Disruption of the Interaction between a7 Nicotinic Acetylcholine … · 2019. 7. 28. · (CDC) demonstrate that between the years 2009 and 2012, 7.6% of American had experienced depression

Disruption of the Interaction between a7 Nicotinic Acetylcholine Receptor and Glutamate N-Methyl-D-

Aspartic Acid Receptor Exerts an Anti-depressive Effect

by

Anlong Jiang

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Physiology University of Toronto

© Copyright by Anlong Jiang, 2019

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Disruption of the Interaction between a7 Nicotinic Acetylcholine

Receptor and Glutamate N-Methyl-D-Aspartic Acid Receptor

Exerts an Anti-depressive Effect

Anlong Jiang

Master of Science

Department of Physiology University of Toronto

2019

Abstract

Major depressive disorder (MDD) has become a serious mental health problem within society,

imposing a heavy burden on patients and their families. However, our current anti-depressive

treatments provide limited benefits and are associated with adverse effects. Previous research has

shown direct protein-protein interactions between a7 nicotinic acetylcholine receptor (nAChRs)

and glutamate N-Methyl-D-Aspartic Acid (NMDA) Receptors. Since both receptors are involved

in the pathophysiology of MDD, we hypothesized that disruption of this protein interaction, via a

novel interfering peptide, would demonstrate an anti-depressive effect in animals. Hence, this

receptor interaction may have a crucial role in MDD, and interfering peptide intervention could

present a novel therapeutic for the treatment of patients with MDD.

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Acknowledgments Firstly, I would like to thank my supervisor Dr. Fang Liu who has always steered me in the right

direction whenever I became lost and confused. The completion of this project and thesis would

not be possible without her ongoing support and guidance.

I would like to express my sincere gratitude to my committee member, Dr. Zhengping Jia and

Dr. Shuzo Sugita. My research would not have been successful without their expertise and

helpful insights.

I would also thank my lab mates, Dongxu Zhai, Ping Su, Haiyin Li, Terrence Lai, Frankie Lee

and Charlie Campbell; it has been my great pleasure to work alongside them. I would like to

send my special thanks to Ping Su for her help with my biochemical experiments and Terrence

Lai for his counseling regarding animal surgery and behavioural tests.

Finally, I would like to thank my family and friends. Their unfailing support and encouragement

have made my Master’s a great time in my life.

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Table of Contents Acknowledgments .......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Figures ............................................................................................................................... vii

List of Abbreviations ................................................................................................................... viii

Introduction ................................................................................................................................ 1

1.1 Major depressive disorder ................................................................................................... 1

1.1.1 Epidemiology of MDD ........................................................................................... 1

1.1.2 Diagnosis and impact .............................................................................................. 2

1.1.3 Current treatments of MDD .................................................................................... 2

1.1.4 Animal models of MDD ......................................................................................... 6

1.2 Acetylcholine receptor biology ......................................................................................... 11

1.2.1 Structure of nAChRs ............................................................................................. 11

1.2.2 Diversity and distribution of nAChRs in brain ..................................................... 12

1.2.3 Function of nAChRs ............................................................................................. 15

1.2.4 nAChRs and depression ........................................................................................ 15

1.3 Glutamate receptor biology ............................................................................................... 17

1.3.1 Structure and diversity of NMDA receptors ......................................................... 18

1.3.2 Function of NMDA receptors ............................................................................... 18

1.3.3 Regulation of NMDA receptors ............................................................................ 21

1.3.4 NMDA receptor and depression ........................................................................... 22

1.4 Rationale ........................................................................................................................... 24

1.5 Hypothesis ......................................................................................................................... 26

Materials and methods ............................................................................................................. 27

2.1 Animals ............................................................................................................................. 27

2.2 Drugs ................................................................................................................................. 27

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2.2.1 TAT peptide .......................................................................................................... 27

2.2.2 TAT-a7-peptide .................................................................................................... 27

2.2.3 Imipramine ............................................................................................................ 28

2.3 Drug administration .......................................................................................................... 28

2.3.1 Surgical implantation of cannula .......................................................................... 28

2.3.2 Intracerebroventricular (ICV) injection ................................................................ 28

2.3.3 Intraperitoneal (IP) injection ................................................................................. 29

2.4 Behavioural Tests .............................................................................................................. 29

2.4.1 Body weight measurement .................................................................................... 29

2.4.2 Forced swim test ................................................................................................... 29

2.4.3 Locomotor test ...................................................................................................... 32

2.5 Biochemical analyses ........................................................................................................ 32

2.5.1 Tissue harvesting and sample preparation ............................................................ 32

2.5.2 Gel electrophoresis and Western blotting ............................................................. 32

2.5.3 Co-immunoprecipitation ....................................................................................... 33

2.6 Statistical analyses ............................................................................................................ 33

2.7 Experimental design .......................................................................................................... 34

2.7.1 Anti-depressive effect of TAT-α7-peptide ........................................................... 34

2.7.2 Dose response of TAT-α7-peptide ........................................................................ 34

Results ...................................................................................................................................... 35

3.1 TAT-α7-peptide showed an anti-depressive effect in FST ............................................... 35

3.2 High dosage of TAT-α7-peptide may alter the locomotor activity .................................. 35

3.3 Single injection of TAT-α7-peptide can still exert an anti-depressive effect ................... 38

3.4 Single injection of TAT-α7-peptide does not alter locomotor activity ............................. 38

3.5 Dose response of the TAT-α7-peptide anti-depressive effect .......................................... 38

3.6 Disruption of α7nAChR-NR2A interaction in different brain regions ............................. 45

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3.6.1 Disruption of α7nAChR-NR2A interaction in hippocampus by TAT-α7-peptide ................................................................................................................... 45

3.6.2 TAT-α7-peptide does not change α7nAChR-NR2A interaction in PFC .............. 45

3.6.3 TAT-α7-peptide does not change α7nAChR-NR2A interaction in PFC .............. 45

3.7 Expressions of α7nAChR and NR2A in different brain regions ...................................... 49

3.7.1 Expression of α7nAChR and NR2A were not changed in rat hippocampus ........ 49

3.7.2 Expression of α7nAChR and NR2A were not changed in rat PFC ...................... 49

3.7.3 Expression of α7nAChR and NR2A were not changed in rat striatum ................ 49

Discussion ................................................................................................................................ 53

