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ACTIVATION OF DOPAMINERGIC PATHWAYS MEDIATE RAPID ONSET ANTIDEPRESSANT EFFECTS OF KETAMINE [ROSA SUMMER]

CANCER THERAPEUTICS - THE FUTURE IS NOW [CHRISTOPHER KNOX]

A REVIEW ON THE TOXICITY OF COPPER:

AN ESSENTIAL MICRONUTRIENT, BUT HIGHLY TOXIC IN EXCESS [JONATHAN CHOW]

INTERVIEW WITH MASTERS STUDENT, DAVE BODENSTEIN

The Future is Now Cancer Therapeutics – The Future is Now Prepared by: Christopher Knox

What if we could genetically manipulate viruses to do our own bidding to fight disease, rather than cause it? Or re-infuse patients with their own cultured immune cells, creating superhuman immunity? How about if there was a magic drug that could help a patient’s immune system target pathogens that have evolved over years of development to hide from it? What was once considered only possible by scientists decades ago in sci-fi movies, has now finally become a reality. Recently, there has been huge progress in the development of innovative therapeutics, including oncolytic viruses, immune checkpoint inhibitors, and adoptive cell transfer therapies. Markets for some immunotherapeutics are expected to rise 20-fold over the next ten years, transforming it into a multi-billion dollar industry.1 The reason being that these therapeutics have huge potential for treating one of the most deadly and pervasive diseases: cancer. The cancer epidemic is more widespread than ever. According to the annual statistics published by the Government of Canada, over 200,000 Canadians were diagnosed with cancer in 2017, and is one of the leading causes of death in Canada next to heart disease, stroke and diabetes. Many standard treatments that exist right now can often seem less than ideal, putting the patient’s health at risk. For example, chemotherapy is a cancer treatment that revolves around the idea that cancer cells multiply faster than regular cells – and therefore, this gives us a way to preferentially target cancer cells, even at the cost of damage to the patient. One example is

Paclitaxel (Figure 1), a common chemotherapeutic used to treat breast cancer.2 This drug can inhibit microtubule structures and result in a failure to complete mitosis, ultimately resulting in cell death. However, Paclitaxel doesn’t discriminate and will attack both friend and foe. As a result, it unfortunately comes with a variety of side effects common to other kinds of chemotherapy, like nausea, vomiting and hair loss due to targeting the patient’s own healthy cells. Sometimes, cancer cells can also develop chemo-resistance, and therefore giving a higher dose of chemotherapeutics may not be an option due to the risk it poses on the patient’s health.

Figure 1: Chemical structure of Paclitaxel, a chemother apeutic derived from the Pacific Yew tree and on WHO’s list of most essential medicines. While effective at treating various cancers, it may induce common chemotherapy side effects, such as hair loss.

One ultimate question remains: is there some way we can add selectivity to this process? One of the most recent and innovative technologies out there are the oncolytic viruses. Essentially, these are genetically engineered viruses that can seek out and infect cancer cells while avoiding the healthy ones.3 After infection, the virus can replicate or interfere with host cell processes and eventually kill the cancer cell. However, it doesn’t stop there – the death of the cancer cell releases its contents, including many potential cancer antigens, which can be sensed by the immune system and continue the attack against the cancer. In fact, this is exactly the mechanism of how T-Vec works, an HSV-1 based virus which is the first oncolytic virus approved in North America and is used for the treatment of melanoma.4 Tactically, it uses the fact that cancer cells may have defects in interferon or protein kinase R (PKR) signalling, so that the virus replicates in only these defective cancer cells, and not healthy ones. In addition, its engineered genome has two extra copies of the GM-CSF molecule, and so when T-Vec kills cancer cells it releases GM-CSF near the cancer site. This molecule is important for the recruitment of dendritic cells and other members of the immune system, furthering the anti-tumor response! Therefore, these viruses are not only killing the cancer cells but also alerting the immune system of its presence. Given that oncolytic viruses have only entered the market in 2015, there is still huge potential to be explored and it’s undoubtedly going to grow for years to come. The next class of promising immunotherapeutics includes the immune checkpoint inhibitors, or ICIs, which intricately utilizes what we know about how the immune system functions. T-cells are a specific class of immune cells that can be thought of as soldiers – they patrol the body and destroy cells

Figure 2: A schematic of the key players involved in interactions between T-cells and antigen presenting cells, in this case a tumor cell. Often, tumor cells increase expression of ligands for inhibitory receptors on T-cells, like CTLA-4 and PD-1, resulting in their ability to evade the immune system. Therapeutics that inhibit this interaction allow for an increased ability of T-cells to be activated and to continue to keep up the fight. Figure source from Dine et al, 2015.

that may potentially be considered threats to the body. What decides whether a T-cell will attack or not? It turns out that it depends not only on the presence of a specific antigen, but also certain signalling molecules, which can activate or inhibit the T-cell from attacking. This includes CTLA4 and PD-1, which are two well known inhibitory receptors expressed on T-cells. Often, cancer cells are tricky and increase expression of ligands for these receptors on their cell surfaces. As a result, it deactivates the T-cells and allows the cancer to escape detection. Therapeutics like Ipilimumab, a monoclonal antibody, binds CTLA-4 specifically and prevents it from binding its ligand, thus allowing the T-cells to keep up the attack.5 The immune system now gets much better odds at fighting the cancer, although there is a risk that the immune system becomes over-activated, which is the main adverse effect of this drug. While a seemingly simple concept, these checkpoint inhibitors have proven useful, significantly improving outcomes in clinical trials for patients with solid tumors and melanoma.

Lastly, one of the newest and most unique therapy forms is that of ACT, or adoptive cell transfer therapy.6 ACT is really just a broad term for a therapy that involves the transfer of cells into an individual. One notable form is known as CAR (chimeric antigen receptor) T-cell therapy. This involves making a custom antibody and inserting it into the membranes of T-cells. The antigen binding, extracellular part can be targeted at practically anything with amazing specificity, including cancer antigens. Meanwhile, the intracellular domain has various “activating” signalling motifs grafted onto it, turning on the T-cell when the custom receptor is activated. Another similar approach to this method is to isolate TILs (tumor-infiltrating lymphocytes) from a patient’s tumor.7 These are essentially T-cells that are specific for the tumor and are trying to fight it, but because cancer often creates a very immunosuppressive environment, these T-cells are unable to attack the cancer effectively. Once these TILs have been isolated from the patient however, they can be activated and expanded 1000-fold in-vitro, before being injected back into

the patient (Figure 2). While currently used for the treatment of melanoma, TIL therapy has been implicated as a possible treatment for a whole variety of cancers as well, including lung, ovarian and breast cancer, and could have potential to treat even more types of cancer than currently imagined. One of the great challenges in cancer therapeutics is trying to target the cancer while avoiding the healthy, normal cells. With the advent of innovative therapeutic approaches like oncolytic viruses, immune checkpoint inhibitors, and adoptive cell transfer, we have been able to add certain selectivity to this process that could have never been imagined before. Although these treatments are currently expensive, as they become more effective over the next few years and more start to enter the market, it may be only a matter of time before they soon become the dominant force for treating cancer.

