) in PD mice and wild type. They observed are significant because it showed that chronic mild gut inflamsignificant motor dysfunction, alpha-synuclein aggregation and mation leads to loss of dopaminergic neurons in alpha-synuclein degeneration in dopaminergic neurons in the DSS treated PD mutant transgenic mice mice compared to the control group (Kishimoto et al., 2019). To confirm that DSS was the only cause for inflammation, they tested serum cytokines levels in all four groups, and they revealed Motor Tests Performance result no significant variations. Next, the authors studied immunoassayed colon and brain tissue with an antibody specific for microglia and macrophages for local tissue inflammation; and discovered induced inflammation in the gut and brain tissue. Their result showed that chronic mild gut inflammation can speed up the onset of PD (Kishimoto et al., 2019). Although the mechanism and physiological functions of alpha-synuclein is not clear (Bernal-Conde et al., 2020), Kishimoto et al., (2019) concluded that their result was consistent with chronic mild gut inflammaFigure. 1 a b c tion exacerbating alpha synuclein in the brain and destroy dopaminergic neurons by travelling retrogradely via vagus nerve Retrieved images from Kishimoto et al., 2019.Figure 1. a The (Kishimoto et al., 2019). Therefore, understanding the cause of graph shows the result of grip strength test. b The graph shows complex neurodegenerative disorders is critical for developing the result of stride length test. c Image show ink footprints effective therapies. Modern therapies for PD offer no cure and (Kishimoto et al., 2019). These results are significant because PD 106

mice treated with DSS developed discernable motor dysfunc- et al., 2018). Although we have seen that chronic mild inflamtion (Kishimoto et al., 2019). mation in the gut lead to an increase in alpha-synuclein aggregation in the brain and degradation of dopaminergic neurons, these authors did not discuss the exact cellular mechanism.

Chronic gut inflammation exacerbates alpha- synuclein pa- Their research concentrated more on the link between gut inthology flammation and motor dysfunction. A limitation to this paper is failing to present specific mechanistic evidence in how inflammation in the gut travels via the vagus nerve. Although their result complied in the research study supports their hypothesis, further research required in this developing field.

Future studies Figure. 2 a

b

Retrieved images from Kishimoto et al., 2019. Figure. 2 a) The images show tyrosine hydroxylase (TH) in substantia nigra. b Is a sagittal section from mice showing alpha synuclein and immediately below each image is a high magnification micrograph image of the midbrain. 200um scale bar. The top image shows substantia nigra WT/H2O and PD/H2O (upper) and WT/DSS and PD/DSS are (lower) region.

Discussions Section Kishimoto et al. (2019) concluded that their findings are important in treatment and prevention of PD by finding a link between chronic inflammation in the gut and neurodegenerative disorders. Alpha-synuclein mutant transgenic PD mice displayed discernable motor deficits, as they could not move at the end of the 12-week study period. Using immunoblot technique, it allowed the author to visualize the increased levels of alpha-synuclein protein and decrease tyrosine hydroxylase dopaminergic neurons compared to the wild type. This allowed them to link the chronic mild gut inflammation to increase in alpha-synuclein aggregation in the brain and degeneration of dopaminergic neurons by a mechanism that aggregates travel retrogradely via the vagus nerve to different regions of the brain. Kishimoto et al. (2019) recognized that their results are consistent with current literature, particularly when explaining that PD may first develop in the peripheral neurons, enteric branches of the vagus nerve. In an experiment conducted in mice by Johns Hopkins Medicine, researchers showed there is reliable evidence that Parkinsonâ&#x20AC;&#x2122;s disease develops in the gut and migrates up to the brain (Kim et al., 2019).

Future studies should involve understanding the alphasynuclein proinflammatory role in association to diet. One proposed study can investigate the change in alpha-synuclein expression using a mouse model with and without PD, and the effects of a high-fat diet on motor function. Models will include neurotoxin 6-hydroxy dopamine (6-OHDA) rat to induce PD and transgenic rats that do not exhibit PD. The Rats will randomly be assigned to a normal diet or ketogenic for 8 weeks. During the 8-week period, different motor function tests will take place weekly. At the end of the 8-week study period, rats will be euthanized to examine the brain and the gut tissues. Techniques such as immunohistochemistry and immunoblot can will be performed to observe tissue changes and measure alphasynuclein expression and dopaminergic neurons in rats with PD and without PD. Immunostaining the brain and gut tissue using an antibody that can bind selectively to alpha-synuclein will help identify primary protein of interest. Overall, this study should show the effects of a high-fat diet on motor and nonmotor symptoms in PD, and if ketogenic diet can aid PD.

Critical analysis Further experiments should concentrate on understanding the role of diet and exercise and its effect on alpha-synuclein as a preventative measure. The study suggests that alpha-synuclein presence in the gut could become a valuable biomarker for the diagnosis of Parkinson's disease. However, a study by Recasens et al., (2018) argues against the presumptive capacity of peripheral nervous system to cause Parkinsonâ&#x20AC;&#x2122;s disease (Recasens 107

REFRENCES

1.

Antony, P., Diederich, N., Krüger, R., & Balling, R. (2013). The hallmarks of Parkinson's disease. FEBS Journal, 280(23), 5981 -5993. https://doi.org/10.1111/febs.12335

2.

Bernal-Conde, L., Ramos-Acevedo, R., Reyes-Hernández, M., Balbuena-Olvera, A., Morales-Moreno, I., & Argüero-Sánchez, R. et al. (2020). Alpha-Synuclein Physiology and Pathology: A Perspective on Cellular Structures and Organelles. Frontiers In Neuroscience, 13. https://doi.org/10.3389/fnins.2019.01399

3.

Borghammer, P., & Hamani, C. (2017). Preventing Parkinson disease by vagotomy. Neurology, 88(21), 1982-1983. https:// doi.org/10.1212/wnl.0000000000003969

4.

Chen, Q., Haikal, C., Li, W., & Li, J. (2019). Gut Inflammation in Association With Pathogenesis of Parkinson’s Disease. Frontiers In Molecular Neuroscience, 12. https://doi.org/10.3389/fnmol.2019.00218

5.

de Lau, L., & Breteler, M. (2006). Epidemiology of Parkinson's disease. The Lancet Neurology, 5(6), 525-535. https:// doi.org/10.1016/s1474-4422(06)70471-9

6.

Dickson, D. (2012). Parkinson's Disease and Parkinsonism: Neuropathology. Cold Spring Harbor Perspectives In Medicine, 2 (8), a009258-a009258. https://doi.org/10.1101/cshperspect.a009258

7.

Fitzgerald, E., Murphy, S., & Martinson, H. (2019). Alpha-Synuclein Pathology and the Role of the Microbiota in Parkinson’s Disease. Frontiers In Neuroscience, 13. https://doi.org/10.3389/fnins.2019.00369

8.

Hu, Q., & Wang, G. (2016). Mitochondrial dysfunction in Parkinson’s disease. Translational Neurodegeneration, 5(1). https://doi.org/10.1186/s40035-016-0060-6

9.

Kalia, L., & Lang, A. (2015). Parkinson's disease. The Lancet, 386(9996), 896-912. https://doi.org/10.1016/s0140-6736(14) 61393-3

10.

Kim, S., Kwon, S., Kam, T., Panicker, N., Karuppagounder, S., & Lee, S. et al. (2019). Transneuronal Propagation of Pathologic α-Synuclein from the Gut to the Brain Models Parkinson’s Disease. Neuron, 103(4), 627-641.e7. https:// doi.org/10.1016/j.neuron.2019.05.035

11.

Kishimoto, Y., Zhu, W., Hosoda, W., Sen, J., & Mattson, M. (2019). Chronic Mild Gut Inflammation Accelerates Brain Neuropathology and Motor Dysfunction in α-Synuclein Mutant Mice. Neuromolecular Medicine, 21(3), 239-249. https:// doi.org/10.1007/s12017-019-08539-5

12.

Pickrell, A., Huang, C., Kennedy, S., Ordureau, A., Sideris, D., & Hoekstra, J. et al. (2015). Endogenous Parkin Preserves Dopaminergic Substantia Nigral Neurons following Mitochondrial DNA Mutagenic Stress. Neuron, 87(2), 371-381. https:// doi.org/10.1016/j.neuron.2015.06.034

13.

Recasens, A., Carballo-Carbajal, I., Parent, A., Bové, J., Gelpi, E., Tolosa, E., & Vila, M. (2018). Lack of pathogenic potential of peripheral α-synuclein aggregates from Parkinson’s disease patients. Acta Neuropathologica Communications, 6(1). https://doi.org/10.1186/s40478-018-0509-1

14.

Xu, L., & Pu, J. (2016). Alpha-Synuclein in Parkinson’s Disease: From Pathogenetic Dysfunction to Potential Clinical Application. Parkinson's Disease, 2016, 1-10. https://doi.org/10.1155/2016/1720621

108

Positive Effects of Probiotic Treatment on Spatial Cognitive Performance and Synaptic Plasticity in a β-amyloid rat model Amna Noor

Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by the presence of amyloid plaques and tau tangles in the brain. Recently, dysbiosis of gut microbiota has been implicated in etiology of various brain dysfunctions, including AD. Currently, there is no effective treatment to stop or slow down the progression of AD. As a result, therapeutic strategies focus on treating various behavioural and cognitive symptoms. The research conducted by Rezaei Asl, Sepehri and Salami (2019) addresses the lack of treatment and hypothesizes that supporting the gut microbiome with probiotics will reverse some of the negative effects of the dysbiosis. An animal model of AD was made by injecting β-amyloid intracerebroventricularly into male Wistar rats. The probiotic treatment was made up of encapsulated Lactobacillus acidophilus, Bifidobacterium bifidum, and Bifidobacterium longum. The rats were further divided into five groups: rats that received water (Con), rats that received probiotics and water (Pro + Con), rats that received the injection (Alz), rats that received injection and probiotics (Alz + Pro), and rats that underwent sham surgery (Sham). Evaluation of different behavioural and electrophysiological aspects of AD via conducting a plethora of tests confirmed restoration of synaptic plasticity (LTP), enhancement of spatial cognitive performance, and an increase in antioxidant to oxidant ratio in rats that received the probiotic concoction. Therefore, the study served as evidence for a novel treatment of AD through probiotic support of gut microbiome.

Keywords: Neurodegeneration, neurodegenerative disease, Alzheimer’s disease, long term potentiation, spatial memory, animal model, gut microbiota, dysbiosis, probiotics

109

INTRODUCTION

to oxidant factors ratio by measuring plasma content of total antioxidant capacity (TAC) and malondialdehyde (MDA).

Alzheimer’s disease (AD) is a neurodegenerative disease that was first discovered in 1906. It is now a leading cause of death and dementia in older adults. Currently, AD is characterized by the deposition of β-amyloid plaques, the formation of neurofibrillary hyperphosphorylated tau protein tangles, neuroinflammation, and progressive impairment of neuronal synapses (Selkoe 2001; Scheltens et al. 2016). However, many of its pathophysiological facets are still being examined (Viña and Sanz‐ Ros 2018). There is no effective treatment to halt or slow down the progression of AD. Available options include combination therapies to manage behavioural symptoms and cognitive functions associated with AD such as memory deterioration (Selkoe Figure 1. Summary of methods and major findings are summa2001; Viña and Sanz‐Ros 2018). rized in the figure above. The β-amyloid injection was given Gut microbiota refers to the population of commensal intracerebroventricularly and probiotics and water were adminmicroorganisms residing in the human gastrointestinal (GI) istered via an intragastric gavage. Probiotics significantly imtract. The gut microbiota is unique to every individual but has proved spatial cognitive performance in the Morris water maze common dominant bacteria, such as Firmicutes and Bacteroide- test and formation of LTP in the hippocampus in (Alz + Pro) and tes, whose composition changes in diseased individuals. In (Pro + Con) mice while also increasing antioxidant to oxidant healthy hosts, microbiota serves to maintain a protective barri- factors ratio which reduced apoptosis. er against pathogens and lives in a state known as eubiosis whereas in diseased subjects the healthy balance of microorganisms in the GI tract is compromised and microbiota enters a MAJOR RESULTS state of dysbiosis (Angelucci et al. 2019; Franceschi et al. 2019).

Behavioural Performances In recent years, there have been numerous reports of a gutbrain axis (GBA). Specifically, GBA is a two-way signalling path- Rezaei Asl, Sepehri and Salami (2019) used a Morris water way between the gut and the brain via the vagus nerve (Collins, maze test to assess task learning and formation of recent memSurette, and Bercik 2012). A dysbiosis in the gut has been linked ories. The test consisted of an acquisition phase whereby anito various disorders of the central nervous system, such as AD mals were given time to locate the platform followed by a (Westfall et al. 2017). Some of these studies have established probe trial test whereby animals searched the maze. This was links between the gut and the hippocampus which is implicated in accordance with previously established methods (Vorhees in the formation of memories and establishment of long-term and Williams 2006). Alz rats required 50% more time than othdepression (LTD) and long-term potentiation (LTP) via the CA1- er groups to locate the platform but no difference was found in CA3 pathway. The gut microbiome is also subject to changes the probe trial test stage. Pro + Alz rats showed improvement that can increase gut permeability and promote bacterial trans- and were able to locate the platform faster and finally, Pro + location due to aging, which is a risk factor for AD (Jiang et al. Con mice located the platform earliest.

2017). In particular, an increase in serum levels of inflammatory cytokines and deposition of β-amyloid plaques have been linked to the direct and indirect effects of dysbiosis in different capacities (Angelucci et al. 2019). Thus, manipulation of the microbiome serves as a good therapeutic target because if the cause-and-effect relation holds, a reverse, that is the introduction of ‘good bacteria’ into diseased individuals, should alleviate symptoms of the disease.

Inspired by this connection, Rezaei Asl, Sepehri and Salami (2019) researched positive effects of a probiotic concoction composed of encapsulated Lactobacillus acidophilus, Bifidobacterium bifidum, and Bifidobacterium longum in rat models of AD made by injecting β-amyloid intracerebroventricularly. Probiotics refer to bacteria that induce favourable changes in host health (Angelucci et al. 2019). The study assessed influence of probiotic treatment on spatial learning and memory via Morris water maze test, basic synaptic transmission and LTP in the hippocampus via recording field excitatory postsynaptic potentials (fEPSPs), and change in the antioxidant

Figure 2. Curves summarize the impact of probiotic administration on behavioural performances of the rat Alz, Con, and Sham models. Probiotics significantly improved the performance of rats in both Alz and Con models which is indicative of better spatial cognitive functioning and formation of memories.

(Figure derived from Rezaei Asl, Sepehri and Salami (2019)).

Synaptic Transmission

110

Rezaei Asl, Sepehri and Salami (2019) stimulated Schaffer collaterals and measured baseline fEPSPs in the CA1 pathway to conclude the effect of probiotic administration on synaptic transmission. Application of high-frequency stimulation increased LTP in Alz + Pro as well as Pro + Con rats. Alz rats showed significantly decreased LTP compared to their counterparts. Overall, the β-amyloid injection reduced hippocampal LTP, but the effects were reversed upon administration of probiotics. This showed that Alz rat models had difficulty forming new memories and learning.

tively links to increased synaptic plasticity in a β-amyloid rat model of AD, hinting a potential therapeutic strategy.

Their results are in line with current literature in that accumulation of β-amyloid impairs learning and memory functions and dysbiosis is implicated in the process. Probiotics have previously been used to show similar results (Schneider et al.

2020; Yang et al. 2020). Literature suggests that the underlying mechanism might be linked to various pathways such as immunological, hormonal, and neuronal, but since the field is relatively new, a definitive link is yet to be established. Authors believe that this link is via a reduction in gut inflammation.

Dysbiosis is linked to inflammation of the gut which causes increased penetrance of the microbiota outside the tract leading to a compromised immune barrier. Probiotics help to reduce this inflammation by increasing the amount of short fatty acid chains that release brain-derived neurotrophic factor (BDNF) and thus reduce inflammation (Rezaei Asl, Sepehri and Salami 2019).

There is a limited number of studies that examine the hippocampal dependant synaptic plasticity vis-à-vis probiotics. β-amyloid plaques have been shown to cause abnormal NMDA receptor activation, which is the main CA1 - CA3 pathway receptor. Through inference from previous literature, authors determined that β-amyloid attenuates LTP by disturbing normal NMDA receptor and BDNF functions. However, the underlying Figure 3. fEPSPs observed in the CA1 area are represented by mechanism remains undetermined (Rezaei Asl, Sepehri and curves in the figure. Baseline fEPSP was recorded and then 10 Salami 2019). recordings were made and averaged every 30 minutes at time marks 30, 60, and 90 minutes. The increased amplitude of fEPSP Lastly, the increase in antioxidant factors and a derepresents enhanced LTP. (Figure derived from Rezaei Asl, crease in oxidant factors is also supported by the literature Sepehri and Salami (2019)).

(Mehta et al. 2017). AD is linked to oxidative stress as βamyloid plaques can stress mitochondria, which results in the generation of reactive oxidative species and free radicals. ProPlasma levels of antioxidant/ oxidant factors biotics increase antioxidant enzymes and reduce inflammation Rezaei Asl, Sepehri and Salami (2019) measured the plasma which reverses this phenomenon (Rezaei Asl, Sepehri and Salalevels of TAC and MDA (oxidant) to conclude the ratio of the mi 2019). factors. This is a very reliable way of measuring oxidative stress (Katerji, Filippova, and Duerksen-Hughes 2019). Alz rat models showed increased MDA levels which were decreased by probiotic administration but remained higher than normal. They also showed decreased TAC plasma levels which were efficiently increased by probiotics. Pro + Con mice showed enhanced TAC to MDA ratio. The results indicated that Alz rat models were more prone to neuronal apoptosis and probiotics could reverse this.

Figure 4. Major implications of the study are highlighted in the figure above.

CONCLUSIONS/ DISCUSSIONS Through this experiment, the authors were able to demonstrate that the probiotics supplement can be used to reverse behavioural and electrophysiological symptoms of AD and thus, serve as a potential therapy. This was due to their observations that AD rat models that received the probiotics supplement demonstrated improved hippocampal dependant cognitive functioning and had increased plasma levels of antioxidant species reducing apoptosis (Rezaei Asl, Sepehri and Salami 2019).

The research is significant because it is the first study that posi-

CRITICAL ANALYSIS The purpose of this study was to supplement gut microbiota with a probiotics concoction to alleviate the negative effects of dysbiosis on AD patients. Although probiotics showed great promise as a potential treatment, the authors used a combination of different bacterial genera to construct the probiotic sup-

111

plement and the effects cannot be attributed to a specific genus (Rezaei Asl, Sepehri and Salami 2019). A study by Magistrelli et al. in 2019 showed a similar decrease in oxidative species with the use of probiotics constructed from lactobacillus and bifidobacterium genus in patients with Parkinsonâ&#x20AC;&#x2122;s Disease.

However, they showed the most improvement in patients that received probiotics composed of L. salivarius and L. acidophilus.

Bubnov et al. 2015 also report the use of Akkermansia muciniphila to maintain the integrity of the gut lining which can help reduce the negative effects of cytokines on learning and memory.

As a follow-up, the authors should focus on specific genera, as well as the optimal composition of these species in the concoction, to have more confidence in conclusions. The underlying mechanisms for their conclusions were unclear due to the lack of evidence, so although they seem logical via inference, a definitive mechanism could not be determined. Furthermore, even though LTD has been linked to a decrease in memory and cognitive functioning as well, the results only account for LTP. This pattern can be seen in previous literature as well. The severity of AD, sex, and age of the patient have all been shown to be confounding variables for probiotics treatment (Agahi et al.

2018; Kim et al. 2020). The authors should have had controls for these variables as well. The rest of the methods used in the study are well-established methods used extensively in the literature (Katerji, Filippova, and Duerksen-Hughes 2019; Vorhees and Williams 2006).

tioned directions, molecules secreted by gut microbiota such as vascular endothelial growth factor B (VEGF-B) and transforming growth factor alpha (TGF-Îą) that regulate neuroinflammation via recruitment of microglia and astrocytes should also be studied as potential mechanisms for the role of probiotics (Giau et al. 2018). Similarly, other factors that could be used in conjunction with probiotics supplement, such as exercise, should be studied, Abraham et al. in 2019 showed that probiotics and exercise therapy significantly increased water-maze performance in mice models of AD. Music has also been linked to an increase in memory retention in patients with AD (Lord and Garner 1993). These results could theoretically be used to model potential combined therapy without the need for invasive procedures. Lastly, it is also important to consider and explore the role of probiotics in reducing LTD. Successful experimentation should show the probiotics categorically reduce the induction of LTD in animal models. In conclusion, the authors provided the first proof of the positive effects of probiotics on hippocampal-dependent synaptic plasticity and several future studies can develop this further.

FUTURE DIRECTIONS Authors of this paper were the first ones to demonstrate that probiotic supplementation has a positive influence on hippocampal-dependent synaptic plasticity in a rat model of AD and as such there was a lack of previous literature to support or contradict their results as well as to identify underlying mechanisms (Rezaei Asl, Sepehri and Salami 2019). As discussed earlier, age, sex, and severity of AD can all influence the impacts of probiotics on the gut microbiota (Agahi et al. 2018; Kim et al.

2020). The sample sizes for different groups in this study are from 7 to 10 rats which are not large enough to control for all the different variables (Rezaei Asl, Sepehri and Salami 2019).

Future researchers should either include a bigger sample size with a wider range of controls in their study or study the effects of probiotics on each of the different type of patient (young female with mild AD vs old female with mild AD et cetera) to determine the efficacy of probiotics as a potential treatment. If the results show no significant differences between the groups and support the results of the study at hand, then probiotics can be used as a successful intervention or vice versa. Adding on to that, the researchers used bacteria from different species to draw their results which makes it difficult to replicate the study (Rezaei Asl, Sepehri and Salami 2019). As a follow-up to this study, the effects of individual bacterium species can be studied. Future studies should also explore multiple models of AD as it is a multifactorial disease. For example, in 2017, Mehta et al. were able to produce similar oxidative stress results in Dgalactose animal models of AD. In addition to the aforemen112

REFRENCES

1.

Abraham, Dora, Janos Feher, Gian Luca Scuderi, Dora Szabo, Arpad Dobolyi, Melinda Cservenak, Janos Juhasz, et al. 2019.

“Exercise and Probiotics Attenuate the Development of Alzheimer’s Disease in Transgenic Mice: Role of Microbiome.”

Experimental Gerontology 115: 122–31. https://doi.org/10.1016/j.exger.2018.12.005.

2.

Agahi, Azadeh, Gholam Ali Hamidi, Reza Daneshvar, Mostafa Hamdieh, Masoud Soheili, Azam Alinaghipour, Seyyed Mohammad Esmaeili Taba, and Mahmoud Salami. 2018. “Does Severity of Alzheimer’s Disease Contribute to Its Responsiveness to Modifying Gut Microbiota? A Double Blind Clinical Trial.” Frontiers in Neurology 9. https://doi.org/10.3389/ fneur.2018.00662.

3.

Angelucci, Francesco, Katerina Cechova, Jana Amlerova, and Jakub Hort. 2019. “Antibiotics, Gut Microbiota, and Alzheimer’s Disease.” Journal of Neuroinflammation 16 (1): 108. https://doi.org/10.1186/s12974-019-1494-4.

4.

Bubnov, Rostyslav V., Mykola Ya Spivak, Liudmyla M. Lazarenko, Alojz Bomba, and Nadiya V. Boyko. 2015. “Probiotics and Immunity: Provisional Role for Personalized Diets and Disease Prevention.” EPMA Journal 6 (1): 14. https:// doi.org/10.1186/s13167-015-0036-0.

5.

Collins, Stephen M., Michael Surette, and Premysl Bercik. 2012. “The Interplay between the Intestinal Microbiota and the Brain.” Nature Reviews Microbiology 10 (11): 735–42. https://doi.org/10.1038/nrmicro2876.

6.

Franceschi, F, V Ojetti, M Candelli, M Covino, S Cardone, A Potenza, B Simeoni, et al. 2019. “Microbes and Alzheimer’ Disease: Lessons from H. Pylori and GUT Microbiota,” no. 23: 426–30.

7.

Giau, Vo Van, Si Ying Wu, Angelo Jamerlan, Seong Soo A. An, Sang Yun Kim, and John Hulme. 2018. “Gut Microbiota and Their Neuroinflammatory Implications in Alzheimer’s Disease.” Nutrients 10 (11). https://doi.org/10.3390/nu10111765.

8.

Jiang, Chunmei, Guangning Li, Pengru Huang, Zhou Liu, and Bin Zhao. 2017. “The Gut Microbiota and Alzheimer’s Disease.” Journal of Alzheimer’s Disease 58 (1): 1–15. https://doi.org/10.3233/JAD-161141.

9.

Katerji, Meghri, Maria Filippova, and Penelope Duerksen-Hughes. 2019. “Approaches and Methods to Measure Oxidative Stress in Clinical Samples: Research Applications in the Cancer Field.” Review Article. Oxidative Medicine and Cellular Longevity. Hindawi. March 12, 2019. https://doi.org/10.1155/2019/1279250.

10.

Kim, Yong Sung, Tatsuya Unno, Byung-Yong Kim, and Mi-Sung Park. 2020. “Sex Differences in Gut Microbiota.” The World Journal of Men’s Health 38 (1): 48–60. https://doi.org/10.5534/wjmh.190009.

11.

Lord, Thomas R., and Joann E. Garner. 1993. “Effects of Music on Alzheimer Patients.” Perceptual and Motor Skills 76 (2): 451–55. https://doi.org/10.2466/pms.1993.76.2.451.

12.

Magistrelli, Luca, Angela Amoruso, Luca Mogna, Teresa Graziano, Roberto Cantello, Marco Pane, and Cristoforo Comi.

2019. “Probiotics May Have Beneficial Effects in Parkinson’s Disease: In Vitro Evidence.” Frontiers in Immunology 10.

13.

Mehta, Varshil, Kavya Bhatt, Nimit Desai, and Mansi Naik. 2017. "Probiotics: An Adjuvant Therapy For D-Galactose Induced Alzheimer's Disease". Journal Of Medical Research And Innovation 1 (1): 30-33. doi:10.15419/jmri.15.

14.

https://doi.org/10.3389/fimmu.2019.00969.

15.

Rezaei Asl, Zahra, Gholamreza Sepehri, and Mahmoud Salami. 2019. “Probiotic Treatment Improves the Impaired Spatial Cognitive Performance and Restores Synaptic Plasticity in an Animal Model of Alzheimer’s Disease.” Behavioural Brain Research 376 (December): 112183. https://doi.org/10.1016/j.bbr.2019.112183.

16.

Scheltens, Philip, Kaj Blennow, Monique M. B. Breteler, Bart de Strooper, Giovanni B. Frisoni, Stephen Salloway, and Wiesje Maria Van der Flier. 2016. “Alzheimer’s Disease.” The Lancet 388 (10043): 505–17. https://doi.org/10.1016/S01406736(15)01124-1.

113

17.

Selkoe, Dennis J. 2001. “Alzheimer’s Disease: Genes, Proteins, and Therapy.” Physiological Reviews 81 (2): 741–66. https://doi.org/10.1152/physrev.2001.81.2.741.

18.

Viña, José, and Jorge Sanz‐Ros. 2018. “Alzheimer’s Disease: Only Prevention Makes Sense.” European Journal of Clinical Investigation 48 (10): e13005. https://doi.org/10.1111/eci.13005.

19.

Vorhees, Charles V, and Michael T Williams. 2006. “Morris Water Maze: Procedures for Assessing Spatial and Related Forms of Learning and Memory.” Nature Protocols 1 (2): 848–58. https://doi.org/10.1038/nprot.2006.116.

20.

Westfall, Susan, Nikita Lomis, Imen Kahouli, Si Yuan Dia, Surya Pratap Singh, and Satya Prakash. 2017. “Microbiome, Probiotics and Neurodegenerative Diseases: Deciphering the Gut Brain Axis.” Cellular and Molecular Life Sciences 74 (20): 3769–87. https://doi.org/10.1007/s00018-017-2550-9.

21.

Yang, Xueqin, Dongke Yu, Li Xue, Hui Li, and Junrong Du. 2020. “Probiotics Modulate the Microbiota–Gut–Brain Axis and Improve Memory Deficits in Aged SAMP8 Mice.” Acta Pharmaceutica Sinica B 10 (3): 475–87. https://doi.org/10.1016/ j.apsb.2019.07.001.

114

The Molecular Mechanisms Underlying Sleep Deprivation and Impaired Fetal Neurodevelopment Pearse Oâ&#x20AC;&#x2122;Malley

Sleep deprivation (SD) has been implicated in several short-term and long-term cognitive deficits in organisms. While most research has focused on adult organisms, there is increasing concern surrounding the effects of pregnant individualsâ&#x20AC;&#x2122; SD on the neurodevelopment of their fetus. A relationship between sleep, stress, and the immune system has previously been established, but the role of SD in causing inflammation was unclear. Baratta et al. (2020) examined the molecular pathways affected by maternal SD and the consequences for the fetus by inducing SD in pregnant mice and obtaining tissue and plasma samples from the mother and fetus. Corticosterone and kynurenic acid (KYNA) were used as markers of stress and impaired metabolism due to SD, respectively, with greater corticosterone levels in the sleep-deprived mice that did not recover from SD and greater KYNA accumulation, correlating to increased KYNA accumulation in the fetal brain. The results indicate that the stress response elicited from maternal SD is related to fetal brain KYNA accumulation, which is associated with weakened memory and learning abilities in the offspring, thus highlighting the dangerous effects of maternal SD on fetal neurodevelopment.

Key words: Sleep deprivation (SD), kynurenine pathway (KP), maternal stress, corticosterone, pregnancy

115

BACKGROUND AND INTRODUCTION

accumulation in the fetal brain, leading to impaired learning and memory (Baratta et al., 2020). Together, these results corSleep deprivation (SD), or experiencing consistently inadequate roborate the role of maternal SD in impaired fetal neurodevelsleep, has long been associated with cognitive and physiological opment. dysfunction in organisms. While previous studies have demonstrated the observable effects of SD on adults—such as increased risky decision-making and decreased memory during MAJOR RESULTS recall tasks (Chen et al., 2017)—less is known regarding the molecular mechanisms through which hormone changes in Maternal corticosterone levels increase in sleep-deprived pregnant individuals affect the developing fetus. Peng et al. mothers (2016) established that SD in pregnant rats contributed to inCorticosterone, a hormone involved in the stress and immune creased susceptibility of sleep disorders in the mothers, but responses, was obtained from the maternal and fetal plasma noted that the biochemical pathways underlying the offspring’s and used as a marker in examining the adverse effects of SD. SD subsequent cognitive impairment were unclear. led to stress in the pregnant rats, as evidenced by the drastic increase in maternal corticosterone serum levels measured in the rats, with the rats whose plasma samples were taken imAn association between SD in pregnant individuals and their mediately after one or three SD sessions having the highest serum pro-inflammatory cytokine levels was established by serum corticosterone (Baratta et al., 2020). The rats who were Okun & Coussons-Read (2006), who also suggest that the lack able to recover following SD had corticosterone levels that of sleep these individuals experience may contribute to inwere comparable to the control rats that did not experience SD creased cytokine levels and systemic inflammation. Neverthe(Fig 1), indicating that the spike in serum corticosterone due to less, the authors state that future research should consider SD is a short-term consequence (Baratta et al., 2020). This rehow these elevated serum cytokine levels could affect the fesult is consistent with other studies examining the effects of SD tus, as their study focused only on the maternal serum levels -induced stress, such as that of Ito et al. (2014), who also used (Okun & Coussons-Read, 2006). SD has further been known to serum corticosterone levels as a metric of stress in murine induce a stress response in sleep-deprived individuals, which models. While an increase in maternal corticosterone plasma can be monitored by the serum corticosterone levels (Wynnedue to SD was observed, fetal corticosterone plasma did not Edwards, Edwards, & Hancock, 2013). Lastly, kynurenine—a seem to be greatly affected by SD (Baratta et al., 2020). metabolite derived from the breakdown of tryptophan, an amino acid obtained from several foods—has been implicated in SD, but its role in inducing cognitive impairments, in the mother and fetus, had yet to be determined (Sforzini, 2019).

With the detrimental impacts that SD can have on individuals, a better understanding of the compromised molecular pathways is crucial to devise therapeutics for pregnant individuals experiencing SD and their newborns, or to advocate for prevention against SD in a public health context. Baratta et al. (2020) sought to elucidate how maternal SD was associated with fetal cognitive impairment, primarily through the kynurenine pathway (KP) through which tryptophan is degraded and did so by inducing SD in pregnant Wistar rats. Following zero, one, or three five-hour SD sessions where the rats' sleep is disturbed every 30 minutes, maternal and fetal plasma, brain, and placental tissue samples were obtained and examined for kynurenic acid (KYNA) accumulation, pro-inflammatory cytokine levels to understand the relationship between sleep and the immune system, and corticosterone levels as a measurement of the stress response in sleep-deprived mice (Baratta et al., 2020). Tissue samples of some mice were either taken immediately after the SD session, or 24 hours after the SD session, during which the mice were allowed to recover with uninterrupted sleep, resulting in a total of six treatment groups (Baratta et al., 2020). Analysis of the blood plasma and tissues indicated a greater stress response in the sleep-deprived mice with no recovery compared to the mice with recovery and control mice, which seemed to be associated with increased KYNA

Figure 1. Increased maternal serum corticosterone in sleepdeprived rats without recovery; SD treatment did not significantly affect fetal serum corticosterone. Figure adapted from Baratta et al. (2020)

Role of SD in KP and KYNA accumulation Tryptophan is obtained from the diet and metabolized to kynurenine in the KY by tryptophan-2,3-dioxygenase, but a buildup of kynurenine from excess tryptophan leads to KYNA accumulation in the serum and brain (Baratta et al., 2020). Serum tryptophan levels are known to increase during SD (Davies et al., 2014), which is why the increased maternal tryptophan serum levels led to greatly increased fetal brain KYNA levels immediately following one session of sleep deprivation (Fig 2) (Baratta et al., 2020). Placental tissue analysis did not provide evidence of increased placental kynurenine or KYNA accumulation, but of most salience is the fetal brain KYNA, which is associated with impaired neurogenesis and decreased cognitive performance (Rudzki et al., 2019).

116

ment of fetuses following maternal SD, a molecule whose function in irregular fetal development had not previously been clear (Baratta et al., 2020).

Figure 2. Maternal brain KYNA levels are relatively unaffected

by SD, while SD led to a drastic increase in fetal brain KYNA levels after one SD session, regardless of recovery time. Figure adapted from Baratta et al. (2020)

SD increases pro-inflammatory cytokine levels in the fetal and maternal brain An increase in pro-inflammatory cytokines following SD was hypothesized, as this elevated immune response is associated with tryptophan breakdown through the KP (Baratta et al., 2020). Therefore, with this immune response elicited by SD, it was assumed that cytokine levels would increase in the sleepdeprived rats compared to the control, consequently increasing KYNA as a by-product of the KP. Placental tissue analysis showed increased IL-1β and IL-6 following SD, with recovery time and sleep condition having no main individual effects (Baratta et al., 2020). Fetal brain tissue analysis also showed a great increase in IL-1β and IL-6, with recovery time having no clear effect (Fig 3) (Baratta et al., 2020).

The authors do not implicate increased maternal corticosterone as a direct correlate to greater kynurenine metabolism and KYNA accumulation, but rather as a correlate to increased proinflammatory cytokine levels (Baratta et al., 2020). This aligns with other studies which assert that there is increased tryptophan degradation following activation of the immune system (Farhangi et al., 2018; Maes et al., 2002). Unlike other studies focusing on the KP and its effects on cognition, however, the study by Baratta et al. (2020) is novel in its focus on the adverse effects that KYNA accumulation has on the fetus rather than the sleep-deprived organism itself.

KYNA accumulation due to SD is of particular concern, given the various consequences it has in individuals. KYNA has been identified as an antagonist to NMDA receptors, which are involved in synaptic plasticity and memory (Plitman et al., 2017); with an excess of KYNA in the brain—as seen in the fetuses of sleepdeprived mothers—glutamate, an important excitatory neurotransmitter, cannot bind to the NMDA receptors (Dornbierer et al., 2019). In addition to impaired memory and learning, the inactivation of the NMDA receptors is related to schizophrenia, so the identification of KYNA as an aggregate in the fetal brain may thus make these newborns more susceptible to these neuropsychiatric disorders (Koola et al., 2018; Wichers et al., 2005).

This information could be used in researching therapeutics that degrade or block excess KYNA in the fetal brain from acting as an NMDA receptor antagonist and inducing these adverse effects (Koola et al., 2018).

CRITICAL ANALYSIS

Figure 3. Fetal brain tissue analysis showed increased IL-1β and IL-6 following SD; recovery time and sleep condition had no main individual effect. Figure adapted from Baratta et al. (2020)

CONCLUSION/DISCUSSION The results of this study indicate a positive association between maternal corticosterone and fetal brain KYNA levels, such that increased maternal serum corticosterone correlates to increased KYNA accumulation in the fetal brain (Baratta et al., 2020). Additionally, maternal corticosterone appears to be positively associated with increased placental IL-1β, as IL-1β levels were higher when maternal serum corticosterone was higher (Baratta et al., 2020). These relationships substantiate the authors’ hypothesis that the maternal stress and immune responses caused detrimental effects in the fetus, thus highlighting KYNA as a key mediator in the impaired neurodevelop-

Although the study by Baratta et al. (2020) is novel in examining pregnant model organisms, the study did not focus on the stage of pregnancy at which the mice were presently—all samples were taken for analysis on either the 18th embryonic day (ED) or 19th ED, or the duration of the rats’ pregnancy up to that point (Baratta et al., 2020). However, the severity of the consequences of maternal SD for the fetus will vary by the extent of their development. For example, the effects of SD on the fetuses of pregnant mice in their first trimester would likely be more severe than those in their third trimester (Lees et al., 2015), as the central nervous system (CNS) largely develops in the early embryonic stages of pregnancy and could be hindered by KYNA accumulation (Salihagic-Kadic et al., 2005).

The three treatment groups the authors had for each recovery time of zero or 24 hours following SD was used to distinguish between the effects of SD on rats that had not been sleep deprived (control), had been sleep deprived for a short amount of time (one SD session), and those that had consistently been sleep deprived (three SD sessions) (Baratta et al., 2020). How-

117

ever, three SD sessions may not be sufficient to represent consistent SD, as another study examining the effects of SD on memory formation used a range of one to seven nights of SD in subjects as a treatment (Lo et al., 2016). Additionally, a 24-hour recovery after SD may also be insufficient to represent the lasting effects of maternal SD on fetuses, despite the short gestation period in mice of 18-22 days (Murray et al., 2010). Given that Baratta et al. (2020) took the plasma and tissue samples only one day following the SD, they were unable to determine if the KYNA accumulation persisted. Therefore, a treatment with many more than three SD sessions and taking samples after the 24-hour recovery period may be required to examine the longterm effects of SD on both the mother and fetus, especially given that Baratta et al. (2020) did not find that the mice’s sleep condition independently had a significant effect on corticosterone, pro-inflammatory cytokine, or KYNA levels (Baratta et al., 2020). This study’s results would likely show that proinflammatory cytokine levels in the mice with recovery was similar to the control mice (Toulmonde et al., 2018), while the mice without recovery had significantly higher proinflammatory cytokine levels (Baratta et al., 2020). The lack of KYNA accumulation later in development may suggest that its effects on fetal neurodevelopment are not as severe as purported to be. Pregnant individuals who are consistently sleep deprived during their pregnancy may be so for multiple days or weeks, representing more sleep conditions in this study, which may have more severe effects on their fetus.

FUTURE DIRECTIONS Future experiments should focus on understanding the effect, if any, of SD on rats at different stages of their pregnancy. As mentioned, SD could be more detrimental to organisms and their offspring earlier on in their pregnancy, as this is primarily when fetal neurodevelopment is occurring (Salihagic-Kadic et al., 2005). Similar to the original study, the proposed study must have a control and two other sleep conditions, with all three groups containing mice at the same stage of pregnancy; these conditions would allow the researchers to examine SD effects at multiple stages of pregnancy if there were three groups of mice for each trimester of pregnancy. It is anticipated that tissue analysis would show similar levels of KYNA accumulation across groups, but an underdeveloped CNS and lack of neurogenesis in the fetuses’ brains of mothers who were in their first trimester of pregnancy (Rajendiran et al., 2015).

These results would indicate that maternal SD is more detrimental if it occurs earlier in the mother’s pregnancy. If the impaired neurodevelopment appears to be consistent across groups, then it is likely that the effects of SD are unrelated to the stage at which an organism is pregnant.

ples could be taken from an additional group of mice who experience more than five SD sessions, as this will allow for comparison between groups on the basis of sleep condition (Khalyfa et al., 2015). Baratta et al. (2020) found that tissues in the group that underwent three SD sessions had comparable proinflammatory cytokine and KYNA levels, and lower levels than the mice that underwent one SD session, regardless of recovery, implying a compensatory effect in these mice with additional SD to maintain homeostatic cytokine and KYNA levels.

Nevertheless, Rosansky et al. (1996) determined that SD does not lead to the body physiologically compensating for the observed decrease in blood pressure, so having more SD conditions may dispute that the results observed by Baratta et al.

(2020) are due to compensation and instead due to a presently unknown molecular mechanism.

Finally, examining the long-term effects of maternal SD on the fetus is possible by performing the tissue and plasma collection and analysis more than 24 hours after recovery. If collected later into the mother’s pregnancy, lasting effects would be observed and may show that KYNA accumulation persists despite recovery, as another study examining embryonic exposure to nicotine demonstrated that nicotine-induced disruption to sleep homeostasis persisted in the offspring into adulthood (Borniger et al., 2017). While Baratta et al. (2020) found KYNA levels were comparable across groups for the mice with and without recovery after SD, Davies et al. (2014) found that plasma levels of metabolites, including tryptophan, remained high 48 hours following SD. With more tryptophan, more KYNA accumulation will occur, potentially leading to further impaired fetal brain neurodevelopment, as Vohra et al. (2018) show that depleting KYNA in the brains of the Caenorhabditis elegans model improves memory and learning ability indicating KYNA’s role in impaired neurogenesis. If there is no clear disparity between mice that have more time to recover and those that do not, this would suggest that SD may not have the long-term fetal consequences that it is speculated to have. Regardless, this information is essential to establish public health policies and advocate against SD in pregnant individuals to offset its adverse fetal effects.

Given that the sleep conditions used by Baratta et al. (2020) did not have a significant main effect by themselves on KYNA accumulation, it is prudent to run the experiment with more sleep conditions to mimic the persistent SD that some pregnant individuals may experience. For example, tissue and plasma sam118

REFRENCES

1.