4.1 Overall findings ................................................................................................................ 53

4.2 Anti-depressive effect of TAT-α7-peptide ....................................................................... 53

4.3 ICV delivery of the peptide ............................................................................................... 54

4.4 Animal model of MDD ..................................................................................................... 54

4.5 Possible mechanism of action ........................................................................................... 55

4.5.1 Anti-depressive effect ........................................................................................... 55

4.5.2 Elevation of locomotor activity ............................................................................ 55

4.6 Expression of receptors and interruption of receptor interaction in different brain regions ............................................................................................................................... 56

4.6.1 Disruption of α7nAChR-NR2A interaction was only observed in the hippocampus ......................................................................................................... 56

4.6.2 Receptor expression in different brain regions ..................................................... 57

4.7 TAT-α7-peptide as a peptide drug .................................................................................... 57

4.7.1 Benefits of TAT-α7-peptide .................................................................................. 57

4.7.2 Limitation of the interfering peptide ..................................................................... 57

4.8 Future directions ............................................................................................................... 58

Conclusion ................................................................................................................................ 60

References ..................................................................................................................................... 61

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List of Figures Figure 1-1. Representative behaviours of animal in FST. .............................................................. 9 Figure 1-2. Schematic representation of homo- and heteropentameric nAChRs and their ligand binding site. ................................................................................................................................... 13 Figure 1-3. Schematic representation of distribution of different nAChRs in the brain. .............. 14 Figure 1-4. Samples of diversified NMDA receptors in the brain. ............................................... 19 Figure 1-5. Distribution of NMDA receptor subunits in the developing mouse brain. ................ 20 Figure 2-1. FST setup and three-dose regimen. ............................................................................ 30 Figure 2-2. FST setup and single-dose regimen. .......................................................................... 31 Figure 3-1. Immobility time after three ICV drug injections. ....................................................... 36 Figure 3-2. Locomotor activity after three ICV drug injections. .................................................. 37 Figure 3-3. Immobility time after single ICV drug injection. ....................................................... 39 Figure 3-4. Weight of the animals when performing FST. ........................................................... 40 Figure 3-5. Locomotor activity after single ICV drug injection. .................................................. 41 Figure 3-6. Immobility time of different dosage of interfering peptide. ...................................... 42 Figure 3-7. Dose response curve of the interfering peptide. ......................................................... 43 Figure 3-8. Weight of the animals when performing FST for dose responsiveness. .................... 44 Figure 3-9. TAT-α7-peptide is able to decrease the α7nAChR-NR2A interaction in rat hippocampus. ................................................................................................................................ 46 Figure 3-10. TAT-α7-peptide does not change the level of α7nAChR-NR2A interaction in rat PFC. .............................................................................................................................................. 47 Figure 3-11. TAT-a7-peptide does not change the level of a7nAChR-NR2A interaction in rat striatum. ........................................................................................................................................ 48 Figure 3-12. TAT-α7-peptide does not alter the expression of α7nAChR and NR2A in the rat hippocampus. ................................................................................................................................ 50 Figure 3-13. TAT-α7-peptide does not alter the expression of α7nAChR and NR2A in the rat PFC. .............................................................................................................................................. 51 Figure 3-14. TAT- α7-peptide does not alter the expression of α7nAChR and NR2A in the rat striatum. ........................................................................................................................................ 52

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List of Abbreviations 5-HT Serotonin 5-HTT Serotonin transporter ACh Acetylcholine AChE Acetylcholine esterase AChR Acetylcholine receptor ADHD Attention deficit hyperactivity disorder AMPA Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid BBB Blood-brain barrier BCA Bicinchoninic acid BDNF Brain derived neurotropic factor CaM Calmodulin CaMKII Ca2+/calmodulin-dependent protein kinase II cAMP Cyclic adenosine monophosphate CBP Cognitive-behavioral therapy CDC Centres for Disease Control and Prevention Cdk5 Cyclin-dependent kinase-5 CKII Casein kinase II CNS Central nervous system CPP Cell-penetrating peptide CSF Cerebrospinal fluid C-terminal Carboxy-terminal DA Dopamine DSM-5 Diagnostic and Statistical Manual of Mental Disorders, fifth edition ECT Electroconvulsive treatment eEF-2K eukaryotic elongation factor 2 kinase EPSC Excitatory post-synaptic current ERK Extracellular-signal-regulated kinase FST Forced swim test GABA Gamma-aminobutyric acid GluR Glutamate receptor GPCR G-protein coupled receptor HIV Human immunodeficiency virus HPA Hypothalamus-pituitary-adrenal HRP Horseradish peroxidase ICV Intracerebroventricular IgG Immunoglobulin G IN Intranasal IPT Interpersonal therapy IV Intravenous kD kiloDalton LH Learned helplessness LTP Long-term potentiation mAChR Muscarinic acetylcholine receptor MAO Monoamine oxidase MAOI Monoamine oxidase inhibitor MAPK Mitogen activated protein kinase

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MDD Major depressive disorder MRS Magnetic resonance spectroscopy mTOR Mechanistic target of rapamycin nAChR Nicotinic acetylcholine receptor NE Norepinephrine NMDA N-methyl-D-aspartate PBS Phosphate buffered saline PFC Prefrontal cortex PKA Protein kinase A PKB Protein kinase B PKC Protein kinase C PST Problem-solving therapies POD Pressurized olfactory device SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SE Side effect SEM Standard error of the mean SNRI Selective norepinephrine reuptake inhibitors SSRI Serotonin selective reuptake inhibitor STAR*D Sequenced treatment alternatives to relieve depression clinical trial TCA Tricyclic anti-depressant TM Transmembrane TST Tail suspension test

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Introduction

1.1 Major depressive disorder Major depressive disorder (MDD) is a highly prevalent mood disorder which has become a

leading cause of morbidity and mortality, however, the exact etiology of MDD is still poorly

understood. Many different hypotheses including the serotonin hypothesis, dopamine hypothesis,

cholinergic hypothesis and glutamatergic hypothesis have attempted to explain the symptoms

and the underlying pathophysiology of the disorder, but no consensus has been reached. Though

none of these theories has been able to completely solve the issue, each provides a different

component which adds to our understanding; this includes an individual’s genetic susceptibility,

current health state, medications and environmental factors. Here, I will give a brief review

regarding MDD, the current treatments and both the cholinergic and glutamatergic hypotheses.