WORKS CITED 1) CAR-T Cell Therapy Market – A Revolution in Cancer Treatment. https://www.coherentmarketinsights.com/market-insight/car-t-cell-therapy-market-102. Published May 2018. Accessed July 25, 2018. 2) Paclitaxel (Taxol, Onxal). http://chemocare.com/chemotherapy/drug-info/ Paclitaxel.aspx. Accessed July 25, 2018. 3) Oncolytic Virus Therapy: Using Tumor-Targeting Viruses to Treat Cancer. https://www.cancer.gov/news-events/cancer-currents-blog/2018/oncolytic-viruses-to-treat-cancer. Published February 9, 2018. Accessed July 25, 2018.

Figure 3: A general outline for a procedure involving TIL therapy. A small segment of tumor is harvested to extract potential TILs. They are rapidly expanded with IL-2 and put through assays that increase specificity of tumor recognition before being injected back into the patient.One of the added benefits of this model is that the cells being injected are autologous; as in, they origi nated from the donor, and thus any potential negative immune responses, such as rejection, are avoided. Figure from Lee and Margolin, 2012.

4) Rehman H, Silk AW, Kane MP, Kaufman HL. Into the clinic: Talimogene laherparepvec (T-VEC), a first-in-class intratumoral oncolytic viral therapy. J Immunother Cancer. 2016; 4:53. 5) Dine J, Gordan R, Shames Y, Kasler MK, Barton-Burke M. Immune Checkpoint Inhibitors: An Innovation in Immunotherapy for the Treatment and Management of Patients with Cancer. Asia Pac J Oncol Nurs. 2017; 4(2): 127-135. 6) Perica K, Varela JC, Oelke M, Schneck J. Adoptive T Cell Immunotherapy for Cancer. Rambam Maimonides Med J. 2015; 6(1): e0004. 7) Lee S, Margolin K. Tumor-infiltrating lymphocytes in melanoma. Curr Oncol Rep. 2012; 14(5): 468474.

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A REVIEW ON THE TOXICITY OF COPPER:

AN ESSENTIAL MICRONUTRIENT, BUT HIGHLY TOXIC IN EXCESS Jonathan Chow

ABSTRACT Copper is one of many transition metals that is an essential micronutrient and has many physiological functions, most notably as components of intracellular redox enzymes. Alterations in endogenous copper homeostasis can have negative consequences to the body. Those that skew copper homeostasis towards excessive amounts can have toxic outcomes such as hepatotoxicity, thyrotoxicity and neurotoxicity. Copper toxicity is also implicated in the progression of numerous disease states (such as Alzheimer and Parkinson), and the inverse remains true resulting in a bidirectional relationship. The aim of this review it to discuss the consequences, outcomes and underlying mechanisms of copper toxicity.

1. INTRODUCTION 1.1 Importance of Copper in the body Copper is a trace element that has a fundamental role in many cellular processes within the body, thus making it an essential micronutrient. The World Health Organization (WHO) has set the minimum daily intake to avoid a deficiency in normal adults at 11 μg/kg of body weight (600 μg/ day for females and 700 μg/day for males). WHO suggests that the recommended daily adult copper intake to be about 15% above the minimum: 700 μg/day (females) and 800 μg/day (males) (1). It has previously been shown that many enzymes require copper ions as cofactors, the majority of which participate in redox cycling reactions (2). Copper can cycle between its two oxidation states (+1, +2) and these copper-dependent enzymes use this cycling as a medium to pass electrons between substrates to. An example of this cycling is in the mitochondrial electron transport chain: Cytochrome c (cyt c) oxidase uses two copper centres to transfer one electron from cyt c to molecular oxygen. The resulting free energy released allows protons to be moved across the inner-mitochondrial membrane (3) creating a proton gradient needed for eventual ATP production via ATP synthase. Without these copper centres, cyt c oxidase will be 9

rapidly degraded and unable to properly assemble (4). Copper has also been found to be necessary components of copper,zinc-superoxide dismutase (Cu,Zn-SOD) enzymes (5) which scavenge and remove superoxide anions (a reactive oxygen species/ROS) (2). Many studies have also shown that copper plays a key role in the development and activation of the immune system. It was originally found that copper systemically increased during the inflammatory response and accumulated at sties of infection (8,9). This gave rise to the hypothesis that bodily copper reserves allow immune cells to produce ROS to mediate their bactericidal activity (9). Murine models later showed that while the above may be true, copper accumulation occurred long after the initial infection (10). Thus, the complete role of copper in the immune system remains to be fully elucidated.

1.2 Copper Homeostasis Research on copper homeostasis within the body and its mechanisms is an active field. Copper is primarily absorbed from the small intestine and transported to the liver bound to albumin and transcuperin (5). The liver is considered to be the primary site of storage and distribution; copper that is released from the liver is almost always bound to ceruloplasim (5). Copper excretion from the body is primarily through the bile (5). Yeast studies have demonstrated that copper is taken up by cells via the Ctr1 membrane bound transporter (human orthologue of the yeast CTR1), which has a high affinity for (+1) copper (10,11). CTR1 deletion mutant yeast cell cultures were found to have defects in growth when plated on low copper media, confirming the role that CTR1 plays in copper uptake in yeast cells (Ctr1 in human cells) (10). Relative to the other aspects of copper homeostasis, not as much is known with regards to intracellular storage in eukaryotes. Other studies in yeast have suggested that the vacuoles play an important role in the storage and mobilization of cellular copper (10,11). Functional screens found that deletion of several genes that maintain vacuolar pH resulted in growth defects associated with lack of cellular respiration. These defects were reversed with the addition of copper to the media supporting the idea that the vacuole is important in copper storage within yeast cells (10). The vacuole is thought to be the yeast orthologue of eukaryotic lysosomes which suggests that lysosomes may mediate eukaryotic copper storage and mobilization (10,11). The mitochondria have a surprisingly higher than predicted amounts of localized copper despite the known role of copper in the function of many mitochondrial enzymes. Previous studies demonstrated that the cytochrome c oxidase chaperone protein (Cox) 17 is responsible for facilitating the transport of cytosolic copper to the mitochondria (10,11). Interestingly, Cox17 is not the sole mediator of this process as it rarely translocates from the intermembrane space of the mitochondria (11). Rather, these studies show that Cox17 mediates mitochondrial uptake of copper from the cytosol via the sequential transfer of copper ions between Cox17, and Cox11 and Sco1 (10,11). Cox11 and Sco1 are responsible for transferring copper ions to the two copper centres found in cytochrome c oxidase (10). Transport of copper to intracellular secretory compartments and excretion from the cell is mediated by Atox1, ATP7A and ATP7B; the latter two being key ATPase transporters involved in copper related diseases (11). 10

2. COPPER TOXICITY 2.1 Sources of Toxicity Despite being an essential micronutrient in humans, copper can have toxic effects when present in excessive amounts. WHO has reported that the upper limit for copper intake in adults is 10 mg/day (females) and 12 mg/ day (males) (1). These values represent what WHO has deemed as the highest amount of daily copper intake that is safe and non-toxic; those taking in higher amounts will be more likely to experience toxicity. Because the upper limit of copper intake is very high, copper toxicity is usually very rare and if it does occur, it is usually due to the ingestion of gram quantities (1). It is this disruption in copper homeostasis that mediates its toxicity in the form of tissue damage and subsequent disease states (5). However, there are several diseases that can contribute to the development of copper toxicity without having to ingest said amounts. These diseases skew the homeostatic balance towards excess copper retention (5). For example, Wilson Disease (WD) is an autosomal recessive disease which results in copper accumulation primarily in the liver and brain, causing extensive tissue damage (12). WD is caused by a mutation in the ATP7B gene on chromosome 13 which results in excessive copper retention within hepatocytes (where ATP7B is primarily expressed) due to the lack of excretion into bile (10,12).