Baratta, A.M., Kanyucha, N.R., Coleb, C.A., Valafarb, H., Deslauriers, J., & Pocivavsek, A. (2020). Acute sleep deprivation during pregnancy in rats: rapid elevation of placental and fetal inflammation and kynurenic acid. Neurobiology of Stress, 2 (100204), 1-9. doi:10.1016/j.ynstr.2019.100204

2.

Borniger, J. C., Don, R. F., Zhang, N., Boyd, R. T., & Nelson, R. J. (2017). Enduring effects of perinatal nicotine exposure on murine sleep in adulthood. American journal of physiology. Regulatory, integrative and comparative physiology, 313(3), R280–R289. https://doi-org.myaccess.library.utoronto.ca/10.1152/ajpregu.00156.2017

3.

Chen, J., Liang, J., Lin, X., Zhang, Y., Zhang, Y., Lu, L., & Shi, J. (2017). Sleep deprivation promotes habitual control over goal -directed control: behavioral and neuroimaging evidence. The journal of neuroscience: the official journal of the society for neuroscience, 37(49), 11979–11992. https://doi-org.myaccess.library.utoronto.ca/10.1523/JNEUROSCI.1612-17.2017

4.

Davies, S.K., Ang, J.E., Revell, V.L., Holmes, B., Mann, A., Robertson, F.P., Cui, N., Middleton, B., Ackermann, K., Kayser, M., Thumser, A.E., Raynaud, F.I., & Skene, D.J. (2014). Effect of sleep deprivation on the human metabolome. Proceedings of the National Academy of Sciences of the United States of America, 111(29), 10761–10766. https://doiorg.myaccess.library.utoronto.ca/10.1073/pnas.1402663111

5.

Dornbierer, D.A., Boxler, M., Voegel, C.D., Stucky, B., Steuer, A.E., Binz, T.M., Baumgartner, M.R., Baur, D.M., Quednow, B.B., Kraemer, T., Seifritz, E., Landolt, H.P., & Bosch, O.G. (2019). NocturnalGamma-Hydroxybutyrate Reduces CortisolAwakening Response and Morning Kynurenine Pathway Metabolites in Healthy Volunteers. The international journal of neuropsychopharmacology, 22(10), 631–639. https://doi-org.myaccess.library.utoronto.ca/10.1093/ijnp/pyz047

6.

Farhangi, M.A., Javid, A.Z., Sarmadi, B., Karimi, P., & Dehghan, P. (2018). A randomized controlled trial on the efficacy of resistant dextrin, as functional food, in women with type 2 diabetes: Targeting the hypothalamic-pituitary-adrenal axis and immune system. Clinical nutrition, 37(4), 1216–1223. https://doi-org.myaccess.library.utoronto.ca/10.1016/ j.clnu.2017.06.005

7.

Ito, T., Maeda, T., Goto, K., Miura, T., Wakame, K., Nishioka, H., & Sato, A. (2014). Enzyme-treated asparagus extract promotes expression of heat shock protein and exerts antistress effects. Journal of food science, 79(3), H413–H419. https:// doi-org.myaccess.library.utoronto.ca/10.1111/1750-3841.12371

8.

Khalyfa, A., Carreras, A., Almendros, I., Hakim, F., & Gozal, D. (2015). Sex dimorphism in late gestational sleep fragmentation and metabolic dysfunction in offspring mice. Sleep, 38(4), 545–557. https://doiorg.myaccess.library.utoronto.ca/10.5665/sleep.4568

9.

Koola, M.M., Sklar, J., Davis, W., Nikiforuk, A., Meissen, J.K., Sawant-Basak, A., Aaronson, S.T., & Kozak, R. (2018).

Kynurenine pathway in schizophrenia: Galantamine-memantine combination for cognitive impairments. Schizophrenia research, 193, 459–460. https://doi-org.myaccess.library.utoronto.ca/10.1016/j.schres.2017.07.005

10.

Lees, C.C., Marlow, N., van Wassenaer-Leemhuis, A., Arabin, B., Bilardo, C.M., Brezinka, C., Calvert, S., Derks, J.B., Diemert, A., Duvekot, J.J., Ferrazzi, E., Frusca, T., Ganzevoort, W., Hecher, K., Martinelli, P., Ostermayer, E., Papageorghiou, A.T., Schlembach, D., Schneider, K.T., Thilaganathan, B., … TRUFFLE study group (2015). 2 year neurodevelopmental and intermediate perinatal outcomes in infants with very preterm fetal growth restriction (TRUFFLE): a randomised trial. Lancet, 385(9983), 2162–2172. https://doi-org.myaccess.library.utoronto.ca/10.1016/S0140-6736(14)62049-3

11.

Lo, J. C., Chong, P. L., Ganesan, S., Leong, R. L., & Chee, M. W. (2016). Sleep deprivation increases formation of false memory. Journal of sleep research, 25(6), 673–682. https://doi-org.myaccess.library.utoronto.ca/10.1111/jsr.12436

12.

Maes, M., Verkerk, R., Bonaccorso, S., Ombelet, W., Bosmans, E., & Scharpé, S. (2002). Depressive and anxiety symptoms in the early puerperium are related to increased degradation of tryptophan into kynurenine, a phenomenon which is related to immune activation. Life sciences, 71(16), 1837–1848. https://doi-org.myaccess.library.utoronto.ca/10.1016/s0024 -3205(02)01853-2

13.

Murray, S. A., Morgan, J. L., Kane, C., Sharma, Y., Heffner, C. S., Lake, J., & Donahue, L. R. (2010). Mouse gestation length is genetically determined. PloS one, 5(8), e12418. https://doi.org/10.1371/journal.pone.0012418

14.

Okun, M.L. & Coussons-Read, M.E. (2007). Sleep disruption during pregnancy: how does it influence serum cytokines?

Journal of Reproductive Immunology. 73(2), 158-165.

119

15.

Peng, Y., Wang, W., Tan, T., He, W., Dong, Z., Wang, Y.T., & Han, H. (2016). Maternal sleep deprivation at different stages of pregnancy impairs the emotional and cognitive functions, and suppresses hippocampal long-term potentiation in the offspring rats. Molecular Brain, 9(17), 1-10. https://dx-doi-org.myaccess.library.utoronto.ca/10.1186%2Fs13041-016-0197 -3

16.

Plitman, E., Iwata, Y., Caravaggio, F., Nakajima, S., Chung, J. K., Gerretsen, P., Kim, J., Takeuchi, H., Chakravarty, M. M., Remington, G., & Graff-Guerrero, A. (2017). Kynurenic Acid in Schizophrenia: A Systematic Review and Meta-analysis.

Schizophrenia bulletin, 43(4), 764–777. https://doi-org.myaccess.library.utoronto.ca/10.1093/schbul/sbw221

17.

Rajendiran, S., Swetha Kumari A, Nimesh, A., Soundararaghavan S, Ananthanarayanan, P. H., & Dhiman, P. (2015). Markers of Oxidative Stress in Pregnant Women with Sleep Disturbances. Oman medical journal, 30(4), 264–269. https://doiorg.myaccess.library.utoronto.ca/10.5001/omj.2015.53

18.

Rosansky, S.J., Menachery, S.J., Whittman, D., & Rosenberg, J.C. (1996). The relationship between sleep deprivation and the nocturnal decline of blood pressure. American journal of hypertension, 9(11), 1136–1138. https://doiorg.myaccess.library.utoronto.ca/10.1016/0895-7061(96)00300-7

19.

Rudzki, L., Ostrowska, L., Pawlak, D., Małus, A., Pawlak, K., Waszkiewicz, N., & Szulc, A. (2019). Probiotic Lactobacillus Plantarum 299v decreases kynurenine concentration and improves cognitive functions in patients with major depression: A double-blind, randomized, placebo controlled study. Psychoneuroendocrinology, 100, 213–222. https://doiorg.myaccess.library.utoronto.ca/10.1016/j.psyneuen.2018.10.010

20.

Salihagic-Kadic, A., Kurjak, A., Medić, M., Andonotopo, W., & Azumendi, G. (2005). New data about embryonic and fetal neurodevelopment and behavior obtained by 3D and 4D sonography. Journal of perinatal medicine, 33(6), 478–490. https://doi-org.myaccess.library.utoronto.ca/10.1515/JPM.2005.086

21.

Sforzini, L. Nettis, M.A. Mondelli, V., & Pariante, C.M. (2019). Inflammation in cancer and depression: a starring role for the kynurenine pathway. Psychopharmacology, 236(10), 2997–3011. https://dx-doiorg.myaccess.library.utoronto.ca/10.1007%2Fs00213-019-05200-8

22.

Toulmonde, M., Penel, N., Adam, J., Chevreau, C., Blay, J. Y., Le Cesne, A., Bompas, E., Piperno-Neumann, S., Cousin, S., Grellety, T., Ryckewaert, T., Bessede, A., Ghiringhelli, F., Pulido, M., & Italiano, A. (2018). Use of PD-1 targeting, macrophage infiltration, and IDO pathway activation in sarcomas: A phase 2 clinical trial. JAMA oncology, 4(1), 93–97. https:// doi-org.myaccess.library.utoronto.ca/10.1001/jamaoncol.2017.1617

23.

Vohra, M., Lemieux, G. A., Lin, L., & Ashrafi, K. (2018). Kynurenic acid accumulation underlies learning and memory impairment associated with aging. Genes & development, 32(1), 14–19. https://doi-org.myaccess.library.utoronto.ca/10.1101/ gad.307918.117

24.

Wichers, M. C., Koek, G. H., Robaeys, G., Verkerk, R., Scharpé, S., & Maes, M. (2005). IDO and interferon-alpha-induced depressive symptoms: a shift in hypothesis from tryptophan depletion to neurotoxicity. Molecular psychiatry, 10(6), 538– 544. https://doi-org.myaccess.library.utoronto.ca/10.1038/sj.mp.4001600

25.

Wynne-Edwards, K.E., Edwards, H.E., & Hancock, T.M. (2013). The human fetus preferentially secretes corticosterone, rather than cortisol, in response to intra-partum stressors. PLoS One, 8(6). https://dx-doiorg.myaccess.library.utoronto.ca/10.1371%2Fjournal.pone.0063684

120

Aβ aggregation and Tau phosphorylation suggest PhIP correlation to Alzheimer’s disease Bhavya Patel

PhIP, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine, is a dietary heterocyclic aromatic amine that is chemically released when cooking meat at increased temperatures. Syeda et al. (2020) examines the correlation between PhIP exposure and the neuropathology of AD. The authors experimented on C57BL/6 mice and divided them into three groups that experienced differing levels of PhIP exposure. A 6-7 week old male and female group of mice were treated with 100/200 mg PhIP/kg for 8 hours once a week. The next group consisted of 8-9 week old male mice exposed to 75mg PhIP/kg for a period of 4 weeks(3x/week) and the last group was injected with 75mg PhIP/ kg for 16 weeks(3x/week). The control for each group was a vehicle treated group injected only with corn oil. Mice were then decapitated, brains were divided into two halves and stored in liquid nitrogen for further processing. Assessments included measuring oxidative damage through ChAT positive cells in the striatal region, synaptic alterations in the hippocampus, and inflammation via GFAP intensity. BACE1, APP, Aβ and tau protein phosphorylation was compared between each group of mice and control. An accumulation of Aβ and tau-phosphorylation are major characteristics of AD and are prominently seen in the 16-week group. Furthermore, oxidative stress is increased in the hippocampus suggesting that the mechanism of Aβ aggregation via PhIP may be a result of BACE1 and APP upregulation. A summary of results is indicated in Figure 1.

121

Introduction: PhIP is found in beer, wine, smoke from cigarettes and produced when cooking meat at high temperatures.

Being a carcinogen, it has increased exposure to humans and harmful consequences. After oxidising to cytochrome p450, PhIP leads to neurotoxicity.

AD consequently is the leading cause of Dementia and leads to loss of declarative memory and cognition. Initial symptoms include poor retention of memory that ultimately intensifies to impaired speech and difficulty in processing thought. There are two subtypes of this disease namely the early-onset AD and the late-onset AD. Likely caused by a combination of genetics and environmental factors, APP is one gene that is identified to be associated with early-onset AD. Hence, it is clinically important to assess the neurogenerative and neurotoxic properties of AD for future treatments.

Major Results In Mann-Whitney test, Kruskal-Wallis nonparametric test and Dunn’s post-hoc test, p < 0.05 is considered statistically significant.

Oxidative damage in ChAT positive cells and negative cells

Nikon A1R inverse scanning confocal microscopes were used to measure oxidative damage in the striatal regions of the Hippocampus. Nitrotyrosine intensity was measured in ChAT positive and negative cholinergic cells to assess for oxidative damage.

Statistical significance compared to control shows the hippocampus is susceptible to oxidative damage in both positive and Furthermore, amyloid plaques are a characteristic of AD. Beta- negative cholinergic ChAT cells post PhIP exposure. (Figure 2) Amyloid is formed by the proteolytic cleavage of Amyloid Precursor Protein, a type I transmembrane glycoprotein, by enzymes β-secretase and γ-secretase. Increased formation of Aβ Increased Synaptic Proteins is seen with increased Synaptic Proteins such as V-GLUT1 and PSD-95 increase is seen BACE1, the β-site cleaving enzyme. One of the major characteronly in the 8hr group of mice. 4 weeks and 16 weeks had no istics of brain lesions of AD involves the abnormal phosphorylaeffect on the upregulation or downregulation of the synaptic tion of tau protein. Firstly, phosphorylated tau will tangle and proteins in the Hippocampus. subsequently cause aggregation in the CA3 regions of the hippocampus that are associated with declarative memory. The CA1 and CA3 regions of the Hippocampus are essential for spatial learning and long-term spatial memory acquisition. This mechanism of phosphorylation is responsible for a cytoskeleton disruption and microtubule disassembly.

Syeda et al. (2020) assessed whether differing levels of PhIP leads to correlation between exposure intensity and symptomatic AD. PhIP effects on the brain are not well studied and thus, this article brings forth some light as to possible mechanisms of impact and related consequences.

Fig.2. Nitrotyrosine staining assessing for oxidative damage in ChAT positive and negative cells of the dorsal striatal region. (A) PhIP exposure to 8hr group, p < 0.01, dose dependent increase in oxidative damage for ChAT positive cells. No difference in ChAT negative cells. (B) PhIP exposure to 4 weeks group, p < 0.01 for ChAT positive cells. In ChAT negative cells, p < 0.001.

(C) PhIP exposure to 16 weeks group, p < 0.001 for ChAT posiFig.1. A detailed visual summary of the primary research article tive neurons. In ChAT negative cells, p <0.001.

by Syeda et. al. (2020).

No Inflammation Microglia activation was studied using GFAP intensity markers at the CA1 hippocampal regions. No signifi122

No significant changes in microglia activation and no inflamma- genic mice and consequently silencing thereafter. tion is seen in hippocampal regions pertaining to all 3 groups.

BACE1 upregulation Mossy fiber projections were quantified in the hippocampus to assess for BACE1 changes. BACE1 is linked to oxidative stress and is positively correlated with PhIP exposure and AD.

Fig. 9. ROI’s drawn around CA3 regions for quantification of APP and Aβ1-42. (A) 8hrs, PhIP-200, p < 0.05 for APP staining. (B) Insignificant results. (C) 16 weeks, p < 0.05 for APP staining; p < 0.05 for Aβ1-42 staining. (D) DAB staining, p < 0.001; Immunofluorescence staining p < 0.05 for Aβ1-42.

Tau Phosphorylation An increase in Tau Phosphorylation in the CA3 St. Pyramidale region of the Hippocampus was seen in the 16 weeks group.

Abnormal tau-protein phosphorylation can also affect normally functioning tau proteins. This directly interacts with microtubules causing disassembly of tubulin in AD patients.

Fig.7. BACE1 assessments by drawing ROIs around mossy fiber regions of the hippocampus. Western Blot analysis for each group were completed to relate BACE1 upregulation with PhIP exposure. (A) 8hrs, PhIP-200, p < 0.01. (B) 4 weeks, p < 0.01. (C) 16 weeks, p <0.05.

Fig.8. BACE1 activity measured in three groups: 1) CTR- control, 2) NA- cognitively normal subjects, 3) Sporadic Alzheimer’s Disease. Results indicate 47% increase of BACE1 activity in AD. Upregulation of APP and Aβ The St. Pyramidale region in the CA1 and CA3 regions were assessed for APP changes while the St.Pyramidale region of CA3 was only imaged for Aβ changes. There was an increase in APP for both 8 hrs and 16 weeks as predicted by BACE1 upregulation. 16 weeks also resulted in an increase of Aβ1-42 aggregation. Aβ formation leads to sequential activation of BACE1 by an increase in oxidative stress that positively feedbacks to increase Aβ aggregation. Previous studies conclude that increased Aβ plaques result in neuronal hyperactivity in trans-

Fig. 10. Staining for p-Tau Ser 199/202 to assess tau phosphorylation in the hippocampus. (A, B) No change. (C) 16 weeks, p < 0.05 for p-tau in hippocampal region; no change in DG. Conclusion PhIP shows relevance to AD neuropathology by inducing oxidative damage in ChAT positive neurons, Aβ aggregation and tauphosphorylation. Amyloid plaques are a predominant charac-

123

teristic of AD that is characterised by its insolubility and fibrillar ied. It is important to include female neuropathology in the sub structure.

-acute and sub-chronic levels of exposure as well as females are more susceptible to AD. The correlation of PhIP with neurotoxicity is also seen in prior reports that suggest downregulation of Dopaminergic neurons Furthermore, the time frame of exposure should be increased in its presence.. Furthermore, the authors agree that PhIP self- with increasing groups of experimental models. Natural expometabolizes to N-OH- PhIP and 4’-OH-PhIP resulting in oxidative sure to PhIP is higher for humans and thus, longer time periods stress as seen is Chen et al. (2007). of study would convey naturally occurring results. For accurate results, the time should be 24 hrs, 2 weeks, 4 weeks, 8 Oxidative stress without inflammation and microglia activation weeks,16 weeks and 32 weeks. With 6 groups of models consuggest these are early neurotoxic events in the presence of taining both male and female, this type of experimental set-up PhIP. PhIP exposure leading to aggregation of Aβ and tau phoswould create more efficient analysis of results. Furthermore, phorylation at 16 weeks suggest exposure time is positively the mechanisms of Aβ aggregation, tau phosphorylation, incorrelated with enhanced symptoms of AD. creased oxidation and BACE1 upregulation may be better interAssessment of PhIP and its effects on glutaminergic synapses preted with group specificity. was studied via measuring levels of PSD-95 and VGLUT-1. StaDopaminergic neurons were not assessed in this literature. tistical significance is quite low, but a mild increase concludes Furthermore, significances of studying synaptic proteins were that acute PhIP exposure leads to increased glutaminergic sigbriefly mentioned. Evaluations of both Dopaminergic neurons nalling. and Glutamatergic neurons by staining with their respective BACE1 cleavage of APP leads to the formation of Aβ. BACE1 antibodies should be completed. Newer findings may have arisupregulation was seen in acute, sub-acute and sub-chronic ex- en if this had been done. If PhIP induces neurotoxicity, there posure to PhIP. Lastly, Tau phosphorylation was dominant in should be a decline in dopaminergic cells and a rise in glutasub-chronic exposure levels of PhIP but the mechanisms of PhIP minergic cell activation. AD results in hyperactivity of synapses induced activation remains unknown. before hypoactivity.

Syeda et al. (2020) also conclude that PhIP is sensitive to CA3 Lastly, learning and memory were not assessed in this paper. regions of the hippocampus whereas previous studies indicated The authors should have included behavioural tests for the variations in CA1 region and the Dentate Gyrus. rodent models to assess for cognition and memory as disturbances to these are seen in AD positive individuals. A group of Critical Analysis injected mice should be kept alive while the other decapitated Most findings of Syeda et. Al are consistent with previous rec- to assess both behavioural changes and molecular changes. A ords; however, many causal relations have not been estab- swim test, adhesive removal test, reach and grasp test, corner lished. Exact mechanisms of tau-phosphorylation, BACE1 up- test, cylinder tests or open-field test could be used to assess regulation and direct AD related neurotoxicity are not con- sensorimotor stability in PhIP-injected mice vs. control. A defirmed. Previous papers mention similar ways of PhIP mediated cline in learning and memory is associated with decreased senneurotoxic events, however, the authors have found no results sorimotor function. Therefore, the mice injected with PhIP will that accurately prove AD relation to PhIP. take longer time to complete tasks compared to control. E.g.

PhIP injected mice would take longer times to remove adhesive Increased studies must be done to form a causal relationship stickers from their paws in the Adhesive Removal Test combetween HAAs, and AD. Limitations of this study include experipared to control. mentation on mostly male mice and a smaller group of mice models.

Overall, this study will include behavioural tests, assess for both sexes, and allows a longer time frame to gather results. This Previous studies of rat embryos suggest PhIP inducing harm to experimental setup will allow for increased statistical signifidopaminergic cells via mediating oxidative stress. A decrease in cance to study the correlation of PhIP exposure and AD neuroneurite length is also seen in the presence of PhIP and N-OHpathology.

PhIP(Griggs et al, 2014). Furthermore, PhIP also decreases dopamine metabolite turnover in striatal regions of the hippocampus and increases oxidative damage in the Substantia Nigra. Such results are key to understanding exact mechanisms of AD neuropathology and thus, should also have been studied.

Future Directions The authors have created a valid technique to study the effect of PhIP exposure by creating three groups of C57BL/6 mice.

PhIP injected for 8hrs, 4 weeks and 16 weeks exposure are appropriate models to study exposure effects. However, female exposure to PhIP was only measured in the acute exposure 8hr group and long-term effects on female rodents were not stud124

REFRENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

“2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine (PhIP) Is Selectively Toxic to Primary Dopaminergic Neurons In Vitro | Toxicological Sciences | Oxford Academic.” Accessed June 6, 2020. https://academic.oup.com/toxsci/ article/140/1/179/1673985.

Agim, Zeynep Sena, and Jason R. Cannon. “Alterations in the Nigrostriatal Dopamine System after Acute Systemic PhIP Exposure.” Toxicology Letters 287 (May 1, 2018): 31–41. https://doi.org/10.1016/j.toxlet.2018.01.017.

Alonso, A. C., T. Zaidi, I. Grundke-Iqbal, and K. Iqbal. “Role of Abnormally Phosphorylated Tau in the Breakdown of Microtubules in Alzheimer Disease.” Proceedings of the National Academy of Sciences 91, no. 12 (June 7, 1994): 5562–66. https://doi.org/10.1073/pnas.91.12.5562.

Bekris, Lynn M., Chang-En Yu, Thomas D. Bird, and Debby W. Tsuang. “Review Article: Genetics of Alzheimer Disease:” Journal of Geriatric Psychiatry and Neurology, November 2, 2010. https://doi.org/10.1177/0891988710383571.

Bharadwaj, Prashant, and Ralph Martins. “A Rapid Absorbance-Based Growth Assay to Screen the Toxicity of Oligomer A Beta(42) and Protect against Cell Death in Yeast.” Neural Regeneration Research 15, no. 10 (October 2020): 1931–36. https://doi.org/10.4103/1673-5374.280318.

Blazquez-Llorca, Lidia, Virginia Garcia-Marin, Paula Merino-Serrais, Jesús Ávila, and Javier DeFelipe. “Abnormal Tau Phosphorylation in the Thorny Excrescences of CA3 Hippocampal Neurons in Patients with Alzheimer’s Disease.” Journal of Alzheimer’s Disease 26, no. 4 (January 1, 2011): 683–98. https://doi.org/10.3233/JAD-2011-110659.

Borghi, Roberta, Stefania Patriarca, Nicola Traverso, Alessandra Piccini, Daniela Storace, Anna Garuti, Gabriella Cirmena, Patrizio Odetti, and Massimo Tabaton. “The Increased Activity of BACE1 Correlates with Oxidative Stress in Alzheimer’s Disease.” Neurobiology of Aging 28, no. 7 (July 1, 2007): 1009–14. https://doi.org/10.1016/ j.neurobiolaging.2006.05.004.

Busche, Marc Aurel, Xiaowei Chen, Horst A. Henning, Julia Reichwald, Matthias Staufenbiel, Bert Sakmann, and Arthur Konnerth. “Critical Role of Soluble Amyloid-β for Early Hippocampal Hyperactivity in a Mouse Model of Alzheimer’s Disease.” Proceedings of the National Academy of Sciences 109, no. 22 (May 29, 2012): 8740–45. https://doi.org/10.1073/ pnas.1206171109.

Chen, Chi, Xiaochao Ma, Michael A. Malfatti, Kristopher W. Krausz, Shioko Kimura, James S. Felton, Jeffrey R. Idle, and Frank J. Gonzalez. “A Comprehensive Investigation of 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine (PhIP) Metabolism in the Mouse Using a Multivariate Data Analysis Approach.” Chemical Research in Toxicology 20, no. 3 (March 1, 2007): 531–42. https://doi.org/10.1021/tx600320w.

Florian, Cédrick, and Pascal Roullet. “Hippocampal CA3-Region Is Crucial for Acquisition and Memory Consolidation in Morris Water Maze Task in Mice.” Behavioural Brain Research 154, no. 2 (October 5, 2004): 365–74. https://doi.org/10.1016/ j.bbr.2004.03.003.

Griggs, Amy M., Zeynep S. Agim, Vartika R. Mishra, Mitali A. Tambe, Alison E. Director-Myska, Kenneth W. Turteltaub, George P. McCabe, Jean-Christophe Rochet, and Jason R. Cannon. “2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine (PhIP) Is Selectively Toxic to Primary Dopaminergic Neurons In Vitro.” Toxicological Sciences 140, no. 1 (July 1, 2014): 179–89. https://doi.org/10.1093/toxsci/kfu060.

Jarrett, Joseph T., and Peter T. Lansbury. “Seeding ‘One-Dimensional Crystallization’ of Amyloid: A Pathogenic Mechanism in Alzheimer’s Disease and Scrapie?” Cell 73, no. 6 (June 18, 1993): 1055–58. https://doi.org/10.1016/0092-8674(93)90635 -4.

Nunan, Janelle, and David H Small. “Regulation of APP Cleavage by α-, β- and γ-Secretases.” FEBS Letters 483, no. 1 (October 13, 2000): 6–10. https://doi.org/10.1016/S0014-5793(00)02076-7.

Probst, Alphonse, Dominique Langui, and Jürg Ulrich. “Alzheimer’s Disease: A Description of the Structural Lesions.” Brain Pathology 1, no. 4 (1991): 229–39. https://doi.org/10.1111/j.1750-3639.1991.tb00666.x.

Seabrook, Guy R., and Thomas W. Rosahl. “Transgenic Animals Relevant to Alzheimer’s Disease.” Neuropharmacology 38, no. 1 (January 1, 1999): 1–17. https://doi.org/10.1016/S0028-3908(98)00170-1.

Syeda, Tauqeerunnisa, Rachel M. Foguth, Emily Llewellyn, and Jason R. Cannon. “PhIP Exposure in Rodents Produces Neuropathology Potentially Relevant to Alzheimer’s Disease.” Toxicology 437 (May 15, 2020): 152436.

125

Apelin-36: An Overlooked Peptide with Promising Effects on Parkinson’s Disease Andrea Pinto

Parkinson’s Disease (PD) is the second most prevalent progressive neurodegenerative disease. It is attributed to a gradual loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) due to the accumulation of misfolded α-synuclein proteins. As a result, common symptoms of PD include rigidity of the muscles, a resting tremor, a shuffling gait and bradykinesia, all of which vary in severity among diagnosed individuals. This variance produces a lack of understanding on how to reverse the disease pathology. Zhu et al. (2019), attempts to study the mechanism by which Apelin -36 prevents endoplasmic reticulum stress (ERS) to ameliorate the common symptoms associated with PD. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP) was injected into C57BL/6 mice to induce neurotoxicity mirroring the hallmark effects of PD. Injection of Apelin-36 into the SNpc of MPTP-treated mouse models improved motor deficits. The treatment also decreased levels of αsynuclein expression but rescued the progressive loss of dopaminergic neurons, SH-SY5Y cells, and tyrosine hydroxylase (TH) expression. Furthermore, a suppression of GRP78, CHOP and the cleaved caspase-12 is detected, indicating an aversion of ERS. Considering these results, Apelin-36 establishes as a promising treatment for PD by improving the major clinical symptom, motor disturbances.

This approach can also be considered as a treatment option for other neurodegenerative disorders with similar symptoms such as Alzheimer's Disease and amyotrophic lateral sclerosis. Moreover, the results reveal a greater understanding of the mechanism by which Apelin-36 can induce neuroprotective effects.

Key words: Parkinson’s disease, neurodegeneration, MPTP-induce mouse models, SH-SY5Y cells, substantia nigra, endoplasmic reticulum stress (ERS), Apelin-36, treatment

126

INTRODUCTION Parkinson’s Disease (PD) is becoming an increasingly concerning neurodegenerative disease due its forecasted prevalence. The disease is further compounded by its complexity, involving a multitude of risk factors as well as affecting both motor and psychiatric function at varying severities (Bietz, 2014). Due to this complexity and variability in symptoms, there has been a low yield in promising treatments. Motor dysfunctions such as bradykinesia, have been associated with the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) as result of α-synuclein aggregation. Zhu et al. (2019) attempts to inhibit endoplasmic reticulum stress (ERS) to alleviate both the loss of dopaminergic neurons and α-synuclein aggregation, thus improving motor deficits. The mechanism by which this is achieved, involves examining the neuroprotective effects of Apelin-36 on MPTP-treated mice, which reveals its potentiality in treating PD (Zhu et al., 2019).

An arduous, yet crucial step to developing a treatment, is to establish a model for the disease. A common chemically induced model for PD is an intranigral injection of MPTP into C57BL/6 mice. MPTP is capable of crossing the blood brain barrier due to its lipophilic properties, where it is then modified into its toxic form, 1-methyl-4-phenylpyridinium, also known as MPP+. The toxic metabolite has a strong selectivity for dopamine receptors, causing an efficient uptake into dopaminergic neurons. Upon its uptake, MPP+ concentrates within the mitochondria to produce reactive oxygen species (ROS), becoming toxic to the neurons (Langston, 2017). Loss of dopaminergic neurons, consequently decreases levels of tyrosine hydroxylase (TH), a rate-limiting enzyme involved in the synthesis of dopamine (Wang et al., 2017). These effects are also demonstrated in SH-SY5Y cells, a neuroblastoma cell line commonly used to study neurodegenerative diseases (Ko et al., 2018; Xicoy et al., 2017).

Similarly to the development of PD, MPTP conveniently targets dopaminergic neurons of the SNpc exclusively (Langston, 2017).

Although this selectivity remains unclear, it is apparent that MPTP-induced mice models are a valuable model for PD by mirroring its pathophysiology.

Another significant contributor to the loss of dopaminergic neurons, are the consequences of endoplasmic reticulum stress (ERS) (Martinez et al., 2017). ERS is triggered by an accumulation of α-synuclein within the ER which activate ER transmembrane protein kinases, PERK and IRE1α (Ryu et al., 2002). A signalling cascade is then initiated, producing transcription factors to promote apoptosis. ERS serves an important interference point to which loss of dopaminergic neurons can be averted, thus decelerating neurodegeneration within individuals.

To reverse the effects of neurodegeneration, Apelin-36 emerges as a potential treatment option. Apelin-36 is a neuropeptide derived from Apelin, an endogenous ligand of a Gprotein coupled receptor (GCPR) (Sakamoto et al., 2016). Most studies are currently examining the ability of Apelin to protect brain injury caused by ischemia. Apelin is naturally produced within the nervous system and has associated neuroprotective properties (Ishimaru et al., 2017).

Examination of these neuroprotective aspects highlights its importance in reversing Parkinsonian symptoms. The study of Zhu et. al consisted of four groups, which received in-

jections of saline, MPTP and/or Apelin-36 for 5 days. The control group was injected with saline. The Apelin-36 group was injected with Apelin-36 and saline. The MPTP group was injected with MPTP and saline. Finally, the Apelin-36+MPTP group was injected with Apelin-36 and MPTP. Motor disturbances were measured using a rotarod test. Immunohistochemistry and immunofluorescence were used to evaluate loss of dopaminergic neurons. Western Blotting was conducted to detect expression of TH and α-synuclein in SNpc. Lastly, cell viability was assessed using CCK-8 assay whereas cell apoptosis was measured by flow cytometry and TUNEL staining. The results of these experiments indicated, intranigral injection of Apelin-36 was capable of dissipating neurotoxic effects by promoting survival of MPP+- treated SH-SY5Y cells, preventing further loss of dopaminergic neurons, restoring TH expression, and improving motor functions of MPTP -induced mice models. Apelin-36 also had the added benefit of inhibiting α-synuclein expression thus preventing further neurodegeneration. Furthermore, a mitigation of ERS was observed in Apelin-36 treated MPTP-induced mouse models. Effects on ERS were concluded by noting a decrease in GRP78, CHOP and cleaved caspase-12 in SH-SY5Y cells, which overall inhibited apoptosis (Zhu et al., 2019).

MAJOR RESULTS Dissipation of Neurotoxicity Apelin-treated MPTP-induced mouse models displayed a dissipation in neurotoxicity, as measured by the survival of SH-SY5Y cells, dopaminergic neurons, TH expression, and motor function.

SH-SY5Y cells were initially treated with Apelin-36 followed by MPP+. CCK-8 assay shows higher dosages of Apelin-36 (1µM) is correlated with a greater cell viability. On the other hand, quantitative analysis of flow cytometry (Fig. 1) reveals decreased levels of cell apoptosis, corroborating the increased cell viability revealed by the CCK-8 assay. Although the effects of Apelin-36 on SH-SY5Y cells have yet to be studied by other groups, the study of Jiang et al. (2018) also noted a gradual increase in the survival of MPP+-treated SH-SY5Y cells after treatment with its isoform Apelin-13.

Fig. 1: Quantitative analysis of flow cytometry indicates a decrease in cell apoptosis in MPP+ treated SH-SY5Y cells, occurring in a dose-dependent manner.

Figure Adapted from Zhu et al. (2019) Brain Research, 1721.

An attenuation of neurotoxicity is also indicated by an increase in dopaminergic neurons in the Apelin-36+MPTP mice group

127

compared to the MPTP group, as presented by immunohistochemistry and immunofluorescence of TH. Increased levels of TH expression reflects increased prevalence of dopaminergic neurons. Likewise to the previous experiment, Apelin-13 treatment on 6-hydroxydopamine treated mice also revealed an attenuation of dopaminergic neuron death (Haghparast et al, 2019). Apelin-36+MPTP mice further demonstrated increased TH expression compared to the MPTP mice group. Due to the lack of experiments conducted using Apelin-36, these results are unable to be confirmed by current literature. However, the results align with the improved motor function of Apelin36+MPTP group, as a significantly longer time was spent on the rotarod compared to MPTP group (Fig. 2). Considering these results, Apelin-36 displays an ability to dissipate neurotoxicity and improve Parkinsonian symptoms.

er, this study involved Apelin-13 treatment as opposed to the Apelin-36. Nevertheless, a reduction in GRP78, CHOP and cleaved caspase-12 mitigated ERS, emphasizing the relevance of the ER in cell viability.

Fig 2: MPTP group shows significant loss in motor function, which was reversed after Apelin-36 treatment, as seen by an increased time spent on the rotarod.

Figure Adapted from Zhu et al. (2019) Brain Research, 1721.

Regulation of α-synuclein A decelerating loss in dopaminergic neurons suggests an involvement between Apelin-36 and α-synuclein. Western blotting reveals increased α-synuclein in the MPTP group which was significantly repressed after Apelin-36 treatment in the mice models and SH-SY5Y cells. This repression in α-synuclein is consistent with a previous study conducted by Zhu et. al.

(2019) using identical experimental procedures. The ability for Apelin-36 to prevent α-synuclein aggregation in SNpc, suggests ERS may also be mitigated.

Fig 3: Quantitative analysis of the Western Blotting reveals decreased expression of GRP78 (A), CHOP (B), and cleaved capase -12 (C) in SH-SY5Y cells treated with MPP+, indicating an overall mitigation of ERS. Figure Adapted from Zhu et al. (2019) Brain Research, 1721.

Mitigation of ERS

DISCUSSION

Effects of Apelin-36 on ERS was the focus in the study of Zhu et al. (2019), as it suggests to be the main target in preventing progressive neurodegeneration. Expression of GRP78, CHOP and cleaved caspase-12 were used as to reflect the severity of ERS. Both Western Blotting and immunofluorescent staining revealed increased expression of GRP78 (Fig. 3-A), CHOP (Fig.3-B), and cleaved capse-12 (Fig. 3-C) in the MPTP group, but expression of all three indicators were inhibited by Apelin-36 treatment. Jian et. al. (2017) also found decreased levels of GRP78, CHOP and cleaved caspase-12 in MPTP-treated mice which was associated with an attenuation of ERS. Howev-

From the experiments conducted by Zhu et. al. (2019), intranigral injection of Apelin-36 into MPTP-treated mouse models was capable of dissipating neurotoxicity by promoting survival of SH-SY5Y cells, presence of dopaminergic neurons, expression of TH, and motor function. Furthermore, the treatment prevented aggregation of α-synuclein in not only the mouse models but as well as the MPP+ treated SH-SY5Y cells.

Most importantly, Apelin-36 mitigated ERS by decreasing expression of its indicators, GRP78, CHOP, and cleaved caspase12. Taking these results into consideration, the authors highlight the neuroprotective abilities of Apelin-36 as well as the

128

inhibition of ERS, causing an overall reversal of Parkinsonian symptoms. Previous studies have only investigated the role of Apelin (Ishimaru et. al, 2017) or Apelin-13 (Jiang et. al, 2018) in neurodegenerative disorders. Ishimaru et al. (2017) reveal an acceleration in retinal neuronal degeneration in Apelin deficient mice, as a result of downregulation in its GCPR signaling.

On the other hand, Jiang et al. (2018), demonstrates the protective effects of Apelin-13 on MPP+-treated SH-SY5Y cells by promoting its survival. Both studies strengthen the correlation between presence of Apelin and the prevention of neuronal death. However, there exists a lack of knowledge whether the success of Apelin and Apelin-13 in decelerating neurodegeneration also extends to Apelin-36. Zhu et al. (2019) attempt to fill this gap by introducing Apelin-36 as a possible treatment option for PD. The treatment has produced promising results both in vitro using MPP+-treated SH-SY5Y cells and in vivo using MPTP-induced mouse models. This neuropeptide sets itself apart from other treatments such as Amantidine, by its ability to combat several mechanisms involved PD pathobiology, as opposed to a single target (Paquette et al., 2013). This is significant considering individuals diagnosed with PD experience several symptoms at varying severities. Although significant research has been published on the molecular pathway of ERS (Ryu et al., 2002) and its consequences (Martinez et al., 2017), Apelin-36 appears to be one of the few drugs able to attenuate this response. As a result, this study creates an opportunity to identify certain targets to suppress within the ERS. Additionally, it shifts the focus of researchers to an entirely unexplored area of neuroscience requiring further attention to the specific molecular mechanisms by which Apelin-36 induces these neuroprotective effects. By considering this avenue for treatment, it generates a new perspective through which researchers can approach other neurodegenerative diseases as well.

CRITICAL ANALYSIS As alluded to previously, the results of the experiment examining the effects of Apelin-36 on dopaminergic neurons, α -synuclein, TH levels, motor function, SH-SY5Y cells and ERS, are all heavily supported by existing literature. This becomes extremely crucial as Zhu et al. (2019) are one of the few groups investigating Apelin-36 treatment through the lens of PD. By performing experiments on already heavily researched topics, the authors rationale for the peptide’s neuroprotective effects becomes increasingly plausible. Another favourable aspect of the study was that its experimental procedures were conducted both in vitro and in vivo. An in vitro experiment performed on MPP+-treated SH-SY5Y cells, allows to examine the effects of the treatment on a molecular basis as well as its relevance to human cells (Xicoy et al., 2017). An in vivo experiment was also performed using the MPTP-induced mouse models, allowing to observe the effects of the treatment in the context of the whole organism. Furthermore, the experiment itself involved a localized injection of Apelin-36 into the SNpc, ensuring only the cells of the SNpc are affected (Machado et al., 2011). Contrastingly, MPTP was systemically introduced via an intraperitoneal injection. As a result, the neurotoxin is able to naturally reproduce the pathophysiology similarly displayed in individuals diagnosed with PD. This includes the aggregation of α-

synuclein, the pattern of dopaminergic neuron loss, and some behavioural deficits (Meredith et al., 2011).

Despite the benefits of the MPTP-induced mouse model, much controversy persists surrounding its use to accurately recapitulate PD pathology as observed in humans. Zeng et. al (2018) contest the benefits of this model due to its inability to generate Lewy-body inclusions, a major hallmark of PD. This issue becomes most prominent if MPTP is introduced intraperitoneally, as performed by the Zhu et al (Gibrat et al, 2009). Beyond this, PD is often diagnosed in humans approximately at the age of 60 years or older. Unfortunately, the life span of mice make it challenging to replicate this onset naturally and is not feasible for researchers to experiment using aged mice (Potashkin et al, 2011) To overcome this issue, a future study should examine alternative models that are more representative of human PD pathophysiology.

A major limitation in the study of Zhu et. al, was the lack of reasoning for the use Apelin-36 over other isoforms of Apelin such as Apelin-12, Apelin-13, or Apelin-17 (Luo et al, 2019).

Ishimaru et al. (2017), specifically emphasizes the biological potency of Apelin-13 in comparison to Apelin-36. This claim suggests if Apelin-13 demonstrates identical neuroprotective effects, then a smaller dose of Apelin-13 could be equivalently effective as a larger dose of Apelin-36. If true, Apelin-13 can be a more beneficial treatment in terms of cost-effectiveness and the safety of the patient.

Additionally, to improve the plausibility of the results from Zhu et al. (2019), the timeline of the study must be extended. Effects of the Apelin-36 treatment models were only studied 5 days after the first injection on mouse models and a 24h incubation period on SH-SY5Y cells, making it unclear if there was a retention in neuroprotective effects. Future investigations should consider monitoring the consequences of Apelin -36 on mice and SH-SY5Y cells periodically over months as opposed to days. Another potential dilemma is the method of administration. Although no significant concerns arise from intranigral injections into mouse models, this method lacks specific targeting of dopaminergic neurons. Accordingly, Apelin -36 is also taken up by surrounding cell types such as astrocytes and microglia. Considering the potency Apelin isoforms, this form of administration may lead to potential off-target effects (Ishimaru et al., 2017). Therefore, alternative techniques of administration must be explored to ensure only dopaminergic neurons are targeted.