1.1.1 Epidemiology of MDD

A recent epidemiological study has revealed that MDD affects approximately 216 million people

worldwide, or 3% of the global population, this is an increase of 17.8% over ten years from 2005

to 2015 (Vos et al. 2016). Unlike other mental illnesses whose age-of-onset is usually during

childhood or adolescence, MDD tends to have a later age-of-onset. The median age-of-onset is

typically in the early to mid 20s and the distribution of MDD age-of-onset is greater than that of

other mental disorders (Kessler and Bromet 2013).

In the United States alone, data published by the Centers for Disease Control and Prevention

(CDC) demonstrate that between the years 2009 and 2012, 7.6% of American had experienced

depression and among these individuals, approximately 3% had developed severe depressive

symptoms. In Canada around 4.9% of adults are suffering from MDD annually and 11.3% of the

total population experience depressive episodes (Lam et al. 2016).

MDD is associated with various risk factors including age, gender and socioeconomic status.

MDD tends to have lower prevalence in both the elderly and married populations while those

who are separated or divorced have significantly higher rates (Weissman et al. 1996; Andrade et

al. 2003). In addition, gender difference also impact the occurrence of MDD, and women are

twice as likely to develop the disorder than men (Van de Velde et al. 2010). Nevertheless, most

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of these epidemiological studies have been done in Western countries, and therefore require

continued investigation and more comprehensive data obtained from low- to middle- income

countries.

1.1.2 Diagnosis and impact

According to Diagnostic and Statistical Manual of Mental Disorders DSM-5 (American

Psychiatric Association 2013), MDD is characterized by at least one discrete depressive

symptom that lasts for a minimum of two weeks. The primary symptoms include depressed

mood, loss of interest or pleasure, severe weight loss or weight gain, insomnia or hypersomnia,

fatigue, inability to think or focus, recurring thoughts of death and even suicide attempts.

These symptoms seriously compromise a patient’s quality of life, and also impose a heavy

economic burden on one’s family and society. A sum of more than 210 billion US dollars is

associated with the overall expense for treating MDD; and this plus the other associated costs of

suicide and workplace related payments has inflated the total treatment expense to 21.5% more

than that in 2005 (Greenberg et al. 2015).

In addition to the economic expenses, MDD can also impair other social activities of patients.

Early onset of depressive episodes can result in an early termination of education, especially in

developed country (Breslau et al. 2008; Lee et al. 2009; Breslau et al. 2011; Porche et al. 2011).

The disease is also related to unemployment and work disability which may be associated with

job loss (Dooley and Fielding 1996; Kawakami et al. 2012).

Furthermore, MDD is also associated with a higher death rate which is accounted for by two

factors: the first being suicide, and the second is the high comorbidity of MDD with other

disorders such as cardiovascular disease, stroke, chronic pain and diabetes mellitus (Elderon and

Whooley 2013; Fiore et al. 2014; Robinson and Jorge 2016; Sheng et al. 2017).

1.1.3 Current treatments of MDD

1.1.3.1 Current guideline of treatments

Based on the patient’s detailed clinical assessments - including evaluation of suicidality,

bipolarity, comorbidity, concomitant medications, and symptoms - a first-line anti-depressant is

chosen and prescribed. If the first-line treatment exerts an early improvement after two to four

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weeks, the treatment continues for another six to eight weeks (Kennedy et al. 2016). Afterwards,

according to the remission of symptoms and risk factors for recurrence, the physician may decide

to maintain the treatment for a half year, 2 years or even longer. If the initial treatment shows

little or no improvement, it is recommended that the dosage be increased for another two to four

weeks. However, after eight weeks, if the non-improvers continue to show tolerance and low

therapeutic effect, switching to another anti-depressant is advised (Kennedy et al. 2016). If the

patients do not respond to any medications, then electroconvulsive treatment (ECT) might be

considered.

1.1.3.2 Medications

Currently, there are many pharmaceutical agents on the markets and being prescribed to the

MDD patients. Nevertheless, they are all sharing the same target, the monoamine system in the

brain, since reduced level of monoamines such as serotonin (5-HT), dopamine (DA) and

norepinephrine (NE) were observed in MDD patients (Eternity 2008; Belujon and Grace 2017).

Therefore, the MDD is linked to a hypoactive monoamine transmission in the brain and a

number of current anti-depressants are targeting this neural transmission. Here, I will briefly

introduce several past and currently-in-use anti-depressants.

1.1.3.2.1 Selective serotonin reuptake inhibitors (SSRIs)

SSRIs are the most widely used anti-depressant worldwide, and they are currently used as first-

line anti-depressants. The most commonly used SSRIs are fluoxetine, paroxetine, sertraline,

citalopram and escitalopram. SSRIs are usually used as first-line treatment since they have

reduced side effect (SE) profiles and better tolerability than other anti-depressants (Mace and

Taylor 2000; RA et al. 2005). As their name suggests SSRIs can hinder the reuptake of the

serotonin released into the synaptic cleft. They bind the serotonin transporter (5-HTT) at high

affinity and block its ability to reuptake 5-HT (Walker 2013). Thus, 5-HT remains in the synapse

for a prolonged period of time. The increased 5-HT level stimulates 5-HT1A receptors which can

downregulate expression of itself on the serotonergic neurons. The 5-HT1A receptors are

autoreceptors that can inhibit the firing rate of the neurons, and the downregulation of 5-HT1A

receptors leads to increased release of the 5-HT into the synapse (Savitz et al. 2009; Kaufman et

al. 2016). Since downregulation of receptor expression is via protein turnover, transcription and

translation, anti-depressive effects are often observed over the course of multiple weeks.

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SSRIs also have a limited level of inhibition towards NE transporter and therefore, SSRI’s are

often used in conjunction with other selective norepinephrine reuptake inhibitors (SNRIs) to

achieve better therapeutic effects.