2.2 Consequences of Toxicity 2.2.1 Hepatotoxicity Because copper is predominantly stored in the liver, this is where the majority of manifestations of copper toxicity have been observed. However, the liver has a great capacity to store copper, and clinical manifestations of toxicity are only observed when this capacity has been exceeded (1). In WD, it has been reported that the liver pathologically progresses in stages from steatosis, to interface hepatitis, fibrosis and finally cirrhosis over time (13) due to the accumulation of copper within the liver. WD has also been characterized by acute liver failure in contrast to the more chronic progression to cirrhosis previously mentioned. Nonetheless, the hepatotoxicity that arises from WD can result in hepatic encephalopathy which further worsens the prognosis of patients (12). Indian childhood cirrhosis (ICC) is a disease with genetic predisposition that affects children aged 1 to 3 and causes liver cirrhosis . This has been primarily attributed to the deposition of excess copper in the liver of such patients. The role of copper was discovered by several groups who observed positive orcein staining in liver biopsy of children with ICC. However, the exact mechanism for this copper deposition remains unclear (14,15). ICC has been reported to differ from WD in all aspects, except for the similar occurrence of copper deposition in the liver (16). Another condition related to copper toxicity is Idiopathic copper toxicosis (ICT). This is a condition that is similar to WD and ICC in that excessive copper deposition is observed along with hepatic lesions, however the etiology is currently unknown (hence the â&#x20AC;&#x153;idiopathicâ&#x20AC;? label) (17). ICT also differs from ICC with regards to global incidence: ICC is primarily seen in the Indian subcontinent, whereas ICT occurs primarily and sporadically elsewhere worldwide. Interestingly, it has been reported that there may be some familial predisposition in individuals in Northern Germany which may provide a greater understanding of the etiology of ICT upon further research (18). ICT has been reported to be characterized by more severe intrahepatic lesions and greater changes in liver histology. However, unlike in WD, there are no reports of any extrahepatic lesions or symptoms. It is noted that due to the lack of extrahepatic lesions, ICT diagnosis is difficult to distinguish from cirrhosis/hepatic lesions caused by other forms of copper toxicity (17). It is worth mentioning that because the exact pathological mechanism of ICC is not fully elucidated, some consider it as a form of ICT with a high incidence in the Indian population.

WHO has reported that the upper limit for copper intake in adults is

10 mg/day (females) & 12 mg/day (males) 1

2.2.2 Thyrotoxicity

While most studies on copper toxicity focus on the liver due to its copper storage capacity, one study focused on the effects of copper-mediated endocrine toxicity. This study showed that female Sprague-dawley rats had significantly reduced serum thyroid hormone (T3 and T4) levels with increased thyroid stimulating hormone levels with pubescent exposure to the higher doses of thiodiazole copper (19). Only four doses were used to in this study (19), and thus caution should be used in assuming a dose-response relationship between thiodiazole copper and the effects on serum T3 and T4. It was also observed that there were hypertrophic/hyperplastic histological changes to the thyroid glands in rats with the higher doses (19).

2.2.3 Neurotoxicity

Hepatic encephalopathy is brain dysfunction due to liver insufficiencies/failure that is a common complication in WD patients (12,20). Copper toxicity has also been implicated in the progression of several neurodegenerative diseases such as Alzheimer and Parkinson (PD) (20,21). Copper can be neurotoxic because it is found in several brain areas such as the basal ganglia and several synaptic membranes (21), where it is involved in catalyzing norepinephrine synthesis via the copper dependent dopamine Î˛-monooxygenase enzyme (2,6,7).

3. SUGGESTED MECHANISMS OF TOXICITY 3.1 Fenton (Haber-Weiss) Reaction As previously mentioned, copper normally participates in many enzymatic reactions because of its ability to cycle between its two oxidation states. This redox potential of copper is in fact one of the most heavily supported mechanisms by copper exerts its toxicity. Copper and other transition metals that are capable of cycling between oxidation states, can participate in the formation of ROS via the infamous Fenton (Haber-Weiss) reaction (5). It has been suggested that cycling of copper ions between their (+2) and (+1) states helps catalyze the formation of hydroxyl radicals from superoxide anions via the intermediate production of hydrogen peroxide (H2O2) (Figure 1) (22). As previously noted, copper ions are a key component in Cu,Zn-SOD (5) which use copper ions in the (+2) state as electron acceptor and two hydrogen ions to detoxify superoxide anion to H2O2, while the copper is reduced to its (+1) state (22,23). Alternatively, it has also been found that copper ions are capable of facilitating the same reaction spontaneously with similar efficiencies (22). Copper in its reduced, (+1) state can catalyze the production of hydroxyl radicals from H2O2 via the Fenton (Haber-Weiss) reaction. This reaction can proceed spontaneously, but is extremely slow and the reaction rate is sped up by the presence of cop-

per or other transition metals. (+1) copper ions can donate an electron to H2O2 and become oxidized to its (+2) state. The H2O2 undergoes fission upon reception of the electron resulting in the formation of hydroxide and a hydroxyl radical (22). Hydroxyl radicals are considered the most reactive ROS in the body and can cause oxidative DNA damage, and the formation of protein adducts and lipid radicals (via lipid peroxidation) (5, 24).

Figure 1. Schematic of the endogenous formation of hydroxyl radical via the Fenton (Haber-Weiss) reaction. Starting from molecular oxygen, a series of partial reductions occur catalyzed by both enzymes and copper. In this representation, both iron and copper are shown to catalyze this reaction, however other transition metals are also capable of this catalysis. Adapted from: Barbusinski, K. (2009) Fenton reaction - Controversy concerning the chemistry. Ecol. Chem. Eng. 16, 347-58.

At its core, the Fenton (Haber-Weiss) reaction is simply the reaction of transition metals with two oxidation states such as iron or copper, with H2O2 (23). Despite a general consensus on the reaction, there seems to be some controversy in regard to the exact chemical mechanism by which the Fenton (Haber-Weiss) reaction occurs. There has been some debate as to which electron shell is the electron donor to the H2O2. It was originally accepted that the donated electron came from the outer shell, however, more recent studies have been contradictory and suggested that the donated electron came from the inner shell (23). It may be that both can occur, but at different levels of the catalytic copper/transition metal. Since it is less energetically favourable to transfer an electron from the inner shell than from the outer shell, it may in fact be that when copper is excess (ie. at toxic levels), the energy produced from the Fenton (Haber-Weiss) reaction is sufficient to supplement the energy required donate an inner shell electron. There has also been some debate as to whether the reaction mechanism is radical or non-radical. Again, traditionally it has been accepted that the outcome of this reaction is the production of a hydroxyl radical. Others have recently proposed a reaction mechanism that does not involve the formation of a radical, but rather species with high oxida-

tive abilities that are just as damaging (23). However, it is more likely that the proposed radical mechanisms occur in vivo. Electron spin trapping experiments have identified the production of hydroxyl radicals in normal and vitamin-E/selenium deficient rats when given excess copper. In the deficient rats, hydroxyl radical formation was increased relative to normal rats, suggesting the potential protective role of vitamin-E and selenium in rats (24). The generation of ROS from excess copper has also been suggested as a mechanism of neurotoxicities. It is thought that oxidative stress and copper toxicity can mediate neurodegenerative diseases by causing cell death in associated brain areas. PD is attributed to loss of the dopaminergic system in the brain which has been suggested to be partly caused by oxidative cellular injury (21). Chronically excessive copper amounts may then contribute to diseases that have an underlying neurodegenerative pathological basis (12,21). However, it is important to note that these neurotoxic mechanisms are not confirmed, and currently still under investigation (12).