FUTURE DIRECTIONS Using the appropriate mouse model is an ongoing and heavily debated issue. Commonly used mouse models are treated with environmental toxins such as 6-hydroxydopamine (6-OHDA), MPTP, paraquat or rotenone (Potashkin et al., 2011).

As mentioned earlier, MPTP is most capable, amongst these options, of reflecting PD pathophysiology as seen in humans.

However, they lack the ability of reproducing an important hallmark of PD, Lewy Body inclusions. A study conducted by Gibrat et al (2009), revealed a 14-day chronic intraperitoneal infusion of MPTP into mice is able to generate these inclusions. Therefore, identical experimental procedures can be conducted on chronically administrated MPTP-induced mouse models to de-

129

termine if Apelin-36 is also capable of attenuating Lewy Body inclusions. As for the other limitations such as age of onset, nonhuman primates appear to be a more representative model. These primates not only have similar brain anatomies to humans but also display the most accurate pathophysiology for PD, especially when administered MPTP. Nonhuman primates clearly exhibit muscular rigidity and bradykinesia, behavioural deficits not demonstrated in mouse models or other simple organisms (Zeng et al., 2018). Due to the astounding success of Apelin-36 in MPTP-induced mouse models, this treatment should also be investigated in nonhuman primates to study its extent in alleviating behavioural deficits.

To compare the efficacy of neuroprotective effects between Apelin-36 and Apelin-13 a study must be conducted such that it is identical to the standards of Zhu et al. (2019). All experimental procedures will be identical, except MPTP-induced mouse models and SH-SY5Y cells will be treated with Apelin-13 as opposed to Apelin-36. From the results presented by Jiang et al., Apelin-13 is expected to reverse Parkinsonian symptoms in a similar manner to Apelin-36. Accordingly, it is reasonable to expect the dissipation of neurotoxicity as demonstrated by an increased survival of SH-SY5Y cells, appearance of dopaminergic neurons, expression of TH, and improved motor function.

An enhanced regulation of α-synuclein should also be expected to be displayed by both the mouse model and SH-SY5Y cells.

Additionally, Apelin-13 will likely downregulate the expression of GRP78, CHOP, and cleaved caspase-12, thus mitigating ERS. As mentioned earlier, Ishimaru et al. (2017) showed Apelin-13 is biologically more potent compared to Apelin-36, suggesting these results would be amplified under identical dosage levels.

Although these results are foreseeable, a possibility exists where contradictory results are received. If this is the case, the results then suggest Apelin-13 activates an alternative pathway that does not contribute to the neuroprotective effects of neurons in SNpc. In order to accurately examine the efficacy of the Apelin36 treatment, a similar study to Zhu et al. (2019) must be conducted but with an extended timeline. This prospective study would involve injecting Apelin-36 or saline once during the beginning of the experiment and monitoring the appropriate markers for approximately 2 months. Due to the potency of Apelin-36, it suggests a single injection produces long-lasting effects (Ishimaru et al, 2017). Therefore, it would be expected that there will be a significant increase in neuroprotective effects before reaching a plateau. On the other hand, a decline in survival of dopaminergic neurons, TH expression, and motor function, or an increase in α-synuclein aggregation, GRP78, CHOP, and cleaved caspase-12, may also occur. These results imply a continuous re-administration of the treatment is required in order to maintain the neuroprotective effects.

The method of administration is also another point of contention, as intranigral injections lacks specificity to dopaminergic neurons. Introduction of cre-dependent adenosineassociated virus (AAV) vectors in MPTP-induced mouse models may be able to overcome this hurdle. The gene encoding for Apelin-36 will be initially inverted and be flanked by 2 loxP sites orientated towards each other. Only cells exposed to a Crerecombinase will recognize the orientation of the loxP sites,

reverting the position of the gene such that it is no longer inverted, thus resulting in its transcription driven under the elongation 1α promoter. This construct will be introduced by AAV serotype 9 by intranigral injection. The purpose of this injection is to avoid possible transduction of the AAV9 into cells of other brain regions, minimizing off-target effects. A major advantage of this administration, is the high tropism AAV9 vectors innately possess for the SNpc. To ensure the vector is expressed in dopaminergic neurons of the SNpc, only these neurons will exclusively contain a Cre-recombinase capable of inverting the gene to allow for its transcription, which can be achieved by developing Cre-transgenic animals (Grames et al., 2018). Before Apelin36 can be introduced, transfection into dopaminergic neurons can be confirmed by replacing the Apelin-36 gene with a green fluorescent protein (GFP) gene, in a separate mice group. The results can then by analyzed by immunohistochemistry. Other factors studied by Zhu et al. can also be examined to determine if this method alters the effectiveness in the reversal of PD symptoms.

Due to the similarity of the experiment performed by Grames et al. (2018), it is expected that the immunohistochemistry reveals localized expression of GFP and Apelin-36 in the mice receiving AAV9 vectors to exclusively dopaminergic neurons of SNpc. The MPTP group will not show any expression as no treatment was introduced. Based on the results presented

by Zhu et al. (2019), it would be hypothesized that the MPTPinduced mice models treated with the AAV9, will have increased TH expression but decreased GRP78, CHOP and cleaved caspase-12 expression. Also, these groups will show improved motor function as indicated by longer times spent on the rotarod. On the other hand, the MPTP group will expect to show contrasting results in all experiments performed. If these expectations are not observed, it suggests other cells of the SNpc are also contributing to the neuroprotective effects through its interaction with Apelin-36. As a result, subsequent studies can then examine the involvement of other cells to provide a more reliable and effective treatment for the future.

130

REFRENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

Beirtz J. M. (2014) Parkinson’s disease: a review. Front Biosci (Schol Ed), 1(6), 65-74 Gibrat, C., Saint-Pierre, M., Bousquet, M., Levesque, D., Rouillard, C., Cicchetti, F. (2009). Differences between subacute and chronic MPTP mice models: investigation of dopaminergic neuronal degeneration and α‐synuclein inclusions. J Neurochem, 109(5), 1469-82 doi:10.1111/j.1471-4159.2009.06072.x Grames, M. S., Dayton, R. D., Jackson K. L., Richard, A. D., Lu, X., Klein, R. L. (2018) Cre-dependent AAV vectors for highly targeted expression of disease-related proteins and neurodegeneration in the substantia nigra. FASEB J, 32(8), 4420-4427 doi:10.1096/fj.201701529RR Haghparast, E., Sheibani, V., Abbasnejad, M., Esaeili-Mahani, S. (2019) Apelin-13 attenuates motor impairments and prevents the changes in synaptic plasticity-related molecules in the striatum of Parkinsonism rats. Peptides, 117, 170091 Ishimaru, Y., Sumino, A., Kajioka, D., Shibagaki, F., Yamamuro, A., Yoshioka, Y., Maeda, S. (2017) Apelin protects against NMDA-induced retinal neuronal death via an APJ receptor by activating Akt and ERK1/2, and suppressing TNF-α expression in mice. J Pharmacol Sci, 133(1), 34-41 doi:10.1016/j.jphs.2016.12.002 Jian, Q., Wang, X., Wu, F., Wan, L., Cheng, B., Wu, Y., Bai, B. (2017). Low Dose of Apelin-36 Attenuates ER StressAssoicated Apoptosis in Rats with Ischemic Stroke. Front Neurol, 8, 556 doi:10.3389/fneur.2017.00556 Jiang, Y., Liu, H., Bingyuan, J., Wang, Z., Wang, C., Yang, C., Pan, Y., Chen, J., Cheng, B., Bai, B. (2018) Apelin‑13 attenuates ER stress‑associated apoptosis induced by MPP+ in SH‑SY5Y cells. Int J Mol Med, 42(3), 1732-1740 doi:10.3892/ ijmm.2018.3719 Ko, J. H., Lee, J. H., Choi. B., Park, J. Y., Kwon, Y. W., Jeon, S., Park, S. D., Kim, S. N. (2018). Neuroprotective Effects of Gagam-Sipjeondaebo-Tang, a Novel Herbal Formula, against MPTP-Induced Parkinsonian Mice and MPP+-induced Cell Death in SH-SY5Y Cells. Evid Based Complement Alternat Med doi:10.1155/2018/2420809 Langston, J. W. (2017). The MPTP Story. J Parksinons Dis, 7 (S1), S11-S19 doi:10.3233/JPD-179006 Luo, H., Han, L., Xu, J. (2019). Apelin/APJ system: A novel promising target for neurodegenerative diseases. J Cell Physiol. 1 -20 Machado, A., Herrera, A. J., Venero, J. L., Santiago, M., de Pablos, R. M., Villaran, R. F., Espinosa-Oliva, A. M., Arguelles S., Sarmiento, M., Delgado-Cortes, M. J., Maurino, R., Cano, J. (2011). Inflammatory Animal Model for Parkinson's Disease: The Intranigral Injection of LPS Induced the Inflammatory Process along with the Selective Degeneration of Nigrostriatal Dopaminergic Neurons. ISRN Neurology Martinez, B. A., Petersen, D. A., Gaeta, A. L., Stanley, S. P., Caldwell, G. A., Caldwell, K. A. (2017) Dysregulation of the Mitochondrial Unfolded Protein Response Induces Non-Apoptotic Dopaminergic Neurodegeneration in C. elegans Models of Parkinson’s Disease. J Neurosci, 37(46), 11085-11100 doi:10.1523/JNEUROSCI.1294-17.2017 Meredith, G. E., Rademacher, D. J. (2012). MPTP Mouse Models of Parkinson’s Disease: An Update. J Parkinsons Dis, 1(1), 19-33. doi:10.3233/JPD-2011-11023 Paquette, M. A., Martinez A. A., Macheda T., Meshul, C. K., Johnson, S. W., Berger, S. P., Giuffrida, A. (2013) Antidyskinetic mechanisms of amantadine and dextromethorphan in the 6-OHDA rat model of Parkinson’s disease: role of NMDA vs. 5-HT1A receptors. Eur J Neursci, 36(9), 3224-3234 doi:10.1111/j.1460-9568.2012.08243.x Potashkin, J. A., Blume, S. R., Runkle, N. K. (2011). Limitations of Animal Models of Parkinson's Disease. Parkinsons Dis, doi: 10.4061/2011/658083 Ryu, E. J., Harding, H. P., Angelastro, J. M., Vitolo, O. V., Ron, D, Greene, L. A. (2002) Endoplastic Reticulum Stress and he Unfolded Protein Response in Cellular Models of Parkinson’s Disease. J Neurosci, 22(24), 10690-10698 doi:10.1523/ JNEUROSCI.22-24-10690.2002 Sakamoto, K., Murakami, Y., Sawada, S., Hiroko U., Mori, A., Nakahara, T., Ishii, K. (2016) Apelin-36 is protective against Nmethyl-D-aspartic-acid-induced retinal ganglion cell death in the mice. Eur J Pharmacol, 791, 213-220 doi:10.1016/ j.ejphar.2016.08.036 Wang, Q., Ren, N., Cai, Z., Lin, Q., Wang, Z., Zhang, Q., Wu, S., Li, H. (2017). Paraquat and MPTP induce meurodengeneration and alteration in the expression profile of microRNAs: the role of transcription factor Nrf2. NPJ Parkinsons Dis, 3, 31 doi:10.1038/s41531-017-0033-1 Xicoy, H., Wieringa, B., Martens, G. J. (2017). The SH-SY5Y cell line in Parkinson’s disease research: a systematic review.

Mol Neurodegener, 12(1), 10 doi:10.1186/s13024017-0149-0 Zeng, X. S., Geng, W. S., Jia, J. J. (2018) Neurotoxin-Induced Animal Models of Parkinson Disease: PathogenicMechanism and Assessment. ASN Neuro, doi:10.1177/1759091418777438 Zhu, J., Dou, S., Wang, C., Jiang, Y., Wang, C., Cheng, B. (2019). Apelin-36 mitigates MPTP/MPP+-induced neurotoxicity: Involvement of α- synuclein and endoplasmic reticulum stress. Brain Res, 1721, 146334 doi:10.1016/ j.brainres.2019.146334 131

Restoration of the UPS through enhancement of UBA1 levels Elaine Pityn

Spinal Muscular Atrophy (SMA) is a disease caused by mutations in survival motor neuron 1 gene (SMN1), inherited through an autosomal recessive pattern. Mutations in SMN1 reduce levels of survival motor neuron (SMN) protein, which causes muscle atrophy and death in severe cases. The reduction of SMN results in aberrant molecular pathways that may be targeted for therapeutic approaches like the ubiquitin pathway. Research shows the depletion of ubiquitin activating enzyme 1 (UBA1) across all SMA models and patientâ&#x20AC;&#x2122;s cells (Powis et al. 2016). Replenishment of UBA1 prevents SMA pathogenesis; for instance in SMA zebrafish, motor axon and motor performance were improved (Powis et al. 2016) . UBA1 levels were replenished by a gene therapy approach with adeno-associated virus serotype 9-UBA1 (AAV9-UBA1). In mouse models it was shown that mice could withstand AAV-UBA1 levels over long periods and secondly, systemic restoration of UBA1 resulted in less organ pathology and improved neuromuscular outcomes, overall weight, survival, and motor performance (Powis et al. 2016). The findings suggest UBA1 restoration has potential as a therapy for SMA.

132

provide a convincing argument for this potential new therapeutic approach to SMA.

3. Major Results: This study verifys targetting UBA1 in SMA models. They showed that manipulating levels of this enzyme had therapeutic effects and offers a potential therapeutic approach.

3.1 UBA1 is a valid target for SMA

Figure 1: Visual Summary for the paper under review (Powis et al. 2016)

2. Introduction: Spinal Muscular Atrophy is a disorder in which genetic mutations of motor neurons lead to a decrease in the amount of SMN protein (Chang et al 2004). This disorder is a leading cause in infant deaths, occurring about 1 in 6,000 to 1 in 10,000 live births (Bowerman et al. 2017). SMA results in deterioration of the lower motor neuron which leads to muscle weakness, atrophy and progressive decline in motor function. SMN has ubiquitous expression, across all cells and tissue types (Chaytow et al.

2018). It is also important for mRNA trafficking, cytoskeleton dynamics, endocytosis and autophagy (Chaytow et al. 2018).

SMN levels have pathological consequences for organs like the heart and liver. SMA may range in severity as modulated by a second duplicate gene SMN2 which can modify severity levels.

In 95% of SMA cases mutations in SMN1 are the cause, but there are forms of X-linked SMA which result from mutations in the ubiquitin-like modifier activating enzyme (UBA1) (Dlamini et al. 2013, Chang et al. 2004). Since the UBA1 pathway has been associated with X-linked SMA it may be implicated in other forms of SMA pathology, as theorized in this paper. The UBA1 enzyme is necessary for protein homeostasis; it is the initial step of the ubiquitin cascade which results in protein catabolism. Since the ubiquitin proteasome system (UPS) marks proteins for degradation, modifications to this pathway can have pathological consequences. This enzyme has not only been connected with x-linked SMA, but has also been implicated in other neurodegenerative conditions (Groen and Gillingwater 2015).

The paper by Powis et al. entitled Systematic restoration of UBA1 ameliorates disease in SMA demonstrates that UBA1 is a target for SMA therapy. The authors show that UBA1 is not only implicated in the x-linked SMA, but also reduces levels in other types of SMA. They used an iPSC from a SMA patient to verify decreased UBA1 levels in humans. Then with SMA-zebra fish they showed restoration of UBA1 levels that could decrease pathology. Finally, in SMA mouse models they injected AAV9UBA1 in mice to replenish UBA1 levels. This rescued mice from declining organ pathology of the heart, liver, promoting better survival, overall health outcomes and weight. These findings

The neurological disorder SMA has been investigated using disease relevant phenotype for iPSC-derived motor neurons.

The same approach was used to verify UBA1 levels. In Figure #2 the UBA1 levels were found to be reduced by 40% in Type I SMA patient derived iPSC compared to control patients UBA1 levels (Powis et al. 2016). Figure 2 also shows that decreasing levels of UBA1 in healthy zebrafish will produce abnormalities in motor axon growth, like the pathology of SMA (Wishart et al.

2014, McWhorter et al. 2003) . Powis et al. quantified protein levels using western blot analysis and found a reduction in SMN levels. They verified this in both SMA zebrafish and SMA mice by demonstrating UBA1 and SMN were reduced and resulted in SMA pathology.

Figure 2:Treatment of Zebrafish with MO, a pharmacological inhibitor of UBA1 produced abnormal axon growth, a pathological feature of SMA (Wishart et a. 2014)`

3.2 UBA1 restorations will recover SMA models The UBA1 levels were recovered in both SMA models for zebrafish and mice via an AAV9-UBA1 injection. In zebrafishSMA the motor phenotype and motor axon morphology was rescued (Powis et al. 2016). Various other authors have shown that inhibition of downstream UBA1 targets in SMA mice will produce similar neuromuscular outcomes (Shorrock et al. 2018, 1; Wishart et al. 2014). In mouse models, AAV9UBA1 injection via the facial vein produced systemic improvements. Organ outcomes, weight, survival, and motor performance all improved. Studies showed a reversal of the neuromuscular pathology and restored muscle fibre diameter. The work of Shorrock et al. 2018 in figure 3 shows AAV9-UBA1 injection increased UBA1 levels which later rescued sensory neurons.

133

3.2 UBA1 restorations will recover SMA models The UBA1 levels were recovered in both SMA models for zebrafish and mice via an AAV9-UBA1 injection. In zebrafishSMA the motor phenotype and motor axon morphology was rescued (Powis et al. 2016). Various other authors have shown that inhibition of downstream UBA1 targets in SMA mice will produce similar neuromuscular outcomes (Shorrock et al. 2018, 1; Wishart et al. 2014). In mouse models, AAV9-UBA1 injection via the facial vein produced systemic improvements. Organ outcomes, weight, survival, and motor performance all improved. Studies showed a reversal of the neuromuscular pathology and restored muscle fibre diameter. The work of Shorrock et al. 2018 in figure 3 shows AAV9-UBA1 injection increased UBA1 levels which later rescued sensory neurons.

3.3 a

Figure 4 Rescuing neuromuscular pathology in SMA Zebrafish though pharmacological inhibition of B-catenin a downstream effect of UBA1 upregulation by quercetin (Wishhart et al. 2018)

and survival. Powis et al. conclude that UBA1 is necessary for maintenance of the neuromuscular system and can be used as a therapeutic target to treat neuromuscular and systemic issues of SMA. UBA1 may be implicated in other neurodegenerative conditions by disruption of protein degradation (Groen and Gillingwater 2015). This paper is critical for the advancement of new therapies treating SMA. Although there are already drugs UBA1 and gene therapies, there are none that target the UBA1 syssafe tem (David et al. 2020). UBA1 restoration whether through AAV ther- mediated injection or pharmacological intervention by increasing UBA1 levels improve SMA models (Shorrock et al.

2018; Wishart et al. 2014).

5. Critical Analysis Figure 3 Protein Levels of SMN, UBA, and GARS a downstream UBA1 target in SMA and SMA+AAV9-UBA1 treated mice. The authors used the same type of treatment. (Shorrock et al. 2018)

5.1 Powis et al. limitations and success

apeutic approach Powis et al. monitored adverse effects of UBA1 systemic increase, through human UBA1 mRNA levels. Using western blot analysis the levels of UBA1 and SMN were increased in both control and SMA mice. They found no adverse responses to the protein levels. The authors suggest that UBA1 is in the top 2% of cellular proteins; cells may have capacity to withstand high levels. In earlier studies, one of which appears in figure 4, SMA progression could be ameliorated by changing UBA1 downstream targets (Wishart et al. 2014).

4. Discussion UBA1 levels are significantly reduced in SMA models which implies that changes to this system contribute to SMA progression. Replenishing UBA1 levels and a subsequent decrease in SMA pathology provides substantial verification. UBA1 influences organ pathology, muscle atrophy, overall size, health,

The authors conducted extensive research on the influence of the UBA1 protein and mediating SMA progression. The authors were correct in their conclusion that UBA1 is a valid target for SMA treatment since other papers produced similar findings (Powis et al. 2016). Although this paper recognizes UBA1 mediated SMA progression, it did not address the mechanisms in which UBA1 is implicated, rather it showed changes in levels of the enzyme; further investigators researched this. The authors reported a conserved response of UBA1 decline among SMA patients and models. The only issue in this paper was evident in Figure 5 (figure 4 in their paper), which compared survival, motor performance and body weight of mice. This figure compares three groups of mice, but does not include the true control group. The caption refer back to a different figure for the controlâ&#x20AC;&#x2122;s results. Since the groups are being compared they all should be presented one graph. In figure 5D the control was injected with AAV9-UBA in the original caption, but does not contain the information in the figure.

134

5.2 Why this treatment works â&#x20AC;&#x201C; at a molecular level 5.2.1 UBA1/GARS Pathway

and see subsequent responses. The combined therapeutic approach should improve the outcomes of SMA models through a systemic restoration of SMN and UBA1 levels. If the injection fails to ameliorate disease this would indicate that packaging of the AAV does not work or there are competing protein interactions. They could also deliver two separate injections and compare to the single injection to verify that packaging is not the issue, rather than upregulation of the gene and enzyme.

The levels of UBA1 in SMA models effect the levels of GARS, an important enzyme for protein synthesis (Shorrock et al. 2018).

This target underlies the molecular pathways for the deafferentation of motor neurons in SMA (Shorrock et al. 2018). They employed the same technique with AAV-9-UBA1 injection which corrected the sensory-motor connectivity at the level of the spinal cord, verifying this method of gene therapy and molecular targets like the original paper.

5.3.2 UBA1 can be used to treat other neurodegenerative conditions

A hallmark of neurodegenerative conditions is protein clumping. UBA1 marks protein for degradation, therefore it may contribute to disease progression. For instance, UBA1 is implicated in polyglutamine protein toxicity in mice with Huntingtonâ&#x20AC;&#x2122;s Disease (HD) (Groen and Gillingwater 2015). Therefore, using the systemic increase of UBA1 in different mouse models of neurodegenerative conditions like HD may reveal a novel treatment.

If administration of UBA1 blocks disease progression this means it could be a broader therapeutic approach than just SMA. Neurodegenerative conditions are complex disease though, so if the UBA1 administration does not work, it may indicate different enzyme in the UPS system that would need further investigation.

Figure 5 Comparing outcomes between treatment groups for SMA. A:Weight, B: Survival, C: Behavioural reflex. D: Appearance (Powis et al. 2016)

5.2.2 UBA1 and B-catenin Disruption of UBA1 in SMA models is due interactions in the cytoplasm involving physical interactions between SMN and UBA1 proteins, and splicing of UBA1 mRNA (Wishart et al.

2014). Accumulation of B-actin resulted from UBA1 dysregulation and blocking B-catenin reduced SMA pathology (Wishart et al. 2014). B-catenin signalling pathways regulate motor neuron differentiation, stability, synaptic function, and structure. Lower levels of UBA1 contribute to neuromuscular issues by ubiquitin impairment and subsequent B-catenin signalling; this information re-enforces the main objectives by Powis et. al that enhancing UBA1 will ameliorate neuromuscular issues.

5. 3 Future Directions 5.3.1 UBA1 as a combined therapeutic approach UBA1 replenishment has been proposed as a novel therapeutic approach for SMA. Current treatments use AAV9 to delivery intact copy SMN1, referred to as Zolgenesma (Schorling, Pechmann, and Kirschner, 2020). A combined therapeutic approach which uses SMN1 and UBA1 levels should be tried in future.

The authors should conduct an experiment which delivers both UBA1 mRNA and SMN1 in one injection to mice and assess therapeutic effects. The authors could use the technique of packaging recombinant AAV vector with a cleavage site placed between the coding sequence of UBA1 and SMN1, for both to be expressed (Foti, Samulski, and McCown 2009). The authors could then administer the virus via the facial vein to SMA mice 135

REFRENCES

1.

Shorrock, Hannah K, Dinja van der Hoorn, Penelope J Boyd, Maica Llavero Hurtado, Douglas J Lamont, Brunhilde Wirth, James N Sleigh, et al. “UBA1/GARS-Dependent Pathways Drive Sensory-Motor Connectivity Defects in Spinal Muscular Atrophy.” Brain 141, no. 10 (October 2018): 2878–94. https:// doi.org/10.1093/brain/awy237

2.

Bowerman, Melissa, Catherina G. Becker, Rafael J. Yáñez-Muñoz, Ke Ning, Matthew J. A. Wood, Thomas H. Gillingwater, and Kevin Talbot. “Therapeutic Strategies for Spinal Muscular Atrophy: SMN and Beyond.” Disease Models & Mechanisms 10, no. 8 (August 1, 2017): 943–54. https://doi.org/10.1242/ dmm.030148.

3.

McWhorter, Michelle L., Umrao R. Monani, Arthur H.M. Burghes, and Christine E. Beattie.

“Knockdown of the Survival Motor Neuron (Smn) Protein in Zebrafish Causes Defects in Motor Axon Outgrowth and Pathfinding.” The Journal of Cell Biology 162, no. 5 (September 1, 2003): 919–32. https://doi.org/10.1083/jcb.200303168.

4.

Wishart, Thomas M., Chantal A. Mutsaers, Markus Riessland, Michell M. Reimer, Gillian Hunter, Marie L. Hannam, Samantha L. Eaton, et al. “Dysregulation of Ubiquitin Homeostasis and β-Catenin Signaling Promote Spinal Muscular Atrophy.” The Journal of Clinical Investigation 124, no. 4 (April 1, 2014): 1821 –34. https://doi.org/10.1172/JCI71318.

5.

Chang, Hui-Chiu, Wen-Chun Hung, Yen-Ju Chuang, and Yuh-Jyh Jong. “Degradation of Survival Motor Neuron (SMN) Protein Is Mediated via the Ubiquitin/Proteasome Pathway.” Neurochemistry International 45, no. 7 (December 1, 2004): 1107–12. https://doi.org/10.1016/j.neuint.2004.04.005.

6.

Groen, Ewout J.N., and Thomas H. Gillingwater. “UBA1: At the Crossroads of Ubiquitin Homeostasis and Neurodegeneration.” Trends in Molecular Medicine 21, no. 10 (October 2015): 622–32. https:// doi.org/10.1016/j.molmed.2015.08.003.

7.

Foti, SB, RJ Samulski, and TJ McCown. “Delivering Multiple Gene Products in the Brain from a Single Adeno-Associated Virus Vector.” Gene Therapy 16, no. 11 (November 2009): 1314–19. https:// doi.org/10.1038/gt.2009.106.

8.

C, David C., Astrid Pechmann, and Janbernd Kirschner. “Advances in Treatment of Spinal Muscular Atrophy – New Phenotypes, New Challenges, New Implications for Care.” Journal of Neuromuscular Diseases 7, no. 1 (2020): 1–13. https://doi.org/10.3233/JND-190424.

9.

Dlamini, Nomazulu, Dragana J. Josifova, Simon M. L. Paine, Elizabeth Wraige, Matthew Pitt, Amanda J. Murphy, Andrew King, et al. 2013. “Clinical and Neuropathological Features of X-Linked Spinal Muscular Atrophy (SMAX2) Associated with a Novel Mutation in the UBA1 Gene.” Neuromuscular Disorders 23 (5): 391–98. https://doi.org/10.1016/j.nmd.2013.02.001.

10.

Chaytow, H., Huang, Y. T., Gillingwater, T. H., & Faller, K. (2018). The role of survival motor neuron protein (SMN) in protein homeostasis. Cellular and molecular life sciences : CMLS, 75(21), 3877–3894. https://doi.org/10.1007/s00018-018-2849-1

136

Omega-3 Improves cognitive Dysfunction in Schizophrenia Via CREB S133 Phosphorylation Victoria Radburn

Schizophrenia (SZ) is a complex mental health disorder associated with social, behavioural and cognitive impairments. It is found to produce reduced levels of brain-derived neurotrophic factor (BDNF), which is key for learning and memory function. ω-3PUFAs have been shown to increase BDNF levels and improve SZ symptoms through the upregulation for the BDNF/CREB pathway, however the mechanism of action still needs to be understood. Guo, et al. (2020) tested ω3PUFAs on MK801-induced SZ rat models, and found increased BDNF and CREB levels, along with restored hippocampal neuron damage. This led to decreased anxiety, restored cognitive-behavioural functions, such as social ability and spatial memory, increased synaptic plasticity and increased dendritic spine density1. They hypothesized that S133 phosphorylation of CREB was responsible for mediating this beneficial outcome of ω-3PUFA treatment. Using the S133A inactivated CREB mutation, they found decreased BDNF levels and a reduction in cognitive function even when treated with ω-3PUFAs. This indicates the importance of phosphorylated S133 CREB in the pathway to producing improved SZ symptoms. With this understanding, future studies and clinical trials can be implemented to test the efficacy of ω-3PUFAs on humans with SZ. Below is an original visual abstract summary of the Guo, et al. (2020) paper and the results found when treating MK801 rats with ω-3PUFAs

137

the SZ model of MK-801 rats9. They continued working to further understand how this treatment functions specifically and published their findings three years later in 2020.

INTRODUCTION Schizophrenia (SZ) is a serious psychiatric neurodisorder affecting 20 million people world-wide2. It is commonly diagnosed in early adulthood, however mild cognitive symptoms can begin much earlier on. SZ can present in many different ways, but symptoms typically fall under one of three distinct categorizations. Psychotic symptoms involve an altered perception of reality, i.e. hallucinations, negative symptoms involve social withdrawal and lack of emotion, and cognitive symptoms include anxiety, learning and memory dysfunction 3.

Here, Gao, et al. (2020) focuses their testing and treatment on improving behavioural and cognitive symptoms, as well as underlying neuronal functions.

The first step into uncovering therapeutics for SZ is understanding the mechanisms of which it affects. One of the mechanisms involved is the brain-derived neurotrophic factor (BDNF) found in the CNS, which plays a key role in neurogenesis, synaptic plasticity and brain development by regulating neuronal differentiation and growth4. A meta-analysis conducted by Green et al. (2010) demonstrated individuals with SZ tend to have reduced plasma BDNF levels, and aimed to further clarify the role BDNF levels have as a biomarker for related disorders4. The cAMP response element binding protein (CREB) has a similar role in neurogenesis and synaptic plasticity, as it is a transcription factor which increases expression of both the BDNF gene and BCL-2: an anti-apoptotic gene1. Evidently, this CREB/BDNF pathway presents as a sufficient target for a treatment or therapy towards SZ. The brain has a very high metabolic rate, and requires lots of ATP, therefore effective neuronal activity is co-dependent on mitochondria function and energy metabolism. Recently investigating the link between diet and cognitive function has risen in popularity, as findings have shown energy metabolism can influence neuronal function and synaptic plasticity 5. For example, Gao, et al. (2016) found that an Omega-6 poly-unsaturated fatty acid (PUFA) precursor, alpha-linolenic acid, enhanced the CREB/BDNF/TrkB pathway by activating ERK and Akt signals in the hippocampus6. Omega-3 (ω-3) PUFA has also been recognized for its major health benefits found in a variety of foods worldwide, with many proven brain contributions including treatment for depression7 and Alzheimer’s disease8. The XiaoChuan Wang Lab previously found, in 2017, that ω-3PUFAs worked to upregulate the CREB/BDNF pathway, by introducing

Guo et al. (2020) investigate the underlying mechanisms of how ω-3PUFAs work to improve cognitive deficits in SZ. They use rats injected with MK-801 as the SZ animal model, conducting various behavioural tests to ensure these animals do have the expected social, learning and memory deficits1. When treated with ω-3PUFAs, the MK-801 rats showed alleviation of these SZ -induced cognitive impairments. They found that ω-3PUFAs show improvements by inducing CREB Ser133 phosphorylation, which in turn activates the necessary CREB/BDNF pathway 1.

The upregulation of this pathway increases synaptic plasticity, prevents hippocampal neuron loss and overall improved cognitive function in the SZ model.

MAIN RESULTS Ω - 3P UF A S IM PR OV E S C OGN I TI V E AN D N EU RO NA L FU NC TI ON IN SZ M O D E L

To explore the cognitive effects of ω-3PUFAs on MK801 SZ-induced impairments, various behaviour tests were conducted with control, MK801 only (Mod) and MK801 treated with ω-3PUFA (Pre) test groups. First the novel object recognition (NOR) test showed the Pre group had a higher curiosity than the Mod group, since they spent more time exploring the novel object1. In the high plus maze test, the time spent in the closed arm was decreased in the Pre group compared to the Mod group (Fig. 2A), indicating a decrease in symptomatic anxiety when treated1. The three boxes social experiment demonstrates a restored social ability in Pre group compared to the Mod group (Fig. 2B)1. In the Morris water maze (MWM), the Pre group was able to find the hidden platform quicker (Fig. 2C) and spent more time in the target quadrant than the Mod group, indicating improved spatial memory with treatment1.

Overall, these findings show that preventative treatment with ω-3PUFAs restores cognitive-behavioural functions such as socialization, learning, memory, and anxiety to a healthy level in MK801 SZ-model rats.

Next, to determine the internal neuronal benefits from ω3PUFA treatment, testing was carried out on hippocampal slices following the behavioural tests. The NMDAR/AMPAR ratio was calculated and demonstrated an increase in the Pre group compared to the Mod group (Fig. 2D), indicating increased synaptic transmission1. When observing spine density, an increase in dendritic spine density was found in the hippocampal neurons of the Pre group (Fig. 2E), indicating that ω-3PUFAs prevent hippocampal neuron damage. Furthermore, western blotting showed significant increases in both CREB and BDNF levels of the hippocampus, in the Pre group compared to the Mod group (Figure 2F,G)1. This indicates that ω-3PUFAs do work to increase the CREB/BDNF pathway, as hypothesized in previous papers.

138

group, curiosity was increased compared to the Mod group (Fig. 2B), which resembles similar levels to the Pre or healthy control groups1. In the MWM, the S133A-Pre group showed a decrease in number of times they crossed the platform, as well as a decrease in time spent in the target quadrant, compared to the Pre group (Fig. 2C) 1. In the S133D group, there was greatly increased time spent in the target quadrant and an increased number of platform crossings, compared to the Mod group (Fig. 2D). There results firstly indicate that ω-3PUFA treatment is rendered ineffective when S133 is not phosphorylated, and secondly that phosphorylated CREB S133 improves cognitive deficits in MK801-induced Schizophrenia.

Electrophysiology and morphology experiments were done to test the role of Ser133 CREB in synaptic plasticity and neurogenesis. Slope of excitatory post-synaptic potential (fEPSP) was lower in the S133A-Pre group than the Pre group, and that the Pre group treated with ω-3PUFAs had a higher slope compared to the Mod group1. The fEPSP slope in the S133D group was found to be much higher than the Mod group as well (Fig. 2E).

Dendritic spine density was found to be decreased in the S133A -Pre group compared to the Pre group, through Golgi staining (Fig. 2F)1. On the other hand, S133D has a decrease in dendrite complexity compared to Mod group hippocampal neurons.

Western blotting BDNF and CREB levels were much lower in S133A-Pre group, compared to the S133D group1. Overall, this demonstrates that phosphorylated CREB at S133 is essential in generating the positive treatment outcomes, such as increased plasticity and prevention of hippocampal neuron loss in MK801 -induced SZ rats.

Figure 1: ω-3PUFAs restore cognitive abilities and neuronal functioning in MK801-induced SZ model. (A-C) Behavioural testing conducted between control, MK801 and MK801+ω3PUFAs groups. The high plus maze test shows number of closed-arm entries (A). The three boxes social experiment shows the time spent interacting with a stranger (B). The Morris water maze test measures the latency to find a hidden platform over 5 days (C). (D) 12 cells from each group were used to determine the NMDA/AMPA receptor ratio to quantify EPSCs. (E) Golgi stain is used to visualize dendritic spines of hippocampal neurons for each group and measure synaptic spine density. (F-G) ω-3PUFAs regulate BDNF and CREB levels in MK801 rats. Treated rats show protein levels of both BDNF and CREB similar to healthy control rats. Significant increase in BDNF (F) and CREB (G) levels in MK801 rats treated with ω3PUFAs compared to the untreated group. Significance level: *p<0.05, **p<0.01, ***p<0.001.

Citation: Guo et al., “ω-3PUFAs Improve Cognitive Impairments Through Ser133 Phosphorylation of CREB Upregulating

CR EB S ER 13 3 P H OS P H OR YL A TI ON M E DI A T ES T H E B EN E FI T S OF Ω - 3 PU F AS

To test the hypothesis that S133 phosphorylation of CREB mediates the beneficial outcome of ω-3PUFA treatment in the SZ model, both unphosphorylated and phosphorylated S133 were tested under the same behavioural tests and neuronal analyses as previously conducted. Unphosphorylated CREB was mimicked at the Ser133 site using a virally injected S133A mutation, and phosphorylated CREB was mimicked using an S133D mutation. Five groups were used for these tests: control, Mod, Pre, MK801+ω-3PUFA+Unphosphrylated CREB (S133A-Pre) and MK801+Phosphorylated CREB (S133D).

Firstly, the NOR test showed a significant decrease in the S133A -Pre group compared to the Pre group, when spending time exploring the novel object (Fig. 2A). However, for the S113D 139

Figure 2: Phosphorylated CREB at S133 mediates cognitive improvements in ω-3PUFA treatment of MK801-induced SZ model. (A-D) Cognitive testing in the unphosphorylated and phosphorylated CREB S133. NOR tests the amount of time spent with the novel object in a 24h period in the S133A-Pre group (A) and the S133D group (B) compared to control, Mod and Pre groups. MWM test spatial memory with the number of times the hidden platform was crossed in the S133A-Pre group (C) and the S133D group (D). (E) The CA3-CA1 Hippocampal LTP was measured and the fEPSP slope was recorded from the CA1 dendritic region, in S133D rats. (F) Golgi staining used to visualize dendritic spines in the hippocampus and calculate the average density. Significance level: *p<0.05,

**p<0.01, ***p<0.001.

only using a certain dose, measurement and model. If the treatment does prove to be effective within a specific dose for a feaCitation: Guo et al., “ω-3PUFAs Improve Cognitive Impairments sible group of patients, then these guidelines can be used to Through Ser133 Phosphorylation of CREB Upregulating BDNF/ implement human testing and eventually a clinical trial.

TrkB Signal in Schizophrenia.”

Future Directions

Discussion

Looking forward, the future goals would be to test the efficacy of ω-3PUFA treatment in humans with SZ. The first clinical trial was conducted by Pawelczyk et al. (2019), composed of a double-blind randomized placebo-controlled study testing observing the effects of n-3 PUFAs on BDNF levels13. They acknowledge the limitations derived from not fully understanding the underlying mechanisms, and inconsistencies in the method of measuring BDNF levels. However, this is an exceptional start in the right direction and can be further tailored using the specific experimental guidelines found through extended research as outlined above. The next clinical trial should include fMRI scans of learning and memory regions such as the hippocampus, when preforming cognitive tasks, as well as spinal CSF levels of BDNF to ensure greater accuracy. Another aspect of the trial could include a long-term study of children or adolescents genetically pre-disposed to Schizophrenia who take ω-3PUFAs to test the ω-3PUFAs have been known to have major health and brain preventative effects, and measure age of diagnosis, if they ever benefits for quite some time. Previous literature had found links even become diagnosed. and interpretations of the beneficial effects ω-3PUFAs have on Through implementing a more specific clinical trial, we would the CREB/BDNF pathway, however the mechanism still rehope to see a same regulation of SZ-induced cognitive sympmained unknown up until this point. These new findings behind toms, and increased BDNF levels, when patients are treated ω-3PUFAs mechanisms, produced by Guo et al. (2020), are pavwith ω-3PUFA compared to a placebo, as observed in MK801 ing the way for ω-3PUFA use as a preventative measure in the rats. If these future experiments fail to provide similar outcomplex disorder, Schizophrenia. comes, a number of factors could be involved including levels of dosage, strength of initial symptoms, how long they have been Critical Analysis diagnosed, age, or simply the fact that humans are not rats.

Firstly Guo, et al. effectively tested their hypothesis surrounding Overall, the successful evidence gained of ω-3PUFAs improving CREB Ser133, by not only testing the positive effects of phos- cognitive and neuronal function through phosphorylated CREB phorylated S133 but also the impact that an unphosphorylated S133 in MK801-induced SZ rats, will propel future experiments S133 mutation would have on ω-3PUFA treatment. However, into Schizophrenia treatment and prevention. other experimental groups the authors should include in testing is S133A with a mutation in BDNF gene expression and a TrkB antagonist with S133A, to determine the sequentiality of mechanistic action and rule out any other possible factors. Additionally, testing the S133D group with enhanced BDNF gene expression would determine the role of BDNF compared to CREB in this mechanism. Another pathway could look into other treatments that may be effective involving direct target of Ser133 phosphorylation or use of other CREB/BDNF pathway enhancers delivered similarly through the diet, such as polyphenols11.

Cognitive impairments caused by Schizophrenia have been shown to pose a major burden on occupational performance, social activities and the patient’s quality of life10. In understanding the underlying mechanisms of these symptoms specifically, fundamental treatment and prevention methods can be further developed. Here Guo et al. (2020) looked into the therapeutic effects of ω-3PUFAs, and their mechanism of action of the important CREB/BDNF pathway. They found ω-3PUFAs restored all of the cognitive-behavioural impairments caused by MK801incduced SZ, by working to upregulate the CREB/BDNF pathway.

Diving deeper into this mechanism, phosphorylated S133 CREB was found to be essential in mediating the ω-3PUFA treatment outcome. Phosphorylated S133 has shown an increase in learning and memory, as well as increased synaptic plasticity and dendritic spine density in the hippocampus.

Given an animal model study, there is almost always the question of ‘will this be just as effective in humans?’. In order to get there, the next experiments needed to be done are expanding the criteria of treatment by testing different ω-3PUFA dosages on different ages, different severity of symptoms, and in both male and female MK801 rats. This should also be tested in a larger number of models, as well as conducted by different labs, to ensure reliability. These same experiments should also be tested on other SZ animal models, such as the ketamine-induced model12. This will account for any biases or restrictions from 140

REFRENCES

1.

1. Guo C, Liu Y, Fang M-S, Li Y, Li W, Mahaman YAR, Zeng K, Xia Y, Ke D, Liu R, et al. ω-3PUFAs Improve Cognitive Impairments Through Ser133 Phosphorylation of CREB Upregulating BDNF/TrkB Signal in Schizophrenia. Neurotherapeutics: The Journal of the American Society for Experimental NeuroTherapeutics. 2020 May 4. doi:10.1007/s13311-020-00859-w

2.