1.1.3.2.2 Tricyclic anti-depressants

Tricyclic antidepressants are another type of classical anti-depressant which are named for their

three ringed molecular structure. To date there are a number of TCAs that have been marketed

(amitriptyline, nortriptyline, clomipramine, dothiepin, doxepin, imipramine) for use as

antidepressants. TCAs have a similar mechanism to SSRIs and SNRIs, inhibiting the reuptake of

5-HT and NE which in turn increases the synaptic transmission by 5-HT and NE (Nutt et al.

2007). In addition, some TCAs are also high affinity antagonists for other 5-HT receptors such as

mAChRs and NMDA receptors which may contribute to their drug efficacy and/or SE profile

(Sills and Loo 1989; Richelson and Souder 1994). Adverse effects related to the antagonism of

mAChRs include blurry vision, lowered gastrointestinal motility/constipation, urinary retention,

cognitive and/or memory impairment, and increased body temperature (Santarsieri and Schwartz

2015). Thus, after SSRIs entered the market, TCAs were no longer used as first-line anti-

depressants.

1.1.3.2.3 Monoamine oxidase inhibitors (MAOIs)

There are many MAOIs which have been marketed for decades, including: isocarboxazid,

phenylzine, tranylcypromine and selegiline. Their therapeutic target is the monoamine system

and the mechanism of action is through inhibition of monoamine oxidase A (MAO-A),

monoamine oxidase B (MAO-B) or a combination of both (Youdim et al. 2006). MAOs are

enzymes which are tightly associated to the outer membrane of the mitochondria. Their function

is to break down the excess exogenous monoamines and terminate the activity of the monoamine

neurotransmitters serotonin, dopamine, epinephrine, and norepinephrine by an oxidative

deamination reaction (Owens 1996; Youdim et al. 2006). Administering MAOIs can inhibit the

breakdown of the monoamines in the CNS allowing more 5-HT, NE and dopamine to be packed

in the vesicles. Similar to TCAs, MAOIs are also infrequently used due to their limited safety,

tolerability as well as requirement of dietary restriction. The use of MAOIs is associated with

some life-threatening effects, and overdose can lead to serious toxicity (Nutt et al. 2007).

Furthermore, MDD patients that are receiving MAOIs treatment cannot consume foods or

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beverages with high levels of tyramine (things such as aged cheese, processed meats, pickled or

fermented foods and alcoholic drinks) (Swartz 2004).

1.1.3.3 Psychotherapies

Beside these medications, psychotherapy, also known as "talk therapy", is another approach used

to help patients with MDD. Cognitive-behavioral therapy is the most commonly seen

psychotherapy in practice. CBT is a short-course (usually 12-20 sessions) and well-structured

intervention. During each session, the therapist will gradually identify the distortion of the

patients thoughts and help the client to interpret the conditions and interactions in a more

positive and meaningful way (Craighead and Dunlop 2014). Other forms of psychotherapies are

interpersonal therapy (IPT) and problem-solving therapies (PST) (Markowitz, John and

Weissman, Myrna 2004; Bell and D’Zurilla 2009). Although psychotherapies have beneficial

effects for depressed individuals, they are mainly suited for mild to moderate depression. For

more severe depression, a combination of medications and psychotherapy might be a better

option.

1.1.3.4 Brain stimulation therapy

Brain stimulation therapy is often considered as a last option if patients are not responding to

medication. Electroconvulsive therapy (ECT) is one of the most studied brain stimulation

therapies employed to treat severe, treatment-resistant depression (Lisanby 2008). During ECT,

electrodes are positioned at precise locations on the head and an electric current triggers a seizure

that usually lasts for less than a minute. The entire procedure is carried out under anesthesia and

muscle relaxant. Though the ECT has proven effective on MDD, the mechanism remains poorly

understood. Recent studies have implied that brain function in the frontal and temporal lobes, the

hypothalamus-pituitary-adrenal (HPA) stress axis, and the mesocorticolimbic dopamine system

are involved in ECT success (Haskett 2014). In addition to the therapeutic benefits, ECT also

displays adverse effects such as headache, muscle aches and severe memory impairment. To

address the memory issue, unilateral ECT where the electrode is only placed on the right side of

the head, has been shown to have improved effects on learning and memory function when

compared to the conventional bilateral ECT (Lisanby 2008).

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1.1.3.5 Limitations of current MDD treatments

In terms of the current MDD medications, though all contemporary anti-depressants have

exhibited promising therapeutic effects when compared to placebo (Cipriani et al. 2018),

limitations are still obvious.

The sequenced treatment alternatives to relieve depression (STAR*D) study in 2008 evaluated

current feasible treatments to improve MDD outcomes. There were four treatment levels and the

patients proceeded to the next level if they did not demonstrate remission for 14 weeks. In the

study, only 30% of patients with MDD showed improvements after being treated with the first-

line anti-depressant, citalopram (Gaynes 2008). Even after all four levels, the cumulated

remission rate showed that only 67% of patients responded to the current treatments and that

33% of MDD patients did not demonstrate any sign of remission (Gaynes 2008).

The monoamine-targeting drugs usually have a delayed onset. It takes approximately two to four

weeks until we see the relief of the symptoms. Although this problem is not fully understood,

people think the drugs mediate their effect by affecting transcription and translation processes.

This might indicate that current anti-depressive treatments are not impacting the core pathology

of MDD.

Furthermore, many current anti-depressants are associated with various adverse effects since

they lack specificity. Among the five most frequently used anti-depressants, the most common

side effects include headache and nausea; with adolescents appearing to have higher risk for

these adverse effects (Anderson et al. 2012). Even though SSRI’s are safer to use compared to

other medications, these drugs still show a discontinuation symptoms after withdrawal of the

drug (Wilson and Lader 2015).

Due to these limitations, developing a novel, effective and more rapidly acting medication with a

safer SE profile is necessary.

1.1.4 Animal models of MDD

In the development of novel anti-depressants, animal models of MDD are a vital part of the

research. Animal models aim to copy the phenotype and pathophysiology of the disease,

allowing researchers to test novel approaches while attempting to ameliorate disease symptoms

7

and improve our understanding. However, due to the genetic and environmental complexity of

MDD, establishing an ideal animal model can be challenging.