At its core, the Fenton (Haber-Weiss) reaction is simply the reaction of transition metals with two oxidation states such as iron or copper, with

H2O2 23

3.2 Endocrine Disruption In the study that identified the reduction in serum T3 and T4 in female Sprague-dawley rats, it was also found that there was an increase of 4-nitrophenol uridinediphosphate-glucuronosyltransferase (UDPGT) in the S9 liver microsome fraction of treated subjects. However, only a statistically significant increase was observed at the highest dose (30mg/ kg) (19). T4 is the most abundant form of thyroid hormone in the serum, and is excreted in the bile via UDPGT-mediated glucuronidation. Upregulation of UDPGT is thought to be the mechanism by which thiodiazole copper caused the observed reduction in serum thyroid hormone (19).

3.3 Altered Lipid Metabolism In studies using a mouse model of WD (ATP7B deficient mice), lipid metabolism was found to be significantly altered before hepatotoxic symptoms were observed. The most apparent alteration was the significant downregulation cholesterol biosynthesis resulting in the alteration of the makeup of serum lipids, most notably in serum triglycerides (25). It was later observed that the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA, had decreased mRNA levels in the WD mouse model. This suggests that copper accumulation in early WD may downregulate the transcription of HMG-CoA and other key genes involved in cholesterol biosynthesis (25).

In fact, it is noted that (+2) copper has the ability to bind to â&#x20AC;&#x153;zinc fingerâ&#x20AC;? DNA binding motifs of nuclear proteins and inhibit their interaction with target DNA sequences. This may be the mechanism by which copper accumulation in WD results in the downregulation of cholesterol biosynthesis and lipid metabolism alterations, although it remains to be confirmed (25).

4. FUTURE RESEARCH/DIRECTIONS Currently, there lacks sensitive biomarkers to excess, non-toxic amounts of copper in the body (12). However, this is likely in part due to the liverâ&#x20AC;&#x2122;s capacity to store large amounts of copper, and toxicity is usually only observed when this capacity is overwhelmed (1). It is then important to investigate and find biomarkers of high copper levels within the body that are not yet toxic enough to be seen clinically. As mentioned above, studies in mouse models have identified alterations in lipid metabolism before hepatotoxicity is observed (25), which indicates that even marginally excessive copper levels can already begin to negatively affect the body. However, the fact that these changes were detectable at pre-symptomatic stages of WD may serve as a potential avenue for finding a sensitive biomarker for copper levels. It is important to note that while this may be promising, these changes were only observed in WD patients who have a mutation in their ATP7B gene (10,12) and may only be limited to this population. Nevertheless, it may prove worthwhile to observe analogous changes in normal individuals with high but non-toxic levels of copper. The gold standard for measuring excess copper accumulation in the body is a liver biopsy, however, it is generally only used when justified by less sensitive biomarkers (12). As most copper in the plasma is bound to ceruloplasim (5), the unbound (free) portion is currently used as an acceptable diagnostic tool to measure copper levels in the body (12). However, it was found that the free portion of copper in the plasma can be raised by any number of disease states, in addition to intake of toxic levels of copper (12). This greatly reduces its sensitivity to detecting toxic amounts of copper in the body, since its use alone would not be able to differentiate between intake toxic amounts, or a disease state that has causes liver necrosis and the release of stored copper. Thus, there is also a need for more sensitive biomarkers that not only senses marginally copper excess, but also can appropriately distinguish sources of excess copper. It may also be worth noting that because current biomarkers of endogenous copper levels lack sensitivity, the WHO recommended dietary intakes have been suggested to be solely estimates (12). Investigating new biomarkers that are more sensitive will then also allow for a greater degree of accuracy in determining recommended dietary intakes for copper. In regards to the debate on the mechanism of the Fenton (HaberWeiss) reaction, another direction of future research could be further elucidation of the reaction mechanism(s). Because it has a fundamental role in the oxidative damaging potential of copper and several transition metals, it may be beneficial to understand the underlying mechanism(s) (5). This would allow potential targeted therapies to prevent the formation of the hydroxyl radical and other oxidative species in cases of copper toxicity.

5. CONCLUDING SUMMARY In conclusion, copper is a key micronutrient that is needed for many physiological enzymatic functions in the body. Many of these functions exploit the ability for copper to undergo redox cycling between its two oxidation states. However, it is this same key ability that mediates much of copperâ&#x20AC;&#x2122;s toxic potential. Via the Fenton (Haber-Weiss) reaction, copper and other transition metals are thought to mediate the generation of the reactive hydroxyl radical, although the exact mechanism remains a point of debate. This hydroxyl radical can cause oxidative damage to many cellular macromolecules, not limited to DNA, proteins and lipids (such as membranes). Ultimately, this oxidative damage can lead to cellular death: This has been implicated in the progression neurodegenerative diseases. Copper has also shown to be thyrotoxic by increasing the elimination of T4 and causing thyroid gland hyperplasia. Changes in lipid metabolism have been observed in early stage WD patients that do not show hepatotoxicity. This may serve as a route for further investigation for its use as a biomarker for excessive copper. There currently is no sensitive biomarker for high copper levels and should be the focus of much future research moving forward.

Acknowledgements

The author would like to thank Dr. Denis Grant and the editors of PharmaChronicle for the help and advice.

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White,C. Lee,J., Kambe,T., Fritsche,K. and Petris,M.J. (2009) A role for the ATP7A copper-transporting ATPase in macrophage bactericidal activity. J. Biol. Chem., 284, 33949-56.

10) Achard,M.E.S., Stafford,S.L., Bokil,N.J., Chartres,J., Bernhardt,P.V., Schembri,M.A., Sweet,M.J. and McEwan,A.G. (2012) Copper redistribution in murin macrophages in response to Salmonella infection. Biochem. J., 444, 51-57. 11) Schlecht,U., Suresh,S., Xu,W., Aparicio,A.M., Proctor,M.J., Davis,R.W., Scharfe,C. and St Onge,R.P. (2014) A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system. BMC Genomics., 15, 1-14. 12) Ferenci,P., Litwin,T., Seniow,J. and Czlonkowska,A. (2015) Encephalopathy in Wilson disease: Copper toxicity or liver failure? J. Clin. Exp. Hepatol., 5, 88-95. Johncillia,M. and Mitchell,K.A. (2011) Pathology of the liver in copper overload. Semin. Liver Dis., 31, 239-44.