2. Schizophrenia. [accessed 2020 Jun 17]. https://www.who.int/news-room/fact-sheets/detail/schizophrenia

3.

3. NIMH » Schizophrenia. [accessed 2020 Jun 17]. https://www.nimh.nih.gov/health/topics/schizophrenia/index.shtml

4.

4. Green MJ, Matheson SL, Shepherd A, Weickert CS, Carr VJ. Brain-derived neurotrophic factor levels in schizophrenia: a systematic review with meta-analysis. Molecular Psychiatry. 2011;16(9):960–972. doi:10.1038/mp.2010.88

5.

5. Gomez-Pinilla F, Tyagi E. Diet and cognition: interplay between cell metabolism and neuronal plasticity. Current opinion in clinical nutrition and metabolic care. 2013;16(6):726–733. doi:10.1097/MCO.0b013e328365aae3

6.

6. Gao H, Yan P, Zhang S, Huang H, Huang F, Sun T, Deng Q, Huang Q, Chen S, Ye K, et al. Long-Term Dietary Alpha-Linolenic Acid Supplement Alleviates Cognitive Impairment Correlate with Activating Hippocampal CREB Signaling in Natural Aging Rats. Molecular Neurobiology. 2016;53(7):4772–4786. doi:10.1007/s12035-015-9393-x

7.

7. Mozaffari-Khosravi H, Yassini-Ardakani M, Karamati M, Shariati-Bafghi S-E. Eicosapentaenoic acid versus docosahexaenoic acid in mild-to-moderate depression: a randomized, double-blind, placebo-controlled trial. European Neuropsychopharmacology: The Journal of the European College of Neuropsychopharmacology. 2013;23(7):636–644. doi:10.1016/ j.euroneuro.2012.08.003

8.

8. Belkouch M, Hachem M, Elgot A, Lo Van A, Picq M, Guichardant M, Lagarde M, Bernoud-Hubac N. The pleiotropic effects of omega-3 docosahexaenoic acid on the hallmarks of Alzheimer’s disease. The Journal of Nutritional Biochemistry.

2016;38:1–11. doi:10.1016/j.jnutbio.2016.03.002

9.

9. Fang M-S, Li X, Qian H, Zeng K, Ye M, Zhou Y-J, Li H, Wang X-C, Li Y. ω-3PUFAs prevent MK-801-induced cognitive impairment in schizophrenic rats via the CREB/BDNF/TrkB pathway. Journal of Huazhong University of Science and Technology.

Medical Sciences = Hua Zhong Ke Ji Da Xue Xue Bao. Yi Xue Ying De Wen Ban = Huazhong Keji Daxue Xuebao. Yixue Yingdewen Ban. 2017;37(4):491–495. doi:10.1007/s11596-017-1762-4

10.

10. Bell M, Bryson G, Greig T, Corcoran C, Wexler BE. Neurocognitive enhancement therapy with work therapy: effects on neuropsychological test performance. Archives of General Psychiatry. 2001;58(8):763–768. doi:10.1001/archpsyc.58.8.763

11.

11. Moosavi F, Hosseini R, Saso L, Firuzi O. Modulation of neurotrophic signaling pathways by polyphenols. Drug Design, Development and Therapy. 2015;10:23–42. doi:10.2147/DDDT.S96936

12.

12. Zugno AI, Chipindo HL, Volpato AM, Budni J, Steckert AV, de Oliveira MB, Heylmann AS, da Rosa Silveira F, Mastella GA, Maravai SG, et al. Omega-3 prevents behavior response and brain oxidative damage in the ketamine model of schizophrenia. Neuroscience. 2014;259:223–231. doi:10.1016/j.neuroscience.2013.11.049

13.

13. Pawełczyk T, Grancow-Grabka M, Trafalska E, Szemraj J, Żurner N, Pawełczyk A. An increase in plasma brain derived neurotrophic factor levels is related to n-3 polyunsaturated fatty acid efficacy in first episode schizophrenia: secondary outcome analysis of the OFFER randomized clinical trial. Psychopharmacology. 2019;236(9):2811–2822. doi:10.1007/ s00213-019-05258-4

141

Hypersocialization, Social Blindness and Motor Deficits: the Role of PSD95 and PSD93 Rebecca Rocco

PSD95 and PSD93 are both membrane-associated guanylate kinase (MAGUK) proteins which are prominent and essential to glutaminergic postsynaptic terminal efficacy and function. Mutations of these proteins have been linked to the development of synaptopathies such as schizophrenia and autism (Yang et al., 2019, Coley et al., 2019). While PSD95 knockout animal models have shown that lack of PSD95 results in strange behaviours such as immobility, repetitive grooming, and heightened anxiety (Zhang et al., 2014), overall behaviour of these animal models has been difficult to assess due to motor impairment. In addition, the majority of genetic mutations within humans are in the heterozygous form, where a deficiency of a gene product is observed rather than a complete absence. Therefore, Winkler et al. (2018) investigated modified behaviour of PSD95 deficient mice.

Furthermore, PSD93 knockout and deficient mice also had their behaviour investigated in order to assess whether PSD93 expression impairment results in similar behavioural phenotypes as in PSD95 deficiency, pointing to a potential shared functional and/or biological redundancy of PSD93/ PSD95. PSD95 deficient and wildtype C57BL/6J mice were raised and then subjected to a wide range of behavioural tests. PSD93 wildtype, deficient and knock-out mice were also investigated using a similar battery of behavioural tests. Mice were taken from each cohort to confirm genetic makeup and expression of PD genes using qPCR methods. This study found that PSD95 deficient mice showed hypersocial behaviours, only mild motor impairment and increased PSD93 expression within the hippocampus; males had increased aggression responding to foreign mice and females had increased vocalization responding to an anesthetized mouse. PSD93 knock-out mice showed comparable hypersocial behaviour to that of PSD95 deficient mice, as well as severe motor impairment. These results suggest that both PSD95 and PSD93 are implicated in social processing and behaviour. As PSD protein impairment has been implicated in the development of neuropsychiatric diseases (Gao et al., 2013), the use of PSD95 deficient mice as a model to study neuropsychiatric diseases which have social disinhibition symptoms associated with them may be beneficial.

Key Words: Membrane-associated guanylate kinases (MAGUKs), Post synaptic density, PSD95, PSD93, schizophrenia, autism, synaptopathies, hippocampus, synaptic plasticity, genetic redundancy, social behaviour, social processing, social disinhibition, social blindness, hypersocialization

142

Introduction The neuronal membrane-associated guanylate kinase (MAGUK) family of proteins are essential scaffolding proteins which act as central building blocks of the glutaminergic postsynaptic density (PSD) framework (Won et al., 2017; Zheng et al., 2011;).

The PSD is a congregation of proteins at the post-synaptic terminal - its structure and its composition allows transmission through glutaminergic excitatory processes to be effective (Scannevin and Huganir, 2000). MAGUKs serve to organize and structurally uphold this PSD framework by organizing protein complexes within the PSD, clustering, moving and modifying glutamate receptors, linking these receptors to intracellular mediators, as well as dynamically enforcing synaptic maturation (Bustos et al., 2017; Chen et al., 2015). Alteration of MAGUK function has been implicated with synaptopathies, such as schizophrenia, autism and Alzheimerâ&#x20AC;&#x2122;s disease (Coley and Gao, 2018; Feyder et al., 2010; Proctor et al., 2010).

PSD95 is a MAGUK and a major constituent of the postsynaptic density framework. PSD95 has been implicated in synapse maturation, plasticity, as well as in shaping the protein composition within glutaminergic post-synaptic terminals (Mardones et al., 2019). These proteins have been implicated in learning and consolidation of memory as well. (Li et al., 2018; Bustos et al., 2017; Zheng et al., 2011) Expression of PSD95 dynamically changes throughout the life cycle of dendritic spines (Lambert et al., 2017). It has also been suggested that the presence of PSD95 is essential for later stages of synaptic development and neurogenesis rather than early on (McGee et al., 2001). PSD95 overexpression studies have shown that overabundance of PSD95 leads to increased postsynaptic clustering, increased number and size of dendritic spines and activity of glutamate receptors within the hippocampus (El-Husseini et al., 2000).

Due to this, it is strongly believed that PSD95 plays a large role in the stabilization of synapses as well as in synaptic plasticity.

Genetic studies have associated MAGUK impairment, such as that of PSD95, within schizophrenia and autism (Yang et al., 2019; Coley and Gao, 2019). Genome sequencing has shown that psychiatric patients show impairment of PSD constituents, such as PSD95 and animal studies have shown that disruption of PSD95 leads to cognition and learning impairment (Coley and Gao, 2018). However, prior behavioural studies have shown that complete PSD95 knockout within mice has resulted in very strange phenotypes, such as severe motor impairment, limb clasping, variable social preferences, in addition to learning and working memory impairment (Coley and Gao, 2019; Zhang et al., 2014). Severe motor phenotypes have made behavioural deduction regarding these knockout models difficult to investigate and interpret. In addition, humans tend to be heterozygous for genetic mutations, leading to a deficiency of protein product rather than a complete lack thereof (Winkler et al. 2018). Due to this, the authors of this study decided to investigate the social behaviour and social processing of PSD95 mutant (+/-) mice relative to PSD95 wildtype (+/+) mice.

nance of the PSD (Favaro et al., 2018; Won et al., 2017). In contrast with PSD95, PSD93 accumulates quicker within developing dendritic spines (Lambert et al., 2017). In addition, PSD93 knockout within Purkinje neurons, where PSD93 is the only MAGUK expressed, has been shown to cause no structural or functional abnormalities, nor changes within the localization of PSD93 interacting proteins (McGee et al, 2001). Both PSD95 and PSD93 had been thought to play similar roles in the maturation of glutaminergic synapses, however, recent studies have argued against this (Favaro et al., 2018). Studies which have investigated PSD95 and PSD93 knockout mice found that glutamate receptors, AMPARs and NMDARs, had reduced synaptic transmission as well as postsynaptic density (PSD) congregation, in addition to an increased number of silent synapses and a sharp reduction in the size of PSD with no significant changes to the pre/post synaptic membrane (Chen et al., 2015). PSD93 knockout (-/-), deficient (+/-) and wildtype (+/+) had their social behaviour investigated.

This paper (Winkler et al., 2018), aimed to investigate the social behaviour and processing in PSD95(+/-) partial knock out mice.

In addition, PSD93(-/-) knockout, PSD93(+/-) partial knock out and PSD93(+/+) wildtype mice had their social behaviour and phenotypes investigated in order to assess whether PSD95/ PSD93 are functionally redundant and whether loss of PSD93 results in similar behavioural consequences as seen in PSD95 knockouts. All male and female mice were kept in different cabinets, where they were group housed or single housed depending on what was required for testing conditions. Mice underwent an extensive battery of tests; sexes were always tested separately, and significant results were prioritized for extensive publication. Observational tests included: dyadic social interactions test, resident/intruder test, ultrasonic vocalization test, the chimney test and behaviour in home cage as well as in the open field. Prior to testing, some mice cohort members had their genotypes verified at 4 weeks of age where hippocampal samples were taken, and their PSD95/93 protein level deficiency or normalcy was verified using qPCR methods.

Main findings of this study included: Both male and female PSD95(+/-) mice demonstrated hypersocialization behaviour within the dyadic social interaction tests. Non-social learning and processing of non-social stimuli appeared to be unaffected in these mice. Expression of NMDA and AMPA glutamate receptor were found to be normal. Male PSD95(+/-) mice were found to be more aggressive than PSD95(+/+) mice within resident/intruder testing. Female PSD95(+/-) mice had increased vocalization when exposed to an anesthetized mouse. PSD95 (+/-) mice moderately showed hypoactivity within the open field, corresponding to mild motor impairment. PSD93(-/-) knockout mice showed comparable hypersocialization behaviour with that of PSD95(+/-). This may indicate that PSD93 is also essential for the processing of social stimuli, but less so than PSD95, as PSD93 heterozygosity does not result in hypersocialization as seen in PSD95 mutants. PSD93(-/-) mice showed a severe motor deficit phenotype. In addition, PSD93 PSD93 is a closely related protein to PSD95. Both arose from also had increased expression within the hippocampus of the duplication and independent alteration of a common anPSD95(+/-) mice, which may be evidence for a partial substitucestral gene. Both of these genes play roles within the maintetion effect of PSD93 for PSD95 deficiency. These results suggest 143

that both PSD95 and PSD93 are involved in social processing and behaviour. In addition, PSD95 deficient mice, which have a milder motor deficit phenotype associated with them, could potentially be used to study neuropsychiatric disorders which feature social disinhibition or â&#x20AC;&#x153;social blindnessâ&#x20AC;? as a symptom.

Major Results PSD95(+/-) Mice: increased socialization, aggression and vocalization

social processing and behaviour may be impaired within PSD95 mice.

Female PSD95 mutants showed increased vocalization when faced with an anesthetized female mouse within an ultrasonic vocalization test. This test has been known to induce vocalization within mice (Winkler et al., 2018). Female PSD95 mutants were individually housed for 3 days prior to the test and were then introduced to an anesthetized female mouse. The number of vocalizations/calls as well as the time it took to initiate vocalization when faced with the anesthetized mouse was recorded [Fig. 2, K-L]. PSD95 mutant females were significantly faster to begin calling out when faced with the incapacitated mouse, and they also expressed more calls in total relative to PSD95 wildtype females. This is also evidence that normal social processing and social behaviour is impaired within PSD95 deficient mice.

Both male and female PSD95(+/-) mice showed increased socialization behaviour within dyadic social interaction testing.

Under this test, same sex/genotype pairs were introduced into a neutral testing cage and were allowed to interaction for 10 minutes. Socialization was then quantified in review of interaction tapes via the display of socially motivated actions within the pairings, such as: time in contact [Fig. 1, A-E], chasing, as well as snout-snout and anogenital sniffing behaviour. PSD95 (+/-) mouse pairs displayed significantly higher bouts of social interaction than that of PSD95(+/+) pairings. The social behavioural profile of PSD95 mutants are shown to be distinct from PSD95 knockout mice. Knockouts have been shown to have impaired motor performance, strange grooming behaviours, increased anxiety-related responses with a variable preference for social stimuli (Coley and Gao, 2019; Feyder et al., 2010).

This study shows that while PSD95 mutants display mild motor impairment, they display fairly normal behaviour in non-social testing conditions, but distinct altered behaviours within social situations. This may be due to the fact that PSD95 is still at significant levels within PSD95 mutants to allow for normal funcFigure 2. Male PSD95 mice underwent a resident/intruder test tioning in non-socially motivated circuits. where inter-male aggression was investigated; PSD95 mutant males were (I) quicker to attack a foreign intruder and (J) more likely to attack overall compared to PSD95 wildtype males.

Female PSD95 underwent an ultrasonic vocalization test, after being presented with a foreign anesthetized female. PSD95 mutant females (K) called out more and (L) took less time to express an initial call when presented with the incapacitated female, relative to PSD95 wildtype females. (Winkler et al., 2018) PSD93(-/-) Mice: increased socialization and motor impairment Figure 1. Time spent in contact within dyadic social interaction testing, same sex/genotype. Both PSD95(+/-) (A) male pairs and PSD93 knockout mice, rather than PSD93 mutants, were found (E) female pairs spent significantly more time in contact with to display hypersocial behaviour comparable to that of PSD95 mutants within this study. PSD93 knockout mice tended to be one another than PSD95(+/+) pairs. (Winkler et al., 2018) in contact with their same sex, same genotype partner more Male PSD95 mutants also showed heightened aggression tooften within testing relative to PSD93 mutants and PSD93 wards foreign males within the resident-intruder test. Within wildtype pairs [Fig. 3, A]. PSD93 knockout mice also displayed a this test, previously group housed PSD95 mutant males were prominent motor deficit phenotype which made observations housed in a single cage for four weeks. The cage was heated in regarding their behaviour more difficult. The finding that PSD93 order to increase the basal level of aggression as C57BL/6 mice knockout mice display motor deficits comes in contrast with have a low standard of inter-male aggression (Winkler et al., the findings of a prior study, which stated that PSD93 knockout 2018). Intruder males were introduced into the cage, and the results in no structural or functional abnormalities, including pairing was observed for 10 minutes or until first sign of attack motor impairment within mice (McGee et al., 2001). The results (biting). Latency of attack as well as number of attack events of of the originally reviewed paper potentially serve as evidence PSD95 mice were recorded [Fig. 2, I-J]. Male PSD95 mutants that both PSD95 and PSD93 are proteins implicated in the prowere significantly more likely to attack quicker and attack more cessing of social stimuli. relative to PSD95 wildtype mice. This is evidence that normal 144

creased vocalization when faced with an anesthetized mouse relative to PSD95 wildtypes. PSD93 knockout mice showed comparable increased socialization when compared to PSD95 mutants, but in addition to severe motor deficits.

Figure 3. (A) PSD93(-/-) knockout mice showed increased social interaction within same sex/genotype pairs. PSD93 knockout pairings spent more time in contact relative to both PSD93(+/-) mutants and PSD93(+/+) wildtype mice. (Winkler et al., 2018) PSD95(+/-): increased hippocampal PSD93 expression PSD95 mutant mice were found to have increased expression of PSD93 within the hippocampus. At 4 weeks of age, some PSD95 cohort mice were taken and had their relative PSD95 and PSD93 protein expression quantified using hippocampal enrichment preparations and qPCR. PSD95 mutants had a greater level of PSD93 protein expressed within the hippocampus relative to PSD95 wildtype mice [Fig. 4, B]. This could be potential evidence of functional or biological redundancy concerning PSD93 under PSD95 deficient conditions. Prior literature has argued that because these related proteins share common structural domains, they may operate in a similar fashion (McGee et al., 2001). Many more recent studies have shown, however, that PSD93 and PSD95 play some complimentary, but opposing roles, within the regulation and development of synapses (Favaro et al., 2018).

These results suggest that both PSD95 and PSD93 are involved in the processing and coordination of social behaviour. PSD95 mutants were shown to demonstrate hypersocial behaviour in the form of reduced social distance, which could be described as “social blindness”- these mice appeared to act more familiar with conspecifics, act more aggressively towards foreign intruders and vocalize more frequently when exposed to incapacitated mice. These mice may have the inability to process specific social signals and react appropriately with regard to other mice.

According to the authors, social recognition and memory were affected in PSD95 mutant mice, but non-social learning, general memory and behaviour towards non-social stimuli were found to be unaffected. Due to this, the authors argue that this evidence points to PSD95 as a prominent component of the processing of social stimuli and control of social behaviour. Much of the literature concerns itself with the observation of PSD95 knockout mice and the change of biomarkers within the brain in addition to severe learning and motor phenotypes. The investigation into PSD95 deficiency with regard to social control and behaviour seen within this study is novel in this respect.

This study also highlights the relative sensitivity of PSD95 levels within the brain as both PSD95(+/-) mice and PSD95(-/-) show distinct behavioural profiles. PSD93 knockout mice displayed similar hypersocial behaviour to that of PSD95 mutants; indicating that PSD93 may have a similar role with regard to the processing of social stimuli as PSD95. They found that PSD93 knockouts also had impaired motor movement, which is at odds with the findings of other studies which stated that PSD93 knockout mice showed no motor coordination problems (McGee et al., 2001).

PSD95 and PSD93 may show some level of functional and/or biological redundancy for one another, as PSD93 protein expression is increased within PSD95 mutant mice. The authors have argued that the increase of PSD93 expression within the hippocampus of PSD95 deficient mice may be due to PSD93 trying to compensate for PSD95 function. They do state however, that PSD95 and PSD93 are likely both implicated in the processing of social stimuli in their own unique ways. Increased PSD93 expression within PSD95 mutants probably cannot take over for all of PSD95’s roles, and overexpression of PSD93 may actually impair social neuronal circuits more. This falls in line with the current thinking, that although PSD93 and PSD95 are Figure 4. (B) Increased PSD93 protein expression within PSD95 structurally very similar, greater evidence supports that these (+/-) mutants relative to PSD95 wildtype mice. Hippocampal proteins act very differently in many contexts (Favaro et al., enrichment preparations were from PSD95 mutant mice at 4 2018). weeks of age, PSD93 protein expression was normalized to The authors conclude that this study’s findings show that GAPDH. (Winkler et al., 2018) PSD95(+/-) and PSD93 (-/-) mice demonstrate behavioural pheConclusions notypes which are the complete opposite as those seen in This study found that PSD95(+/-) mice had increased socializa- mouse models of monogenic autism. Within these models of tion behaviour as well as increased PSD93 expression within autism, mice display reduced social interaction, reduced interthe hippocampus. Male PSD95 mutants showed increased ag- male aggression, reduced vocalization and mild motor impairgression towards foreign intruders and females showed in- ment. PSD95 mutant mice in particular, show increased social 145

interaction, increased inter-male aggression and increased vocalization. In contrast, studies investigating complete knockout of PSD95 within mice, have found that PSD95(-/-) mice show strange behaviours that emulate autistic-like behaviour, such as those mentioned previously (Coley and Gao et al., 2019, Feyder et al., 2010). The mechanisms which underlie the strong behavioural distinction between PSD95 mutant and PSD95 knockout mice are still unknown. Nonetheless, the authors conclude that PSD95 mutant mice, which feature only mild motor impairment, may have the potential to be used as model systems for the investigation of neuropsychiatric conditions which feature symptoms such as hypersocialization and issues with social cue processing.

Critical Analysis This study featured a wide array of behavioural tests, which were impeccably done. Every test was performed multiple times, which supported the validity of their results. Statistics of all the tests performed were featured within the article document. Visualization methods were used in order to summarize the primary findings beautifully. Comparisons between knockout animals, mutant animals and wildtype animals were concisely stated. Genetic testing done prior to the commencement of behavioural tests ensured that PSD proteins were causally implicated within the results. Throughout the paper, the authors rely on other reports of PSD95 knockout behaviour in order to compare the behaviour of PSD95(+/-) mutants. The authors should have investigated PSD95 knockout animals in this study as well. This is because, reports regarding the social likability of PSD95 mice vary. Feyder et al. (2010), argues that PSD95 knockout animals show normal social interactions in pair testing, but increased preference for social stimuli. Coley and Gao (2019), a more recent study published after the study under review, operates under the assumption that PSD95 knockout mice display a complete lack of sociability. If the authors had included PSD95 knockout animals within the study, comparing the behavioural profiles of PSD95 mutant and PSD95 knockout mice may have been more valid. The results of this study also feature conflict. McGee et al (2001), found that the knockout of PSD93 within Purkinje neurons resulted in no structural or functional abnormalities, and no motor coordination issues within these mice. The paper under review however, states that PSD93 knockout animals within this study demonstrated distinct motor impairment phenotypes - so much so, that PSD93 knockout animals were unable to complete every test within the study. In addition, observing animals and discussing their behaviour can only further science so far.

Causal mechanisms explaining how PSD95 or PSD93 may be implicated in social processing networks must be elucidated.

This paper did not emphasize how they believe these proteins contribute to the processing of social cues or the processing of behaviour. The authors merely stated that causal mechanisms can only be speculated upon. Furthermore, one of the main conclusions of this paper featured this idea that because PSD95 (+/-) demonstrated hypersocialization, apparent social blindness and only mild motor deficits, these mice would make adequate models in order to study neuropsychiatric conditions which feature similar symptoms. The authors should hold off

this recommendation prior to understanding functionally how PSD95 deficiency may cause behaviours emulating social blindness within mice. After reviewing this paper, experiments which look at the social processing functionality of PSD95 and PSD93 should be done. Experiments which look at localization of PSD proteins within the development of young mice into adulthood should be looked at. Objective biomarkers should be used in order to describe the dynamic nature and function of the PSD proteins under study. By understanding the native function of PSD95 and PSD93, impairments of their function can be better understood, and resulting behavioural symptoms can be better treated.

Future Directions In order to further this area of research, the biological mechanisms which account for hypersocialization within PSD95 mutant and PSD93 knockout mice should be investigated. During qPCR testing, the researchers of this study found that expression of the glutamate receptor NMDA subunits NR1, NR2A, and NR2B, along with that of AMPA GluR1 within PSD95 mutants were unaffected. Other research has also found that reduction of PSD95 levels does not impact NMDA expression, rather, it impacts NMDA modulation and clustering. (Chen et al., 2015; McGee et al., 2001; Scannevin and Huganir, 2000). Reduction in glutamate receptor congregation within the postsynaptic density framework has been associated with an increased number of silent synapses within the brain of PSD95 and PSD93 knockout mice (Chen et al., 2015). Visualization studies, which would be tailored to the region of the brain under investigation, could be done to observe the congregation of NMDA and AMPA receptors in glutaminergic areas associated with the government of social behaviour. If congregation of receptors is impaired within PSD95(+/-) or PSD93(-/-) mice, this may explain the malfunction of social processing as synaptic connections overall are impaired. If congregation of NMDA and AMPA receptors are normal, then this could mean that the PSD95 levels within PSD (+/) or PSD93 levels within PSD93(-/-) may be sufficient in order to allow for normal congregation of these essential receptors. Age of the mice may also be a confounding factor: presence of PSD95/93 and their role in synaptic plasticity and development varies through development (Lambert et al., 2017; McGee et al., 2001).

In addition, research produced within the same year as the original review articleâ&#x20AC;&#x2122;s publication, supports a causal pathway between PSD95 and conditioned fear. Li et al. (2018), found that NMDAR stimulation can cause an increase in nitric oxide (NO), which must link with PSD95, in order for fear memory formation to occur. Nitric oxide formed must link to PSD95, as PSD95 is associated with neuronal oxide synthase(nOS), which is implicated further on in the pathway. If this NO/PSD95 interaction is impaired, then fear memory is selectively reduced.

Reduction in the available PSD95 to engage with this key interaction may explain why PSD95(+/-) mice display social blindness and hypersocialization features: their social fear consolidation may be impaired. Visualization studies could also be done in order to see the relationship between nitric oxide and PSD95 within the amygdala, which is a key part of the brain associated

146

with fear-based memory construction. If this pathway is implicated in the hypersocialization behaviour seen within PSD95 mutant mice, then the NO/PSD95 relationship should be investigated to see if it struggles, or malfunctions, under reduced PSD95 conditions. This could explain the social behaviour of PSD95 mutant within the original study under review. If the interaction between NO and PSD95 appear to be normal, other members of the pathway may be implicated instead, such as nOS which is directly associated with PSD95.

147

REFRENCES 1.

Bustos, F. J., Ampuero, E., Jury, N., Aguilar, R., Falahi, F., Toledo, J., Ahumada, J., Lata, J., Cubillos, P., Henríquez, B., Guerra, M. V., Stehberg, J., Neve, R. L., Inestrosa, N. C., Wyneken, U., Fuenzalida, M., Härtel, S., Sena-Esteves, M., Varela-Nallar, L., … van Zundert, B. (2017). Epigenetic editing of the Dlg4/PSD95 gene improves cognition in aged and Alzheimer’s disease mice. Brain: A Journal of Neurology, 140(12), 3252–3268. https://doi.org/10.1093/brain/awx272

2.

Chen, X., Levy, J. M., Hou, A., Winters, C., Azzam, R., Sousa, A. A., Leapman, R. D., Nicoll, R. A., & Reese, T. S. (2015). PSD95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density. Proceedings of the National Academy of Sciences of the United States of America, 112(50), E6983-6992. https://doi.org/10.1073/ pnas.1517045112

3.

Coley, A. A., & Gao, W.-J. (2018). PSD95: A synaptic protein implicated in schizophrenia or autism? Progress in NeuroPsychopharmacology & Biological Psychiatry, 82, 187–194. https://doi.org/10.1016/j.pnpbp.2017.11.016

4.

Coley, A. A., & Gao, W.-J. (2019). PSD-95 deficiency disrupts PFC-associated function and behavior during neurodevelopment. Scientific Reports, 9(1), 9486. https://doi.org/10.1038/s41598-019-45971-w

5.

El-Husseini, A. E., Schnell, E., Chetkovich, D. M., Nicoll, R. A., & Bredt, D. S. (2000). PSD-95 involvement in maturation of excitatory synapses. Science (New York, N.Y.), 290(5495), 1364–1368.

6.

Favaro, P. D., Huang, X., Hosang, L., Stodieck, S., Cui, L., Liu, Y.-Z., Engelhardt, K.-A., Schmitz, F., Dong, Y., Löwel, S., & Schlüter, O. M. (2018). An opposing function of paralogs in balancing developmental synapse maturation. PLoS Biology, 16 (12), e2006838. https://doi.org/10.1371/journal.pbio.2006838

7.

Feyder, M., Karlsson, R.-M., Mathur, P., Lyman, M., Bock, R., Momenan, R., Munasinghe, J., Scattoni, M. L., Ihne, J., Camp, M., Graybeal, C., Strathdee, D., Begg, A., Alvarez, V. A., Kirsch, P., Rietschel, M., Cichon, S., Walter, H., Meyer-Lindenberg, A., … Holmes, A. (2010). Association of Mouse Dlg4 (PSD-95) Gene Deletion and Human DLG4 Gene Variation With Phenotypes Relevant to Autism Spectrum Disorders and Williams’ Syndrome. American Journal of Psychiatry, 167(12), 1508– 1517. https://doi.org/10.1176/appi.ajp.2010.10040484

8.

Gao, C., Tronson, N. C., & Radulovic, J. (2013). MODULATION OF BEHAVIOR BY SCAFFOLDING PROTEINS OF THE POSTSYNAPTIC DENSITY. Neurobiology of Learning and Memory, 105, 3–12. https://doi.org/10.1016/j.nlm.2013.04.014

9.

Lambert, J. T., Hill, T. C., Park, D. K., Culp, J. H., & Zito, K. (2017). Protracted and asynchronous accumulation of PSD95family MAGUKs during maturation of nascent dendritic spines. Developmental Neurobiology, 77(10), 1161–1174. https:// doi.org/10.1002/dneu.22503

10.

Li, L.-P., Dustrude, E. T., Haulcomb, M. M., Abreu, A. R., Fitz, S. D., Johnson, P. L., Thakur, G. A., Molosh, A. I., Lai, Y., & Shekhar, A. (2018). PSD95 and nNOS interaction as a novel molecular target to modulate conditioned fear: Relevance to PTSD. Translational Psychiatry, 8(1), 155. https://doi.org/10.1038/s41398-018-0208-5

11.

Mardones, M. D., Jorquera, P. V., Herrera-Soto, A., Ampuero, E., Bustos, F. J., van Zundert, B., & Varela-Nallar, L. (2019).

PSD95 regulates morphological development of adult-born granule neurons in the mouse hippocampus. Journal of Chemical Neuroanatomy, 98, 117–123. https://doi.org/10.1016/j.jchemneu.2019.04.009

12.

McGee, A. W., Topinka, J. R., Hashimoto, K., Petralia, R. S., Kakizawa, S., Kauer, F., Aguilera-Moreno, A., Wenthold, R. J., Kano, M., & Bredt, D. S. (2001). PSD-93 Knock-Out Mice Reveal That Neuronal MAGUKs Are Not Required for Development or Function of Parallel Fiber Synapses in Cerebellum. Journal of Neuroscience, 21(9), 3085–3091. https:// doi.org/10.1523/JNEUROSCI.21-09-03085.2001

13.

Proctor, D. T., Coulson, E. J., & Dodd, P. R. (2010). Reduction in post-synaptic scaffolding PSD-95 and SAP-102 protein levels in the Alzheimer inferior temporal cortex is correlated with disease pathology. Journal of Alzheimer’s Disease: JAD, 21 (3), 795–811. https://doi.org/10.3233/JAD-2010-100090

14.

Scannevin, R. H., & Huganir, R. L. (2000). Postsynaptic organisation and regulation of excitatory synapses. Nature Reviews Neuroscience, 1(2), 133–141. https://doi.org/10.1038/35039075

15.

Winkler, D., Daher, F., Wüstefeld, L., Hammerschmidt, K., Poggi, G., Seelbach, A., Krueger-Burg, D., Vafadari, B., Ronnenberg, A., Liu, Y., Kaczmarek, L., Schlüter, O. M., Ehrenreich, H., & Dere, E. (2018). Hypersocial behavior and biological redundancy in mice with reduced expression of PSD95 or PSD93. Behavioural Brain Research, 352, 35–45. https:// doi.org/10.1016/j.bbr.2017.02.011 148

16.

Won, S., Levy, J. M., Nicoll, R. A., & Roche, K. W. (2017). MAGUKs: Multifaceted synaptic organizers. Current Opinion in Neurobiology, 43, 94–101. https://doi.org/10.1016/j.conb.2017.01.006

17.

Yang, Y., Geng, Y., Jiang, D., Ning, L., Kim, H. J., Jeon, N. L., Lau, A., Chen, L., & Lin, M. Z. (2019). Kinase pathway inhibition restores PSD95 induction in neurons lacking fragile X mental retardation protein. Proceedings of the National Academy of Sciences of the United States of America, 116(24), 12007–12012. https://doi.org/10.1073/pnas.1812056116

18.

Zhang, J., Saur, T., Duke, A. N., Grant, S. G. N., Platt, D. M., Rowlett, J. K., Isacson, O., & Yao, W.-D. (2014). Motor Impairments, Striatal Degeneration, and Altered Dopamine-Glutamate Interplay in Mice Lacking PSD-95. Journal of Neurogenetics, 28(1–2), 98–111. https://doi.org/10.3109/01677063.2014.892486

19.

Zheng, C.-Y., Seabold, G. K., Horak, M., & Petralia, R. S. (2011). MAGUKs, synaptic development, and synaptic plasticity.

The Neuroscientist: A Review Journal Bringing Neurobiology, Neurology and Psychiatry, 17(5), 493–512. https:// doi.org/10.1177/1073858410386384

149

How a Hormone that Helps with Sleep can Reduce the Effects of Diabetes Towards the Hippocampus Angenelle Eve Rosal

Diabetes is a metabolic disease that has been studied to cause cognitive decline as it can damage and deregulate brain regions that are important for learning and memory, like the hippocampus. While there is still no explanation why diabetes influences hippocampal damage and dysfunction, researchers are aiming to find solutions to ameliorate these changes. Melatonin, a hormone important for sleep, has been previously investigated to be a promising neuroprotective agent. The present study

by Wongchitrat et al addresses the potential benefits of melatonin towards hippocampal impairment caused by diabetes. Male Wistar Rats were first divided into a normal fat diet group or a high-fat diet group injected with streptozotocin (HFD-fed/STZ) , which induced a diabetic phenotype. At 7 weeks, three groups were produced when some HFD-fed/STZ rats were given melatonin injections for 4 weeks daily(HFD-fed/STZ with melatonin). After treatment ended, the three groups were tested on the Morris Water Maze, where the HFD-fed/STZ group with melatonin had rescued memory impairments, and not the HFD-fed/STZ group. All rats were then immediately anesthetized and their hippocampal tissues were homogenized for Western Blot Analysis. The HFD-fed/STZ group with melatonin were analyzed to have an increase in proteins involved in hippocampal neurogenesis,synaptic plasticity, as well as brain insulin signalling and a decrease in proteins involved in astrocytic activity. This was not observed in the HFD-fed/STZ group. Overall, this study demonstrated that melatonin helps attenuate hippocampal damage and may be a promising therapeutic for diabetic individuals who are suffering from cognitive deficits.

Key Words: Diabetes, hippocampus, diabetic encephalopathy, brain damage, dysfunction, neurogenesis, synaptic plasticity, astrocytes, insulin signalling, cognition, memory.

150

Background and Introduction Diabetes is a metabolic disease that is known to cause hyperglycemia and promote a lack of insulin production 1. Along with its notable associations with obesity, numerous studies have shown great interest in investigating its effects towards the brain1-3. Termed to be “diabetic encephalopathy”, diabetic individuals were first observed to have poor performance when completing cognitive tasks 3. However, though many publications regarding this matter have been well documented, understanding why diabetes damage brain regions that aid learning and memory is still poorly investigated, like in the hippocampus3-5.In diabetic-animal models, lesions in the hippocampus due to increased astrocytic activity and oxidative stress were analyzed, causing significant memory deficits 6. Additionally, diabetes induced through a high-fat diet (HFD) caused a deregulation of hippocampal neurogenesis7.Due to these occurrences, numerous research have tried to understand how these impairments can be counteracted4,8. Melatonin, a hormone that aids sleep, has been investigated to provide neuroprotection, as observed to reduce oxidative damage in mice with traumatic brain injury9-10. Currently, melatonin has also been determined to participate in glucose metabolism 10. In this context, the absence of melatonin in the blood was observed to induce insulin resistance in peripheral muscles, producing diabetic symptoms in studied animals10. Based on this evidence, it is suggested that reduced melatonin levels may be correlated with the development of diabetes. Recently, melatonin treatment was found to be beneficial in attenuating insulin resistance in obese rats by increasing insulin receptors (IRs) and downstream insulin signalling proteins such as phosphorylated extracellular signal-regulated kinases ( p-ERK) in the hypothalamus11. However, its benefits in hippocampal damage caused by diabetes has not been well-studied thus far. Wongchitrat et al examined the potential effects of melatonin towards the hippocampus in diabetic-like models. Male Wistar Rats were first divided into a normal fat diet group or a HFD group injected with streptozotocin (HFD-fed/STZ) that caused a diabetic-like phenotype. Some of the HFD-fed/STZ rats were then given injections of melatonin 4 weeks daily(HFD-fed/STZ with melatonin), producing three groups overall. After treatment, the groups were trained and tested in the Morris Water Maze. The HFD-fed/STZ group with melatonin had a significant rescue in spatial memory deficits than the HFD-fed/STZ group. All rats were then immediately anesthetized, and their hippocampal tissues were homogenized for Western Blot Analysis. Proteins involved in astrocytic activity, hippocampal neurogenesis, and synaptic plasticity were analyzed to observed how diabetes alter these processes. Additionally, proteins associated with brain insulin signalling were also investigated due to high levels of IRs in the hippocampus 4. An increase in proteins involved in

hippocampal neurogenesis, synaptic plasticity, as well as brain insulin signalling and a decrease in astrocytic proteins were examined in the hippocampal tissues of the HFD-fed/STZ group with melatonin.This outcome was not observed in the HFD-fed/ STZ group. Comprehensively, this study outlined that melatonin reduces hippocampal dysfunction caused by diabetes and may be a potential therapeutic for diabetic individuals experiencing memory deficits.

Major Results Spatial Memory Testing The HFD-fed/STZ group had a longer escape time than the control in the Morris Water Maze. The HFD-fed/STZ group with melatonin had reduced memory deficits, leading to a shorter escape time and enhanced retention memory instead. These results validated an existing hypothesis that diabetes does cause cognitive dysfunction and that melatonin is able to reduce this 3,5.

Astrocytic Activity Expressions of the glial fibrillary acidic protein (GFAP), a protein marker for astrocytes, was inspected in all homogenized hippocampal tissues of the three groups to assess alterations in astrocytic activity. The HFD-fed/STZ group had increased levels of GFAP than the HFD-fed/STZ group with melatonin, validating an existing hypothesis that diabetes induces memory impairments by increasing astrogliosis 4,6.

Figure 1: Normalization of Western Blot data using actin illustrated that the HFD-fed/STZ group had an increased expression of GFAP than the HFD-fed/STZ group with melatonin. Figure adapted from “Melatonin attenuates the high-fat diet and streptozotocin-induced reduction in rat hippocampal neurogenesis.” by Wongchitrat et al. Neurochemistry International 2016;100:97-109.

151

Hippocampal Neurogenesis Three neurogenesis protein markers were investigated in all homogenized hippocampal tissues of the three groups to assess changes in hippocampal neurogenesis; nestin, to visualize neural stem cells, doublecortin(DCX), to visualize migrating neurons, and β-III tubulin, to visualize mature neurons 4. A decrease in all three markers in the HFD-fed/STZ group was observed while the HFD-fed/STZ group with melatonin had an increase. These observations support the idea that diabetes has a negative impact towards hippocampal neurogenesis and melatonin can attenuate this by increasing neuronal proliferation and migration7,12.

Figure 3: A-B) Normalization of Western Blot data using actin illustrated that the HFD-fed/STZ group with melatonin had an increase in Synaptophysin and PSD-95 expression in comparison to the HFD-fed/STZ group. C) NR2A subunits that aid NMDA receptor expression increased in the HFD-fed/STZ group with melatonin. Figure adapted from Wongchitrat et al. Melatonin attenuates the high-fat diet and streptozotocin-induced reduction in rat hippocampal neurogenesis. Neurochemistry International. 2016;100:97-109.

Insulin Receptors and p-ERK signalling.

Since deficits in brain insulin signalling was studied to aid cognitive decline and there are high IRs levels in the hippocampus, changes in proteins involved in the IR/ERK signalling pathway in all homogenized hippocampal tissues were investigated 4,14.

The HFD-fed/STZ group had a decrease in IR-β levels, a subunit in IRs, and p-ERK levels in comparison to the HFD-fed/STZ group with melatonin that had an increase instead 4. These results validate the existing hypothesis that protein structures Figure 2: Normalization of Western Blot data using actin illus- involved in insulin signalling are down-regulated by diabetes trated that the HFD-fed/STZ group with melatonin had an in- and melatonin can counteract this by enhancing their expression4,14. creased expression of protein markers involved in neurogenesis in comparison to the HFD-fed/STZ group ; nestin(A), DCX(B) and β- III tubulin(C). Figure adapted from “Melatonin attenuates the high-fat diet and streptozotocin-induced reduction in rat hippocampal neurogenesis.” by Wongchitrat et al. Neurochemistry International. 2016;100:97-109.

Synaptic Proteins and NMDA Receptors Expression of two presynaptic and postsynaptic proteins in all homogenized hippocampal tissues were examined to study changes in synaptic plasticity; synpatophysin and PSD-95 respectively. The HFD-fed/STZ group had reduced levels of these two proteins in comparison to the HFD-fed/STZ group with melatonin that had enhanced expressions. This validates the existing hypothesis that neuronal synapses are disrupted by diabetes13. Further, NMDA receptors were also studied as they play a role in synpatogenesis4. The HFD-fed/STZ group also had a reduction in the NR2A protein subunit of NMDA receptors, reducing NMDA receptor expression.These results verify that diabetes deregulates synaptic functions and melatonin is suggested to counteract this4,13.

Figure 4: Normalization of Western Blot data using actin and ERK 1/2 illustrated that the HFD-fed/STZ group with melatonin had an increase in IR-β and p-ERK expression in comparison to the HFD-fed/STZ group. Figure adapted from “Melatonin attenuates the high-fat diet and streptozotocin-induced reduction in rat hippocampal neurogenesis.” by Wongchitrat et al. Neurochemistry International. 2016;100:97-109.