An ideal animal model of MDD should be considered from the following set of criteria: face

validity, construct validity and predictive validity. Face validity is defined as the ability of an

animal model to demonstrate an analogous phenotype and pathophysiology of the diseased state

found in humans. Construct validity corresponds to the features and etiology of the animal model

and should be directly comparable to human MDD. Predictive validity determines whether the

animal model can share a common treatment to that of human patients (Willner and Mitchell

2002; Anisman and Matheson 2005; Vollmayr et al. 2007; Yan et al. 2010). The following is a

brief review of the typical animal models of MDD, including ones that I have employed in my

own studies.

1.1.4.1 Forced swim test

Forced swimming test (FST) was initially developed by Porsolt et al. (Porsolt et al. 1977) and it

is an acute despair-based test. It has become a commonly used behavioural assay for assessing

anti-depressive effects in rodents.

The test can be carried out using both mouse and rat species, and it is based on the rodents

behaviour in water. There are four basic behaviours in water which are climbing, swimming,

diving and immobile state (see Figure 1-1 for detailed illustrations). Climbing behaviour is

described as the rats actively moving in a vertical direction against the wall of the cylinder in an

effort to escape. Swimming behaviour is any horizontal movement at the surface of the water.

Diving is characterized by rodents attempting to reach the bottom of the cylinder and the

immobile state is where the rats are only making the necessary actions to maintain buoyancy in

the water.

Unlike the mouse FST which is a one-day paradigm, the rat FST follows a two-day protocol. On

day one, rats will go through a training session during which they will be placed into an

inescapable water-filled cylinder for 15 minutes. 24 hours later, the procedure repeats for a five-

minute session and immobility of the animal will be recorded and assessed (Slattery and Cryan

2012). After the initial stage where the animals demonstrate vigorous escape behaviour - such as

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climbing and swimming - their struggling declines and they begin to demonstrate an immobile

condition. The length of the immobile state is used to indicate how depressed the animal is.

Animals used in FST have little similarity between the human depression symptoms and

swimming behaviour of the animals in the test. In addition, animals are not exposed to any stress

which may alter their physiological state prior to the test. Strictly speaking, the FST is not an

animal model of MDD, and FST lacks face and construct validity (Nestler and Hyman 2010).

Moreover, since swimming heavily relies on motor activity, some non-antidepressants are also

able to alter the time of immobility. For instance, sedatives or motor-stimulating substances

could result in a false-positive or false-negative result. Therefore, motor activity tests should

accompany the FST to rule out any false results. Other factors can also influence the results of

the FST such as body weight, housing conditions, and surgical manipulation (Bogdanova et al.

2013). However, despite these complications, the FST is still considered a robust screening test

for anti-depressants.

1.1.4.2 Tail suspension test

Tail suspension test (TST) is another despair-based behavioural test which was originally

proposed by Steru et al. in 1985 (Steru et al. 1985). Similar to FST, the TST is not an animal

model of MDD. The primary rodent species examined with TST is the mouse since its tail can

bear the smaller body weight without imposing any injury. In the behavioural test, the mouse

will be suspended by its tail and two behaviours will be recorded, active escaping action and

immobile state. There are three major difference between FST and TST: firstly, since the TST

does not require water immersion, animals will not be affected by hypothermic states (Steru et al.

1985). Secondly, the objective measure in TST may be more precise, and lastly, the TST is more

sensitive to lower dosage of the anti-depressants (Steru et al. 1985).

The assessment system for TST is identical to that for the FST and due to this similarity, the TST

shares similar criticism to FST. TST also lacks construct and face validity and limits the

utilization of the TST model beyond its ability to screen for antidepressants (Nestler and Hyman

2010; Yan et al. 2010).

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Figure 1-1. Representative behaviours of animal in FST.

There are four characteristic behaviours in the FST. From top left to bottom right: Swimming, Animals perform lateral movement in the water; climbing, animals vigorously move vertically against the wall of the cylinder and try to escape from the top of the cylinder; diving, animals immerse in the water and try to touch and escape from the bottom of the cylinder; immobility, animals only perform necessary movement to keep them floating on the water. First three behaviours are classified as mobile state and the immobility is classified as immobile state.

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1.1.4.3 Social defeat

The social defeat model, also known as resident-intruder test, is based on social conflict and

stress. Different from FST and TST, the social defeat model is a chronic stress model. The model

is induced by placing a mild rodent referred to as a “intruder” into a home cage of a more

aggressive and dominant male rodent referred as “resident” (Hollis and Kabbaj 2014). The

resident will quickly attack the intruder and force it to be subordinate. The intruder will become

submissive, supine posture, emit frequent calls of distress and demonstrate freezing behaviour.

Social defeat procedure exhibits feature of construct, face and predictive validity (Nestler and

Hyman 2010), and therefore it is a better model of MDD than FST and TST.

However, there is a major drawback of the social defeat model. The model is based on the

behaviour of male rodents making it difficult to examine anti-depressive effects in females

(Hollis and Kabbaj 2014). Furthermore, social defeat is initiated in adult rodents and thus,

addressing the developmental factors of childhood depression can be challenging.

1.1.4.4 Learned helplessness

Learned helplessness (LH) is an animal model of depression which was first introduced in 1976

(Overmier and Seligman 1967). Different from FST and TST, LH possesses construct and face

validities, and is thought to recapitulate MDD in a better way.

LH takes longer to establish and validate as a predictive model, and usually follows a five-day

protocol timeline. The LH model involves short-term exposure to unpredictable and

uncontrollable noxious stress, such as electric shock. The animals subsequently develop coping

deficits for aversive but escapable conditions. The anti-depressive effects of drugs can be

assessed by the number of successful escapes following drug administration.

More importantly, the LH model not only works on rodents, but can also be extended to human

subjects. Similar to the animal LH paradigms, human LH may use the same or a different

aversive stimulus in both the pre-exposure and the test phase. For example, the aversive stimulus

could be finger electric shock, noxious noises or high-demand cognitive test (Pryce et al. 2011).

In the subsequent test phase, the subjects who have received the inescapable or unsolvable pre-

exposed stimulus, performed significantly worse compared to the native group which did not

receive any stimulus (Pryce et al. 2011).

11

1.2 Acetylcholine receptor biology The acetylcholine receptors (AChRs) are crucial membrane receptors in the body. They are

expressed not only on neuronal cells but also non-neuronal cells (Dani and Bertrand 2007;

Albuquerque et al. 2009). They are a vital component of the central nervous system (CNS)

responding to the endogenous neurotransmitter, acetylcholine (ACh).