15) Nayak,N.C. and Chitale,A.R. (2013) Indian childhood cirrhosis (ICC) & ICClike diseases: The changing scenario of facts versus notions. Indian J. Med. Res., 137, 1029-1042. 16) Hayashi,H., Shinohara,T., Goto,K., Fujita,Y., Murakami,Y., Hattori,A., Tatsumi,Y., Shimizu,A. and Ichiki,T. (2012) Liver structures of a patient with idiopathic copper toxicosis. Med. Mol. Morphol., 45, 105-9. 17) Muller,T., Schafer,H., Rodeck,B., Haupt,G., Koch,H., Bosse,H., Welling,P., Lange,H., Krech,R., Feist,D., Muhlendahl,K.E., Bramswig,J., Feichtinger,H. and Muller,W. (1999) Familial clustering of infantile cirrhosis in Northern Germany: A clue to the etiology of idiopathic copper toxicosis. J. Pediatr., 135, 189-96. 18) Zhang,L. Wang,J. Zhu,G.N. and Su,L. (2010) Pubertal exposure to thiodiazole copper inhibits thyroid function in juvenile female rats. Exp. Toxicol. Pathol., 62, 163-9. 19) Pal,A. (2014) Copper toxicity induced hepatocerebral and neurodegenerative diseases: An urgent need for prognostic biomarkers. Neurotoxicology, 40, 97101. 20) Desai,V. and Kaler,S.G. (2008) Role of copper in human neurological disorders. Am. J. Clin. Nutr., 88, 855S-8S. 21) Goldstein,S. and Czapski,G. (1986) The role and mechanism of metal ions and their complexes in enhancing damage in biological systems or in protecting these systems from the toxicity of O2-. J. Free Radic. Biol. Med., 2, 3-11. 22) Barbusinski,K. (2009) Fenton reaction – Controversy concerning the chemistry. Ecol. Chem. Eng., 16, 347-58. 23) Kadiiska,M.B., Hanna,P.M., Jordan,S.J. and Mason,R.P. (1993) Electron Spin Resonance Evidence for free radical generation in copper-treated vitamin Eand selenium-deficient rats: In vivo spin-trapping investigation. Mol. Pharmacol., 44, 222-7. 24) Huster,D. and Lutsenko,S. (2007) Wilson disease: not just a copper disorder. Analysis of a Wilson disease model demonstrates the link between copper and lipid metabolism. Mol. Biosyst., 3, 816-24.

KETAMINE

ACTIVATION OF DOPAMINERGIC PATHWAYS MEDIATE RAPID ONSET ANTIDEPRESSANT EFFECTS OF KETAMINE By: Rosa Summer

Abstract The current treatment for major depressive disorder (MDD) shows delayed onset of therapeutic action and many MDD patients become treatment resistant. A sub-anesthetic dose of ketamine, an antagonist at the NMDA receptor, has been shown to elicit rapid onset therapeutic effects that persist beyond its half-life. Current proposed mechanisms of action involve activation of the glutaminergic system, resulting in increased synaptogenesis and neural activation in the prefrontal cortex and hippocampus. The mesolimbic dopaminergic circuit has also been shown to be dysregulated in MDD, as it mediates reward, pleasure, and mood. Witkin et al. show that ketamine and LY341495, a mGlu2/3 inhibitor, both increase glutamate release and stimulate dopaminergic firing in the ventral tegmental area. They also show increased extracellular dopamine in the medial prefrontal cortex and nucleus accumbens, and these compounds also induces dopamine hypersensitivity, assessed using the locomotion assay. Lastly, they demonstrate that ketamine and LY341495 can induce antidepressant effects in forced swim test and tail suspension test, in a dose dependent manner. Treatment with the AMPA receptor antagonist NBQX is able to attenuate the antidepressant effects elicited by ketamine and LY341495, confirming the antidepressant effects are AMPA-dependent. This data suggests an indirect activation of the dopamine system by ketamine or LY341495 administration, as dopamine antagonists have little to no effect on antidepressant effects. 20

INTRO

Background and Introduction Major depressive disorder (MDD) is one of the most prevalent psychiatric disorders and a leading cause of disability worldwide.1,2 Symptoms for MDD include persistent low mood, anhedonia, weight changes, sleep disturbances, and suicidal ideation.3 MDD is currently treated with selective serotonin reuptake inhibitors (SSRIs) or serotonin-norepinephrine reuptake inhibitors (SNRIs); however, the onset of action is 4-6 weeks and only a third of the patients respond. In addition to the delayed onset of action and lack of response on a majority of the population, many responders often develop resistance and often relapse.1,2 When ketamine was shown to have rapid antidepressant effects within a few hours of treatment and lasting several days,1,2,6 special interest arose in determining the mechanism of action of the rapid onset of effects in search of

Figure 1: Network of inputs and outputs to the mesolimbic dopa minergic circuits. The cell bodies of the mesolimbic pathway originate in the VTA and project to NAc. Many glutaminergic and monoaminergic pathways interact with this system.22 Figure retrived from

Arias-CarriĂłn et al., 2010.

another drug with equally potent antidepressant effects without the psychomimetic effects and addictive potential. The antide pressant effects of ketamine persist beyond its half-life of 1-3 hours, sug-

gesting its mechanism of action extends beyond its interaction to the target receptor, the NMDA glutamate receptor (NMDAR). Ketamine binds 21

preferentially to the extrasynaptic NMDARs.2 While synaptic NMDAR activation leads to activation and promote neuronal survival, extrasynaptic NMDAR activation promotes synapse atrophy and excitotoxicity leading to neuronal death.1 Therefore, ketamine antagonism at the extrasynaptic NMDARs have a neuroprotective effect, in line with its antidepressant effects. Recent research has also shown that inhibition of metabotropic glutamate receptors 2/3 (mGlu2/3) can also elicit antidepressant effects by decreasing Gi-coupled inhibition of glutamate signaling to postsynaptic AMPA and NMDA receptors. There are currently many hypotheses on ketamineâ&#x20AC;&#x2122;s mechanism of action. Many theories describe the blockade of NMDA receptor, leading to AMPA activation downstream, resulting in increased BDNF release, mTOR activation, synaptogenesis, LTP formation, and overall increased neural activation in prefrontal cortex and hippocampus.1,2,5 The mesolimbic circuit plays a role in reward, pleasure, mood, and addiction and is often dysregulated in MDD.7,8,14 Figure 1 shows a simplified schematic of the neurotransmitter systems involved in regulating the mesolimbic circuit of dopaminergic neurons originating from the ventral tegmental area (VTA) projecting to the nucleus accumbens (NAc).7 As shown, the VTA is under control by many glutaminergic pathways. Previous research has also shown that lesion of the VTA dopaminergic neurons were able to induce depression in animal models, while stimulation of the medial forebrain bundle (MFB) of the VTA by deep brain stimulation was able to elicit antidepressant effects.13 Thus, the researchers of this paper hypothesized that ket-

amine elicits its antidepressant effects through activation of the mesolimbic dopamine circuit. They tested their hypothesis by assessing the effect of ketamine and a mGlu2/3 inhibitor, LY341495, on the firing activity of the dopaminergic neurons in the VTA using electrophysiology and measuring the output of extracellular dopamine in NAc and medial prefrontal cortex, two regions of mesolimbic projections downregulated in depression (Figure 2).14 Lastly, they assessed the antidepressant effects of ketamine and LY341495 in two animal models of depression, forced swim test and tail suspension test, to identify the role of AMPA glutamate receptors in mediating these effects. In each assay, the effects of ketamine and LY341495 were compared to a standard SSRI, citalopram, to show the difference between the two mechanisms of action and the ability for these drugs to elicit antidepressant effects in treatment resistant depression.

Figure 1: Schematic of the mechanism of action of ketamine and LY341495 proposed in this study. Ketamine and LY341495 inhibit NMDAR and mGluR2/3, respec tively. The result is increased glutamate release in the VTA, stimulating postsyn aptic AMPA and NMDA receptors and stimulate dopaminergic neuron firing. These dopaminergic neurons project to the PFC and NAc to increase dopamine release, leading to antidepressant effect.