Conclusion and Discussion Evidently, Wongchitrat et al demonstrated that melatonin is able to reduce hippocampal dysfunction caused by diabetes.

Hippocampal damage was attenuated in the HFD-fed/STZ group with melatonin due to the fact that astrocytic activity was reduced and hippocampal neurogenesis, synaptic plasticity and brain insulin signalling was enhanced, as observed through changes in specific protein marker expressions. The authors suggested a novel hypothesis where a deregulation of the insulin pathway may explain why a dysfunction in hippocampal neurogenesis and synaptic plasticity is associated with diabetes. Overall, melatonin was suggested to be a potential thera-

152

peutic for diabetic individuals experiencing memory loss. Taking into consideration that numerous researchers have previously tested potential treatments that may reduce diabetes-induced hippocampal damage, this paper provides additional insight on how melatonin can also be a promising remedy. Melatonin was observed in this study to counteract the dysfunction of important hippocampal mechanisms, which are normally needed for the proper functioning of the hippocampus. These results were consistent with previous literature that assessed the beneficial role of melatonin towards the brain9,11. Further, the HFDfed/STZ group had significant cognitive decline and deficits in hippocampal processes needed for learning and memory. This is also consistent with previous research that studied the influence of diabetes towards the hippocampus and cognition 6,7,12-14.

The observed impact that melatonin had in this paper is currently relevant in research as the number of diagnosed individuals with diabetes have grown over the past years3,15. Diabetes associated with cognitive decline has been reviewed to possibly cause global future health problems due to an aging population3,15. Thus, the neuroprotective functions of melatonin explored in this study may help reduce this future complication ahead of time.

Critical Analysis The authors of this study identified that melatonin has beneficial effects in attenuating hippocampal dysfunction caused by diabetes. As a result, it has been implicated that it can be a potential treatment in diabetic individuals with cognitive decline.

However, it should be noted that the study did not provide insight regarding the long-term effects of melatonin treatment, as observed when daily melatonin injections were given for only 4 weeks. This should be taken into account considering that diabetes is well known to be a chronic disorder in humans that induces long-term complications 16-18. Not only that, but longitudinal studies have also demonstrated that chronic hyperglycemia in elderly due to type 2 diabetes had rapid cognitive decline17-18. Thus, authors should conduct longer studies to further examine the effects of long-term melatonin treatment, which can benefit human clinical trials. Using male Wistar rats is also a caveat in this study. Several researchers have investigated that the effects of diabetes towards the hippocampus are more prominent in females19-20. For instance, severe cognitive impairment and bigger brain injuries following ischemic stroke was examined in some reports to be prominent in diabetic female animals than males19-20. Therefore, the authors should investigate the effects of melatonin in both sexes to prevent gender bias. Further, although the hippocampus plays a major role in learning and memory, there are also other brain regions affected by diabetes that may also influence cognitive decline. A study utilizing magnetic resonance imaging on diabetic aged

patients revealed that poor cognitive performance in the Stroop test was associated with thalamus lesions, a brain region known to aids cognitive function 21.Thus, future experiments should investigate the effects of melatonin in other deregulated brain regions influenced by diabetes.

Future Directions To elaborate on future experiments based on the critical analysis above, the authors should consider in examining the longterm effects of melatonin as it was not justified in their paper.

Longitudinal studies with the same experimental design of the original study should be performed to fulfill this investigation.

However, both adult male and female rats will be randomized into the three groups to prevent gender bias. Additionally, melatonin treatment should also be given for a extended period before hippocampal tissues are homogenized. The HFD-fed/STZ group with melatonin are still expected to have better memory and an enhancement of proteins in particular hippocampal mechanisms studied in the original study after prolonged melatonin treatment. This implicates that melatonin can be used long-term to effectively attenuate diabetic-induced hippocampal dysfunction. A decrease in the proteins and prominent cognition decline in these rats despite long-term melatonin treatment may suggest that melatonin is not beneficial for chronic use and can only induce short term benefits. Further, the authors should assess the beneficial effects of melatonin in other damaged brain regions caused by diabetes. Since thalamic damage was linked to memory impairments in diabetic elderly, an experiment that examines the effects of melatonin in the thalamus of diabetic-like rats should be performed. Two groups should be assessed, where diabetic-like rats are either given melatonin or saline injections. Using the Morris Water Maze to investigate memory deficits, diabetic-like rats treated with melatonin are expected to have a shorter escape time than rats treated with saline, implicating rescued cognitive deficits. MRI imaging and staining for myelin and axon cells in the thalamus should then be conducted considering that a loss of these cells indicate thalamic damage, as observed in multiple sclerosis 22.

The thalamus of diabetic-like rats treated with melatonin are expected to have less damage due to more thalamic axons and myelin cell staining visualized, suggesting that this may have contributed to the rescued memory deficits. A lesioned thalamus in diabetic-like rats given melatonin treatment may suggest that melatonin only attenuates hippocampal damage, in which rescued memory was only due to an enhancement of hippocampal processes that aid cognition. Ultimately, these experiments further investigate the benefits of melatonin towards diabetic encephalopathy in various circumstances.

153

REFRENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

Piero M. Diabetes mellitus – a devastating metabolic disorder. Asian Journal of Biomedical and Pharmaceutical Sciences.

2015;4(40):1-7. doi:10.15272/ajbps.v4i40.645.

Roriz-Filho JS, Sá-Roriz TM, Rosset I, et al. (Pre)diabetes, brain aging, and cognition. Biochimica et Biophysica Acta (BBA) Molecular Basis of Disease. 2009;1792(5):432-443. doi:10.1016/j.bbadis.2008.12.003.

Moheet A, Mangia S, Seaquist ER. Impact of diabetes on cognitive function and brain structure. Annals of the New York Academy of Sciences. 2015;1353(1):60-71. doi:10.1111/nyas.12807.

Wongchitrat P, Lansubsakul N, Kamsrijai U, Sae-Ung K, Mukda S, Govitrapong P. Melatonin attenuates the high-fat diet and streptozotocin-induced reduction in rat hippocampal neurogenesis. Neurochemistry International. 2016;100:97-109. doi:10.1016/j.neuint.2016.09.006.

Neves G, Cooke SF, Bliss TVP. Synaptic plasticity, memory and the hippocampus: a neural network approach to causality.

Nature Reviews Neuroscience. 2008;9(1):65-75. doi:10.1038/nrn2303.

Moghaddam HK, Baluchnejadmojarad T, Roghani M, et al. Berberine Ameliorate Oxidative Stress and Astrogliosis in the Hippocampus of STZ-Induced Diabetic Rats. Molecular Neurobiology. 2013;49(2):820-826. doi:10.1007/s12035-013-85597.

Boitard C, Etchamendy N, Sauvant J, et al. Juvenile, but not adult exposure to high-fat diet impairs relational memory and hippocampal neurogenesis in mice. Hippocampus. 2012;22(11):2095-2100. doi:10.1002/hipo.22032.

Muriach M, Bosch-Morell F, Alexander G, et al. Lutein effect on retina and hippocampus of diabetic mice. Free Radical Biology and Medicine. 2006;41(6):979-984. doi:10.1016/j.freeradbiomed.2006.06.023.

Ding K, Wang H, Xu J, et al. Melatonin stimulates antioxidant enzymes and reduces oxidative stress in experimental traumatic brain injury: the Nrf2–ARE signaling pathway as a potential mechanism. Free Radical Biology and Medicine.

2014;73:1-11. doi:10.1016/j.freeradbiomed.2014.04.031.

Cipolla-Neto J, Amaral FG, Afeche SC, Tan DX, Reiter RJ. Melatonin, energy metabolism, and obesity: a review. Journal of Pineal Research. 2014;56(4):371-381. doi:10.1111/jpi.12137.

Zanuto R, Siqueira-Filho MA, Caperuto LC, et al. Melatonin improves insulin sensitivity independently of weight loss in old obese rats. Journal of Pineal Research. 2013;55(2):156-165. doi:10.1111/jpi.12056.

Ho N, Sommers MS, Lucki I. Effects of diabetes on hippocampal neurogenesis: Links to cognition and depression. Neuroscience & Biobehavioral Reviews. 2013;37(8):1346-1362. doi:10.1016/j.neubiorev.2013.03.010.

Abbas T, Faivre E, Hölscher C. Impairment of synaptic plasticity and memory formation in GLP-1 receptor KO mice: Interaction between type 2 diabetes and Alzheimers disease. Behavioural Brain Research. 2009;205(1):265-271. doi:10.1016/ j.bbr.2009.06.035.

Mcnay EC, Ong CT, Mccrimmon RJ, Cresswell J, Bogan JS, Sherwin RS. Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance. Neurobiology of Learning and Memory. 2010;93(4):546-553. doi:10.1016/ j.nlm.2010.02.002.

Zeytinoglu M, Huang ES. Diabetes and Aging: Meeting the Needs of a Burgeoning Epidemic in the United States. Health Systems & Reform. 2015;1(2):128-141. doi:10.1080/23288604.2015.1037042.

Lloyd A, Sawyer W, Hopkinson P. Impact of Long-Term Complications on Quality of Life in Patients with Type 2 Diabetes not Using Insulin. Value in Health. 2001;4(5):392-400. doi:10.1046/j.1524-4733.2001.45029.x.

Kanaya AM, Barrett-Connor E, Gildengorin G, Yaffe K. Change in Cognitive Function by Glucose Tolerance Status in Older Adults. Archives of Internal Medicine. 2004;164(12):1327. doi:10.1001/archinte.164.12.1327.

Logroscino et al. “Prospective study of type 2 diabetes and cognitive decline in women aged 70–81 years. Obstetrics & Gynecology. 2004;103(6):1339. doi:10.1097/01.aog.0000128995.34224.7c.

Sakata A, Mogi M, Iwanami J, et al. Female exhibited severe cognitive impairment in type 2 diabetes mellitus mice. Life Sciences. 2010;86(17-18):638-645. doi:10.1016/j.lfs.2010.03.003.

Li W, Ward R, Valenzuela JP, Dong G, Fagan SC, Ergul A. Diabetes Worsens Functional Outcomes in Young Female Rats: Comparison of Stroke Models, Tissue Plasminogen Activator Effects, and Sexes. Translational Stroke Research. 2017;8 (5):429-439. doi:10.1007/s12975-017-0525-7.

Akisaki T, Sakurai T, Takata T, et al. Cognitive dysfunction associates with white matter hyperintensities and subcortical atrophy on magnetic resonance imaging of the elderly diabetes mellitus Japanese elderly diabetes intervention trial (JEDIT). Diabetes/Metabolism Research and Reviews. 2006;22(5):376-384. doi:10.1002/dmrr.632.

Minagar A, Barnett MH, Benedict RHB, et al. The thalamus and multiple sclerosis: Modern views on pathologic, imaging, and clinical aspects. Neurology. 2013;80(2):210-219. doi:10.1212/wnl.0b013e31827b910b.

154

The effects of oxytocin administration in the attenuation of autistic symptoms in ASD mice models Maureen Sahar

Autism Spectrum Disorder, or ASD, is characterized as an early-onset neurodevelopment condition that results in a decline of social interaction and consistent repetitive practices. With no treatment available, alongside an expanding prevalence in diagnoses, current research is directed towards treating symptomatic behaviours and neuronal irregularities. More specifically, recent studies have begun exploring the use of oxytocin as a therapeutic agent in alleviating social cognitive behaviours in ASD models (Wang et al., 2018. Matsuo et al., 2020). Research conducted by Wang et al.

(2018) investigates how the administration and therapeutic use of oxytocin, a hormone associated with neuromodulation and social interactions, may help improve symptoms synonymous with autism in autistic mice models (Wang et al., 2018). The results indicate that after oxytocin was administered to ASD mice, their overall social cognitive behaviours, as seen through their stress responses, improved significantly compared to autistic mice that did not receive doses of oxytocin (Wang et al., 2018). In addition to these results, the autistic mice models who received oxytocin displayed a significant decrease in oxidative stress, inflammation, and activated microglia in their hippocampal and amygdala tissue samples in comparison to their autistic counterparts which did not receive oxytocin (Wang et al., 2018). In essence, these findings depict promising novel evidence for the use of oxytocin as a therapeutic agent for the mitigation of autistic symptoms in ASD models.

Key Words: Autism Spectrum Disorder, ASD, Oxytocin, oxidative stress, inflammation, microglia, social cognition

155

BACKGROUND AND INTRODUCTION Autism Spectrum Disorder, or ASD, is defined as an early -onset neurodevelopmental condition that results in attenuated social cognitive behaviours and increased repetitive actions.

Typically seen before the age of three, ASD symptoms are most notably observed through a severe deficit of attention, communication, and eye contact, and an increase in anxiety and restricted behaviours (Zachor et al., 2020).

The prevalence of ASD has been definitively increasing throughout the decades, with recent studies asserting higher contingencies of male diagnoses compared to female diagnoses at a 4:1 ratio (Irding et al., 2015. Halladay et al., 2015). Although the cases of autism are increasing significantly, there is currently no available treatment for ASD, due to the lack of understanding in the initiation of ASD development. This in part can be attributed to its variance in initial components that may be caused by one or several genetic, epigenetic, or environmental factors (Neal et al., 2012).

Despite this gap in knowledge, there has been growing evidence that hormones, such as oxytocin, can be used as potential treatments for alleviating ASD symptoms (LoParo et al., 2015). Oxytocin is a hormone encoded by the OXT receptor gene, OXTR, and is primarily released by the hypothalamus; its fundamental function has been shown to be strongly associated with social behaviour and the knockout of the OXTR gene can cause malfunctions in the neuroendocrine system which assists in the control of an organism’s social interaction (Lazzari et al., 2019). Sequencing of the OXTR gene has revealed the attenuation of distinct SNPs that encode varying social phenotypes that can cause distinct alterations in temperaments and behavioural impairments (Tost et al., 2010). In addition to these findings, ASD patients also display greater levels of reactive oxidative species, ROS, which is theorized to cause neuroinflammatory responses (Osredkar et al., 2019. Pangrazzi et al., 2020. PopaWagner et al., 2013). Consecutive research, in regards to the modulation of neuroinflammation, has shown oxytocin’s beneficial role as an anti-inflammatory regulator due to its ability in diminishing pro-inflammatory cytokine production (Inoue et al., 2019). As a result of oxytocin’s dynamic role in social interaction, it has been suggested that the low levels of the hormone found in ASD patients may play a constitutional role in ASD symptoms (LoParo et al., 2015).

Corresponding to the promising role that Oxytocin may play in alleviating ASD symptoms, more research is required to understand its benefits as a therapeutic treatment. Accordingly, in a study conducted by Wang et al. (2018), the researchers observed the effects of oxytocin administration in autistic mice models in order to determine whether or not oxytocin can mitigate ASD symptoms. With the use of several stress-inducing tests and subsequent examination of hippocampal and amygda-

la tissue samples, Wang et al. (2018) reported that ASD mice that were administered oxytocin displayed significantly lower ASD behavioural and cognitive symptoms and reduced inflammatory and oxidative stress responses in neural tissue samples.

Similarly, the ASD mice which received a dose of oxytocin more closely resembled the control group rather than the ASD group who did not receive oxytocin (Wang et al., 2018). Ultimately, these results implicate that oxytocin is a plausible therapeutic agent for potential treatments of ASD behavioural and cognitive symptoms.

MAJOR RESULTS Oxytocin Administration Reduces ASD In Mice After undergoing four tests that observe behavioural and cognitive dispositions, ASD mice that received oxytocin treatment displayed significantly decreased ASD behaviours when compared to ASD mice that did not receive oxytocin. More specifically, oxytocin treated mice were observed to have significantly elevated social preference indexes as seen by their inclination to interact with other mice placed in neighbouring chambers. These results parallel those of control groups and can be linked to improvements in anxiety and cognitive functioning of the oxytocin treated mice.

Similar experiments conducted additionally exemplify that after Cntnap2 mutant mice models, which have a high prevalence of ASD, were treated with oxytocin, their preference for social interaction with an unfamiliar mouse significantly increased (Peñagarikano et al., 2015). In addition to these findings, results attained by a separate BALB/cByJ ASD mouse model correspondingly display that ASD mice who received oxytocin treatment spent longer periods of time socially interacting with an unfamiliar mouse (Moy et al., 2019).

Figure 1. displays an ASD mouse model undergoing a 3chambered social interaction test, which observes a mouse’s inclination to interact with an inanimate object or an unfamiliar mouse. The results display a significant increase in an ASD mouse model’s preference to interact with an unfamiliar mouse after treatment with oxytocin, OXT, rather than the inanimate object (Peñagarikano et al., 2015).

156

Although Wang et al. (2018) found that there was no significant alteration in the amount of marbles buried by the oxytocin treated ASD mice in a marble burying test that observes anxiety levels, these results may be a reflection of the dosage given to the mice. Moy et al. (2019) observed significant changes in their highly-dosed ASD group which were treated with a 1.0 mg/kg dose of oxytocin, as compared to Wang et al.’s 200 μg/kg treatment. Although the results are conflicting, they can be accredited to the distinctive dosages administered to each ASD model. Nevertheless, oxytocin treatment displays a significant reduction in ASD behavioural symptoms, and increases overall sociability in ASD mice.

Oxytocin Reduces Oxidative Stress In ASD Mice Brain Tissue Oxidative stress has been observed to significantly increase in the brains of patients who suffer from ASD and is theorized to be an instigator of autism neuropathology (Palmieri et al., 2010. Matsuo et al., 2020). Due to the significant correlation found between oxidative stress and ASD, several studies have begun examining the impact that oxytocin may play in potentially diminishing oxidative stress levels in ASD patients (Wang et al., 2018). After obtaining hippocampal and amygdala tissue samples, they observed significantly decreased oxidative stress levels, as measured by various amounts of protein production, in ASD mice that were treated with oxytocin (Wang et al., 2018).

However, these results are conflicting when compared to a more recent study that compared the effects of oxytocin and 5 -aminolevulinic acid treatment on oxidative stress levels in ASD mice (Matsuo et al., 2020). After administering 12 μg/kg of oxytocin and analyzing hippocampal tissue samples, the researchers did not observe a significant decrease in oxidative stress levels in the ASD mice models. However, it should be noted that the dosage of oxytocin treatment differed substantially between the two studies, as seen by the 12 μg/kg dosage in Matsuo et al’s study compared to the 200 μg/kg dosage in Wang et al’s (2018) study, and may be the cause of the contrasting results attained.

Therefore, more controlled dosage studies must be completed before oxytocin’s role in alleviating oxidative stress in ASD patients can be fully understood.

a significant decrease in oxidative stress, as seen with a decrease in immunofluorescence, in the 5-aminolevulinic acid treated ASD mice but not in the oxytocin treated ASD mice (Matsuo et al., 2020).

Oxytocin Reduces Inflammation and The Number of Activated Microglia In ASD Mice Models As a result of the increased oxidative stress levels in ASD patients, neuronal tissue is typically accompanied by higher levels of inflammation as seen by abnormal neuroinflammatory markers in the brain (Lacivita et al., 2017). In order to test whether or not oxytocin treatment can reduce inflammation, Wang et al. (2018) employed the use of immunofluorescence to observe the levels of interleukin, IL, and tumour necrosis factor, TNF in oxytocin treated ASD hippocampal and amygdala samples. Overall, the amount of inflammation in the oxytocin treated mice did decrease.

Further studies correspondingly display that oxytocin has the ability to treat inflammatory responses in the brain (Yuan et al., 2016). By studying murine glial activation models and their cause of subsequent neuroinflammation, which has been positively associated with brain disorders, Yuan et al’s. (2016) results demonstrated that oxytocin treatment has the ability to diminish the release of pro-inflammatory molecules. These results depict an affirming outlook for the use of oxytocin as a potential therapeutic hormone for ASD. Additionally, Wang et al. (2018) demonstrated that after oxytocin was administered to ASD mice, there was a significant decrease in their microglia activation, as seen through immunofluorescence. Microglia are well known for their role in homeostasis and modulation of inflammation within the brain; however, as a result of ongoing neuroinflammation in ASD patients, extensive microglial activation may cause an unstable amount of mediators in the brain and subsequent neuronal damage (Block et al., 2007. Koyama et al., 2015). In a recent study carried out, murine microglia that had been stimulated were suppressed via oxytocin treatment (Inoue et al., 2019); indicating that oxytocin exhibits considerable regulation on neuroinflammatory responses and, likewise, ASD symptoms. Ultimately, more studies are needed to confirm oxytocin’s role in the management of microglia.

DISCUSSION AND CONCLUSIONS Conclusively, the results attained by Wang et al. (2018) establish a concrete framework for the use of oxytocin as a potential treatment for individuals who suffer from ASD. With its promising ability to increase social interaction and reduce oxidaFigure 2. Displays immunofluorescent tissue samples of the hip- tive stress, inflammation, and microglial activation in an ASD pocampal regions of ASD mice models after treatment with oxy- murine model, Wang et al. convey the encouraging capacities tocin, OXT, and 5-aminolevulinic acid, 5-ALA. As shown, there is that oxytocin offers as a therapeutic channel in diminishing ASD symptoms. Furthermore, the results provide enhancing evidence 157

that inflammation, oxidative stress, and microglia may play a part in the initiation of autism.

Wang et al. (2018) do recognize that their results parallel other experiments conducted; specifically with those pertaining to an increase in social interaction. This is a significant feat due to the challenge that most individuals with ASD have to live with; on account of increasing evidence portraying the positive effects of oxytocin on ASD murine models, an increase in social interaction yields affirming outcomes for the future of ASD advancements.

However, not all results match the literature provided, as seen with oxytocinâ&#x20AC;&#x2122;s inability to decrease oxidative stress (Matsuo et al., 2020). As a result, more research is required in order to definitively determine if a significant relationship between oxytocin and oxidative stress reduction exists. Overall, the findings exhibited by Wang et al. (2018) display a promising and appealing future for the amelioration of ASD symptoms and expands the breadth of research for therapeutic alternatives for treating ASD. CRITICAL ANALYSIS Overall, the experiments conducted by Wang et al. (2018) show promising results for the future treatment of ASD; however, there are several areas that can be improved. For instance, experiments conducted by Matsuo et al. (2020) examined the role of working memory in their ASD mouse model treated with oxytocin, since individuals who suffer from ASD are typically observed to have deficits in their working memory (Calhoun et al., 2019). Hence, it would be appropriate for Wang et al. (2018) to additionally examine the role that oxytocin plays in this area, alongside the other ASD symptoms investigated. Nevertheless, the use of appropriate controls alongside tests that effectively examine distinct ASD symptoms allow for sufficient evaluation of the mice models.

Moreover, Wang et al. (2018) lack a discussion on the limitations of their study. It seems that the majority of their discussion was dependent on the findings of other experiments done that reinforce their data. Likewise, the only future direction given was a statement that expressed their confidence to use oxytocin as a possible therapeutic agent. This overspeculation is critical and needs to be ablated.

In essence, more work needs to be carried out before any solid conclusions can be made from this study. With recent conflicting data being published, more studies are essential in determining the actual role of oxytocin treatment in ASD.

to observe any potential discrepancies that oxytocin administration may have on murine ASD models. From the available literature provided, there seems to be varying dosage levels that can dramatically affect the outcomes of experiments; therefore, by administering several doses, Wang et al. (2018) can help determine the most suitable treatment in experimental models of ASD and oxytocin.

As mentioned previously, working memory tests can be included in future work in order to examine any additional and distinct ASD symptoms. This can be done via social preference testing and can examine both working memory and social interactions. Likewise, ultrasonic vocalizations can be tested in the murine ASD models to further identify and determine social interactions (Calhoun et al., 2019). If oxytocin does have conclusive impacts on ASD symptoms, the murine ASD model treated with oxytocin should, in theory, display increased social preferences and ultrasonic vocalizations.

More research is required to determine appropriate oxytocin dosing alongside an expanded testing range for ASD symptoms. Although the tests completed by Wang et al. (2018) displayed acceptable results, three of the four tests specifically tested for anxiety. ASD is accompanied by a multitude of symptoms; therefore, the experiments carried out should not be limited to a few of them, rather they should encompass the wide range of symptoms that ASD can result in.

FUTURE DIRECTIONS There are several steps that Wang et al. can take moving forward. To begin with, a new experimental design that investigates varying dosages of oxytocin can be implemented in order 158

REFRENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

Wang, Yu, Shanshan Zhao, Xu Liu, Yumin Zheng, Lei Li, and Su Meng. "Oxytocin improves animal behaviors and ameliorates oxidative stress and inflammation in autistic mice." Biomedicine & Pharmacotherapy 107 (2018): 262-269.

Zachor, Ditza A., and Esther Ben‐Itzchak. "From Toddlerhood to Adolescence, Trajectories and Predictors of Outcome: Long‐ Term Follow‐Up Study in Autism Spectrum Disorder." Autism Research (2020).

Idring, Selma, Michael Lundberg, Harald Sturm, Christina Dalman, Clara Gumpert, Dheeraj Rai, Brian K. Lee, and Cecilia Magnusson. "Changes in prevalence of autism spectrum disorders in 2001–2011: findings from the Stockholm youth cohort." Journal of autism and developmental disorders 45, no. 6 (2015): 1766-1773.

Halladay, Alycia K., Somer Bishop, John N. Constantino, Amy M. Daniels, Katheen Koenig, Kate Palmer, Daniel Messinger et al. "Sex and gender differences in autism spectrum disorder: summarizing evidence gaps and identifying emerging areas of priority." Molecular autism 6, no. 1 (2015): 36.

Neale, Benjamin M., Yan Kou, Li Liu, Avi Ma’Ayan, Kaitlin E. Samocha, Aniko Sabo, Chiao-Feng Lin et al. "Patterns and rates of exonic de novo mutations in autism spectrum disorders." Nature 485, no. 7397 (2012): 242-245.

LoParo, Devon, and I. D. Waldman. "The oxytocin receptor gene (OXTR) is associated with autism spectrum disorder: a metaanalysis." Molecular psychiatry 20, no. 5 (2015): 640-646.

Lazzari, Virginia Meneghini, Josi Maria Zimmermann-Peruzatto, Grasiela Agnes, Roberta Oriques Becker, Ana Carolina de Moura, Silvana Almeida, Renata Padilha Guedes, and Marcia Giovenardi. "Hippocampal gene expression patterns in oxytocin male knockout mice are related to impaired social interaction." Behavioural brain research 364 (2019): 464-468.

Tost, Heike, Bhaskar Kolachana, Shabnam Hakimi, Herve Lemaitre, Beth A. Verchinski, Venkata S. Mattay, Daniel R. Weinberger, and Andreas Meyer–Lindenberg. "A common allele in the oxytocin receptor gene (OXTR) impacts prosocial temperament and human hypothalamic-limbic structure and function." Proceedings of the National Academy of Sciences 107, no. 31 (2010): 13936-13941.

Inoue, Takayuki, Hajime Yamakage, Masashi Tanaka, Toru Kusakabe, Akira Shimatsu, and Noriko Satoh-Asahara. "Oxytocin Suppresses Inflammatory Responses Induced by Lipopolysaccharide through Inhibition of the eIF-2α–ATF4 Pathway in Mouse Microglia." Cells 8, no. 6 (2019): 527.

Osredkar, Joško, David Gosar, Jerneja Maček, Kristina Kumer, Teja Fabjan, Petra Finderle, Saša Šterpin, Mojca Zupan, and Maja Jekovec Vrhovšek. "Urinary Markers of Oxidative Stress in Children with Autism Spectrum Disorder (ASD)." Antioxidants 8, no. 6 (2019): 187.

Pangrazzi, Luca, Luigi Balasco, and Yuri Bozzi. "Oxidative Stress and Immune System Dysfunction in Autism Spectrum Disorders." International Journal of Molecular Sciences 21, no. 9 (2020): 3293.

Popa-Wagner, Aurel, Smaranda Mitran, Senthilkumar Sivanesan, Edwin Chang, and Ana-Maria Buga. "ROS and brain diseases: the good, the bad, and the ugly." Oxidative medicine and cellular longevity 2013 (2013).

Peñagarikano, Olga, María T. Lázaro, Xiao-Hong Lu, Aaron Gordon, Hongmei Dong, Hoa A. Lam, Elior Peles et al. "Exogenous and evoked oxytocin restores social behavior in the Cntnap2 mouse model of autism." Science translational medicine 7, no.

271 (2015): 271ra8-271ra8.

Moy, Sheryl S., Brian L. Teng, Viktoriya D. Nikolova, Natallia V. Riddick, Catherine D. Simpson, Amy Van Deusen, William P. Janzen, Maria F. Sassano, Cort A. Pedersen, and Michael B. Jarstfer. "Prosocial effects of an oxytocin metabolite, but not synthetic oxytocin receptor agonists, in a mouse model of autism." Neuropharmacology 144 (2019): 301-311.

Palmieri, L., V. Papaleo, V. Porcelli, P. Scarcia, L. Gaita, R. Sacco, J. Hager et al. "Altered calcium homeostasis in autismspectrum disorders: evidence from biochemical and genetic studies of the mitochondrial aspartate/glutamate carrier AGC1."

Molecular psychiatry 15, no. 1 (2010): 38-52.

Matsuo, Kazuya, Yasushi Yabuki, and Kohji Fukunaga. "5-aminolevulinic acid inhibits oxidative stress and ameliorates autistic -like behaviors in prenatal valproic acid-exposed rats." Neuropharmacology 168 (2020): 107975.

Lacivita, Enza, Roberto Perrone, Lucia Margari, and Marcello Leopoldo. "Targets for drug therapy for autism spectrum disorder: challenges and future directions." Journal of medicinal chemistry 60, no. 22 (2017): 9114-9141.

159

20.

21.

22.

23.

Yuan, Lin, Song Liu, Xuemei Bai, Yan Gao, Guangheng Liu, Xueer Wang, Dexiang Liu, Tong Li, Aijun Hao, and Zhen Wang.

"Oxytocin inhibits lipopolysaccharide-induced inflammation in microglial cells and attenuates microglial activation in lipopolysaccharide-treated mice." Journal of neuroinflammation 13, no. 1 (2016): 77.

Block, M. L., and J-S. Hong. "Chronic microglial activation and progressive dopaminergic neurotoxicity." (2007): 1127-1132.

Koyama, Ryuta, and Yuji Ikegaya. "Microglia in the pathogenesis of autism spectrum disorders." Neuroscience research 100 (2015): 1-5.

Calhoun, Susan L., Amanda M. Pearl, Julio Fernandez-Mendoza, Krina C. Durica, Susan D. Mayes, and Michael J. Murray.

"Sleep disturbances increase the impact of working memory deficits on learning problems in adolescents with highfunctioning Autism spectrum disorder." Journal of autism and developmental disorders (2019): 1-13.

160

The Protein Pallidin and its Prognosis in Schizophrenia Ece Su Sayin

The mental disorder schizophrenia has been a key research area for the past few decades considering the high number of people that suffer from it. The etiology of schizophrenia is still not known, but there are many theories on it. One of the theories made by Shi et al. (2017) show that Pallidin promotes transcriptional activity of p38. The overexpression of p38 leads to cell differentiation. The problem is that when Pallidin is present, this inhibits growth proteins such as Coronin 1b and Rab13.

When this overexpression without the important growth factors takes place, this leads to abnormal neuronal development. The authors hypothesize that Pallidin influence growth of nerve processes via p38 and this causes abnormally developed brains, increasing risk of schizophrenia. What the authors could not fully conclude is when this overexpression happens, does it lead to schizophrenia like they assume.

Key Words: Schizophrenia; Neurodevelopment; Pallidin; P38

161

mass from the brain was from the lateral cerebral ventricle, hippocampus and prefrontal cortex. The weight increase was due to a decrease in dendritic cells. Researching a protein found throughout the central nervous system and brain, in which it is seen to affect the development of neuronal cells was next. This is why the authors, Shi et al. (2017), wanted to explore the importance of the protein Pallidin in the brain and the central nervous system.

The proteins involved

Figure 1 Visual summary explaining the paper by Shi et al.

Made on BioRender by Ece Su Sayin Background Information Schizophrenia is a very different and complex mental disorder that is affecting many people in the world today. Even though one percent of the world population experiences schizophrenialike symptoms (Insel, 2010), the reasons people experience schizophrenia are still unknown. Schizophrenia is defined by the collective onsets of psychosis with paranoid illusions and auditory hallucinations. Schizophrenia only affects a few critical regions in the brain and neurotransmitters like dopamine (Javitt, 2010). Since the causes of schizophrenia are not known, there is constant research going on to test various types of proteins, enzymes and complexes in the brain and how changes can lead to schizophrenia.

Previously, it has been found that the protein Pallidin is highly expressed in the hippocampus of the brain, especially in the glutaminergic neurons. Also, Ach neurons exhibit high levels of Pallidin (Larimore et al., 2014). In the hippocampus of a mouse brain, high activity of the Ach neuron suggests that it is involved in the regulation of the synaptic membrane. In addition to Pallidin playing an important role in the regulation of synaptic activity, the protein, p38, has an essential role in apoptosis. In the genome, research by Shao et al. (2015), it has shown that this protein promotes gene expression and is a crucial protein in neuronal development. Since apoptosis is a crucial and regulatory factor for any development in the body, the protein p38 to have apoptotic abilities ensures successful neuronal development.

Research Question

By combining various important key research ideas on the activities that are going on in the brain and the individual function of these proteins, Shi et al. (2017) wanted to test if these two critical proteins are interconnected and play a role in abnormalities in the brain together. They hypothesized that Pallidin and p38 are important factors in neurodevelopment, which has to play Direction for Research an essential role in the prognosis of schizophrenia. The authors When it comes to research in this field, there are many aspects overall wanted to verify if increases in Pallidin and p38 lead to one can tackle. In the past, research has been done about the the onsets of schizophrenia and causes behind it. interactions of neurotransmitters in the brain. These studies show that neurotransmitters due play a role in the development Major Results of schizophrenia (Carlsson et al., 1999). The interaction between the different kinds of neurotransmitters is essential in the role of The authors took three different mammalian cells, HEK293A, neuronal circuits, which ultimately gives insight into antipsy- HCT116 and N2a and treated them with fetal bovine serum, chotic activity. The neurotransmitter theory provides validity as MEM and antibiotic culture medium. Alongside the mammalian there is evidence for genes that play a role in the neurotrans- cell lines, they took wildtype mice and regular sandy mice in mitter system's function. Besides, there is research on the neu- which they extracted the cortical neurons from the cortical tisrotransmitter system abnormality hypothesis (PadeÄ­skaia et al., sues. Depending on the study, they performed various experi1980). This hypothesis focuses on pieces of evidence found from ments on the appropriate cell line or cortical neuron, which is CAT scans that there is some abnormality in the brain structure why there was a variety. The authors in this study concluded from the images. Also, since the structure of the brains looked with three main points in relevance to their hypothesis. different from person to person, they weighed them to see if there were any noticeable differences. They concluded and proved that the mass of a brain with schizophrenia versus a Firstly, the authors concluded that Pallidin does influence tranhealthy brain was, in fact, much lighter. This proves that when scriptional activity on p38, but when HDAC is bound to p38 and people get the onsets of schizophrenia, physical changes due to E2F1, it inhibits the transcriptional factor. Following immunocyoccur in the brain. With the continuation of this area, Huard et tochemistry, the results showed that in the HCT116 cell line, al. (2007) followed up with the research that this decrease in when the transcriptional activity of HDAC was overexpressed, 162

this caused an increase in Pallidin, which in return caused an increase in p38. In the western blot analysis that was done, increased levels of Pallidin also caused an increase in p21, and a qPCR showed increased levels of mRNA (Figure 1). This made the authors conclude that Pallidin influences transcriptional activity on p38 even when it is bound to HDAC, an inhibiting protein.

During cell differentiation, if Pallidin levels decrease, this influences the expression of p38. The reduction in p38 has a direct effect on cell differentiation, causing a reduction, which affects cell differentiation. When p38 was knocked down, using realtime PCR, they were able to detect mRNA levels.

Next, using real-time PCR and a western blot, when p38 was knocked down, they measured the levels of mRNA, which showed that the expression of the two proteins, Coronin 1b and Rab13 decreased (Figure 2). The authors noticed that the overexpression of Pallidin caused a decrease in the growth factors, which were downstream genes of p38. For successful neuronal development, the downstream genes of p38 are required, which are essential growth factors. When the overexpression of Pallidin inhibits these growth factors, this leads to the abnormal development of the neurons as there are not enough growth factors ensuring the proper development of the neurons in the brain.

growth factors.

Adapted from “Pallidin protein in neurodevelopment and its relation to the pathogenesis of schizophrenia” by Shi et al. (2017).

Discussion The primary conclusion that the authors have made in their research is the general idea is that Pallidin via P38 promotes an increase in transcriptional activity. The enzyme, HDAC, inhibits this transcriptional activity by binding to E2F1 and p38. That is why the protein, Pallidin, is required to inhibit HDAC. The overexpression of Pallidin and, in return, continues to the overexpression of p38 when an inhibiting factor is not there, this leads to cell differentiation. The problem is that the overexpression of Pallidin leads to a decrease in the release of growth factors such as Rab1 and coronin 1b. As this cell differentiation occurs in the brain without any growth factors, this affects neuronal development. As the neurons in the brain develop irregularly, the authors hypothesize that this abnormal development of the brain and the central nervous system, ultimately leading to schizophrenia.

The authors' conclusion is relevant since they have a good and robust idea about the causes of schizophrenia. In tackling this field with numerous unknowns, it is very safe first to try to figure out what happens in a person's brain, leading to this mental disorder. The authors' theory about the overexpression of Pallidin and p38 and its effects on neurodevelopment are in alignment with other few pieces of research. In an older research paper by Chen et al. (2017), they were able to do a null mutation on Pallidin in Drosophila. Their ideology was that since Pallidin is one of the subunits of the BLOC-1 complex, they wanted to determine if Pallidin played a role in neuronal and synaptic development. They showed that Pallidin promotes fast synaptic vesicle recycling, which is essential to maintain functional vesicle transport during neuronal activity. Both Chen et al. and Shi et al. prove that "Pallidin" plays a vital role in neuronal development.

A paper by Ryder & Faundez (2009) showed that DTNBP1, the gene encoding for dysbindin, a Pallidin homolog, is also linked to schizophrenia.

Critical Analysis Figure 2 (top) shows the western blot indicating increased levels of Pallidin. Adapted from “Pallidin protein in neurodevelopment Every paper that is written and published has both strengths and and its relation to the pathogenesis of schizophrenia” by Shi et weaknesses, as no research is considered perfect. When analyzing a paper, the strengths and weaknesses identified by the al. (2017) reader are mostly subjective as there are many ways to conduct research and publish a paper. This paper by Shi et al. was overall well done, and the research they have conducted shows excellent validity. Like all research, this paper also had a few weaknesses, which will also be discussed.

One of the strengths of this paper is that the authors followed a chronological order for the experiments they did. The numerous . experiments they have done around the protein Pallidin adds new knowledge to this field of study, building off of previous Figure 3 Shows a western blot. Increased levels of Pallidin lead to research. In science, it is essential to follow a chronological order decreased levels of Coronin 1b and Rab13, which are important and to have a cohesive strategy as this reduces confusion. The 163

authors did a great job explaining in their introduction, why this protein called â&#x20AC;&#x153;Pallidinâ&#x20AC;? with not much previous research, was essential to study and the mechanisms behind it. Their methodologically, for the most part, followed a precise sequence of events. It made much sense that they first concluded what transcription factor Pallidin inhibited (HDAC), followed by determining the growth factors involved to paint the overall picture. Another strength of this paper is that they tested the same mechanisms on numerous different cell lines and mice cortical neurons from two different mice. Instead of picking just one cell line or a cortical neuron from one mouse and showing results favouring the validity of their hypothesis, they showed a variety. This variety that they showed insured and proved that not all cell lines and even cortical neurons from the same mice produce similar results. Another strength of this paper is the great amount of detail they provided in their methods section.

They outlined the ten different steps they took to gather all their data and perform the experiments. In the first part of their methods, they explained where they bought or received all the cell lines and mice that give the places proper credit.

Also, the research was approved by the ethics board. This is crucial for the validity of the experiment, as this proves that the steps taken in the experiment follow the ethical guidelines of the institution. The most important strength of this paper is that the hypothesizing mechanism is true, and their results are similar to other papers. Like previously mentioned, the paper

by Chen et al. (2017) and Ryder & Faundez (2009) both prove that this protein Pallidin is in the BLOC-1 complex, which is involved in synaptic and neuronal development. The authors of this paper go beyond the research of the others by providing further knowledge on other proteins involved like p38, HDAC, p21, rab13 and coronin 1b, which is an addition to the knowledge in this field.

On the other hand, like most papers, this article had a few weaknesses. Even though it was mentioned earlier that the authors had a clear methodology for their experiment, their explanation of their results section was weak. They did not fully explain their results, and their visuals like graphs and western blots were very hard to understand. The legends of the proposed graphs were not clear, and it was hard to make out the comparison they were trying to make. The authors included four to five big illustrations, with each illustration having six to seven images. This created an overwhelming presentation of data, and they did not clearly explain in detail the relevance of each one. A better way to present their results would have been selecting the most important figures and putting an asterisk on what they wanted their audience to focus on. The balance of the number of visuals and their explanation was low, which left it to the reader to understand and interpret. Another suggestion for the results section would have been to explain the names on the axis, what they stand for, and their relevance. For example, they used short forms like Si-NC, Si-p38 and RA, and not explain what it stands for. The point of publishing literature is to provide a clear explanation of their findings and making sure it is understandable by everyone reading it.

Throughout their experiment, the authors do a good job dis-

cussing the importance of each protein and making connections between the concepts. What makes their discussion/ future directions part weak is that they do not necessarily provide full-on support showing that an abnormally developed brain leads to schizophrenia. They provide great detail about Pallidin via p38 influencing neuronal development, which is in line with previous research, but the connection to schizophrenia is not fully there. The entire point of this paper was to find a mechanism and how overexpression/under expression can lead to the mental disorder of schizophrenia. The authors spend a reasonable amount of time proving the importance of Pallidin but do not necessarily connect it to their main argument. In a way, this is not entirely a weakness as it leaves open for future experiments. However, it would have been a more persuasive argument if they spent more time discussing the connection to schizophrenia.

Future Directions So far, the research around Pallidin and its involvement in causing schizophrenia has come a long way, showing that when this protein is increased, so does the prognosis of schizophrenia.

There are still many missing links in this field of study that limit researchers on making keen connections. The missing links on finding even a simple question like its etiology prevents doctors and health care workers from helping to treat these patients.