Sensitivity to certain exogenous compounds classifies AChRs into two major categories,

nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs).

The nAChRs respond to nicotine which is the principle psychoactive substance in tobacco, while

mAChRs are activated by muscarine, a toxin found in the mushroom named Amanita muscaria.

The nAChRs (similar to the GABAA, 5-HT3 serotonin receptors) are members of the Cys-loop

ligand-gated ion channel family that are selectively permeable to Na+, K+ and Ca2+ under

physiological conditions (Dani 2015). The mAChRs belong to the G-protein coupled receptors

(GPCRs) superfamily with seven transmembrane helices. They exert their functions through

activation of a second messenger system and downstream enzymes. Since AChRs have been

linked to various disorders such as MDD, schizophrenia, attention deficit hyperactivity disorder

(ADHD), Alzheimer's disease as well as tobacco addiction, AChRs have become a new

therapeutic target in recent drug development (Taly et al. 2009; Bertrand et al. 2015).

1.2.1 Structure of nAChRs

nAChRs are large protein complexes with an average molecular weight of approximately 290

kDa. They are composed of five subunits each which can be identical or homologous; to date, a

total of 17 variable subunits have been discovered. These subunits include ten α subunits named

α1 to α10 (α8 was only found in avian species but not in mammals), four β subunits named β1 to

β4, one γ, one δ, and one ε subunit (Albuquerque et al. 2009; Dani 2015). Of these subunits, nine

α subunits and three β subunits are expressed in the brain. The subunits of nAChRs share

structural similarities, and all subunits have a conserved extracellular N-terminal domain, three

transmembrane helices, a variable loop in the cytoplasm and a fourth transmembrane domain

with a short extracellular C-terminus (Albuquerque et al. 2009).

The nAChRs found in muscle tissue are comprised of an α1 subunit and four non-α subunits

which are β1, γ, δ and ε. While nAChRs that are found in neuronal cells exclusively contain α

12

(α2-α10) and β (β2-β4) subunits. Only α7-α9 subunits are capable of forming homopentameric

receptors while the heteropentameric receptors consist of both α and β subunits. To date, a

number of high-resolution crystallographical and electromicroscopic analyses have provided

detailed information regarding the structure of the nAChRs. The five subunits are assembled

symmetrically to create a central pore (20 Å in diameters) which extends 60 Å across the

synaptic membrane, allowing ions to flow within (Unwin 2005). The ligand binding sites are

hydrophobic pockets that reside at the boundary between each subunit. A previous study on α4β2

heteroreceptors showed that the α4 and adjacent β2 subunits co-form the binding pockets which

contribute to the ACh binding pharmacology, while the binding pockets for homoreceptors are

found between subunits (Karlin 2002; Celie et al. 2005).

1.2.2 Diversity and distribution of nAChRs in brain

The nAChRs are highly heterogenous and complex, this is due to the number of stoichiometric

combinations of their subunits and their variable localizations (Figure 1-2); and subunit

composition can affect both pharmacology and function of the receptors (Gotti et al. 2006; Gotti

et al. 2007). According to their affinity, the nAChRs are organized into two classes, high affinity

receptors and low affinity receptors. The mammalian high affinity receptors are comprised of α4

and β2 subunits while the low affinity receptor consists of α7 subunits.

Each brain region expresses different nAChRs (Figure 1-3) (Gotti et al. 2006; Gotti et al. 2007):

three types, α4β2, α4α5β2 and α7, are expressed in the cortex and four different types are found

in hippocampus which are α4β2, α4α5β2, α3β5 and α7. The heteropentameric receptors are

expressed throughout the brain. Nevertheless, of the homopentameric receptors, only the α7

receptor is widely distributed in the brain.

13

Figure 1-2. Schematic representation of homo- and heteropentameric nAChRs and their

ligand binding site.

The heteropentameric receptors consist of various subunits and the ligand binding sites are formed between α and β subunit. The homopentameric receptor contains only α subunits and the ligand binding sites are formed in the junction between adjacent subunit. The subunits are arranged in a symmetrical way with the ion channel pore in middle. Figure adopted from Nicotinic receptors: Allosteric transitions and therapeutic targets in the nervous system (Taly et al. 2009).

14

Figure 1-3. Schematic representation of distribution of different nAChRs in the brain.

nAChRs are widely expressed in the brain and different brain regions have different nAChR subtypes. Among the homopentameric receptors, the α7 receptor is the widely distributed in the brain and among the heteropentameric receptors, the α4β2 was found in number of brain regions. Figure adopted from Nicotinic receptors: Allosteric transitions and therapeutic targets in the nervous system (Taly et al. 2009).

15

1.2.3 Function of nAChRs

The nAChRs are ligand-gated ion channels which mediate fast neurotransmission. Upon

activation by two molecules of ACh or other agonists, including nicotine, the receptors undergo

conformational reorganization resulting in the opening of the channel pore (Albuquerque et al.

2009). The downstream effects will divide between two mechanisms: (1) the cation influx will

lead to membrane depolarization and (2) activation of other voltage-gated ion channels will

occur.

Since nAChRs are also permeable to Ca2+, especially α7 receptors (Fucile 2004), an influx of

calcium ions either via nAChRs or activated voltage-gated calcium channels can act as a signal

to exert an effect/initiate second messenger systems which triggers downstream signaling

pathways. In addition to Ca2+ entry by channels, some evidence also suggests that the entry of

Ca2+ by activated of α7nAChRs can lead to a release of Ca2+ from intracellular stores (Tsuneki et

al. 2000; Sharma and Vijayaraghavan 2001). The entry of Ca2+ can directly promote the release

of neurotransmitter at presynaptic terminals. For instance, α7nAChRs at excitatory presynaptic

terminals are shown to facilitate the release of glutamate with the presence of cadmium and

tetrodoxin which were used to block the calcium channels and prevent action potentials (Gray et

al. 1996). Besides the instant events following calcium entry, calcium may also modulate

neurons over both short- and long-term periods. Sustained agonism of nAChRs can lead to a

reversible desensitization of the receptors and these changes are thought to be mediated by

protein kinase C (PKC), calmodulin and CaMKII (Giniatullin et al. 2005). In terms of long-term

effect, nAChR downstream signaling is also involved in the synaptic plasticity and memory. The

nAChR agonist and antagonist are able to enhance or impair the cognitive performance in

animals (Ed and Bb 1998). In this process, calcium dependent extracellular-signal-regulated

kinase (ERK)- mitogen activated protein kinase (MAPK) pathways play a critical role (Nestler

2002; Sweatt 2008) and in hippocampal slices, activation of α7nAChRs was demonstrated to

increase the probability of long-term potentiation (LTP) (Ji et al. 2001).