RESULTS Major Results

Increased VTA dopamine firing

Witkin et. al used electrophysiology, animal behaviour, and neurochemical assays to show the interplay between the glutamate system and dopaminergic circuits. First, they showed that ketamine and LY341495 were both able to increase the number of spontaneously active dopaminergic cells with no effect on firing rate and burst activity. The effects were attenuated with AMPA antagonist NBQX and were not elicited by citalopram, suggesting that the mechanism of increased dopaminergic firing is due to glutaminergic activation. This data was obtained from electrophysiology measurements of the dopaminergic neurons in the VTA of male rats 10 minutes following drug treatment. 22

Increased extracellular dopamine at mPFC and NAc In vivo neurochemical studies also revealed increased extracellular dopamine in two areas VTA dopaminergic neurons project to in the mesolimbic pathway: medial PFC and v. In both areas, ketamine was able to stimulate a greater release of dopamine than LY341495, suggesting the NMDA receptor antagonism was more potent in inducing an antidepressant effect than mGlu2/3 antagonism.

Potentiation of dopamine-dependent locomotion To expand on the notion of dopaminergic activation, the researchers also showed potentiation of dopamine-mediated locomotion. Quinpirole is a D2/D3 agonist that induces locomotor activity in animal models; when combined with ketamine or LY341495, the animals exhibit hyperlocomotion to a greater extent than with either drug alone, suggesting that these compounds induce dopamine hypersensitivity.

AMPA-dependent antidepressant activity Lastly, the antidepressant efficacy of ketamine and LY341495 were assessed in NIH Swiss mice and Sprague-Dawley rats. They used the forced swim test (FST) and tail suspension test to assess antidepressant activity, where decreased immobility represents increased resilience and thus increased antidepressant efficacy. The FST of rats treated with ketamine and LY341495 both showed significant antidepressant effects, comparable to that of the imipramine antidepressant control, in a dose-dependent manner. They also tested ketamine and LY341495 in NIH Swiss mice because they observed a false negative effect of SSRI antidepressant effect in the rats, while it was seen in the mice. All three compounds decreased immobility time in a dose-dependent manner. Similar to previous results, the antidepressant effects of both ketamine and LY341495 were attenuated when coadministered with NBQX, which confirms that the antidepressant effects were indeed mediated by AMPA-R dependent signaling. As expected, NBQX had no effect on citalopram antidepressant effects, since the mechanism of action for SSRIs do not involve the AMPA receptor or dopaminergic pathwaysLastly, the researchers tested ketamine and LY341495 on CD1 mice, an animal model of treatment resistant depression, using the tail suspension assay. Both drugs elicited strong antidepressant effects in a dose-dependent manner \, while citalopram had no antidepressant effects.

CONCLUSION Discussion and Conclusion

The data presented by this paper propose a mechanism of action for ketamine antidepressant activity involving the mesolimbic dopaminergic pathway. The authors describe a clear pathway where inhibition of NMDAR or mGlu2/3 receptors stimulate the release of glutamate into the VTA, which binds and activates AMPA receptors. As a result, ketamine increases dopaminergic firing at the VTA in neurons projecting towards the NAc and mPFC, showing an efflux of dopamine at those areas. The ketamine and LY341495 antidepressant effects seen in the animal models of depression were also shown to be AMPA dependent as the AMPA antagonist was able to attenuate the antidepressant response. These results were further recapitulated in experiments showing increased dendritic spine density and AMPA protein expression in the NAc following ketamine treatment.9 Overall, this paper offers the connection between changes in the glutamate system and mesolimbic dopamine system seen with ketamine treatment and offers mGlu2/3 as a possible therapeutic target for rapid onset antidepressants.

ANALYSIS

Critical Analysis and Further Directions IT IS IMPORTANT TO EMPHASIZE THAT THE EFFECTS OF KETAMINE ON DOPAMINE IS MOST LIKELY INDIRECT

There were several inconsistencies in this paper that were of note. First, they were not consistent in administering a specific enantiomer of ketamine in the different assays. S-(+)-ketamine was used in the microdialysis assay of the mPFC and (+)-ketamine was used for the locomotion assays, while a racemic mixture was used for the other experiments. This is important as other studies have shown a differential effect between the R- and S-ketamine enantiomers.10-12 However, it was shown that the differences are significant in the long-lasting effects,12 rather than the immediate effects assayed in this study.

In addition, the method of administration of drugs varied greatly between different assays. While the FST in mice used only i.p. injections, the FST in rats used i.v., i.p. and po for ketamine, LY341495, and citalopram, respectively. Due to the different rate of absorbance with the different routes, it could explain why there was no effect seen with citalopram treatment, even at high doses. They also only used one dose of ketamine for the electrophysiology and microdialysis studies, while multiple doses were used for LY341495, showing a dose-response effect. Furthermore, the dose chosen for ketamine (10mg/kg) did not

elicit a statistically significant effect in the forced swim test in NIH Swiss mice, which raises the question whether the antidepressant effect they saw were directly correlated with the surge in dopaminergic activity. To combat this ambiguity, ketamineâ&#x20AC;&#x2122;s effects on dopaminergic transmission should also be assayed in a dose-response manner. Similarly, they only performed the dopamine assays on healthy rats rather than rats with induced depression. The data presented may not be accurate in depressive patients, as there are known neurolobiological changes that occur in MDD.1,2 The observation of upregulated mesolimbic stimulation upon ketamine use is not unexpected, as many drugs of abuse increase dopamine levels in the NAc.7,15 It is important to also test ketamine in depression models to confirm these findings also apply in MDD and that the upregulation of dopamine is an antidepressant effect rather than that of addiction. However, by showing that the AMPA antagonist NBQX can similarly attenuate dopamine firing and antidepressant effects suggest the upregulation of dopaminergic pathways is likely to be seen in depression models as well. Further, the researchers used only male animals in their experiments, likely because the hormonal cycles interfere with antidepressant effect. However, this does not capture the clinical reality, where MDD is twice as prevalent in women than men2 and several subtypes of depression are hormonal, such as post-partum depression.3 Therefore, it is crucial that both female and male animals are used in any preclinical drug testing. Recent research has shown that females do respond to ketamine antidepressant effects differently than males. Inuguez et. al showed that the itncreased sensitivity to stress seen in women could be recapitulated in a mouse model, and this system could be used to test sex-specific responses to antidepressants.16 Dossat et. al 24

showed that females indeed are more sensitive to ketamine antidepressant effects, possibly as a result of their estrous cycle.17,18 Interestingly, a recent randomized prospective clinical trial showed that low-dose ketamine does not have an effect in the prevention of post-partum depression.19 The contradicting evidence between preclinical and clinical data suggests even greater complexity in the effect of hormones on antidepressant effect. Early clinical trials for ketamine as an antidepressant have showed much higher response rates compared to traditional antidepressant therapies, up to 70% response with repeated doses.6 However, it would be valuable to understand the pathology behind patients are are resistant to ketamine and whether those patients respond to standard antidepressant therapy. Research has clearly shown that MDD is a heterogenous disorder in terms of pathology and treatment response. Thus far, preclinical and clinical trials with ketamine have only been performed on treatment-resistant. It would be useful to assess response in the responder group as well to better characterize the different pathobiology behind MDD.