So far, up to this point in research, scientists have identified the proteins, enzymes and complexes involved in the development of schizophrenia. What would make advancements in this field is by finding a way to make the knockdown of Pallidin in the brains of live mice and test if they develop schizophrenia-like symptoms. This would be very beneficial to research as the authors have left off saying that they predict this abnormal neurodevelopment will lead to schizophrenia. Pallidin is homologous of dysbindin (Shi et al., 2017), which is another subunit on the BLOC-1 complex. Inhibited levels of Pallidin showed inhibited levels of dysbindin and vice versa (Spiegel et al., 2015), which prove that their functions in the complex are interconnected. In the same paper, it was found that the gene encoding for dysbindin is DTNBP1. This means that if we are successful at knocking down dysbindin, levels of Pallidin will also decrease. The objective of knocking down dysbindin/Pallidin is to verify that experiments in Shi et al. (2017) and to do further testing on the development of schizophrenia. A knockdown in mice can be achieved by RNA interference as this silences the gene of interest for some time. A way to test if this protein has been silenced is by doing an RNA screening similar to DNA screening. This tests to see if the gene of interest has been silenced. The expected results from this experiment would positive, but because it is an unknown field of study, it is hard to come up with definitive answers. If the iRNA does not work, this could mean that there is more to this BLOC-1 complex that researchers do not fully understand. If you cannot induce and cause a mouse to get schizophrenia via the protein Pallidin, there is still another way to test the involvement of Pallidin.

What can also be down to test the protein Pallidin and how it is related to schizophrenia would be to use DNA directed RNA

164

interference. This could be done on mice or humans who already have schizophrenia-like symptoms. This technique of knocking down disease-causing genes has excellent potential in the development of therapeutic drugs (Lisitskaya et al., 2018).

This research would be highly translational in humans because it has been found that Pallidin and dysbindin have similar expressions in both mice and human brains (Spiegel et al., 2015).

In conclusion, the entire study of neuroscience still has much ground to cover to help the millions of people suffering daily.

This protein Pallidin and the entire BLOC-1 complex hold promising results for the etiology of schizophrenia, but there is still much more to uncover and research.

165

REFRENCES

1.

Carlsson, A., Waters, N., & Carlsson, M. L. (1999). Neurotransmitter interactions in schizophrenia—Therapeutic implications. Biol. Psychiatry, 46(10), 1388–1395. https://doi.org/10.1016/S0006-3223(99)00117-1

2.

Chen, X., Ma, W., Zhang, S., Paluch, J., Guo, W., & Dickman, D. K. (2017). The BLOC-1 Subunit Pallidin Facilitates ActivityDependent Synaptic Vesicle Recycling. ENeuro, 4(1). https://doi.org/10.1523/ENEURO.0335-16.2017

3.

Huard, C., Martinez, R. V., Ross, C., Johnson, J. W., Zhong, W., Hill, A. A., Kim, R., Paulsen, J. E., & Shih, H. H. (2007). Transcriptional profiling of C2C12 myotubes in response to SHIP2 depletion and insulin stimulation. Genomics, 89(2), 270–279. https://doi.org/10.1016/j.ygeno.2006.10.006

4.

Insel, T. R. (2010). Rethinking schizophrenia. Nature, 468(7321), 187–193. https://doi.org/10.1038/nature09552

5.

Javitt, D. C. (2010). Glutamatergic theories of schizophrenia. Isr. J. Psychiatry Relat. Sci., 47(1), 4–16.

6.

Larimore, J., Zlatic, S. A., Gokhale, A., Tornieri, K., Singleton, K. S., Mullin, A. P., Tang, J., Talbot, K., & Faundez, V. (2014).

Mutations in the BLOC-1 Subunits Dysbindin and Muted Generate Divergent and Dosage-dependent Phenotypes. J. Biol.

Chem., 289(20), 14291–14300. https://doi.org/10.1074/jbc.M114.553750

7.

Lisitskaya, L., Aravin, A. A., & Kulbachinskiy, A. (2018). DNA interference and beyond: Structure and functions of prokaryotic Argonaute proteins. Nat. Commun, 9(1), 5165. https://doi.org/10.1038/s41467-018-07449-7

8.

Padeĭskaia, E. N., Kutchak, S. N., & Polukhina, L. M. (1980). Comparative activity of depot sulfanilamides in experimental infection in mice caused by K1. Pneumoniae. Antibiotiki, 25(3), 193–198.

9.

Ryder, P. V., & Faundez, V. (2009). Schizophrenia: The “BLOC” May Be in the Endosomes. Science Signaling, 2(93), 1-13. https://doi.org/10.1126/scisignal.293pe66

10.

Shao, M., Tang, S.-T., Liu, B., & Zhu, H.-Q. (2015). Rac1 mediates HMGB1‑induced hyperpermeability in pulmonary microvascular endothelial cells via MAPK signal transduction. Mol. Med. Rep., 13(1), 529–535. https://doi.org/10.3892/ mmr.2015.4521

11.

Shi, Q., Li, C., Li, K., & Liu, Q. (2017). Pallidin protein in neurodevelopment and its relation to the pathogenesis of schizophrenia. Mol. Med. Rep., 15(2), 665–672. https://doi.org/10.3892/mmr.2016.6064

12.

Spiegel, S., Chiu, A., James, A. S., Jentsch, J. D., & Karlsgodt, K. H. (2015). Recognition deficits in mice carrying mutations of genes encoding BLOC-1 subunits Pallidin or Dysbindin. Genes, Brain and Behavior, 14(8), 618–624. https:// doi.org/10.1111/gbb.12240

166

Understanding the Link between Inflammation and Mental Health Alana Sofer

Mental illness is a serious public health crisis in desperate need of more attention and answers. Almost everyone on the planet will be indirectly affected by mental illness. Although some may perceive suicide as rather distant, surprisingly itâ&#x20AC;&#x2122;s the second leading cause of death in people ages 1034; and major risk factors for suicide are depression and just plain old stress. As we know, experiencing prolonged stress increases the levels of cortisol in the body, which in turn can have negative effects on our immune and inflammatory responses. Delving into the molecular processes involved, some studies have also shown that abnormalities in the immune system may contribute to the development of depression. Previous literature shows irregularities in inflammatory cytokines in the serum and cerebral spinal fluids of depressed individuals. However, prior to this research, it was not clear if these irregularities were present in the brains of these individuals. By determining mRNA and protein levels of certain cytokines involved in the inflammatory response within the prefrontal cortices of depressed individuals, this paper uncovers the answer to that question. Their findings suggest an up regulation of proinflammatory cytokines in the brains of individuals suffering from depression, who died by suicide. These answers may be able to pave the path towards more effective diagnoses, treatments and a detailed understanding of the pathogenesis and pathophysiology of various mental illnesses; things we are still quite distant from.

167

Although similar results were shown in the author’s previous study, we know that the pathogenesis of depression may differ in teenagers vs. adults. Similarly, the pathophysiology of teenage depressive suicide may differ from that of adults. This paper sought to determine the mRNA and protein levels of proand anti-inflammatory cytokines in adults who died by suicide, in order to determine if suicide pathology was similar between the two groups. Since the results determined were quite similar, it is clear that various forms of depressive suicide have a similar pathology; this information can hopefully influence treatments and diagnoses in the future.

Major Results

Background Every individual on Earth encounters stress in one way or another, making it a universal phenomenon. Through their studies, Garcia-Bueno et al. 2008 and Goshen et al. 2009, both emphasize stress-related immunosuppression and its impact on various cellular immune processes. Since stress and depression are key components in suicidal behaviour, suicides link to impaired immunity is not surprising (Black and Miller, 2015). Various studies have touched on similar subject matter in this field; further connecting immunity and inflammation with depression and suicide. Through a meta-analysis, Howren et al. 2009 and Valkanova et al. 2013, demonstrated a clear link between compromised immune states and depression, looking specifically at proinflammatory cytokine IL-6 and C-reactive protein. Cytokines, once artificially administered, had also been shown to induce behavioural changes very similar to depression in animals, (Dantzer, 2001). This ‘sickness behaviour’ was further demonstrated in humans by Dantzer et al. 2008, when cancer and melanoma patients were administered IFN-α, and depression was induced. The main evidence showing a link between inflammatory cytokines and depression, which inturn influenced this paper, was the increase in proinflammatory cytokines in the serum of depressed patients shown by Dowlati et al. 2010, Liu et al. 2012, and Schiepers et al. 2012. The work by Lindqvist et al. 2009 also influenced this paper by showing abnormalities in cytokine levels, specifically IL-6, in the cerebrospinal fluid of individuals who died by suicide. Similarly, Janelidze et al. 2011 demonstrated an increase in IL-6 and TNF-α and a decrease in IL-2 in the blood of individuals who has attempted suicide. Overall, based on previous literature, there is a clear link between depression and inflammation, however it was not clear whether these results would be echoed within the brains of these individuals. Shelton et al. 2011 looked at inflammatory gene expression in prefrontal cortex samples of depressed patients, where they found upregulation of IL-1β and IL-2. This suggests that there would be inflammation in these brain areas in depressed and/or suicidal patients. The same authors of this current paper also completed an earlier study that showed an increase in protein and mRNA expression levels of the cytokines Il-1β, IL-6 and TNF-α in the prefrontal cortices of teenagers who died by depressive suicide. They decided to take this information one step further.

This study sought to find out mRNA and protein expression levels of pro- and anti-inflammatory cytokines in the PFCs of individuals who died by suicide vs. non-psychiatric controls.

Using real-time polymerase chain reactions, researchers determined the mRNA expression levels of IL-1β, IL-6, TNF-α, Lymphotoxin A, Lymphotoxin B, IL-8, IL-10, IL-13 and IL-1RA. Using enzyme-linked immunosorbent assay and Western Blots, researchers determined the protein expression levels of the same cytokines. Regarding the mRNA levels, there were significant differences in the levels of IL-6, TNF-α, lymphotoxin A, IL-10, IL1RA, lymphotoxin B and IL-1β in depressed individuals vs. controls (shown in Figure A). Specifically, there was an increased in levels of TNF-α, lymphotoxin A, IL-1β and IL-6, and a decrease in levels of Lymphotoxin B, IL-1RA and IL-10 in depressed individuals when compared to controls. Evidence showed, no major differences in the mRNA levels of cytokines IL-8 and IL-13.

Regarding protein levels, there were significant differences between the two groups in TNF-α, IL-1β, IL-1RA, IL-6, IL-10 and lymphotoxin A (Shown in Figure B). There was an increase in protein expression of TNF-α, lymphotoxin A, IL-1β and IL-6 in the depressed group. IL-1RA and IL-10 levels were significantly lower in the depressed group. Protein levels of IL-8, lymphotoxin B and IL-13 displayed no significant differences between the two groups. Overall this study showed abnormalities in both pro- and anti-inflammatory cytokines and specifically, an upregulation of proinflammatory cytokines in the depressive experimental group. This data strengthens the evidence shown in past literature, proving it’s occurrences in the brain and showing that inflammatory cytokine level abnormalities are consistent with individuals suffering from depression.

168

Figure A: This chart depicts the mean mRNA expression levels of TNF- α, Lymphotoxin A (LTA), Lymphotoxin B (LTB), IL-1β, IL-1RA, IL-8, IL-10 and IL-13 in the post-mortem tissues of the prefrontal cortices of the experimental group in black (depressed individuals that died of suicide – DS) and the control group in white (non-psychiatric individuals – NC).

Pandey, Ghanshyam N., Hooriyah S. Rizavi, Hui Zhang, Runa Bhaumik, and Xinguo Ren. “Abnormal Protein and MRNA Expression of Inflammatory Cytokines in the Prefrontal Cortex of Depressed Individuals Who Died by Suicide.” Journal of Psychiatry & Neuroscience 43, no. 6 (2018): 376–85. https://doi.org/10.1503/jpn.170192.

that the pathophysiology of teenage depressive suicide is similar to that of adults. This new information emphasizes the irregularity in the levels of inflammatory cytokines in suicidal and depressed individuals. Now that this data has been echoed many times, researchers must go the step further and answer the questions why and how this phenomenon occurs.

Critical Analysis It is important to note that one of the limitations of this study was that some individuals from the experimental group were taking antidepressants around their time of death, however this likely had very little effect on the results. Previous studies, including the ones by Eller et al. 2008 and Sluzewska et al. 1995, have suggested that using antidepressants could actually lower levels of proinflammatory cytokines in individuals with depression. However, this current study’s results did not find any significant differences amongst the depressed individuals on antidepressants and those who were not. Furthermore, researchers found an overall increase of cytokines in the entire experimental group, demonstrating contrast with previous literature.

Various confounding variable could have affected the results seen in past studies.

Figure A: This chart depicts the mean mRNA expression levels of TNF- α, Lymphotoxin A (LTA), Lymphotoxin B (LTB), IL-1β, IL-1RA, IL-8, IL-10 and IL-13 in the postmortem tissues of the prefrontal cortices of the experimental group in black (depressed individuals that died of suicide – DS) and the control group in white (nonpsychiatric individuals – NC).

The BMIs of the individuals involved were not taken into account in this study, but has been shown to impact cytokine levels in previous literature. This should be studied more closely, in order to rule out the possibility that these differences in cytokine levels could be attributed to differences in BMIs amongst individuals. There were also several confounding variables that Pandey, Ghanshyam N., Hooriyah S. Rizavi, Hui Zhang, Runa were shown to affect the results. Age was a confounding variaBhaumik, and Xinguo Ren. “Abnormal Protein and ble shown to affect mRNA levels of TNF-α; therefore it should MRNA Expression of Inflammatory Cytokines in the Prefrontal Cortex of Depressed Individuals Who Died by be investigated further, because age on its own also affects cySuicide.” Journal of Psychiatry & Neuroscience 43, no. 6 tokine levels, according to past research. The sample size of this (2018): 376–85. https://doi.org/10.1503/jpn.170192. study was also quite small (24 individuals in each group) so hopefully in the future a similar study of a larger population could be done, in order to reinforce these results.

Conclusions This study showed that the mRNA and protein levels of proinflammatory cytokines IL-1β, IL-6, TNF-α and lymphotoxin A were significantly increased, and levels of anti-inflammatory cytokines IL-10, and IL-1RA were significantly decreased in the PFCs of depressed individuals who died by suicide, compared with controls. The study also showed that there wasn’t any notable difference in the protein and mRNA expression concentrations of cytokines IL-8 and IL-13. Overall, the data from this study suggest that varying levels of cytokines is associated with depressed suicide, and further claims that there is a significant disproportion of pro- and anti-inflammatory cytokines in depressed individuals. Although these findings are not surprising based on previous studies, they do prove these imbalances are in fact happening in the brains of these individuals, not just in their CSF and serum. Studies prior had shown a definite imbalance in cytokine levels; however this study was able prove specific increases in certain pro-inflammatory cytokines and decreases in anti-inflammatory cytokines. This study also showed

Other than discrepancies over the affect of antidepressants on cytokines, this paper fits the current literature completely. Confirming that inflammatory cytokine levels are a major part of the pathophysiology of depressive suicide. However, certain aspects of the research could have been fine tuned, assuring that all other possibilities become ruled out; including age, BMI and sample size.

Future Directions Moving forward, it is important for researchers to develop an understanding of the how and the why. An important question to ask is, now that we know there is a connection between depression and inflammatory cytokines, how do they fit in with other pathophysiologies of the disease? Also, whether their varying levels are a cause or a result of the disease. An interesting future study would be whether or not these levels could

169

be manipulated in real-time to produce a positive effect on behaviour, and how would this impact other areas of the body (essentially doing opposite of inducing ‘sickness behaviour’).

This study would involve the artificial administration of mechanisms to inhibit pro-inflammatory cytokines and their responses, and/or administration of mechanisms to upregulate antiinflammatory cytokines and their responses, in the brains of individuals suffering from depression. I would anticipate the opposite of inflammatory cytokine induced “sickness behaviour” to occur. Where individuals start to feel better, their inflammatory response decreases and their cytokine levels start to match those of a non-depressed individual’s more closely. If this were to not occur, it would show that the mechanisms impacting cytokine levels and its affect on depression are not so cut and dry. This information would impact all areas of mental illness, including possible new treatments and mechanisms for diagnoses.

170

REFRENCES 1.

Black, Carmen, and Brian J. Miller. “Meta-Analysis of Cytokines and Chemokines in Suicidality: Distinguishing Suicidal Versus Nonsuicidal Patients.” Biological Psychiatry 78, no. 1 (2015): 28–37. https://doi.org/10.1016/j.biopsych.2014.10.014.

2.

Clark, Sarah M., Ana Pocivavsek, James D. Nicholson, Francesca M. Notarangelo, Patricia Langenberg, Robert P. Mcmahon, Joel E. Kleinman, et al. “Reduced Kynurenine Pathway Metabolism and Cytokine Expression in the Prefrontal Cortex of Depressed Individuals.” Journal of Psychiatry & Neuroscience 41, no. 6 (2016): 386–94. https://doi.org/10.1503/ jpn.150226.

3.

Dantzer, Robert, Jason C. O'connor, Gregory G. Freund, Rodney W. Johnson, and Keith W. Kelley. “From Inflammation to Sickness and Depression: When the Immune System Subjugates the Brain.” Nature Reviews Neuroscience 9, no. 1 (2008): 46–56. https://doi.org/10.1038/nrn2297.

4.

Dantzer, Robert. “Cytokine-Induced Sickness Behavior: Mechanisms and Implications.” Annals of the New York Academy of Sciences 933, no. 1 (2006): 222–34. https://doi.org/10.1111/j.1749-6632.2001.tb05827.x.

5.

Dowlati, Yekta, Nathan Herrmann, Walter Swardfager, Helena Liu, Lauren Sham, Elyse K. Reim, and Krista L. Lanctôt. “A Meta-Analysis of Cytokines in Major Depression.” Biological Psychiatry 67, no. 5 (2010): 446–57. https://doi.org/10.1016/ j.biopsych.2009.09.033.

6.

Eller, Triin, Veiko Vasar, Jakov Shlik, and Eduard Maron. “Pro-Inflammatory Cytokines and Treatment Response to Escitaloprsam in Major Depressive Disorder.” Progress in Neuro-Psychopharmacology and Biological Psychiatry32, no. 2 (2008): 445–50. https://doi.org/10.1016/j.pnpbp.2007.09.015.

7.

García-Bueno, Borja, Javier R. Caso, and Juan C. Leza. “Stress as a Neuroinflammatory Condition in Brain: Damaging and Protective Mechanisms.” Neuroscience & Biobehavioral Reviews 32, no. 6 (2008): 1136–51. https://doi.org/10.1016/ j.neubiorev.2008.04.001.

8.

Goshen, Inbal, and Raz Yirmiya. “Interleukin-1 (IL-1): A Central Regulator of Stress Responses.” Frontiers in Neuroendocrinology 30, no. 1 (2009): 30–45. https://doi.org/10.1016/j.yfrne.2008.10.001.

9.

Howren, M Bryant, Donald M. Lamkin, and Jerry Suls. “Associations of Depression With C-Reactive Protein, IL-1, and IL-6: A Meta-Analysis.” Psychosomatic Medicine 71, no. 2 (2009): 171–86. https://doi.org/10.1097/psy.0b013e3181907c1b.

10.

Janelidze, Shorena, Daniele Mattei, Åsa Westrin, Lil Träskman-Bendz, and Lena Brundin. “Cytokine Levels in the Blood May Distinguish Suicide Attempters from Depressed Patients.” Brain, Behavior, and Immunity 25, no. 2 (2011): 335–39. https:// doi.org/10.1016/j.bbi.2010.10.010.

11.

Larsson, Anders, Lena Carlsson, Anne-Li Lind, Torsten Gordh, Constantin Bodolea, Masood Kamali-Moghaddam, and Måns Thulin. “The Body Mass Index (BMI) Is Significantly Correlated with Levels of Cytokines and Chemokines in Cerebrospinal Fluid.” Cytokine 76, no. 2 (2015): 514–18. https://doi.org/10.1016/j.cyto.2015.07.010.

12.

Lindqvist, Daniel, Shorena Janelidze, Peter Hagell, Sophie Erhardt, Martin Samuelsson, Lennart Minthon, Oskar Hansson, Maria Björkqvist, Lil Träskman-Bendz, and Lena Brundin. “Interleukin-6 Is Elevated in the Cerebrospinal Fluid of Suicide Attempters and Related to Symptom Severity.” Biological Psychiatry 66, no. 3 (2009): 287–92. https://doi.org/10.1016/ j.biopsych.2009.01.030.

13.

Liu, Yang, Roger Chun-Man Ho, and Anselm Mak. “Interleukin (IL)-6, Tumour Necrosis Factor Alpha (TNF-α) and Soluble Interleukin-2 Receptors (SIL-2R) Are Elevated in Patients with Major Depressive Disorder: A Meta-Analysis and MetaRegression.” Journal of Affective Disorders 139, no. 3 (2012): 230–39. https://doi.org/10.1016/j.jad.2011.08.003.

14.

Miller, Andrew H., Ebrahim Haroon, Charles L. Raison, and Jennifer C. Felger. “Cytokine Targets In The Brain: Impact On Neurotransmitters And Neurocircuits.” Depression and Anxiety 30, no. 4 (2013): 297–306. https://doi.org/10.1002/ da.22084.

15.

Pandey, Ghanshyam N., Hooriyah S. Rizavi, Hui Zhang, Runa Bhaumik, and Xinguo Ren. “Abnormal Protein and MRNA Expression of Inflammatory Cytokines in the Prefrontal Cortex of Depressed Individuals Who Died by Suicide.” Journal of Psychiatry & Neuroscience 43, no. 6 (2018): 376–85. https://doi.org/10.1503/jpn.170192.

16.

Pandey, Ghanshyam N., Hooriyah S. Rizavi, Xinguo Ren, Jawed Fareed, Debra A. Hoppensteadt, Rosalinda C. Roberts, Robert R. Conley, and Yogesh Dwivedi. “Proinflammatory Cytokines in the Prefrontal Cortex of Teenage Suicide Victims.” Journal of Psychiatric Research 46, no. 1 (2012): 57–63. https://doi.org/10.1016/j.jpsychires.2011.08.006.

171

18.

Rizavi, Hooriyah S., Xinguo Ren, Hui Zhang, Runa Bhaumik, and Ghanshyam N. Pandey. “Abnormal Gene Expression of Proinflammatory Cytokines and Their Membrane-Bound Receptors in the Lymphocytes of Depressed Patients.” Psychiatry Research 240 (2016): 314–20. https://doi.org/10.1016/j.psychres.2016.04.049.

19.

Schiepers, Olga J.g., Marieke C. Wichers, and Michael Maes. “Cytokines and Major Depression.” Progress in NeuroPsychopharmacology and Biological Psychiatry 29, no. 2 (2005): 201–17. https://doi.org/10.1016/j.pnpbp.2004.11.003.

20.

Shelton, R C, J Claiborne, M Sidoryk-Wegrzynowicz, R Reddy, M Aschner, D A Lewis, and K Mirnics. “Altered Expression of Genes Involved in Inflammation and Apoptosis in Frontal Cortex in Major Depression.” Molecular Psychiatry 16, no. 7 (2010): 751–62. https://doi.org/10.1038/mp.2010.52.

21.

Stowe, R. P., M. K. Peek, M. P. Cutchin, and J. S. Goodwin. “Plasma Cytokine Levels in a Population-Based Study: Relation to Age and Ethnicity.” The Journals of Gerontology Series A: Biological Sciences and Medical Sciences65A, no. 4 (2009): 429 –33. https://doi.org/10.1093/gerona/glp198.

22.

Służewska, A., J. K. Rybakowski, M. Laciak, A. Mackiewicz, M. Sobieska, and K. Wiktorowicv. “Interleukin-6 Serum Levels in Depressed Patients before and after Treatment with Fluoxetine.” Annals of the New York Academy of Sciences 762, no. 1 (2006): 474–76. https://doi.org/10.1111/j.1749-6632.1995.tb32372.x.

23.

Turnbull, Andrew V., and Catherine L. Rivier. “Regulation of the Hypothalamic-Pituitary-Adrenal Axis by Cytokines: Actions and Mechanisms of Action.” Physiological Reviews 79, no. 1 (1999): 1–71. https://doi.org/10.1152/physrev.1999.79.1.1.

24.

Valkanova, Vyara, Klaus P. Ebmeier, and Charlotte L. Allan. “CRP, IL-6 and Depression: A Systematic Review and MetaAnalysis of Longitudinal Studies.” Journal of Affective Disorders 150, no. 3 (2013): 736–44. https://doi.org/10.1016/ j.jad.2013.06.004.

25.

Zunszain, Patricia A., Nilay Hepgul, and Carmine M. Pariante. “Inflammation and Depression.” Behavioral Neurobiology of Depression and Its Treatment Current Topics in Behavioral Neurosciences, 2012, 135–51. https:// doi.org/10.1007/7854_2012_211.

172

Role of Lateral Hypothalamic Dopaminergic Mechanisms in Feeding Regulation: A Study Caitlin Therence Tejowinoto

The mesolimbic dopaminergic pathway, also known as the reward pathway, has been associated with motivation of certain behaviours in living organisms. Food intake is one of the key components of survival and studies have shown that food consumption results in dopamine release in the reward pathway, suggesting that dopaminergic mechanisms may be important in feeding regulation. The lateral hypothalamus is also thought to be a key player in feeding regulation because it has been linked to motivation of food consumption, innervated by appetite-affecting neurons from the arcuate nucleus, and the presence of neuropeptides that affects feeding behaviour such as melanocortin concentrating hormone and orexin. Presence of dopamine receptors in the lateral hypothalamus has been reported, hence Yonemochi et al. (2019) investigated the role of dopaminergic mechanisms in the lateral hypothalamus in regard to feeding regulation. It was found that the dopaminergic function in the lateral hypothalamus regulates feeding by a negative feedback mechanism, where the release of dopamine from feeding stimulates dopamine receptors in the lateral hypothalamus, which reduces food intake.

This study is important in investigating the homeostasis of food intake and energy balance, which can be applied to energy balance disorders.

Keywords: Dopamine, feeding regulation, lateral hypothalamus, neuropeptides, food intake, dopamine receptors

173

Introduction

take?; 4) What are the roles of dopamine receptors D1 and D2 within the LH on neuropeptides (Orexin, MCH, NPY, AgRP, Feeding is essential as it maintains energy balance, POMC)? Answering these questions would clarify whether doallowing organisms to perform basic bodily functions. Chronic paminergic mechanisms in LH regulates feeding through hypodisruptions of energy balance can lead to energy balance disorthalamic neuropeptides. ders like obesity and anorexia, which can be detrimental to one’s health (Reynolds et al., 2015). Dopaminergic pathways in the central nervous system is a significant component of the Methods brain’s reward system. The reward system motivates organisms To analyze the relationship between food intake, gluto perform certain behaviours such as feeding, drinking, and reproduction (Arias-Carrión et al., 2010). Studies have shown cose, and LH dopamine, mice were fasted for 16 hours, then that feeding, especially the consumption of palatable food, either re-fed or injected by glucose (control mice were fasted results in a release of dopamine in the mesolimbic dopaminer- throughout the process) while monitoring the LH dopamine gic pathway (Hajnal & Norgen, 2001; Schwartz et al., 2010; levels. Dopamine levels were monitored using in-vivo microdiYonemochi et al., 2019). Therefore, the mesolimbic dopaminer- alysis; dialysates were collected every 20 minutes pre- and post gic pathway is thought to play a key role in regulating feeding - refeeding. Dopaminergic neuron projections were visualized behaviour, specifically promoting the intake of preferred food. using the retrograde tracer Fluoro-gold (FG). FG were injected into the LH of rats, which were then left for a week. Following Another key to survival is homeostasis, defined as the the week, the brains were removed, fixed with paraformaldeprocess of maintaining a stable internal environment which is hyde, and then sectioned. Sections were also incubated with an mainly regulated by the hypothalamus. A specific region within antibody against tyrosine hydroxylase (TH), an enzyme responthe hypothalamus, the arcuate nucleus (ARC), is responsible for sible for the synthesis of dopamine. To investigate the role of feeding and metabolism regulation. ARC contains two types of D1 and D2 receptors, mice were fasted for 16 hours. Following neurons: 1) the agouti-related peptide (AgRP) and neuropepfasting, mice were injected with either D1 agonist (SKF 38393 tide Y (NPY) expressing neurons (AgRP/NPY) which are orexihydrochloride), D1 antagonist (SCH 23390 hydrochloride), D2 genic, meaning that they increase appetite, and 2) the proagonist (quinpirole hydrochloride), or D2 antagonist (lopiomelanocortin (POMC) expressing neurons (POMC) which sulpiride) and their cumulative food intake was monitored up are anorexigenic, which suppresses appetite (Timper & Brünto 4 hours following injection. Motor impairments due to dopaing, 2017). These neurons project to the lateral hypothalamus mine deficiency can affect feeding behaviour, hence drugs were (LH), where more neuropeptides that affect feeding behaviour administered at a dose that mice locomotor activity is unaffectsuch as melanocortin concentrating hormone (MCH) and orexin ed (Schwartz et al., 2010). Reverse transcription was also done are found, thus, LH is suggested to contribute to feeding reguto quantify the amount of mRNA of the neuropeptides of interlation as well (Sakurai et al., 1998; Brown et al., 2015; Yonemoest. An hour following drug injections, mice hypothalamus were chi et al., 2019). More reasons that LH may be important in dissected and the RNA were isolated. Reverse transcription was feeding is that lesion of LH in rats caused a loss of motivation to conducted followed by PCR, then analyzed using electrophoreeat or drink even though the rats were still physically able to do sis. so, resulting in starvation and dehydration; and electrical stimulation of LH can motivate feeding (Brown et al., 2015; Stuber & Wise, 2016).

Major Results Dopamine levels in the LH were found to be correlated Food Intake, Glucose, and LH Dopamine with food intake and dopamine receptors have been shown to In a fasted state, LH dopamine levels remained relatively be found within the LH (Meguid et al., 1995; Fetissov et al., 2002; Ikeda et al., 2018). A study by Ikeda et al. (2018) found steady. Food intake and glucose injection were both found to that inhibition of dopamine receptors in LH increases food in- increase LH dopamine levels (Fig. 1). This is consistent with pretake in mice, suggesting the possibility of a negative feedback vious findings by Meguid et al. (1995), where they found that mechanism through these dopamine receptors. Hence, dopa- dopamine release in LH is correlated with meal size. Yonemochi minergic function within the LH is suggested to play a key role et al. (2019) suggests that food intake increases blood glucose levels, which then stimulates glucose-responsive neurons in the in feeding behaviour regulation. hypothalamus.

Yonemochi et al. (2019)’s Study Yonemochi et al. (2019) followed up on Ikeda et al.

(2018)’s findings by conducting a study investigating the role of dopaminergic mechanisms in LH on feeding. The study aimed to answer the following questions: 1) What are the effects of food intake and glucose on LH dopamine levels?; 2) How do dopaminergic neurons project to the LH?; 3) What are the roles of dopamine receptors D1 and D2 within the LH on food in174

Fig. 1. Adapted from Yonemochi et al. (2019). Effect of refeeding (left) and glucose injection (right) on LH dopamine levels.

Both refeeding and glucose injection increases LH dopamine levels.

Projections of Dopaminergic Neurons to LH FG were found to be present in the VTA and SNc, indicating that the neurons projected from VTA and SNc to LH. TH were also found in most FG-positive cells. Since TH is a dopamine precursor, the presence of TH confirms that the neurons were indeed dopaminergic neurons.

rats resulted in a decrease of daily food intake and body weight. It has also been previously reported that striatal D2 receptors were downregulated in obese rats and knocking down of D2 resulted in compulsive feeding (Johnson & Kenny, 2010). These findings suggest that D1 and D2 activation terminates feeding. However, a study by Chen et al. (2014) found that injection of a different D1 agonist, SKF81297, into the perifornical LH led to an increase in food and alcohol intake, which contradicts the findings of the present study. A possible reason for this discrepancy is the possibility of different agonists giving rise to different effects, thus, additional research in this area is still needed.

Role of D1 and D2 Receptors on Hypothalamic Neuropeptides

Role of D1 and D2 Receptors on Food Intake When mice were injected with D1 receptor agonist (SKF 38393) or D2 receptor agonist (quinpirole), cumulative food intake is lower than control (Fig. 2). When the receptors are blocked by the antagonists, no change in food intake was observed. However, when the receptor agonists were administered together with the antagonists, there is no overall effect on food intake.

Stimulation of D1 receptors did not affect the mRNA of preproorexin nor MCH, while stimulation of D2 receptors decreased the amount of preproorexin mRNA but did not affect mRNA of MCH (Fig. 3). It has been discovered that the activity of orexin neurons decreases during refeeding or high blood glucose levels (Brown et al., 2015). This agrees with the present studyâ&#x20AC;&#x2122;s findings that suggests feeding increases LH dopamine levels, which stimulates D2 receptors, which in turn inhibits the activity of orexin neurons hence the low preproorexin levels.

Fig. 3. Adapted from Yonemochi et al. (2019). Effect of activation of dopamine receptors on preproorexin mRNA (left) and pro-MCH mRNA (right). A and B represents activation of D1 receptors, C and D represents activation of D2 receptors. Activation of dopamine D2 receptors (by administration of quiniprole) decreases preproorexin mRNA.

Although not shown in a figure, Stimulation of D1 receptors decreased the mRNA levels of NPY and AgRP, while stimulation of D2 receptors increased the mRNA levels of POMC. These results suggest a possibility that the stimulation of D1 receptors decreases feeding by inhibiting NPY/AgRP neurons, while D2 receptors in the LH decreases feeding by inhibiting orexin neurons and stimulating POMC neurons. This preSame results were found in a study done by Kuo sent study did not uncover the mechanisms behind the ob(2002), where co-administration of SKF 38393 and quinpirole in served phenomena. Yonemochi et al. (2019) suspected that LH Fig.2. Adapted from Yonemochi et al. (2019). Cumulative food intake following manipulation of lateral hypothalamic D1 receptors (left) and D2 receptors (right). Stimulation of D1 and D2 receptors result in decreased food intake.

175

D1 and D2 receptors affect NPY/AgRP and POMC neurons through GABA signalling. This idea is supported by the findings by de Vrind et al. (2019), where the activation of LH GABA neurons that coexpress the leptin receptor resulted in a decrease in body weight and food intake. However, the mechanism is still unclear.

Conclusions The present study by Yonemochi et al. (2019) found that: 1) food intake increases dopamine levels in the LH, 2) dopaminergic neurons project from the VTA and SNc to the LH, 3) stimulation of dopamine D1 and D2 receptors in the LH decreases food intake, and 4) stimulation of D1 receptors in LH inhibits NPY/AgRP neurons while stimulation of D2 receptors inhibits orexin neurons and stimulates POMC neurons. The authors came to a conclusion that feeding stimulates dopaminergic neurons that project to the LH from VTA and SNc, which acts as a negative feedback to terminate food intake through dopamine D1 and D2 receptors, possibly through neuropeptides.

The LH is involved in both motivation and homeostatic pathways. It has been previously thought that hypothalamic dopamine signalling through neurons in dorsomedial and ARC inhibits food intake (Schwartz et al., 2000). Although the present study did not establish a detailed pathway, it confirms that the dopaminergic pathway has inhibitory effects on food intake. The findings serve as a good starting point for future investigation of the mechanisms behind this phenomena. This regulation of feeding behaviour in the LH maintains the body’s energy balance. Looking into the underlying mechanism is important to further understand energy balance disorders and how to restore energy balance through either new medication or therapy methods.

dents. Nts neurons are activated by leptin, and the lateral hypothalamic area is the only site where leptin receptor (LepR) co -expressing Nts neurons are present. These neurons, along with the regular Nts neurons present in the LH, project to the VTA, suggesting that leptin and Nts may play a role in feeding regulation through dopaminergic mechanisms.

The present study discovered that stimulation of D1 and D2 receptors in the LH inhibits NPY/AgRP neurons and stimulates POMC neurons respectively, however, the underlying mechanisms are yet to be thoroughly investigated. Following the findings of this study, the researchers should uncover the precise mechanism on how D1 and D2 receptors affect NPY/AgRP and POMC neurons.

Another thing to note is that Yonemochi et al. (2019)’s study was conducted on male ICR mice and male Wistar rats.

While sex was kept the same for the sake of consistency and reducing variability, the researchers should have included one more (separate) group of female mice and rats. Male biology and female biology are different, hence the effects seen in male models might not be observed in female models. These studies have the potential to be developed further to investigate energy balance homeostasis and treatment for energy balance disorders. Female representation in these studies are greatly needed so that an effective treatment can be achieved for everyone.

Future directions How stimulation of D1 and D2 receptors in the LH inhibits NPY/AgRP neurons and stimulates POMC neurons is still unclear. Yonemochi et al. (2019) proposed a possible mechanism underlying these phenomena through GABA interneurons found in the LH (Fig. 4). This suggested experiment will clarify how the dopamine D1 and D2 receptors affect neuropeptides which are responsible for the regulation of feeding behaviour.

Critical Analysis The present study by Yonemochi et al. (2019) clarified the role of dopaminergic function in the LH in the regulation of feeding. The results of the present study mostly agree with published literature, except for the study by Chen et al. (2014) on the role of D1 and D2 in LH on food and alcohol intake, where they found that stimulation of D1 promotes food and alcohol consumption, whereas stimulation of D2 suppresses alcohol consumption. The present study found that both D1 and D2 suppress feeding behaviour when stimulated. Some possible reasons for this discrepancy are the usage of a different D1 agonist (SKF81297) by Chen et al. (2014), or that Chen et al. (2014) studied a specific part of the LH, the perifornical Fig. 4. Yonemochi et al. (2019)’s proposed mechanism of how lateral hypothalamus (PF/LH) which may have different effects LH D1 and D2 receptors affect NPY/AgRP neurons and POMC neurons through activation and inhibition of GABA. on feeding compared to LH as a whole.

The neuropeptides studied in this present study are: NPY, AgRP, orexin, and POMC. Other neuropeptides that the LH D1 Receptors’ Effect on NPY/AgRP Neurons authors can investigate are neurotensin (Nts) and leptin. NeuExperiments will be done on D2R-knockout mice. Mice rotensin is thought to also play a role in the regulation of feeding, as central Nts has been shown to reduce feeding in ro- will be separated into 3 treatments: 1) cerebral injection of D1 176

agonist; 2) cerebral injection of D1 agonist and GABA antagonist, and 3) control. Procedure will be similar to the present study by Yonemochi et al. (2019). After drug injection, the mice hypothalamus will be dissected, followed by RNA isolation, then reverse transcription. The mRNA present in the samples will be analyzed using electrophoresis. The mice group injected with D1 agonist only would have decreased levels of NPY and AgRP mRNA compared to the control group. If the suggested mechanism in Fig. 4 were to be true, when GABA antagonist is injected along with D1 agonist, there will be no decrease in NPY and AgRP mRNA as GABA interneurons will not inhibit NPY/AgRP neurons.

LH D2 Receptorsâ&#x20AC;&#x2122; Effect on POMC Neurons To investigate the effect of D2 receptors, experiment will be done on D1R-knockout mice. Mice will be separated into 3 groups: 1) cerebral injection of D2 agonist; 2) cerebral injection of D2 agonist and GABA agonist, and 3) control. Just like mentioned above, mice will undergo drug injection, hypothalamus dissection, RNA isolation, reverse transcription, and electrophoresis. Injection of D2 agonist will result in an increase of POMC mRNA compared to the control. If the suggested pathway in Fig. 4 were to be true, when GABA agonist is injected along with the D2 agonist, there will be no increase in POMC as the POMC neurons are inhibited by the GABA agonists.

177

REFRENCES 1.

Arias-Carrión, O., Stamelou, M., Murillo-Rodríguez, E., Menéndez-González, M., & Pöppel, E. (2010). Dopaminergic reward system: A short integrative review. International Archives of Medicine, 3(1), 24. https://doi.org/10.1186/1755-7682-3-24

2.

Brown, J. A., Woodworth, H. L., & Leinninger, G. M. (2015). To ingest or rest? Specialized roles of lateral hypothalamic area neurons in coordinating energy balance. Frontiers in Systems Neuroscience, 9. https://doi.org/10.3389/ fnsys.2015.00009

3.

Chen, Y.-W., Morganstern, I., Barson, J. R., Hoebel, B. G., & Sarah F. Leibowitz. (2014). Differential Role of D1 and D2 Receptors in the Perifornical Lateral Hypothalamus in Controlling Ethanol Drinking and Food Intake: Possible Interaction with Local Orexin Neurons. Alcoholism: Clinical and Experimental Research, 38(3), 777–786. https://doi.org/10.1111/ acer.12313

4.

de Vrind, V. A. J., Rozeboom, A., Wolterink‐Donselaar, I. G., Luijendijk‐Berg, M. C. M., & Adan, R. A. H. (2019). Effects of GABA and Leptin Receptor‐Expressing Neurons in the Lateral Hypothalamus on Feeding, Locomotion, and Thermogenesis.

Obesity, oby.22495. https://doi.org/10.1002/oby.22495

5.

Dong-Yih Kuo. (2002). Co-Administration of Dopamine D1 and D2 Agonists Additively Decreases Daily Food Intake, Body Weight and Hypothalamic Neuropeptide Y Level in Rats. Journal of Biomedical Science, 9(2), 126–132. https:// doi.org/10.1007/bf02256023

6.

Fetissov, S. O., Meguid, M. M., Sato, T., & Zhang, L.-H. (2002). Expression of dopaminergic receptors in the hypothalamus of lean and obese Zucker rats and food intake. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 283(4), R905–R910. https://doi.org/10.1152/ajpregu.00092.2002

7.

Hajnal, A., & Norgren, R. (2001). Accumbens dopamine mechanisms in sucrose intake. Brain Research, 904(1), 76–84. https://doi.org/10.1016/S0006-8993(01)02451-9

8.

Ikeda, H., Yonemochi, N., Ardianto, C., Yang, L., & Kamei, J. (2018). Pregabalin increases food intake through dopaminergic systems in the hypothalamus. Brain Research, 1701, 219–226. https://doi.org/10.1016/j.brainres.2018.09.026

9.

Johnson, P. M., & Kenny, P. J. (2010). Dopamine D2 receptors in addiction-like reward dysfunction and compulsive eating in obese rats. Nature Neuroscience, 13(5), 635–641. https://doi.org/10.1038/nn.2519

10.

Meguid, M. M., Yang, Z.-J., & Koseki, M. (1995). Eating induced rise in LHA-dopamine correlates with meal size in normal and bulbectomized rats. Brain Research Bulletin, 36(5), 487–490. https://doi.org/10.1016/0361-9230(95)92128-3

11.

