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 density 1 . 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

INTRODUCTION

Schizophrenia (SZ) is a serious psychiatric neurother understand how this treatment functions specifically and published their findings three years later in 2020. 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 deficits 1 . When treated with ω-3PUFAs, the MK-801 rats showed alleviation of these SZ 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,

disorder affecting 20 million people world-wide 2 . 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 growth 4 . 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 disorders 4 . 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 gene 1 . 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 hippocampus 6 . 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 depression 7 and Alzheimer’s disease 8 . The XiaoChuan Wang Lab previously found, in 2017, that ω-3PUFAs the SZ model of MK-801 rats 9 . They continued working to fur-induced cognitive impairments. They found that ω-3PUFAs

worked to upregulate the CREB/BDNF pathway, by introducing prevents hippocampal neuron loss and overall improved cognitive function in the SZ model.

MAIN RESULTS Ω-3PUFAS IM PROVES COGNITIVE AND NEURONAL FUNCTION IN SZ M ODEL

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 object 1 . 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 treated 1 . 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 treatment 1 . 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 transmission 1 . 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.

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 group, curiosity was increased compared to the Mod group (Fig. 2B), which resembles similar levels to the Pre or healthy control groups 1 . 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 genesis. Slope of excitatory post-synaptic potential (fEPSP) was plasticity and prevention of hippocampal neuron loss in MK801

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 Impairtest the role of Ser133 CREB in synaptic plasticity and neurolower 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 group 1 . 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 group 1 . Overall, this demonstrates that phosphorylated CREB at S133 is essential in generating the positive treatment outcomes, such as increased

ments Through Ser133 Phosphorylation of CREB Upregulating -induced SZ rats.

CREB SER133 PHOSPHORYLATION M EDIATES THE BENEFITS OF Ω-3PUFAS

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 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. Citation: Guo et al., “ω-3PUFAs Improve Cognitive Impairments Through Ser133 Phosphorylation of CREB Upregulating BDNF/ TrkB Signal in Schizophrenia.”

Discussion

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 MK801- incduced 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.

ω-3PUFAs have been known to have major health and brain benefits for quite some time. Previous literature had found links pre-disposed to Schizophrenia who take ω-3PUFAs to test the

and interpretations of the beneficial effects ω-3PUFAs have on comes, a number of factors could be involved including levels of diagnosed, age, or simply the fact that humans are not rats.

the CREB/BDNF pathway, however the mechanism still remained unknown up until this point. These new findings behind ω-3PUFAs mechanisms, produced by Guo et al. (2020), are paving the way for ω-3PUFA use as a preventative measure in the complex disorder, Schizophrenia.

Critical Analysis

Firstly Guo, et al. effectively tested their hypothesis surrounding CREB Ser133, by not only testing the positive effects of phosphorylated S133 but also the impact that an unphosphorylated S133 mutation would have on ω-3PUFA treatment. However, 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 only using a certain dose, measurement and model. If the treatment does prove to be effective within a specific dose for a feasible group of patients, then these guidelines can be used to implement human testing and eventually a clinical trial.

Future Directions

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 preventative effects, and measure age of diagnosis, if they ever even become diagnosed.

Through implementing a more specific clinical trial, we would hope to see a same regulation of SZ-induced cognitive symptoms, and increased BDNF levels, when patients are treated with ω-3PUFA compared to a placebo, as observed in MK801 rats. If these future experiments fail to provide similar outdosage, strength of initial symptoms, how long they have been delivered similarly through the diet, such as polyphenols11.

Overall, the successful evidence gained of ω-3PUFAs improving cognitive and neuronal function through phosphorylated CREB S133 in MK801-induced SZ rats, will propel future experiments into Schizophrenia treatment and prevention.

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

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