Lastly, it is important to emphasize that the effects of ketamine on dopamine is most likely indirect. A recent study by Can et. al showed that ketamine administration had no direct effect on NAcc evoked dopamine release, and neither ketamine nor its metabolites had any binding affinity for the dopamine receptor (DR) or dopamine transporter (DAT).20 Supporting this, DR antagonists, such as antipsycotic medications, do not interfere with ketamine antidepressant activity and dopamine agonists, such as L-dopa, do not have antidepressant effects. Similarly, a DAT inhibit does not decrease depressive symptoms, unless when combined with modulation of the monoaminergic systems, such as bupropion or the combination of fluoxetine and olanzapine. Combined, this data suggests that while the dopaminergic system is indeed modulated, its activation alone is not effective in alleviating depression. Understanding of the interplay between the different neurotransmitter systems at a molecular and neural network level is crucial in identifying a novel antidepressant with the rapid onset and high efficacy of ketamine.

REFERENCES 1)

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Strasburger, S. E., Bhimani, P. M., Kaabe, J. H., Krysiak, J. T., Nanchanatt, D. L., Nguyen, T. N., … Raffa, R. B. (2017). What is the mechanism of Ketamine’s rapid-onset antidepressant effect? A concise overview of the surprisingly large number of possibilities. Journal of Clinical Pharmacy and Therapeutics, 42(2), 147–154. https://doi.org/10.1111/jcpt.12497

American Psychiatric Association. Diagnostic and statistical manual of mental disorders, (5th edn; DSM–5). Washington, D.C.: American Psychiatric Association Publishing, 2013

Witkin, J. M., Mitchell, S. N., Wafford, K. A., Carter, G., Gilmour, G., Li, J., … Monn, J. A. (2017). Comparative Effects of LY3020371, a Potent and Selective Metabotropic Glutamate (mGlu) 2/3 Receptor Antagonist, and Ketamine, a Noncompetitive N -Methyl-d-Aspartate Receptor Antagonist in Rodents: Evidence Supporting the Use of mGlu2/3 Antagonists, for the Treatment of Depression. Journal of Pharmacology and Experimental Therapeutics, 361(1), 68–86. https://doi.org/10.1124/jpet.116.238121

Liu, B., Liu, J., Wang, M., Zhang, Y., & Li, L. (2017). From Serotonin to Neuroplasticity: Evolvement of Theories for Major Depressive Disorder. Frontiers in Cellular Neuroscience, 11. https://doi.org/10.3389/fncel.2017.00305

Newport, D. J., Carpenter, L. L., McDonald, W. M., Potash, J. B., Tohen, M., Nemeroff, C. B., & The APA Council of Research Task Force on Novel Biomarkers and Treatments. (2015). Ketamine and Other NMDA Antagonists: Early Clinical Trials and Possible Mechanisms in Depression. American Journal of Psychiatry, 172(10), 950–966. https://doi.org/10.1176/appi.ajp.2015.15040465

Nestler, E. J., & Carlezon, W. A. (2006). The Mesolimbic Dopamine Reward Circuit in Depression. Biological Psychiatry, 59(12), 1151–1159. https://doi.org/10.1016/j.biopsych.2005.09.018

Yadid, G., & Friedman, A. (2008). Dynamics of the dopaminergic system as a key component to the understanding of depression. In Progress in Brain Research (Vol. 172, pp. 265–286). Elsevier. https://doi.org/10.1016/S0079-6123(08)00913-8

Strong, C. E., Schoepfer, K. J., Dossat, A. M., Saland, S. K., Wright, K. N., & Kabbaj, M. (2017). Locomotor sensitization to intermittent ketamine administration is associated with nucleus accumbens plasticity in male and female rats. Neuropharmacology, 121, 195–203. https://doi.org/10.1016/j.neuropharm.2017.05.003

10) Yang, C., Shirayama, Y., Zhang, J., Ren, Q., Yao, W., Ma, M., … Hashimoto, K. (2015). R-ketamine: a rapid-onset and sustained antidepressant without psychotomimetic side effects. Translational Psychiatry, 5(9), e632. https://doi.org/10.1038/tp.2015.136 11) Kavalali, E. T., & Monteggia, L. M. (2018). The Ketamine Metabolite 2R,6R-Hydroxynorketamine Blocks NMDA Receptors and Impacts Downstream Signaling Linked to Antidepressant Effects. Neuropsychopharmacology, 43(1), 221–222. https://doi.org/10.1038/ npp.2017.210 12) Fukumoto, K., Toki, H., Iijima, M., Hashihayata, T., Yamaguchi, J., Hashimoto, K., & Chaki, S. (2017). Antidepressant Potential of ( R )-Ketamine in Rodent Models: Comparison with ( S )-Ketamine. Journal of Pharmacology and Experimental Therapeutics, 361(1), 9–16. https://doi.org/10.1124/jpet.116.239228 13) Furlanetti, L. L., Coenen, V. A., & Döbrössy, M. D. (2016). Ventral tegmental area dopaminergic lesion-induced depressive phenotype in the rat is reversed by deep brain stimulation of the medial forebrain bundle. Behavioural Brain Research, 299, 132–140. https://doi.org/10.1016/j.bbr.2015.11.036 14) Vandenheuvel, D., & Pasterkamp, R. (2008). Getting connected in the dopamine system. Progress in Neurobiology, 85(1), 75–93. https://doi.org/10.1016/j.pneurobio.2008.01.003 15) Caffino, L., Piva, A., Mottarlini, F., Di Chio, M., Giannotti, G., Chiamulera, C., & Fumagalli, F. (2017). Ketamine Self-Administration Elevates αCaMKII Autophosphorylation in Mood and Reward-Related Brain Regions in Rats. Molecular Neurobiology. https:// doi.org/10.1007/s12035-017-0772-3 16) Iñiguez, S. D., Flores-Ramirez, F. J., Riggs, L. M., Alipio, J. B., Garcia-Carachure, I., Hernandez, M. A., … Castillo, S. A. (2018). Vicarious Social Defeat Stress Induces Depression-Related Outcomes in Female Mice. Biological Psychiatry, 83(1), 9–17. https:// doi.org/10.1016/j.biopsych.2017.07.014 17) Dossat, A. M., Wright, K. N., Strong, C. E., & Kabbaj, M. (2018). Behavioral and biochemical sensitivity to low doses of ketamine: Influence of estrous cycle in C57BL/6 mice. Neuropharmacology, 130, 30–41. https://doi.org/10.1016/j.neuropharm.2017.11.022 18) Rincón-Cortés, M., & Grace, A. A. (2017). Sex-Dependent Effects of Stress on Immobility Behavior and VTA Dopamine Neuron Activity: Modulation by Ketamine. International Journal of Neuropsychopharmacology, 20(10), 823–832. https://doi.org/10.1093/ ijnp/pyx048 19) Xu, Y., Li, Y., Huang, X., Chen, D., She, B., & Ma, D. (2017). Single bolus low-dose of ketamine does not prevent postpartum depression: a randomized, double-blind, placebo-controlled, prospective clinical trial. Archives of Gynecology and Obstetrics, 295(5), 1167–1174. https://doi.org/10.1007/s00404-017-4334-8 20) Can, A., Zanos, P., Moaddel, R., Kang, H. J., Dossou, K. S. S., Wainer, I. W., … Gould, T. D. (2016). Effects of Ketamine and Ketamine Metabolites on Evoked Striatal Dopamine Release, Dopamine Receptors, and Monoamine Transporters. Journal of Pharmacology and Experimental Therapeutics, 359(1), 159–170. https://doi.org/10.1124/jpet.116.235838 21) Witkin, J. M., Monn, J. A., Schoepp, D. D., Li, X., Overshiner, C., Mitchell, S. N., … Rorick-Kehn, L. M. (2016). The Rapidly Acting Antidepressant Ketamine and the mGlu2/3 Receptor Antagonist LY341495 Rapidly Engage Dopaminergic Mood Circuits. Journal of Pharmacology and Experimental Therapeutics, 358(1), 71–82. https://doi.org/10.1124/jpet.116.233627 22) Arias-Carrión, O. Stamelou, M., Murillo-Rodríguez, E., Menéndez-González, M., Pöppel, E. Dopaminergic reward system: a short integrative review. International Archives of Medicine 2010, 3, 24 doi:10.1186/1755-7682-3-24

INTERVIEW INTERVIEW 27

LEARN ABOUT THE PHARMACHRONICLES OF A RESPECTED ACADEMIC.