O’Connor, E. C., Kremer, Y., Lefort, S., Harada, M., Pascoli, V., Rohner, C., & Lüscher, C. (2015). Accumbal D1R Neurons Projecting to Lateral Hypothalamus Authorize Feeding. Neuron, 88(3), 553–564. https://doi.org/10.1016/ j.neuron.2015.09.038

12.

Reynolds, C., Gray, C., Li, M., Segovia, S., & Vickers, M. (2015). Early Life Nutrition and Energy Balance Disorders in Offspring in Later Life. Nutrients, 7(9), 8090–8111. https://doi.org/10.3390/nu7095384

13.

Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli, R. M., Tanaka, H., Williams, S. C., Richardson, J. A., Kozlowski, G. P., Wilson, S., Arch, J. R. S., Buckingham, R. E., Haynes, A. C., Carr, S. A., Annan, R. S., McNulty, D. E., Liu, W.-S., Terrett, J. A., Elshourbagy, N. A., … Yanagisawa, M. (1998). Orexins and Orexin Receptors: A Family of Hypothalamic Neuropeptides and G Protein-Coupled Receptors that Regulate Feeding Behavior. Cell, 92(4), 573–585. https://doi.org/10.1016/S00928674(00)80949-6

14.

Schwartz, M. W., Woods, S. C., Porte, D., Seeley, R. J., & Baskin, D. G. (2000). Central nervous system control of food intake. Nature, 404(6778), 661–671. https://doi.org/10.1038/35007534

15.

Stuber, G. D., & Wise, R. A. (2016). Lateral hypothalamic circuits for feeding and reward. Nature Neuroscience, 19(2), 198– 205. https://doi.org/10.1038/nn.4220

16.

Timper, K., & Brüning, J. C. (2017). Hypothalamic circuits regulating appetite and energy homeostasis: Pathways to obesity. Disease Models & Mechanisms, 10(6), 679–689. https://doi.org/10.1242/dmm.026609

17.

Yonemochi, N., Ardianto, Chrismawan, Yang, Lizhe, Yamamoto, Shogo, Ueda, Daiki, Kamei, Junzo, Waddington, John L., & Ikeda, Hiroko. (2019). Dopaminergic mechanisms in the lateral hypothalamus regulate feeding behavior in association with neuropeptides. Biochemical and Biophysical Research Communications, 6.

178

The Therapeutic Potential of the Novel Player Kcnn2 in Fetal Alcohol Syndrome Disorder Pathogenesis Sofia Tiu

One of the leading contributors to disability within fetal development is alcohol, affecting 3% of all newborn children.1 Fetal alcohol spectrum disorders (FASD) is an umbrella term that is used for a set of disorders that arise by prenatal alcohol exposure (PAE) administered by a pregnant mother.2 Main FASD treatments currently rely on cognitive-behavioural treatments due to the lack of medications.3 Although the effects of these therapies have been well studied, there is ongoing research for more biological advances into discovering FASD pathogenesis. Currently, FASD pathogenesis has observed that PAE affects certain downstream neurodevelopmental pathways which lead to amplified consequences, such as excessive cell death and proliferation and modified cell signaling affecting gene expression.4 A recent study by Mohammad et al. indicates that increased expression of Kcnn2 is associated with motor learning disability in CD-1 FASD mice models.5 Furthermore, Kcnn2knockdown PAE mice express higher levels of motor learning in earlier adulthood compared to PAEmice with increased Kcnn2 expression.5 Investigation into this Kcnn2-motor pathway has indicated that inhibiting Kcnn2 channels increases motor cortex activation and decreases motor symptoms in a FASD mouse model, indicating that Kcnn2 inhibition may be a novel route for treating FASD motor progression in humans.5 Key words: Fetal Alcohol Syndrome Disorder (FASD), prenatal alcohol exposure (PAE), Kcnn2, motor learning, CD-1 mice model

179

Introduction

embryonic (E) days 16 and 17. These days correlate with human embryonic motor cortical development.5 Using EtOH or phosphate-buffered saline (PBS), fetal mice were administered EtOH/PBS via intraperitoneal injection. To prevent confounds, ratios of EtOH and PBS were created by comparing overall bodyweight at postnatal (P) day 0 for consistency.

Fetal Alcohol Syndrome Disorders (FASD) are caused by maternal administration of alcohol during gestation leading to prenatal alcohol exposure (PAE).2 They are often characterized by various combinations of facial deformations, developmental deficits and neurocognitive deficiencies that gradually worsen overtime.2 No single cause of FASD has been identified, and it is At P28, mice underwent open-field testing to measure likely that several factors contribute to its presentation. locomotion and agility. PAE and control mice failed to show locomotor deficits and anxious behaviour. Thus, suggesting Research indicates that different genetic mutations that PAE does not affect overall motor ability, only the learning can contribute to different aspects of the abnormalities present of motor ability. in FASD. Due to this, there are no effective treatments for all individuals.5 Additionally, research has linked infantile motor Accelerated rotarod tests were performed at P30-31. skills to early brain development.6 Through focusing on motor Initially, PAE-mice did not display significant differences. Howskill development as one of the main indices of cognitive devel- ever, PAE-mice exhibited an inability to learn as the rotarod opment, FASD research suggests that motor improvements in accelerated and increased susceptibility to falling (Figure 1B). early infants can lead to progressive cognitive improvement in Additionally, PAE-mice had lower learning indices compared to early childhood.6 the control-mice. (Figure 1A).

Previous studies have shown that heat shock signaling (HS) may contribute to the complex pathogenesis of FASD. 7,8,9 Evidence from mouse models show repetitive exposure of ethanol (EtOH) activates transcription factor heat-shock-factor-1 (HSF1) in a spontaneous manner.7,8 Further investigation of the HSF1 pathway suggests that activation of HSF1 within the embryonic mouse brain in response to PAE may contribute to FASD consequences.9 This research displayed that HSF1 deficiencies in cortical cells in utero could lead to higher variability in neural progenitor cells (NPC), not only in mice, but in humans as well.10 Therefore, it is possible that HSF1 and its downstream pathways in NPCs are important therapeutic targets for FASD. Further investigation of HSF1 pathways derived multiple targets that are responsible for mature brain abnormalities caused by HSF1. Although many of these targets remain unknown, few studies aim to investigate and analyze their role in FASD mice models. Mohammad et al., show that tracing HSF1 lineage leads to Kcnn2, which increased expression causes deficits in motor skills without affecting overall motor function. 5,11 Ultimately, this study demonstrates that decreased activity of Kcnn2 channels improves motor function in mice and provides therapeutic potential for humans with FASD due to its ability to decrease motor FASD symptoms.511

Major Results Mohammad et al. discovered that predisposed CD-1 mice models of FASD express higher genetic expression of Kcnn2 in cortical layers II and III of the motor cortex (M1). 5 Further analysis suggests that decreased activity of Kcnn2 channels has a causal effect on decreasing FASD motor symptoms and increasing cognitive capabilities, making it a potential target for FASD therapeutics.5

PAE Consequences in FASD-Mice Models

Figure 1.) A The learning indices of the control mice are significantly higher than PAE-mice, who are generally not capable of completing the accelerated rotarod task. Control mice withstand the acceleration of the rotarod more successfully than the PAE-induced mice and undergo motor learning (A-C). Mohammad, Shahid, Stephen J. Page, Li Wang, Seiji Ishii, Peijun Li, Toru Sasaki, Aiesha Basha, et al. â&#x20AC;&#x153;Kcnn2 Blockade Reverses Learning Deficits in a Mouse Model of Fetal Alcohol Spectrum Disorders.â&#x20AC;? Nature Neuroscience 23, no. 4 (April 2020): 533â&#x20AC;&#x201C;43. https://doi.org/10.1038/s41593-020-0592-z.

Previous literature has indicated that self-feeding behaviour is characteristic of FASD12. In the single-pellet reaching task, a successful trial consists of the mouse grasping the pellet and placing it in their mouth without dropping/fumbling. After several trials, control-mice increased their success rate by 25%.

PAE-mice exhibited less than an 8% increase in their success rates (Figure 2E). Comparing the forelimb grip strength of PAE and control was similar suggesting failed trials was not due to muscular weakness, but the impaired learning of grasping the pellet (Figure 2F). Lastly, learning indices of the accelerated rotarod and single pellet reaching tests were compared and indicated a significant correlation (Figure 2G).

Researchers administered EtOH to fetal mice during 180

Figure 2.) An example of the single-pellet reaching box is pictured in D. Those treated with PAE were less likely to be successful in reaching the pellet with only slight increases in success after several days . This failure is not due to muscular weakness, as seen in E-F. Graphical correlation of the learning indices of the accelerated rotarod and single pellet reaching test signifies a connected pathway that controls the motor deficits seen in both tasks in G. Mohammad, Shahid, Stephen J. Page, Li Wang, Seiji Ishii, Peijun Li, Toru Sasaki, Aiesha Basha, et al. “Kcnn2 Blockade Reverses Learning Deficits in a Mouse Model of Fetal Alcohol Spectrum Disorders.” Nature Neuroscience 23, no. 4 (April 2020): 533–43. https://doi.org/10.1038/ s41593-020-0592-z.

Figure 3.) Immunohistochemical analysis at P30 of PAE mice demonstrates the number of Kcnn2+ neurons are increased in PAE mice compared to the control. More so, this increase is not observed in HSF1-KO mice, seen in A-B. Mohammad, Shahid, Stephen J. Page, Li Wang, Seiji Ishii, Peijun Li, Toru Sasaki, Aiesha Basha, et al. “Kcnn2 Blockade Reverses Learning Deficits in a Mouse Model of Fetal Alcohol Spectrum Disorders.” Nature Neuroscience 23, no. 4 (April 2020): 533–43. https:// doi.org/10.1038/s41593-020-0592-z.

Kcnn2 Blockage and Learning in PAE-Mice

To investigate the inhibition of Kcnn2 channels, researchers administered tamapin, a Kcnn2 channel blocker.14 Intraperitoneal injection of labeled-tamapin at P30 decreased Acute Activation of HS Signaling on PAE-Mice Models Kcnn2+ neuron activity in the M1. Results showed that tamapin Mohammad et al. utilized an HSE-RFP reporter system significantly increased motor learning in PAE-mice compared to to lineage trace the cells that are responsible for motor deficits controls.3 in the cortex and its downstream effects. Constructs were also developed with GFP to indicate successful electroporation in the M1.

Constructs were delivered using electroporation at E15, 24-hours prior to PAE. In PAE-mice, most GFP+ neurons (in layers II and II of M1) were RFP+, indicating HS-signaling activation had occurred upon PAE.7 In contrast, there was less than 2% of co-expression of GFP/RFP in control-mice.

Researchers used unicellular RNA sequences to identify differentially expressed genes in PAE-mice to avoid masking other genes. Principal component analysis (PCA) showed genes Figure 4.) Tamapin administration to PAE-mice increases time specific to RFP+ neurons were associated with learning, plastici- spent on the accelerated rotarod, indicating increased motor ty, and long-term potentiation (LTP). learning. Mohammad, Shahid, Stephen J. Page, Li Wang, Seiji Ishii, Peijun Li, Toru Sasaki, Aiesha Basha, et al. “Kcnn2 Blockade Reverses Learning Deficits in a Mouse Model of Fetal AlcoKcnn2 in PAE-Mice Models hol Spectrum Disorders.” Nature Neuroscience 23, no. 4 (April Among 37 genes, Kcnn2 was the only gene that encoded 2020): 533–43. https://doi.org/10.1038/s41593-020-0592-z. a small-conductance calcium-activated potassium channel in the RFP+ group. Previous literature has shown that the Kcnn2 is influential to learning and memory.13 Immunohistochemical analysis of PAE-mice neuronal cells demonstrate increased activation in the M1 at P30.

In addition, researchers confirmed Kcnn2 is endogenous by using HSF1-knockout (KO) mice, where Kcnn2 failed to increase in response to PAE without HS-signaling activation, thereby not a consequence of RFP+ cells (Figure 3A-B).

181

Figure 5.) Tamapin administration to PAE-mice increases the success rate of the single-pellet reaching test, indicating increased motor learning. Mohammad, Shahid, Stephen J. Page, Li Wang, Seiji Ishii, Peijun Li, Toru Sasaki, Aiesha Basha, et al.

“Kcnn2 Blockade Reverses Learning Deficits in a Mouse Model of Fetal Alcohol Spectrum Disorders.” Nature Neuroscience 23, no. 4 (April 2020): 533–43. https://doi.org/10.1038/s41593-020 -0592-z.

ers on older mice (P180+) to examine human adult equivalent effects.16 By doing so, the experimental conditions will increase in external validity and representation of FASD medications being given to all age groups.

Additionally, the researcher confirms that Kcnn2 was 5th out of 37 possible genes that demonstrated interactions with EtOH in the cortex. Despite Mohammad et al. indicating that Kcnn2 and FASD have a causal relationship in the M1, there are other genes indicated by RFP+ neurons that may interfere with FASD pathogenesis. In order to increase the internal validity, future research should ensure there is a precise qualitative measure between all RFP+ genes that are being manipulated.

Therefore, stating that Kcnn2 is the cause of motor deficiency in the mice may not necessarily be true due to confounding genes.

Similarly, researchers created a tamoxifen-inducible Kcnn2 short-hairpin RNA (shRNA) and control shRNA into M1 at E15. Rotarod and single-pellet reaching tasks before and after tamoxifen-injection allows Kcnn2 blockage after 30 minutes of exposure. Increased motor learning skills of the rotarod and single-pellet reaching tasks demonstrated that Kcnn2knockdown in the M1 was sufficient to improve learning defiFurthermore, the discussion states that Kcnn2 channels cits in PAE-mice. were not limited to layers II and III of the M1 which decreases the internal validity that Kcnn2 are solely responsible in motor deficiencies. Kcnn2 channels are found in various parts of the Conclusions central and peripheral nervous system and are largely known for neurodegeneration in Alzheimer’s disease (AD).17 Previous The data presents evidence of a causal relationship beresearch has noted that Kcnn2 channels inhibit synaptic plastictween Kcnn2 expression and the motor-learning deficiencies of ity, therefore, inhibiting Kcnn2 increases synaptic plasticity.

FASD in a mouse model. It was determined that Kcnn2 levels However, many studies on Kcnn2 channels are not human studare increased in PAE-mice. Additionally, motor deficits are alleies and fail to administer specific neuronal Kcnn2 blockers withviated by decreased Kcnn2 activity via tamapin or tamoxifenout transgenic mice.18 Human studies of AD have shown that inducible Kcnn2-shRNA. These findings suggest that the blockhumans cannot survive with minimal Kcnn2 channel activity age of Kcnn2 shows great potential in future FASD therapies. due to their ability to modulate glutamate intake. 19 Research by The discovery of FASD treatments is important due to Allen et al. argue against Kcnn2 blockers, as pharmacological the lack of effective therapies.3 Although Kcnn2 is new in FASD Kcnn2 activation can increase neurorestorative properties and research, further investigation can determine whether Kcnn2 decrease neurodegeneration in AD.19 Therefore, Kcnn2blockers are eligible for human intervention. Previous studies blockage in humans may cause different consequences than in that investigated HS-signaling pathways target other genes that mice, especially in different areas that express Kcnn2. are responsible for cortical deficits, but have yet to see any Lastly, the quantity of the drug and its effects are left improvements in FASD adults.3,6,10 inconclusive. Mohammad et al. based their drug concentrations Mohammad et al. conclude that blocking Kcnn2 expres- on mouse body weight and only gave a singular dose. Although sion in mice should be transferred over to human studies in this singular dose improves motor learning in PAE-mice, these order to investigate its true effectiveness. Although the evi- improvements diminished quickly with a two-week maximum. dence for Kcnn2 blockers is strong in mouse models, research is The research failed to expand on how often this drug would uncertain if these results would directly translate to human need to be administered. Other pharmacological questions that models.5 Despite the strong findings in the mouse model, fur- were not investigated were the dose-dependent consequences, ther research is required to ensure efficiency and safety in for blocking efficiency per dosage given and/or evaluating the marfuture therapeutic uses. gin of safety (MOS). Overall, these are all factors that should be considered in future research.

Critical Analysis The data collected by Mohammad et al. provides evidence for a promising therapeutic approach to FASD. However, several aspects of this study require improvement. Firsly, this study only investigated the effects of Kcnn2 blockade in mice up to P35. Although the most studied age groups of FASD are children, evidence from literature displays that FASD-adults exhibit different biological and behavioural symptoms. 15 By only examining mice at date P35, concluding Kcnn2 blockers are an eligible FASD medication only applies to one age group.

This can be prevented by examining the effects of Kcnn2 block-

Future Directions Future endeavors should begin by studying the significance of Kcnn2 in comparison to the other genes that are functionally relevant to FASD motor deficiencies. This would be done by examining other RFP+ genes which are activated in PAE response and present in HS-signaling of NPCs. If the results demonstrate that Kcnn2 is not affected by coinciding genes activated in HS-signaling, then it would increase causation of Kcnn2. If Kcnn2 results to act comorbidly with another gene, then this would contradict the conclusions of Mohammad et

182

al., potentially guiding researchers to investigate the new gene of interestâ&#x20AC;&#x2122;s downstream effects on motor learning. Another note of importance is investigating whether potential genes activate other brain regions than layers II and III of the M1 via HSF1-lineage trace. As seen in the present study, Kcnn2 upregulation was not exclusive to the M1 layers. If Kcnn2 and the new gene active other areas that influence motor learning, this would prompt a reevaluation of their findings. In the case of a multiple affected regions of FASD, this would prohibit the development of human studies as the FASD pathogenesis in mice remains unclear.

In order to progress to human models, future research must produce a drug that minimizes negative side effects that may cause further motor and cognitive decline. The drug must target M1 Kcnn2 channels without transgenic humans and not target peripheral Kcnn2 that contribute neuroprotective properties.19 Experiments that address these issues should include testing the long-term effects of these drugs and Kcnn2 regulation within the M1. One way to mimic the effects of Kcnn2 blockers and their specificity is to use transgenic-FASD and wild -type mice to analyze which Kcnn2 channels are the ultimate targets for Kcnn2 blockers. To understand the level of Kcnn2 responsiveness and MOS, another factor to consider is the dose-dependency. If the drug is required in higher doses to effectively inhibit, then the MOS decreases and individuals are more prone to negative side effects. Beyond this, testing Kcnn2 blockers on wild-type mice would examine the consequences of Kcnn2 inhibition that are currently unaccounted for.

There are still many conflicts in confirming Kcnn2 is a therapeutic drug to combat FASD in humans. Currently, the research that has been conducted highlights promising genes that aid in creating future therapies for FASD in humans.

183

REFRENCES

1.

Popova, Svetlana, Shannon Lange, Kevin Shield, Alanna Mihic, Albert E Chudley, Raja A S Mukherjee, Dennis Bekmuradov, and Jürgen Rehm. “Comorbidity of Fetal Alcohol Spectrum Disorder: A Systematic Review and Meta-Analysis.” The Lancet 387, no. 10022 (March 5, 2016): 978–87. https://doi.org/10.1016/S0140-6736(15)01345-8.

2.

Mukherjee, Raja A S, Sheila Hollins, and J Turk. “Fetal Alcohol Spectrum Disorder: An Overview.” Journal of the Royal Society of Medicine 99, no. 6 (June 1, 2006): 298–302. https://doi.org/10.1177/014107680609900616.

3.

Doig, Jenna, John D. McLennan, and W. Ben Gibbard. “Medication Effects on Symptoms of Attention-Deficit/Hyperactivity Disorder in Children with Fetal Alcohol Spectrum Disorder.” Journal of Child and Adolescent Psychopharmacology 18, no. 4 (May 9, 2007): 365–71. https://doi.org/10.1089/cap.2007.0121.

4.

Sulik, Kathleen K. “Chapter 26 - Fetal Alcohol Spectrum Disorder: Pathogenesis and Mechanisms.” In Handbook of Clinical Neurology, edited by Edith V. Sullivan and Adolf Pfefferbaum, 125:463–75. Alcohol and the Nervous System. Elsevier, 2014. https://doi.org/10.1016/B978-0-444-62619-6.00026-4.

5.

Mohammad, Shahid, Stephen J. Page, Li Wang, Seiji Ishii, Peijun Li, Toru Sasaki, Aiesha Basha, et al. “Kcnn2 Blockade Reverses Learning Deficits in a Mouse Model of Fetal Alcohol Spectrum Disorders.” Nature Neuroscience 23, no. 4 (April 2020): 533–43. https://doi.org/10.1038/s41593-020-0592-z.

6.

Streissguth, A. P., H. M. Barr, and P. D. Sampson. “Moderate Prenatal Alcohol Exposure: Effects on Child IQ and Learning Problems at Age 7 1/2 Years.” Alcoholism, Clinical and Experimental Research 14, no. 5 (October 1990): 662–69. https:// doi.org/10.1111/j.1530-0277.1990.tb01224.x.

7.

Ishii, Seiji, Masaaki Torii, Alexander I. Son, Meenu Rajendraprasad, Yury M. Morozov, Yuka Imamura Kawasawa, Anna C. Salzberg, et al. “Variations in Brain Defects Result from Cellular Mosaicism in the Activation of Heat Shock Signalling.” Nature Communications 8 (2017). https://doi.org/10.1038/ncomms15157.

8.

Torii, Masaaki, Masanori Sasaki, Yu-Wen Chang, Seiji Ishii, Stephen G. Waxman, Jeffery D. Kocsis, Pasko Rakic, and Kazue Hashimoto-Torii. “Detection of Vulnerable Neurons Damaged by Environmental Insults in Utero.” Proceedings of the National Academy of Sciences of the United States of America 114, no. 9 (February 28, 2017): 2367–72. https:// doi.org/10.1073/pnas.1620641114.

9.

El Fatimy, Rachid, Federico Miozzo, Anne Le Mouël, Ryma Abane, Leslie Schwendimann, Délara Sabéran-Djoneidi, Aurélie de Thonel, et al. “Heat Shock Factor 2 Is a Stress-Responsive Mediator of Neuronal Migration Defects in Models of Fetal Alcohol Syndrome.” EMBO Molecular Medicine 6, no. 8 (August 2014): 1043–61. https://doi.org/10.15252/ emmm.201303311.

10.

Hashimoto-Torii, Kazue, Masaaki Torii, Mitsuaki Fujimoto, Akira Nakai, Rachid El Fatimy, Valerie Mezger, Min J. Ju, et al.

“Roles of Heat Shock Factor 1 in Neuronal Response to Fetal Environmental Risks and Its Relevance to Brain Disorders.”

Neuron 82, no. 3 (May 7, 2014): 560–72. https://doi.org/10.1016/j.neuron.2014.03.002.

11.

Lam, Jenny, Nichole Coleman, April Lourdes A. Garing, and Heike Wulff. “The Therapeutic Potential of Small-Conductance KCa2 Channels in Neurodegenerative and Psychiatric Diseases.” Expert Opinion on Therapeutic Targets 17, no. 10 (October 2013): 1203–20. https://doi.org/10.1517/14728222.2013.823161.

12.

Amos-Kroohs, Robyn M., Birgit A. Fink, Carol J. Smith, Lyanne Chin, Sandra C. Van Calcar, Jeffrey R. Wozniak, and Susan M. Smith. “Abnormal Eating Behaviors Are Common in Children with Fetal Alcohol Spectrum Disorder.” The Journal of Pediatrics 169 (February 2016): 194-200.e1. https://doi.org/10.1016/j.jpeds.2015.10.049.

13.

Lin, Mike T., Rafael Luján, Masahiko Watanabe, John P. Adelman, and James Maylie. “SK2 Channel Plasticity Contributes to LTP at Schaffer Collateral-CA1 Synapses.” Nature Neuroscience 11, no. 2 (February 2008): 170–77. https://doi.org/10.1038/ nn2041.

14.

“Scorpion Venom Research Around the World: Indian Red Scorpion | SpringerLink.” Accessed June 10, 2020. https:// link.springer.com/referenceworkentry/10.1007%2F978-94-007-6647-1_5-1.

15.

Moore, Eileen M., and Edward P. Riley. “What Happens When Children with Fetal Alcohol Spectrum Disorders Become Adults?” Current Developmental Disorders Reports 2, no. 3 (September 2015): 219–27. https://doi.org/10.1007/s40474015-0053-7.

184

16.

Hagan, Catherine, D.V.M., and Ph.D. “When Are Mice Considered Old?” The Jackson Laboratory. Accessed June 11, 2020. https://www.jax.org/news-and-insights/jax-blog/2017/november/when-are-mice-considered-old.

17.

Kuiper, Els F. E., Ad Nelemans, Paul G. M. Luiten, Ingrid M. Nijholt, Amalia M. Dolga, and Uli L. M. Eisel. “KCa2 and KCa3 Channels in Learning and Memory Processes, and Neurodegeneration.” Frontiers in Pharmacology 3 (2012). https:// doi.org/10.3389/fphar.2012.00107.

18.

Stackman, Robert W., Rebecca S. Hammond, Eftihia Linardatos, Aaron Gerlach, James Maylie, John P. Adelman, and Thanos Tzounopoulos. “Small Conductance Ca2+-Activated K+Channels Modulate Synaptic Plasticity and Memory Encoding.” Journal of Neuroscience 22, no. 23 (December 1, 2002): 10163–71. https://doi.org/10.1523/JNEUROSCI.22-2310163.2002.

19.

Allen, Duane, Bernd Fakler, James Maylie, and John P. Adelman. “Organization and Regulation of Small Conductance Ca2+ -Activated K+ Channel Multiprotein Complexes.” Journal of Neuroscience 27, no. 9 (February 28, 2007): 2369–76. https:// doi.org/10.1523/JNEUROSCI.3565-06.2007.

185

Treating Parkinson’s Disease Symptoms Using Optogenic Deep Brain Stimulation in the Subthalamic Nucleus Adam Vanderlaan

Deep brain stimulation has been proven to be effective, however some of the mechanisms for its success are unclear. A patient who has experienced Parkinson’s symptoms for over 4 years may undergo this procedure which involves doctors using a thin wire to send electrical impulses into the brain. This stimulation alleviates Parkinson’s symptoms like muscle stiffness, slowness to movement, and muscle tremors. However, there are other symptoms associated with Parkinson’s that it does not treat and may even worsen issues involving memory and thinking (Michael J. Fox Foundation, 2020). DBS targets the subthalamic nucleus, a node that is critical to motor control from the basal ganglia. In animal models, inhibiting the function of the subthalamic nucleus using DBS has been demonstrated to be an effective method of treatment for Parkinson’s motor symptoms. It is similarly effective in humans but requires high frequency chronic stimulation from an implanted device (Benabid, 2003). The study by Yu et al. further investigated the mechanisms behind this form of treatment and concluded that a higher stimulation rate contributed to reduced abnormal oscillatory activity in the subthalamic nucleus, therefore treating Parkinson’s motor symptoms better than low frequency optogenic treatment. Their main findings stipulated that the kinetic properties of the opsins used are very important to the results of the optogenic deep brain stimulation treatment.

186

turning after high rate DBS, but no change in behaviour after low rate stimulation. The other opsin that was tested was ChR2, which had no change to behaviour after both high rate and low rate stimulation. These results were significant as a measurable improvement in forelimb stepping was observed after high rate optogenic DBS to the STN using the opsin Chronos.

Major Results

The main discovery of the study by Yu et al. was that using an opsin that could keep up with the high rates of stimulation required for DBS effectively reduced the forelimb motor Background and Introduction degradation caused by Parkinson’s. The kinetically slower opsin ChR2 was unable to elicit a response in both high rate and low rate stimulation, which confirms that the kinetics of the opsin Optogenic DBS therapy has many clinical benefits, but are just as important as the rate by which it is stimulated. the mechanisms behind its effect remain somewhat of a mystery. Further research into the subject could potentially optimize treatment, and a broader understanding of its function may lead to its use in the treatment of other neurological disorders. Deep brain stimulation was first introduced in 1997 and was a vast improvement to the treatment of Parkinson’s symptoms over previous ablative methods like thalamotomy (Benabid et al., 2003). DBS is a safer alternative as it is reversible, and the high frequency electrical pulses at 100khz were very effective at inhibiting activity that produces movement from Parkinson’s disease. Benabid et al. found that DBS of the subthalamic nucleus in particular greatly improved the daily activities of patients even after a 12-month period and reThe above graphics from Yu et al. visually demonstrate marked that potential uses for the treatment were “far from the main findings of the paper. On the left in dark blue, fully explored at this time”. This treatment also allowed them Chronos is seen to reduce the turns per minute of the rats unto decrease other dopaminergic treatments by 50%. As this der testing, but only at stimulation rates of 75pps to 130pps. technique is so proficient at getting meaningful results in paConversely, the ChR2 is unable to mitigate the effects of Parkintients and comes at a relatively lower risk than previous treatson’s motor control symptoms at all stimulation rates. These ments, it is justifiable to dedicate further resources to enhance results are in line with other studies which concur that DBS at our knowledge of the underlying processes in DBS. the STN is an effective treatment for Parkinson’s motor sympIn the paper by Yu et al., optogenic deep brain stimula- toms, however there is still debate around the actual reason as tion was used to treat mouse models with induced Hemi- to why this is the case. A study by Lüscher et al. proposes that Parkinson’s disease. Optogenic therapy involves genetically this type of treatment works due to the inhibition of an overacmodifying the neurons in the subthalamic nucleus to produce a tive striatal output pathway, and that the DBS is correcting a type of protein called an opsin. When specific ranges of light pathway that is malfunctioning as a result of dopamine abare received by the opsin, the neuron will activate. In this way, sence, without actually restoring dopamine levels. researchers can specifically target certain neurons in the brain and activate them at will (Frontiers for Young Minds, 2020). Yu et al. compared two different opsins in their approach. A low Conclusion/Discussions frequency, commonly used opsin called ChR2, and an opsin that responded to high frequency stimulation called Chronos.

The key takeaway from this paper is that the kinetic All of the rats were injected with 6-OHDA to induce HemiParkinson’s and implanted with optical fibers in the subthalam- properties of the selected opsin play a large role in its effectiveic nucleus. A circling test and adjusted step test were per- ness in treatment, and as such may be an important avenue of formed to demonstrate the effects of DBS, where the ratio of research for further discoveries. If we can better understand steps with the contralateral forelimb to the ipsilateral forelimb how exactly the treatment works, then perhaps it can be apwas measured over the course of the tests. They found that plied more efficiently or used as treatment in other scenarios. rats injected with Chronos exhibited suppressed ipsilateral With deep brain stimulation it is hard to identify exactly what is 187

affected by the treatment, and even harder to specifically activate neurons without affecting many of the adjacent neuronal regions. For this reason, DBS via optogenics may not be feasible as a treatment in humans since our brains are vastly more complex than the genetically modified ones found in rats currently being used for optogenics (Lüscher et al.). There is also a discussion to be had as to why the subthalamic nucleus is the preferred target for the therapy, as the internal pallidum may be just as effective (Krack et al.). This illustrates another area where there is still much left to investigate.

Critical Analysis

The study by Yu et al. sets out to explore an area of science where there are many questions left to be answered, but exactly how much they achieved to further the research is questionable. It has long been established that deep brain stimulation is able to achieve treatment to Parkinson’s motor symptoms since its discovery in 1997 (Benabid et al., 2003). The researcher’s use of optogenics to implement the DBS is compelling, but the likelihood that that technology will ever be safely utilized in human patients is uncertain. However, they were able to conclude that only ultrafast opsins like Chronos would be effective in treatment, and this is progress nonetheless. It is always hard to see where the next breakthrough will come from, so studies like these are important to fully explore all aspects of a topic that are available to researchers with today’s technology. An example of where this new information may be implemented could be as a follow up to a study by Chiang et al. where the opsin ChR2 was used to treat epileptic symptoms. In this case, they used high frequency stimulation while using ChR2 and achieved a response to the treatment. It would be interesting to see the same experimental procedure carried out using the ultrafast opsin Chronos now that we know that the kinetics of the opsin can play a large role in the outcome of the procedure.

There is certainly a lot to learn about DBS and its applications to treat neurological diseases. Since we don’t quite understand exactly how it treats these parkinsonian symptoms, I think it would be beneficial to use it on a plethora of different rats with various induced neurological diseases. Using the results from the study by Yu et al., we know that high frequency stimulation using ultrafast opsins has a pronounced effect on the brains of the test subjects, so maybe it would have as of yet unknown effects on rats with induced Alzheimer’s, epilepsy, or other neurodegenerative diseases. Knowing that depression relates to dopaminergic imbalances, DBS may even be a method of treatment to explore. It was speculated by Lüscher et al. that DBS treatment corrected a similarly malfunctioning pathway in the case of Parkinson’s. In a field of science where there is still so much to learn, doing as much research as possible into the unknown is sure to yield some fascinating results.

188

REFRENCES

1.

Kondabolu, Krishnakanth, Marek Mateusz Kowalski, Erik Andrew Roberts, and Xue Han. “Optogenetics and Deep Brain Stimulation Neurotechnologies.” Cognitive Enhancement Handbook of Experimental Pharmacology, 2015, 441–50. https:// doi.org/10.1007/978-3-319-16522-6_15.

2.

“What Is Optogenetics and How Can We Use It to Discover ...” Accessed June 18, 2020. https://kids.frontiersin.org/ article/10.3389/frym.2017.00051.

3.

“Deep Brain Stimulation.” The Michael J. Fox Foundation for Parkinson's Research | Parkinson's Disease. Accessed June 18, 2020. https://www.michaeljfox.org/news/deep-brain-stimulation.

4.

Benabid, Alim Louis. “Deep Brain Stimulation for Parkinson’s Disease.” Current Opinion in Neurobiology 13, no. 6 (2003): 696–706. https://doi.org/10.1016/j.conb.2003.11.001.

5.

Yu, Chunxiu, Isaac R. Cassar, Jaydeep Sambangi, and Warren M. Grill. “Frequency-Specific Optogenetic Deep Brain Stimulation of Subthalamic Nucleus Improves Parkinsonian Motor Behaviors.” The Journal of Neuroscience 40, no. 22 (2020): 4323 –34. https://doi.org/10.1523/jneurosci.3071-19.2020.

6.

Krack, Paul, and Marwan I. Hariz. “Deep Brain Stimulation in Parkinson Disease—What Went Wrong?” Nature Reviews Neurology 6, no. 10 (2010): 535–36. https://doi.org/10.1038/nrneurol.2010.141.

7.

Lüscher, C, and P Pollak. “Optogenetically Inspired Deep Brain Stimulation: Linking Basic with Clinical Research.” Swiss Medical Weekly, 2016. https://doi.org/10.4414/smw.2016.14278.

8.

Chiang, Chia-Chu, Thomas P. Ladas, Luis E. Gonzalez-Reyes, and Dominique M. Durand. “Seizure Suppression by High Frequency Optogenetic Stimulation Using In Vitro and In Vivo Animal Models of Epilepsy.” Brain Stimulation 7, no. 6 (2014): 890–99. https://doi.org/10.1016/j.brs.2014.07.034.

189

Chronic Jet Lag and Its Long-Term Effects on Brain Function Michelle Wrona

Frequent transmeridian travel has the potential to have a detrimental effect on circadian rhythms and brain function. This idea is commonly known as jet lag, which often has a greater influence on travelers undergoing a phase advance, which relates to eastbound travel, instead of phase delays, instigated by westbound travel. Researchers are seeking to determine the effects of chronic jet lag simulation on adult male Sprague Dawley rats. Specifically, they speculate that chronic jet lag negatively influences cognitive and affective behaviours, and hippocampal neurogenesis. The three randomly assigned experimental groups utilized included a control group, phase advancement group, where the light period was shortened by 6 hours, and a phase delay group, whose dark period increased by 6 hours. The light-dark (LD) cycle shifts occurred over a span of eight weeks, with each shift occurring once a week. After the eighth shift, the rats underwent a variety of tests, including: sucrose consumption testing, open field testing, elevated plus maze, forced swim test (FST), and object recognition testing. These tests were conducted to evaluate alterations in emotional behaviour, specifically in anxiety and depression-like symptoms, and to examine short-term memory and learning. It was implied that phase advances had a negative effect on the rats, signifying that it enhances depressive behaviours and deficits in memory. Further discussions showcased that phase advance rats had reduced DCX+ cells in the hippocampus, in addition to disrupted object retention memory. Additionally, symptoms of depression were seen after phase advances. Furthermore, phase delays had little to no effect on the measures examined.

Key words: chronic jet lag; light dark (LD) cycle; circadian rhythms; phase advance; phase delay; hippocampal neurogenesis; depression; anxiety

190

Jet lag is a condition experienced by humans shifting time zones due to travel, leading to the lack of synchrony between the time in the new time zone and the body’s clock (Shen et al., 2019). Several factors, including the direction travelled, timing of travel, and number of time zones shifted, have an impact on the severity of this condition (Choy and Salbu, 2011). Symptoms include lack of concentration, lethargy, disorders impacting the gastrointestinal tract, irritability, and difficulties with sleep (Zhang et al., 2020).

This condition impacts the body’s circadian “clock,” which, over time, will likely acclimatize to the new time zone (Choy and Salbu, 2011). When experienced frequently, this condition may become chronic, leading to cognitive deficits and depression.

Depressive behaviours are likely instigated due to the hypothalamic-pituitary-adrenal (HPA) axis causing the exogenous administration of CORT, a corticosteroid which regulates stress (Shen et al., 2019). It is also suggested that the central circadian clock impacts cognitive functions such as learning and memory to the extent that the greater the phase advance, the greater impact on memory (Loh et al., 2010).

Although multiple studies demonstrate the negative effects of jet lag, clinical studies have rarely focused on the decline in neurogenesis in animal models, especially in those sharing similarities in brain structures to humans. Horsey et al.’s study highlighted the decrease in hippocampal neurogenesis through immunohistochemistry, where immature neurons were viewed using DCX, a doublecortin. The quantity of immature DCX + dendate granule cells was assessed by an optical fractionator method, and later analyzed. This is a significant part of the study which highlights that jet lag has a long-lasting effect on an individual, instead of causing short-term symptoms such as fatigue. This suggests that the Horsey et al. study has the potential to be revolutionary, allowing individuals to view the major findings initially seen in rats as something that may affect everyone.

While it is known that jet lag can have chronic effects on brain function, specific tests on animal models have not been conducted to physically view these behaviours. Horsey et al. were aware of the negatively associated impacts of chronic jet lag, though their purpose was to seek physical evidence of deficits in brain function and the decrease of neurogenesis. By viewing decreased neurogenesis in the hippocampus, disrupted object retention memory, body weight changes in phase advance rats, the authors could conclude that jet lag, especially in animals experiencing phase advancements by at least 6 hours, has a detrimental effect. They determined that the consequences of phase advances include an increase in anxiety and depression, memory impairments, and neurogenesis disruption. These findings provide a link between phase advances and increased harmful effects on brain function and overall cognition. This

implies that humans travelling westbound are less likely to experience damaging effects on brain function.

Major Results Memory Impairments in Jet Lag-Influenced Rats

To investigate the effects of experimental chronic jet lag on memory, Horsey and her colleagues performed a novel object recognition test. It was discovered that after a 60-minute retention interval, phase advance rats had a lower discrimination index than control and phase delay rats. They demonstrated that chronic jet lag in rodents induces impairments in object recognition memory, suggesting that the impairments specifically occur in the stabilization of new memories. This stabilization is normally controlled by long-term potentiation and synaptic efficacy, though, due to circadian disruption, instability resulted. This memory impairment is induced by stress level cortisol (Cho et al., 2000), meaning that exposure to this hormone has the potential to result in memory deficits. Phase delays had little to no effect on the rodent’s memory and learning skills.

Other studies also suggest that memory impairments have a relationship with the decline in hippocampal neurogenesis (Gibson et al., 2010). After chronic temporal disruption procedures, rodents were unable to learn a specific task and demonstrate preference amongst a rewarding versus unrewarding chamber.

Adapted from Gibson, Erin M., Connie Wang, Stephanie Tjho, Neera Khattar, and Lance J. Kriegsfeld. 2010. “Experimental ‘Jet Lag’ Inhibits Adult Neurogenesis and Produces Long-Term Cognitive Deficits in Female Hamsters.” Edited by Shin Yamazaki. PLoS ONE 5 (12): e15267. https://doi,org/10.1371/ journal.pone.0015267

Background and Introduction

Figure 1 A) control versus jet lagged (JL) hamsters; control show preference for black chamber, JL show no preference (B) after training; control rodents show preference for rewarding chamber, JL show no preference; JL return to standard LD cycle (C) after more training; control rodents show same preference; JL show no preference; training began in chambers opposite to first test (D) JL did not learn new task; control rodents preferred new chamber Increase in Anxiety and Depressive Behaviours in Jet LagInfluenced Rats It is hypothesized that melatonin has a role in alleviating jet lagrelated symptoms. In individuals with depression, there are aberrations in the secretion of this hormone, leading to low

191

levels (Katz et al., 2001). In many studies, researchers focus on the phase shift hypothesis (PSH), which indicates that a phase shift in the LD cycle can induce depression in an individual (Lewy et al., 2007). In Horsey et al.’s study, evidence from several behavioural measures studying phase shifts in LD cycles support the relationship between the increase in anxiety and depression and disruptions in circadian rhythms. Horsey and her colleagues utilized a sucrose consumption test in their study to test for anhedonia in the male rodents. In the study, decreased sucrose consumption was demonstrated in the rodents subjected to weekly phase advances in comparison to phase delays.

Studies have shown that high stress due to increased cortisol levels and jet lag have a role in the induction of hippocampal atrophy, leading to deficits in learning and memory (Cho, 2000).

It is predicted that circadian disruption has the detrimental effect of reducing neurogenesis and hippocampal cell proliferation, which accounts for the cognitive deficits previously explained. Deficits from the reduction in neurogenesis have been hypothesized to occur after the termination of jet lag, meaning that this condition causes long-term negative effects on the brain (Gibson et al., 2010). The Horsey et al. study also demonstrated the same idea, implying that phase advances have the potential to act as “stressors” that decrease hippocampal neurogenesis. In the study, researchers sought to find the total number of DCX + cells in rodents, and compare the number amongst the experimental groups. In phase advance rats, this number was significantly lower than phase delay rodents or those on a standard LD cycle.

Adapted from Horsey, Emily A., Teresa Maletta, Holly Turner, Chantel Cole, Hugo Lehrman, and Neil M. Fournier. 2020. “Chronic Jet Lag Simulation Decreases Hippocampal Neurogenesis and Enhances Depressive Behaviors and Cognitive Deficits in Adult Male Rats.” Frontiers in Behavioral Neuroscience 13 (January). https://doi.org/10.3389/fnbeh.2019.00272.