1.2.4 nAChRs and depression

1.2.4.1 Cholinergic hypothesis of depression

In 1972 the cholinergic hypothesis was proposed by Janowsky and colleagues, who suggested

that depressive symptoms are associated with the hyperactivity of cholinergic neurotransmission

16

(Janowsky et al. 1972). Early observation noted that the inhibition of acetylcholine esterase

(AChE) by organophosphate poisoning led to depressive-like symptoms and people using these

substances often had higher occurrences of depression. Follow-up experiments also showed that

after administration of physostigmine, a AChE inhibitor that can penetrate the BBB, an increase

in depressive symptoms and decrease in mania were observed in human subjects (Janowsky et al.

1974). On the other hand, the opposite relationship was also observed afterwards. Stressors, such

as electric shock or forced swimming, which can induce depressive-like behaviours can also lead

to the hypersensitive cholinergic system (Dilsaver et al. 1986).

1.2.4.2 Smoking and depression

The primary psychoactive compound in tobacco and cigarettes is nicotine. As described above,

nicotine is an agonist for the nAChRs, creating a complicated relationship between smoking and

depression. As reported in a prospective study done by Klungsøyr (among 2727 adults with 1190

people re-interviewed) depression rates were twice as common in smokers than non-smokers,

and four times as frequent in heavy smokers (Klungsøyr et al. 2006). Subsequent evidence has

suggested that the smoking behaviour is able to provoke depression, and individuals with MDD

tend to have a more difficult time quitting smoking, compared to people without depression

(Wilhelm et al. 2006; Weinberger et al. 2017). In addition, nicotine withdrawal caused by

smoking cessation has been shown to induce depression, dysphoria, and anxiety, which may last

for 10 weeks (Hughes et al. 1991; Hughes 1992). There are several hypotheses attempting to

explain these observations (Prochaska et al. 2017); some studies suggest that the smoking and

depression may have a causal relationship where smoking induces pathological alternations in

neurotransmission. The self-medication hypothesis postulates that smoking behaviour is an

action of self-medication in depressed individuals to ameliorate their current symptoms.

Moreover, nAChRs are upregulated as a result of smoking, and this increase in nAChR is

maintained for at least two weeks after smoking cessation - this may cause depressive symptoms

after abstinence (Staley 2006). Overall, the association between smoking and depression is

compounded by continued nicotine use and this relationship warrants further investigation.

1.2.4.3 Anti-depressants targeting nAChRs

nAChR antagonists and partial agonists have exhibited anti-depressive effects (Mineur and

Picciotto 2010). Since MDD is hypothetically caused by hyperactivity of the cholinergic system,

17

nAChR blockers (such as mecamylamine) can therefore exhibit anti-depressive effects.

Mecamylamine is a non-selective and non-competitive inhibitor of nAChRs, and it has been

tested for its promising anti-depressive ability in FST and TST (Rabenstein et al. 2006).

Nevertheless, Rabenstein and colleagues found that the anti-depressive effect disappeared in

mice with b2 or a7 subunit knock-out (Rabenstein et al. 2006). Hence, the α7nAChR

homopentameric receptor is thought to be a crucial element in MDD pathology.

Nicotine and other partial agonists also show an anti-depressive effect, and this effect may be

mediated by other mechanisms when compared to nAChR blockers. Many people believe that

nicotine is involved in the desensitization of the nAChRs. Previous studies have shown that

smoking 0.13 (1 to 2 puff) of a cigarettes can lead to 50% saturation of high affinity nAChRs for

3.1 hours, while smoking a whole cigarette causes > 88% a4b2 receptor to be occupied in the

human brain (Brody et al. 2006). More importantly, after nicotine binding, there is a long-term

decline in nAChR activity due to desensitization. As for the partial agonist cytisine, which has

low efficacy at high-affinity nAChRs but full agonistic effects on α7nAChR, anti-depressive

effects have been demonstrated in rodents. However, their mechanism of action is complicated,

and partial agonists can activate nAChRs limiting the agonistic effect of endogenous ACh.

Therefore, if symptoms of MDD are induced by elevated ACh, partial agonists may have anti-

depressive effect if administered to individuals who have low level of ACh, therefore increasing

depression-like symptoms (Mineur and Picciotto 2010). However, the exact role of cholinergic

neurotransmission in MDD is still controversial. Studies from Andreason et al. showed that only

nAChR antagonists, not agonists, have anti-depressive effects in mouse FST and TST

(Andreasen et al. 2009), which is inconsistent with the anti-depressive effects of nAChR

agonists. Therefore, the involvement of nAChR in MDD still needs further refinement.

1.3 Glutamate receptor biology Glutamate is a primary excitatory neurotransmitter in the CNS which binds glutamate receptors

(Meldrum 2000). There are two major types of glutamate receptors, ionotropic glutamate

receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). The iGluRs are ligand-gated

ion channels while mGluRs are GPCRs. iGluRs fall into three classes according to their distinct

pharmacology; these are α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type,

N-methyl-D-aspartate (NMDA) type and kainate receptors (Traynelis et al. 2010).

18

1.3.1 Structure and diversity of NMDA receptors

NMDA receptors are vast tetrameric protein complexes with variable composition. There are

seven subunits which have been discovered, with three major subtypes, GluN1, GluN2 (GluN2A,

GluN2B, GluN2C and GluN2D) and GluN3 (GluN3A and GluN3B) (Traynelis et al. 2010). All

subunits share a high level of homology and similar structural organization. Each NMDAR

subunit consists of a large N-terminal extracellular domain, three transmembrane domains, a re-

entry loop that forms the pore-lining region (membrane domain 2) and an intracellular C-

terminal domain.