Q&A

Dave Bodenstein MASTERS STUDENT

Dave is a second year Masterâ&#x20AC;&#x2122;s student working under the direction of Dr. Andreazza. While not doing research, he enjoys photography, playing in a band, and is an experienced mountain-climber! We had a chance to talk to Dave about his experiences both inside and out of the lab.

SO TELL US A LITTLE BIT ABOUT YOURSELF. Hey everyone I’m Dave, and I’m a graduate student at Dr. Andreazza’s lab. My project is about analyzing portem-mortem brain tissue from different brain areas. Specifically I am looking at mitochondrial DNA in bipolar disorder patients. Outside of school, I was a musician for many years: I played guitar my whole life, and delved into various types of arts.

WHAT IS YOUR FAVORITE PART OF GRAD SCHOOL? I love being in the lab and running all kinds of experiments! It’s all about having an idea of what work you want to do, collaborating with other people, and work on things you’re passionate about. I get to make my own protocol and test it out. It’s the pursuit of knowledge that really drives me -that’s really fun.

SINCE YOU DID YOUR UNDERGRAD AT UOFT AS WELL, HOW WOULD YOU COMPARE THE EXPERIENCE BETWEEN THAT AND GRAD SCHOOL? I’d say that in undergrad, you’re focused on going to class, studying, doing your assignments, and doing it all over again. It’s a lot of learning new information; sure some of them are really interesting, but others are just things you got to know. They’re not as fun but they’re important. In grad school, what you’re learning and doing has will go towards what your thesis is going to be; you get this focus of: this is really interesting, this is what I want to do. You’re coming into the lab, doing experiments, analyzing data, and seeing real hard evidence to questions that you might have. Everything you do has real life impact in grad school.

“

Everything you do

has real life impact in grad school. WHAT MAKES YOU INTERESTED IN BRAIN IN PARTICULAR?

I always found mental health to be very interesting. For many years, I’ve struggled with depression, to the point where sometimes I couldn’t get through the day. Having that as my personal experience, and having known some people who suffered bipolar disorder, I became very interested in how mental illnesses are developed. I feel a personal connection to the science that I’m doing.

TALK ABOUT YOUR CLIMBING EXPERIENCE? It’s a very nice to get out of the lab and be in a different environment. Being in that environment is incredible, every time you see these amazing views of the mountains, you think “it can’t get any better than this”, and then you see more, and it gets better and better. I think there is a real draw to going out to nature. You look up at the stars and it doesn’t even look real, like someone made a painting. You’re surrounded by massive mountains and you can sort of just reflect on your life, and think “wow, i’m actually here”! It was an amazing thing I was able to do, and it was one of the hardest things I was able to do. It was exhausting -- physically, mentally -- but it was so amazing, and definitely a trip of the lifetime. I recommend everyone to at least go out and experience this for themselves.

DID YOU EXPERIENCE ANY ALTITUDE SICKNESS THERE?

“

Thankfully no, but I did see some people get these effects. However, another team was rushing up the mountain to quickly, and 4-5 people started getting sick, and some of them even needed oxygen and had to be helicoptered out. We were fortunate that we didn’t get sick, but you take for granted how difficult the trek is. It might not look hard, but it can potentially be life threatening.

Knowled

BEING A GRADUATE STUDENT IN PHARMACOLOGY, HOW DO YOU MANAGE YOUR WORKLIFE BALANCE?

Maybe it’s just me, but I can study and study, but at a certain point I won’t be able to focus anymore no matter how hard I try. When this happens, I close my books, close my notes, I go out, take some photos, make some music, and try to “clean” my mind and then come back later. I found it to be actually necessary to do all these things, and it’s one of the reasons I found some success in this program. By taking a lot of breaks, I find that I actually end up doing better -- it sometimes gives me a new perspective on things, and then I find this new energy to go back to my work.

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sake

- that

WAS

I didn’ friends, school i lab, 31

AFTER YOU DO YOUR MASTERS, WHAT’S NEXT FOR YOU DOWN THE ROAD?

PhD! I really love doing this kind of stuff. Just being able to continue on to research and delve deeper into these questions, figuring out every aspect of it so that I can truly understand this topic is my goal. Knowledge for knowledge’s sake -- that’s the goal.

“

edge for

ledge’s

that’s the

oal.

WHAT’S THE MOST IMPORTANT THING TO KEEP IN MIND WHILE AT GRAD SCHOOL? One thing is to not get too overwhelmed and take things too hard. Research is all about collaborating, and part of collaborating is taking constructive criticism. You need to take criticism at face value, and not personally or to heart. It’s not a comment about you, it’s about what you made. You also got to accept that sometimes, “I don’t know” is the best answer. In meetings, if someone asks you a question you can’t answer, you can’t beat yourself up about it; you just have to say say you don’t know. Take note of it, do some research, try and figure it out and you might just figure out the answer. It’s a learning process.

S THERE ANYTHING YOU DIDN’T EXPECT ABOUT GRAD SCHOOL?

’t expect it to be so fun! I’ve been having lots of fun, I made many , and I still have time to go out for lunch or drinks. People think grad is all work and no play, but it’s really not the case.Getting out of the having fun and relaxing is a a key to succeeding in grad school. 32

IF YOU WERE A PI, WHAT WOULD YOU RESEARCH? Well, I currently have rough idea, but it’s essentially looking at different pathways of brain gene expression and how it affects mood disorders. I’m doing a whole bunch of reading since I just finished up my work with mitochondrial DNA. Now is a time for me to ask myself, “what’s next?” I have a million different ideas, so I’m reading a million different papers to find out how different regulatory pathways are affecting each other in mitochondrial proteins, especially in electron transport chain. I think if I was a PI, I would also explore the different pathways involved in addiction, because right now we have a big issue with addiction and mental health.That could be a very interesting path to go down.

WHAT ADVICE DO YOU WANT TO GIVE TO PEOPLE FOR PEOPLE WHO WANNA GO DOWN THIS GRAD SCHOOL PATH? Get a research position in your undergrad, like an ROP, or even your PCL472/474 research project. That gives you an idea of how research works. It’s a crash course that lets you explore your options. If you get a research opportunity, I would highly recommend that. Projects is how I realize i really love doing research. I was working with Dr. Peter Wells in fourth year, and working with mice and doing all these things, I realized “wow this is really cool”, I really love doing these things. I really love coming up with all these different ideas. If you’re the kind of person with a desire for knowledge, and you like to search and search and figure things out from different angles, this is the path you should go.

Dave Bodenstein

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PHARMACHRONICLE Volume 2. October 2018