Figure 2 A) Change in body weight in rodents of three experimental groups; phase advanced rats weight increased significantly more than standard and phase delays rodents. (B) Phase advanced rats consumed less sucrose after shifts 4 and 6 than standard and phase delay rats. (C) Sucrose consumption over a 24 hr period after shift 8 ended in (B).

Phase advanced rats consumed less sucrose than standard and phase delayed rats.

Adapted from Horsey, Emily A., Teresa Maletta, Holly Turner, Chantel Cole, Hugo Lehrman, and Neil M. Fournier. 2020. “Chronic Jet Lag Simulation Decreases Hippocampal Neurogenesis and Enhances Depressive Behaviors and Cognitive Deficits in Adult Male Rats.” Frontiers in Behavioral Neuroscience 13 (January). https:// doi.org/10.3389/fnbeh.2019.00272.

Similar behavioural measures were studied in the FST, where phase advance rats experienced greater immobility than the other two groups. Similar findings were seen in the open field arena and elevated plus maze, alluding to decreased exploration of areas being related to increased anxiety levels. The study implies that phase advances influence mood regulation and anxiety (Horsey et al., 2020).

Figure 4 Estimation of the quantity of DCX + cells in the dendate subgranular zone. Chronic phase advance rats had a significantly reduced amount of DCX + cells in comparison to standard LD cycle rodents or phase delay rodents.

Adapted from Horsey, Emily A., Teresa Maletta, Holly Turner, Chantel Cole, Hugo Lehrman, and Neil M. Fournier. 2020. “Chronic Jet Lag Simulation Decreases Hippocampal Neurogenesis and Enhances Depressive Behaviors and Cognitive Deficits in Adult Male Rats.” Frontiers in Behavioral Neuroscience 13 (January). https://

Conclusion/Discussion

The authors highlight that DCX is not represented solely during early neuronal differentiation, but throughout the entire maturation (Horsey et al., 2020). Chronic jet lag affects immature (DCX +) neuronal maturation due to phase advances causing a loss of late stage DCX + cells. This indicates that the process of phase advance likely obstructs cell maturation and survival. The loss of these late stage cells in the hippocampus associates with weakened learning and memory. Moreover, Horsey and her colleagues imply that increased stress and corticosteroid levels decrease the number of late stage DCX + cells, reducing dendritic complexity of immature granule cells.

Horsey et al. (2020) concluded that their findings demonstrate a

Figure 3 A) FST; time immobile on day 1 of a 15 min test. (B) Time link between chronic jet lag and detrimental deficits in cogniimmobile on day 2 of a 5 min test. Phase advanced rodents spent more tion, memory, and ones causing anxiety and depressive behavtime immobile than other experimental groups on day 2.

iours. By conducting several tests on adult male rats after inducDecreased Hippocampal Neurogenesis in Jet-Lag Influenced Rats ing chronic jet lag for eight weeks, the authors demonstrated 192

that phase advances lead to a decline in hippocampal neurogenesis, which, in turn, has a negative effect on object retention memory and learning, and increases anxiety and depression. Phase delays have limited effects on behaviour and neurogenesis, emphasizing that chronic jet lag likely contributes to negative long-lasting effects in eastward travel.

evidence. There is a suggestion that phase advances can have differing effects on females due to a contrast in emotional responses to stimuli. While other studies have focused on jet lag simulations on female rodents (Gibson et al., 2010), the differences between male and female behaviours were not studied, thus making this an important experiment to be conducted.

Furthermore, the authors acknowledge that there are other explanations for the increase in cognitive deficits in the rodent brain due to chronic circadian disruption. One is the idea that circadian clock gene expression is likely disturbed due to phase advances of the LD cycle (Horsey et al., 2020). Phase advances are also hypothesized to impact glucocorticoid secretion and the response to stress more than phase delays, signifying that phase advances can induce chronic stress. The authors state that chronic stress has an unfavourable impact on molecular mechanisms, especially those involved in the hippocampus, amygdala and nucleus accumbens, brain regions that impact behaviour, memory and learning. If the hippocampus is directly affected, this implies that stress can instigate the absence of maturation of adult-born neurons, which was seen in this study.

While there is a plethora of evidence suggesting that the hippocampus has an essential role in spatial memory and being a synaptic basis for learning (Iggena et al., 2017; Bliss and Collingridge, 1993), the authors in this study suggest that more evidence of the role of adult hippocampal neurogenesis is required to fully comprehend the implications of decreased neurogenesis due to jet lag. Horsey and her colleagues identified impairments in the hippocampus on a surface level view, though disrupted neurogenesis has a greater impact on brain function than what is currently known.

The theory that hippocampal neurogenesis is specifically impacted by chronic jet lag is one that has not been deeply studied by other researchers. Horsey and her colleagues explore various negative effects of chronic jet lag, though a decline in hippocampal neurogenesis seems to be the spark that affects all the other behaviours seen in the male rodents, including cognitive deficits in memory.

Critical Analysis While Horsey et al. (2020) provide revolutionary results on the negative effects of chronic jet lag on rodents, there is still much to be explored in this field and more experiments to be conducted to make accurate conclusions. After reviewing this paper, one concept to be explored is the influence of chronic jet lag on mood disorder development. While the authors highlighted that chronic phase advances leads to an increase in anxiety and depressive behaviours, there is an absence of information pertaining to the development of these behaviours and what axes or pathways jet lag impacts to instigate the disorders. Studies (Katz et al., 2001) publish the psychiatric aspects of jet lag by reviewing the symptoms of mood disorders, though very few have been able to determine the molecular mechanisms circadian disruptions impair to cause these disorders.

Another possible study to be conducted is one to decide if sex differences exist in behavioural responses to jet lag. In this study, Horsey et al. (2020) explore jet lag simulation effects on adult male rats. However, the authors acknowledge that their data is limited in making conclusions due to the lack of female

Future Directions Researchers should look at performing an identical experiment as to Horsey et al.â&#x20AC;&#x2122;s, but instead, utilize female rats. This will allow for greater focus on potential sex differences that possibly involve emotional responses. By creating an identical study, researchers will eliminate any error and can see if females experience the same effects as male rodents. It is predicted that females will likely experience a similar effect in memory and neurogenesis, but a differing impact of the development of mood disorders. This is an essential study that must be conducted to see if all animal models experience the same effects of jet lag, and if this can be further applied to humans.

Seeking the effects of jet lag on human hippocampal neurogenesis would be the most important part of the study to mimic.

Viewing the impact would allow researchers to learn more about the hippocampus as a brain region in general. By using procedures such as immunohistochemistry, hippocampal brain slices can be viewed. While different tests should be carried out, such as self-report tests indicating anxiety and depression instead of a FST, which would not be effective in humans, the same impairments can be tested for.

This study focused on the increase in depressive and anxiety behaviours in male rodents phenotypically. By genotyping rodents and viewing the alterations in circadian clock gene expression over time, the authors can make concrete conclusions about their results. A way to view the change in gene expression over time is to track mRNA oscillations of clock genes (Waddington Lamont et al., 2007), which present 24-hour rhythms in the SCN. After or during the jet lag simulation of animal models, oscillations can be tracked over time and compared to pre-jet lag levels. It is expected that oscillations will change especially due to phase advances, affecting gene expression.

193

REFRENCES 1.

Cho, Kwangwook, A. Ennaceur, Jon C. Cole, and Chang Kook Suh. 2000. “Chronic Jet Lag Produces Cognitive Deficits.” Journal of Neuroscience 20 (6): RC66–RC66. https://doi.org/10.1523/JNEUROSCI.20-06-j0005.2000.

2.

Cho, Kwangwook. 2001. “Chronic ‘Jet Lag’ Produces Temporal Lobe Atrophy and Spatial Cognitive Deficits.”

Nature Neuroscience 4 (6): 567–68. https://doi.org/10.1038/88384.

3.

Choy, Mary, and Rebecca L. Salbu. 2011. “Jet Lag.” Pharmacy and Therapeutics 36 (4): 221–31.

4.

Davidson, Alec J., Oscar Castanon-Cervantes, Tanya L. Leise, Penny C. Molyneux, and Mary E. Harrington. 2009.

“Visualizing Jet Lag in the Mouse Suprachiasmatic Nucleus and Peripheral Circadian Timing System.” European Journal of Neuroscience 29 (1): 171–80. https://doi.org/10.1111/j.1460-9568.2008.06534.x.

5.

Deacon, S., and J. Arendt. 1996. “Adapting to Phase Shifts, I. An Experimental Model for Jet Lag and Shift Work.” Physiology & Behavior 59 (4–5): 665–73. https://doi.org/10.1016/0031-9384(95)02147-7.

6.

Gibson, Erin M., Connie Wang, Stephanie Tjho, Neera Khattar, and Lance J. Kriegsfeld. 2010. “Experimental ‘Jet Lag’ Inhibits Adult Neurogenesis and Produces Long-Term Cognitive Deficits in Female Hamsters.” Edited by Shin Yamazaki. PLoS ONE 5 (12): e15267. https://doi.org/10.1371/journal.pone.0015267.

7.

Horsey, Emily A., Teresa Maletta, Holly Turner, Chantel Cole, Hugo Lehmann, and Neil M. Fournier. 2020.

“Chronic Jet Lag Simulation Decreases Hippocampal Neurogenesis and Enhances Depressive Behaviors and Cognitive Deficits in Adult Male Rats.” Frontiers in Behavioral Neuroscience 13 (January). https:// doi.org/10.3389/fnbeh.2019.00272.

8.

Iggena, Deetje, York Winter, and Barbara Steiner. 2017. “Melatonin Restores Hippocampal Neural Precursor Cell Proliferation and Prevents Cognitive Deficits Induced by Jet Lag Simulation in Adult Mice.” Journal of Pineal Research 62 (4): 1-12. https://doi.org/10.1111/jpi.12397.

9.

Katz, G. R., Durst, R., Zislin, Y., Barel, Y., and Knobler, H.Y. 2001. “Psychiatric Aspects of Jet Lag: Review and Hypothesis | Elsevier Enhanced Reader.” Medical Hypotheses 56 (1): 20-23. https://doi.org/10.1054/ mehy.2000.1094.

10.

Kolla, Bhanu P., and R. Robert Auger. 2011. “Jet Lag and Shift Work Sleep Disorders: How to Help Reset the Internal Clock.” Cleveland Clinic Journal of Medicine 78 (10): 675–84. https://doi.org/10.3949/ccjm.78a.10083.

11.

Lamont, Elaine Waddington, Francine O. James, Diane B. Boivin, and Nicolas Cermakian. 2007. “From Circadian Clock Gene Expression to Pathologies.” Sleep Medicine, Circadian Rhythms in Sleep Medicine, 8 (6): 547–56. https://doi.org/10.1016/j.sleep.2006.11.002.

12.

Lewy, Alfred J., Jennifer N. Rough, Jeannine B. Songer, Neelam Mishra, Krista Yuhas, and Jonathan S. Emens.

2007. “The Phase Shift Hypothesis for the Circadian Component of Winter Depression.” Dialogues in Clinical Neuroscience 9 (3): 291–300.

13.

Loh, Dawn H., Juliana Navarro, Arkady Hagopian, Louisa M. Wang, Tom Deboer, and Christopher S. Colwell.

2010. “Rapid Changes in the Light/Dark Cycle Disrupt Memory of Conditioned Fear in Mice.” PLoS ONE 5 (9). https://doi.org/10.1371/journal.pone.0012546.

14.

Shen, Qichen, Junli Wu, Yuehan Ni, Xiaoxian Xie, Chunan Yu, Qingfeng Xiao, Jiafeng Zhou, Xia Wang, and Zhengwei Fu. 2019. “Exposure to Jet Lag Aggravates Depression-like Behaviors and Age-Related Phenotypes in Rats Subject to Chronic Corticosterone.” Acta Biochimica et Biophysica Sinica 51 (8): 834–44. https:// doi.org/10.1093/abbs/gmz070.

15.

Zhang, Feifei, Weikai Li, Huiru Li, Shaobing Gao, John A. Sweeney, Zhiyun Jia, and Qiyong Gong. 2020. “The Effect of Jet Lag on the Human Brain: A Neuroimaging Study.” Human Brain Mapping 41 (9): 2281–91. https:// doi.org/10.1002/hbm.24945.

194

Curcumin as a Potential Treatment in Parkinsonâ&#x20AC;&#x2122;s Disease Cathy Xiong

Parkinsonâ&#x20AC;&#x2122;s disease (PD) is a progressive neurodegenerative condition characterized by the misfolded alpha-synuclein proteins, the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), along with motor deficits and nonmotor symptoms (Balestrino & Schapira, 2020; Klingelhoefer & Reichmann, 2015). There is currently no cure for PD and treatments mainly use dopamine substitutes or deep brain stimulation to treat the motor symptoms (Connolly & Lang, 2014; Okun, 2012). Many studies were investigating potential therapeutic uses of the natural substances derived from plants (Scapagnini et al., 2011). Curcumin, a common ingredient in curry as turmeric, is known to be an antioxidizing and anti-inflammatory agent (Sharma, Gescher, & Steward, 2005).

The current study by Khatri and Juvekar (2016) administrated rotenone in mice to produce the Parkinsonian symptoms and tried to combat it with curcumin. The study aimed to investigate the efficacy of curcumin on reversing the damage done by rotenone in mice using behavioural and biochemical examinations. The rotenone treated mice performed poorly on the rotarod test and were less mobile in the actophotometer and open field test compared to the control mice. The administration of rotenone also increased the levels of lipid peroxidation and nitrite, and activity of acetylcholinesterase, and decreased the activities of antioxidant enzymes, and mitochondrial complex enzymes. Results revealed that curcumin treated mice restored the negative effect of rotenone on both motor and cognitive functions and improved the impairment on oxidative defence and enzyme activities in mitochondrial complexes.

Keywords: Parkinsonâ&#x20AC;&#x2122;s disease (PD), rotenone, curcumin, motor deficits, antioxidant, mitochondrial complex, mice model

195

mental groups got the same rotenone injection with curcumin (Cur) oral treatment of 50 mg/kg, 100 mg/kg, or 200 mg/kg, respectively. Before the experiment started, the mice had a week to adapt to the new environment, and then all mice had practiced on the rotarod apparatus for another five days. An initial behavioural test was done and set as day zero. On day one, the mice were treated as mentioned above with the behavioural examination. After repeated for twenty-one times, all mice were sacrificed on day twenty-two for measures in brain tissue.

Major Results Behavioural Observations Figure 1. Visual abstract for the outline of the experiment performed by Khatri and Juvekar (2016) Background and Introduction As the second most common neurodegenerative disease after Alzheimer’s, 10% - 15 % of the Parkinson’s disease (PD) can be attributed to genetics while the rest are due to unknown factors and considered environmental (Emamzadeh & Surguchov, 2018). The known pathology of PD is the accumulation and aggregation of the misfolded proteins called alpha-synuclein into Lewy bodies, which is a hallmark of PD (Balestrino & Schapira, 2020). Oligomers formed by α-synucleins are toxic to the cells and cause the dopaminergic neural death in the substantia nigra pars compacta(SNpc), which results in the disturbances in motor functions (Ciechanover & Kwon, 2015). Patients with PD display slow and rigid movements, resting tremors, and impairments in gait and posture, which precede by nonmotor symptoms (Klingelhoefer & Reichmann, 2015). Furthermore, the significant reduction of mitochondrial complex I is another key feature in PD, and is thought to associated with dementia in PD (Gatt et al., 2016).

There was no difference in all groups for the actophotometer test on day 0. On day 21, ROT treated group showed a significant decrease in locomotion compared to the control group.

For Cur treated groups, as the dosage of curcumin increased, the more locomotive activity was performed, compared to ROT treated group. On the rotarod test, a similar trend was observed as the actophotometer test, where a significant reduction of time remaining on the rotarod was found in ROT treated group compared to the control, and gradual increases were found in Cur treated groups compared to ROT. In the open field test (OFT), line crossing, grooming, and rearing followed the same trend. ROT treated mice showed longer immobility time compared to all other mice on day 21.

Spices and herbs have always been an essential to both the culinary and pharmacological aspects of the human history.

Apart from their contribution to the colouring and flavouring of food, their polyphenolic properties also benefits the brain in preventing conditions associate with age, such as cancer and neurodegenerative diseases (Scapagnini et al., 2011). Curcumin has been used in ancient herbal medicine for its antioxidant, anti-inflammatory, and neuroprotective effects (Witkin & Li, 2013). Study has found curcumin to attenuate cytotoxicity and inhibit apoptosis (Fan et al., 2017).

Figure 2. Behavioural observations for day 0 and day 21 in OFT on (A) line crossing, (B) rearings, (C) grooming, and (D) immobility time in all groups. Adapted from “Neuroprotective effect of curcumin as evinced by abrogation of rotenone-induced motor deficits, oxidative and mitochondrial dysfunctions in mouse model of Parkinson's disease”, by D. K. Khatri, & A. R. The current study by Khatri and Juvekar (2016) used curcumin Juvekar, Pharmacology Biochemistry and Behavior, 150–151, to test its ability against rotenone-induced neuronal death and 39–47. movement disabilities. Thirty Swiss albino male mice were randomly assigned into 5 groups, including the control group, the negative control group, and the treatment groups. The control Biochemical Analysis group received vehicle control, and the negative control group were injected with rotenone (ROT) 1 mg/kg. The three experi- The lipid peroxidation (LPO) and nitrite levels were increased in 196

of oxidation and mitochondrial impairment. Therefore, the findings suggested that curcumin has the potential to be used therapeutically in managing PD (Khatri & Juvekar, 2016). This is an important finding because PD are mostly due to unknown causes and is incurable, and current pharmacological treatments and deep brain stimulation are targeting at reducing motor symptoms. As the result of Khatri and Juvekar (2016) has shown, curcumin not only can improve the behavioural symptoms, but also on the biochemical level. Furthermore, a study discovered that curcumin can bind to oligomers and fibrils and reduce their toxicity both in vitro and in vivo, which suggested that curcumin are relevant in modulating, or even halting, the α -synuclein aggregation in PD (Singh et al., 2013). A similar finding was also revealed by another study, where Gautam, Karmakar, Bose, & Chowdhury (2014) found that a mixture of curcumin and β-cyclodextrin could suppress protein aggregation, and even disaggregate the ones that already formed, making it a potential treatment in preventing PD. As one of the neurodegenerative diseases, PD also has features such as dementia and protein aggregation like Alzheimer’s and Huntington’s (Ciechanover & Kwon, 2015; Gatt et al., 2016). Therefore, if curcumin can help with PD, it might also be beneficial in other Figure 3. Levels of LPO and SDH activity in all groups. Adapted neurodegenerative diseases. from “Neuroprotective effect of curcumin as evinced by abrogation of rotenone-induced motor deficits, oxidative and mitoCritical Analysis chondrial dysfunctions in mouse model of Parkinson's disease”,

by D. K. Khatri, & A. R. Juvekar, Pharmacology Biochemistry and The behavioural results showed impairment in locomotion and Behavior, 150–151, 39–47. coordination due to paralysis of the limbs of the mice, which confirmed the Parkinsonian damage done by rotenone (Khatri Conclusion and Discussion & Juvekar, 2016). However, the current paper did not perform a test examining the dopaminergic neurons or the dopamine The results revealed the neuroprotective role of curcumin on level, which is the reason for the motor deficits. Therefore, the brain of the rotenone-induced Parkinsonian mice. The be- maybe the authors can include the influences of any effects of havioural observations illustrated that long-term administra- rotenone and curcumin on dopamine in the brain in order to tion of rotenone resulted in reduced overall behavioural activi- make the experiment more closely relevant to PD. Also, the ty by actophotometer, impaired motor coordination by authors in the current paper mentioned no limitations about rotarod, and longer immobility duration in the OFT(Khatri & the experiment. As an oral treatment, the authors should menJuvekar, 2016). Analysis showed that rotenone increased the tion the bioavailability of curcumin. Curcumin is poorly soluble level of LPO and nitrite, which meant there was higher oxida- in water and highly unstable in the digestive tract, making its tive stress in rotenone treated mice (Khatri & Juvekar, 2016). protein disaggregating task still challenging in vivo (Gautam et Another increase in AChE level was shown in ROT treated mice, al., 2014). Study using even a high dose of curcumin (12g/day) which indicated a decrease in acetylcholine at synapse and in human showed a weak bioavailability due to its low absorptherefore reduced cognitive functions (Khatri & Juvekar, 2016). tion and quick metabolizing and eliminating from the body On the other hand, Cur treated mice showed higher levels of (Anand, Kunnumakkara, Newman, & Aggarwal, 2007).

SOD, CAT, and GSH compared to ROT treated mice, which revealed the antioxidant ability of curcumin (Khatri & Juvekar, Future Directions 2016). ROT treated mice showed a decrease in both SDH and MTT reduction that led to mitochondrial dysfunction, which curcumin helped improved (Khatri & Juvekar, 2016). These find- Future studies should focus on developing curcumin formulas ings are consistent with the study done by Ramsés García-Niño with enhanced bioavailability for using in PD or other neuro& Pedraza-Chaverrí (2014), which found that curcumin main- degenerative diseases. Ways like pairing curcumin with other tained the levels of SOD, CAT, and GSH, and protect against agents that increase its bioavailability, modifying its structure, mitochondrial alterations under the oxidative stress of heavy or finding a derivative that works better in human body should be studied. Other spices or herbal medicines can be used with metals. curcumin for a synergic effect that boost the efficacy, such as crocin that manages cell death, loganin that decreases neuroinKhatri and Juvekar (2016) found that curcumin conserved the flammation, and icariin that protects mitochondria function cognitive and motor functions as well as attenuated the degree the ROT treated group compared to control and Cur treated groups showed a gradual decrease compared to ROT treated group. The similar pattern was also found in acetylcholinesterase (AChE) activity. For activities of superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH), ROT treated group showed a significant decrease compared to control and Cur increased the activities compared to ROT. The succinate dehydrogenase (SDH) and MTT reduction activities displayed a similar trend.

197

(Chen et al., 2020). There has been study that used piperine from black pepper, which inhibits drug elimination at intestines, to increase the bioavailability of curcumin by 2000% in both rats and humans (Shoba et al., 1998). In the same setting as the current paper, mice can be group into control, negative control, and mice with the three combinations of curcumin and crocin, loganin, and icariin.

The combination of better results can be delivered using intranasal administration for better uptake by the brain tissue (Prasad, Tyagi, & Aggarwal, 2014). Furthermore, several studies had used the nanoparticle approach as a deliver method to overcome the low bioavailability of curcumin (Ipar, Dsouza, & Devarajan, 2019; Umerska et al., 2018; Xie et al., 2011). Overall, more investigations can be done in improving the efficacy and bioavailability of curcumin by trying out different agents and diliveries.

198

REFRENCES 1.

Anand, P., Kunnumakkara, A. B., Newman, R. A., & Aggarwal, B. B. (2007). Bioavailability of curcumin: Problems and promises. Molecular Pharmaceutics. https://doi.org/10.1021/mp700113r

2.

Balestrino, R., & Schapira, A. H. V. (2020, January 27). Parkinson disease. European Journal of Neurology. Blackwell Publishing Ltd. https://doi.org/10.1111/ene.14108

3.

Chen, S. Y., Gao, Y., Sun, J. Y., Meng, X. L., Yang, D., Fan, L. H., … Wang, P. (2020, April 22). Traditional Chinese Medicine: Role in Reducing β-Amyloid, Apoptosis, Autophagy, Neuroinflammation, Oxidative Stress, and Mitochondrial Dysfunction of Alzheimer’s Disease. Frontiers in Pharmacology. Frontiers Media S.A. https://doi.org/10.3389/fphar.2020.00497

4.

Ciechanover, A., & Kwon, Y. T. a. (2015, March 13). Degradation of misfolded proteins in neurodegenerative diseases: therapeutic targets and strategies. Experimental & Molecular Medicine. Nature Publishing Group. https:// doi.org/10.1038/emm.2014.117

5.

Connolly, B. S., & Lang, A. E. (2014, April 23). Pharmacological treatment of Parkinson disease: A review. JAMA - Journal of the American Medical Association. American Medical Association. https://doi.org/10.1001/jama.2014.3654

6.

Emamzadeh, F. N., & Surguchov, A. (2018, August 30). Parkinson’s disease: Biomarkers, treatment, and risk factors. Frontiers in Neuroscience. Frontiers Media S.A. https://doi.org/10.3389/fnins.2018.00612

7.

Fan, C. dong, Li, Y., Fu, X. ting, Wu, Q. jian, Hou, Y. jun, Yang, M. feng, … Sun, B. liang. (2017). Reversal of Beta-AmyloidInduced Neurotoxicity in PC12 Cells by Curcumin, the Important Role of ROS-Mediated Signaling and ERK Pathway. Cellular and Molecular Neurobiology, 37(2), 211–222. https://doi.org/10.1007/s10571-016-0362-3

8.

García-Niño, W. R., & Pedraza-Chaverrí, J. (2014). Protective effect of curcumin against heavy metals-induced liver damage. Food and Chemical Toxicology. https://doi.org/10.1016/j.fct.2014.04.016

9.

Gatt, A. P., Duncan, O. F., Attems, J., Francis, P. T., Ballard, C. G., & Bateman, J. M. (2016). Dementia in Parkinson’s disease is associated with enhanced mitochondrial complex I deficiency. Movement Disorders, 31(3), 352–359. https:// doi.org/10.1002/mds.26513

10.

Gautam, S., Karmakar, S., Bose, A., & Chowdhury, P. K. (2014). β-cyclodextrin and curcumin, a potent cocktail for disaggregating and/or inhibiting amyloids: A case study with α-synuclein. Biochemistry, 53(25), 4081–4083. https:// doi.org/10.1021/bi500642f

11.

Ipar, V. S., Dsouza, A., & Devarajan, P. V. (2019). Enhancing Curcumin Oral Bioavailability Through Nanoformulations. European Journal of Drug Metabolism and Pharmacokinetics. https://doi.org/10.1007/s13318-019-00545-z

12.

Khatri, D. K., & Juvekar, A. R. (2016). Neuroprotective effect of curcumin as evinced by abrogation of rotenone-induced motor deficits, oxidative and mitochondrial dysfunctions in mouse model of Parkinson’s disease. Pharmacology Biochemistry and Behavior, 150–151, 39–47. https://doi.org/10.1016/j.pbb.2016.09.002

13.

Klingelhoefer, L., & Reichmann, H. (2015). Pathogenesis of Parkinson disease - The gut-brain axis and environmental factors. Nature Reviews Neurology. https://doi.org/10.1038/nrneurol.2015.197

199

14.

Okun, M. S. (2012, October 18). Deep-brain stimulation for Parkinson’s disease. New England Journal of Medicine. Massachussetts Medical Society. https://doi.org/10.1056/NEJMct1208070

15.

Prasad, S., Tyagi, A. K., & Aggarwal, B. B. (2014). Recent developments in delivery, bioavailability, absorption and metabolism of curcumin: The golden pigment from golden spice. Cancer Research and Treatment. Korean Cancer Association. https://doi.org/10.4143/crt.2014.46.1.2

16.

Scapagnini, G., Sonya, V., Nader, A. G., Calogero, C., Zella, D., & Fabio, G. (2011). Modulation of Nrf2/ARE pathway by food polyphenols: A nutritional neuroprotective strategy for cognitive and neurodegenerative disorders. Molecular Neurobiology, 44(2), 192–201. https://doi.org/10.1007/s12035-011-8181-5

17.

Sharma, R. A., Gescher, A. J., & Steward, W. P. (2005). Curcumin: The story so far. European Journal of Cancer, 41(13), 1955–1968. https://doi.org/10.1016/j.ejca.2005.05.009

18.

Shoba, G., Joy, D., Joseph, T., Majeed, M., Rajendran, R., & Srinivas, P. S. S. R. (1998). Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers. Planta Medica, 64(4), 353–356. https://doi.org/10.1055/s-2006957450

19.

Singh, P. K., Kotia, V., Ghosh, D., Mohite, G. M., Kumar, A., & Maji, S. K. (2013). Curcumin modulates α-synuclein aggregation and toxicity. ACS Chemical Neuroscience, 4(3), 393–407. https://doi.org/10.1021/cn3001203

20.

Umerska, A., Gaucher, C., Oyarzun-Ampuero, F., Fries-Raeth, I., Colin, F., Villamizar-Sarmiento, M. G., … Sapin-Minet, A. (2018). Polymeric nanoparticles for increasing oral bioavailability of Curcumin. Antioxidants, 7(4), 46. https:// doi.org/10.3390/antiox7040046

21.

Witkin, J., & Li, X. (2013). Curcumin, an Active Constiuent of the Ancient Medicinal Herb Curcuma longa L.: Some Uses and the Establishment and Biological Basis of Medical Efficacy. CNS & Neurological Disorders - Drug Targets, 12(4), 487–497. https://doi.org/10.2174/1871527311312040007

22.

Xie, X., Tao, Q., Zou, Y., Zhang, F., Guo, M., Wang, Y., … Yu, S. (2011). PLGA nanoparticles improve the oral bioavailability of curcumin in rats: Characterizations and mechanisms. Journal of Agricultural and Food Chemistry, 59(17), 9280–9289. https://doi.org/10.1021/jf202135j

200

Anterior Cingulate Cortex as Therapeutic Target for Autism Yunqing Zhu

Autism spectrum disorder is a neurodevelopmental disorder in which social deficits is one of the core symptoms. However, there is currently no effective treatment targeting this symptom. In the study by Guo et al. (2019), the author used Shank3 knockout mice as animal model for autism, and observed the structural and functional changes in the anterior cingulate cortex. They found that Shank3 mutations caused morphological and functional change in pyramidal neurones of the region, and led to social deficits (Guo et al. 2019). They then enhanced the pyramidal neurone activity in anterior cingulate cortex using optogenetics, chemogenetics and a pharmacological compound, and were able to rescue the social impairments (Guo et al. 2019). These findings provide a causal link between hypoactivity of the anterior cingulate cortex and social impairments in autism, and proves that the area could be a potential therapeutic target (Guo et al. 2019).

201

Background and Introduction

Major Findings

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects about 1% of individuals globally (Lai, Lombardo, and Baron-Cohen 2014). Although it greatly impacts the quality of life of affected individuals, there is currently no effective pharmacological treatment for the core symptoms of the disorder. This is in part due to the heterogeneity and lack of understanding of the underlying mechanisms of the disorder. Instead, available treatments focus on socio-behavioural interventions or treating symptoms of comorbid disorders.

Global deletion of Shank3 caused morphological and functional deficits in excitatory pyramidal neurones of the ACC, resulting in social deficits

Social communication deficit is one of the core diagnostic criteria of ASD. Successful social interactions and behaviour require effective processing and integration of social inputs from various sources (Guo et al. 2019). The anterior cingulate cortex (ACC) is an association cortex involved in multiple cognitive processes including learning and error monitoring (Apps, Rushworth, and Chang 2016). Anatomically, the ACC is highly connected to regions in the brain important for social behaviours and information processing (Apps, Rushworth, and Chang 2016). Numerous neuroimaging studies have demonstrated altered neural circuitry in ACC of ASD patients compared to healthy controls ( Balsters et al. 2016; Zhou et al. 2016; Zikopoulos and Barbas 2013). Furthermore, studies in both humans and monkeys found that the ACC is activated when engaging in economic games involving the need for social cooperation, decision making based on social information and making predictions based on othersâ&#x20AC;&#x2122; behaviours (Gabay et al. 2014; Haroush and Williams 2015). Laidi et al. (2019) also found through a magnetic resonance imaging (MRI) study that adults with ASD display decreased cortical thickness in ACC. Thus, it can be concluded that ACC is highly implicated in various social behaviours.

Previous studies have established the important role ACC plays in integration social information and regulating behaviours.

However, the underlying neural mechanisms that cause the social deficits are unknown. Moreover, previous studies had not been able to establish a causal link between specific ACC dysfunctions with ASD-related social deficits. Therefore, Guo et al.

(2019) used a Shank3 mice model for ASD to test whether dysfunctions in ACC directly leads to social deficits. The team found that Shank3 knockout (KO) mice showed aberrations in neuronal morphology as well as synaptic functions in their ACC excitatory pyramidal neurones and that selective deletion of Shank3 in the ACC was sufficient to generate the social deficits shown in KO mice (Guo et al. 2019). Furthermore, they found that increasing the excitatory activities in the ACC by using optogenetic and chemogenomic techniques as well as pharmacological agents successfully rescued the social deficits (Guo et al. 2019). As a result, the author concluded that hypoactivity in the ACC directly causes social impairments in ASD, and that the ACC should be further researched as a potential therapeutic target for ASD (Guo et al. 2019).

Using fluorescent in situ hybridization and immunofluorescent staining, Guo et al.(2019) found that Shank3 mRNA is mainly expressed in the excitatory pyramidal neurones in the ACC. Therefore, the authors focused on the morphological and function changes caused by Shank3 mutation in these ACC pyramidal cells in their study. Green fluorescent protein (GFP) was delivered to the ACC of wild-type (WT) and Shank3 KO mice to visualize the pyramidal neurones in the region(Guo et al. 2019).

It was found that the neurones in KO mice showed decreased dendritic complexity, spine density as well as reduced number of mushroom-shaped spines compared to WT(Guo et al. 2019).

In addition, electron microscopy showed decreased average length and thickness of postsynaptic densities in the pyramidal cells in ACC of KO mice(Guo et al. 2019).

Guo et al. then measured the post-synaptic activities of the ACC pyramidal neurones in KO and WT mice using whole cell patchclamp recording. Results showed weaker a-amino-3-hydroxy-5methylisoxazole-4-propionic acid receptor (AMPAR)-mediated postsynaptic transmissions in KO mice as well as a reduced AMPAR/N-methyl-D-aspartate receptor (NMDAR) ratio(Guo et al.

2019). Higher intensities of presynaptic stimulations were required to generate an action potential postsynaptically(Guo et al. 2019). Western blot analysis also showed reduced expression of the AMPAR subunit GluR1 (Guo et al. 2019). These results suggest that AMPAR dysfunctions are the predominant cause of functional deficits in the ACC pyramidal neurones of KO mice (Guo et al. 2019). Furthermore, paired training paradigm failed to induce long-term potentiation (LTP) in KO mice as it did in the WT (Guo et al. 2019).

Behavioural tests were used on the KO and WT mice. In particular, three-chamber test and home cage social interaction test both showed significant social deficits in KO mice compared to WT (Guo et al. 2019). Elevated plus maze and open field tests also resulted in higher anxiety levels in the KO mice (Guo et al.

2019). Then, the social interaction task was repeated with fiber photometry systems implanted into the mice to measure the real-time calcium activities as the mice approached the novel mice in their home cage. KO mice showed decreased ACC excitatory activity during this task compared to WT (Guo et al.

2019).

Conditional Knockout (CKO) of Shank3 gene in the ACC region alone was sufficient to generate hypoactivity in pyramidal neurones and social deficits Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing technique was used to selectively knock out Shank3 in the ACC of the mice models (Guo et al.

2019). As a result, this generated similar patterns of aberrations

202

in synaptic activity in the pyramidal cells as in KO mice, including decreased postsynaptic activities, decreased AMPAR/ NMDAR ratio, as well as decreased expressions of AMPAR and NMDAR subunits (Guo et al. 2019). Three-chamber test and social interaction tests were again used on the CKO, and both showed significant social deficits (Guo et al. 2019). These results indicate that deletion of Shank3 in ACC alone was sufficient to cause functional and social deficits in mice models, suggesting causal link between decreased excitatory activities in ACC and social deficits seen in ASD (Guo et al. 2019).

Guo et al. (2019) found their results of abnormal morphology in the ACC to be consistent with the previous studies that found altered anatomical characteristics, decreased volume or cortical thickness in the ACC of ASD patients (Haznedar et al. 2000; Laidi et al. 2019). It was well established that ACC plays an important role in social behaviours and that the general region is implicated in ASD. However, Guo et al. (2019) expanded beyond these findings in that they identified the specific neural substrates involved in the ACC changes, and proved experimentally that these dysfunctions directly cause the social impairments seen in ASD. They also demonstrated that these deficits could be rescued by enhancing the activities of ACC pyramidal neurones (Guo et al. 2019).

Critical Analysis In the study by Guo et al. (2019), the authors looked at whether dysfunctions in ACC pyramidal neurones were sufficient to cause ASD-like social communications symptoms in Shank3 model mice and established that reactivation of the ACC pyramidal neurones ameliorated these symptoms. However, Guo et al. (2019) recognised that the level of photostimulation they applied using optogenetics in this study may be outside physiological levels of stimulation in the brain. As they found that lowest levels of photostimulation did not achieve significant improvements in sociability, it might be that effective intensity of Enhancement of ACC excitatory activities rescued synaptic stimulation symptoms of the pyramidal neurones which is functional and social deficits enough to ameliorate symptoms in human brain might not be As the causal link between hypoactivity of ACC and ASD-like attainable (Guo et al. 2019). social deficits were established, the authors then explored whether using optogenetic, chemogenetic and pharmacological methods to enhance the activity of ACC pyramidal neurones Furthermore, Guo et al. (2019) mentioned that AMPAR could would rescue these social deficits. The results showed that be a potential therapeutic target as hypoactivity in the ACC due optogenetically enhancing ACC excitatory activities led to in- to AMPAR dysfunctions lead to the social deficits. However, creased sociability shown in behavioural tests in both KO and ASD is also characterised by over-excitation of neurones in WT mice as well as reduced anxiety (Guo et al. 2019). On the many other brain regions which may lead to symptoms such as other hand, optogenetic inhibition of the same neurones led to hypersensitivity to sensory inputs. Although an AMPAR-PAM social deficits in WT mice similar to those exhibited by KO mice might be useful in enhancing activity of the ACC, if applied to (Guo et al. 2019). Selective activation of ACC pyramidal neu- humans, it would need to be delivered to target the ACC sperones using designer receptors exclusively activated by designer cifically to avoid overstimulating other areas of the brain. drugs (DREADDs) also improved social behaviours (Guo et al.

2019). In addition, both selective re-expression of Shank3 in ACC and the injection of AMPAR-positive allosteric modulator Future Direction (PAM) CX546 improved synaptic functions on top of sociability The study by Guo et al. (2019) points to ACC as an area of inter(Guo et al. 2019). est in ASD research. Now that the causal relationship between ACC under-excitation and social deficits is established, future studies could look at the specific molecular mechanisms inConclusion/Discussion volved in the area. As Guo et al. (2019) pointed out that AMPAR Guo et al. ( 2019) concluded that mutations in Shank3 cause is a main factor in the ACC dysfunctions, genomic sequencing dysfunctions in pyramidal neurones of ACC, directly leads to could be done to screen for potential over- or underexpression hypoactivity in the area and in turn causes social impairments. of genes related to the receptor. It would also be useful to map However, optogenetically and chemogenetically activating the out the circuitry that the pyramidal neurones in ACC are inpyramidal neurones of ACC effectively rescued these social defi- volved in. This would include looking at what other areas and cits. Since AMPAR-related dysfunctions was the main cause of the types of neurones that these pyramidal neurones are consynaptic hypoactivity, using an AMPAR-PAM also significantly nected to. ameliorated the social impairments. Thus, ACC can be considered as a potential therapeutic target for ASD. 203

REFRENCES

1.

Apps, Matthew A.J., Matthew F.S. Rushworth, and Steve W.C. Chang. 2016. “The Anterior Cingulate Gyrus and Social Cognition: Tracking the Motivation of Others.” Neuron 90 (4): 692–707. https://doi.org/10.1016/j.neuron.2016.04.018.

2.

Balsters, Joshua H., Dante Mantini, Matthew A.J. Apps, Simon B. Eickhoff, and Nicole Wenderoth. 2016. “ConnectivityBased Parcellation Increases Network Detection Sensitivity in Resting State FMRI: An Investigation into the Cingulate Cortex in Autism.” NeuroImage: Clinical 11: 494–507. https://doi.org/10.1016/j.nicl.2016.03.016.

3.

Gabay, Anthony S., Joaquim Radua, Matthew J. Kempton, and Mitul A. Mehta. 2014. “The Ultimatum Game and the Brain: A Meta-Analysis of Neuroimaging Studies.” Neuroscience & Biobehavioral Reviews 47 (November): 549–58. https:// doi.org/10.1016/j.neubiorev.2014.10.014.

4.

Guo, Baolin, Jing Chen, Qian Chen, Keke Ren, Dayun Feng, Honghui Mao, Han Yao, et al. 2019. “Anterior Cingulate Cortex Dysfunction Underlies Social Deficits in Shank3 Mutant Mice.” Nature Neuroscience 22 (8): 1223–34. https:// doi.org/10.1038/s41593-019-0445-9.

5.

Haroush, Keren, and Ziv M. Williams. 2015. “Neuronal Prediction of Opponent’s Behavior during Cooperative Social Interchange in Primates.” Cell 160 (6): 1233–45. https://doi.org/10.1016/j.cell.2015.01.045.

6.

Haznedar, M. Mehmet, Monte S. Buchsbaum, Tse-Chung Wei, Patrick R. Hof, Charles Cartwright, Carol A. Bienstock, and Eric Hollander. 2000. “Limbic Circuitry in Patients With Autism Spectrum Disorders Studied With Positron Emission Tomography and Magnetic Resonance Imaging.” American Journal of Psychiatry 157 (12): 1994–2001. https://doi.org/10.1176/ appi.ajp.157.12.1994.

7.

Lai, Meng-Chuan, Michael V Lombardo, and Simon Baron-Cohen. 2014. “Autism.” The Lancet 383 (9920): 896–910. https:// doi.org/10.1016/S0140-6736(13)61539-1.

8.

Laidi, Charles, Jennifer Boisgontier, Amicie de Pierrefeu, Edouard Duchesnay, Sevan Hotier, Marc-Antoine d’Albis, Richard Delorme, et al. 2019. “Decreased Cortical Thickness in the Anterior Cingulate Cortex in Adults with Autism.” Journal of Autism and Developmental Disorders 49 (4): 1402–9. https://doi.org/10.1007/s10803-018-3807-3.

9.

Zhou, Yuanyue, Lijuan Shi, Xilong Cui, Suhong Wang, and Xuerong Luo. 2016. “Functional Connectivity of the Caudal Anterior Cingulate Cortex Is Decreased in Autism.” Edited by Emmanuel Andreas Stamatakis. PLOS ONE 11 (3): e0151879. https://doi.org/10.1371/journal.pone.0151879.

10.

Zikopoulos, Basilis, and Helen Barbas. 2013. “Altered Neural Connectivity in Excitatory and Inhibitory Cortical Circuits in Autism.” Frontiers in Human Neuroscience 7. https://doi.org/10.3389/fnhum.2013.00609.

204