Functional NMDA receptors require two GluN1 subunits with either two GluN2 subunits or a

combination of GluN2 and GluN3 subunits (Traynelis et al. 2010). However, subunit

compositions varies in different brain regions and developmental stages, especially for GluN2

subunits which are the major determinant of receptor homogeneity (Figure 1-4 and Figure 1-5)

(Paoletti et al. 2013). In the embryonic brain, only GluN2B and GluN2D are expressed. After

birth, GluN3A begins expression and significant shaping of GluR occurs in the first two weeks

postnatally. GluN2A subunits are gradually expressed and become both widely spread and the

most abundant receptor subunits. In opposition, GluN2D expression plummets and is expressed

almost exclusively in diencephalon and mesencephalon at a low level.

The subunit composition can also affect the properties of the channels. Depending on which

subunit the assembly has, their deactivation kinetics are dramatically changed. In terms of GluN1

and GluN2 di-heterotetrameric receptors, the GluN2A-consisting receptor deactivates rapidly

compared with other subtypes, while the GluN2D subunit-containing receptor can conduct a

current for a prolonged period of time (Paoletti et al. 2013). In addition, various subunits might

also influence the gating, permeability and sensitivity of Mg2+ blockade (Paoletti et al. 2013).

1.3.2 Function of NMDA receptors

NMDA receptors are considered to be both ligand-gated and voltage-gated ion channels in

nature. Their endogenous ligands are glutamate and glycine and, as their name suggested, they

can also be activated by NMDA. The receptor is critical for synaptic plasticity and memory

functions.

19

Figure 1-4. Samples of diversified NMDA receptors in the brain.

NMDA receptors are expressed in various subtypes including di-heterotetrameric and tri-heterotetrameric receptors. Each NMDA receptor requires two GluN1 subunits with either two GluN2 subunits or a combination of GluN2 and GluN3 subunits. Here are some examples of NMDA receptor that are expressed in the brain. Figure adopted from NMDA receptor subunit diversity: Impact on receptor properties, synaptic plasticity and disease (Paoletti et al. 2013).

20

Figure 1-5. Distribution of NMDA receptor subunits in the developing mouse brain.

NMDA receptor subunits are expressed differently during development (Postnatal day 0 (P0), postnatal day 14 (P14), and adult) in different brain regions. Figure adopted from NMDA receptor subunit diversity: Impact on receptor properties, synaptic plasticity and disease (Paoletti et al. 2013).

21

At resting stage, the channel cavity is blocked by a Mg2+ ion preventing flow through the

receptor. Hence, ligand binding alone is not sufficient to fully activate the receptor but are also

dependent on membrane depolarization. Sufficient depolarization will expel the Mg2+ ion

allowing a high flux of both Na+ and Ca2+ ions into the cells (Traynelis et al. 2010). Ca2+ ions can

trigger a wide range of signaling cascades. For example, higher concentration of Ca2+ will bind

to calmodulin (CaM) which subsequently activates the Ca2+/ calmodulin-dependent protein

kinase II (CaMKII). Activated CaMKII can phosphorylate AMPA receptors on the post-synaptic

membrane to increase their Na+ conductivity. In addition, CaMKII can also facilitate the

exocytosis of the internalized AMPA receptors to the post-synaptic membrane, which leads to

enhanced strength of the synapse.

1.3.3 Regulation of NMDA receptors

NMDA receptors can be regulated in several different methods. Of these, the most important and

heavily studied is receptor phosphorylation. There are numbers of serine/threonine kinases that

can phosphorylate the subunit of NMDA receptors such as cAMP-dependent protein kinase A

(PKA), protein kinase C (PKC), protein kinase B (PKB), CaMKII, cyclin-dependent kinase-5

(Cdk5), and casein kinase II (CKII) (Chen and Roche 2007). Additionally, tyrosine kinases can

also regulate the function of NMDA receptors. Among these kinases, the second messenger

activated protein kinases have undergone the most intensive research. For example, activation of

PKA by cAMP results in increases the NMDA receptor-mediated excitatory post-synaptic

currents. More interestingly, PKA is also accepted to have the ability to regulate NMDA receptor

Ca2+ permeability, since inhibition of PKA causes a reduced fraction of NMDA receptor-

mediated Ca2+ influx (Skeberdis et al. 2006).

In addition to phosphorylation, NMDA receptor can be regulated through direct protein-protein

interactions. In previous research, NMDA receptors have been shown to have direct protein-

protein interaction with dopamine D1 receptors, a GPCR, and this interaction can be used to

regulate the function of NMDA receptors (Lee et al. 2002). The direct interactions between D1

and NR2A subunit of NMDA receptors leads to decreased NMDA receptor-mediated current.

Other than the D1 receptor, the α7nAChRs can also dimerize with NMDA receptors through the

NR2A subunit (Li et al. 2012). Activation of α7nAChRs by ACh can induce synergistic effect on

NMDA receptor mediated current (Li et al. 2013).

22

1.3.4 NMDA receptor and depression

1.3.4.1 Glutamate level in CNS and peripheral

Glutamate abnormalities have been observed in plasma, cerebrospinal fluid (CSF) and brain

tissue of patients with mood disorders. Accumulating evidence suggests that glutamatergic

transmission is linked to the MDD as previous studies have reported that there is an elevated

level of glutamate in the plasma, and CSF of depressed individuals when compared to that of

healthy subjects (Mitani et al. 2006; Kucukibrahimoglu et al. 2009; Madeira et al. 2018).

Furthermore, other research has demonstrated that anti-depressant treatment can lower the

glutamate level found in the plasma of MDD patients (Maes et al. 1998; Kucukibrahimoglu et al.

2009). Although the exact mechanism of glutamate increase in the plasma and CSF is not well-

understood, these findings imply the involvement of the glutamatergic system in MDD and

suggest that glutamate might be a biomarker which can serve as a valuable diagnostic tool for

treatment. Other than examining glutamate levels in plasma and CSF, post-mortem studies show

that glutamate levels are significantly higher in the PFC of people with MDD and bipolar

disorder (Hashimoto et al. 2007). The same increasing trend was also detected in dorsolateral

PFC in bipolar patients (Lan et al. 2009).

Brain imaging using proton magnetic resonance spectroscopy (1H-MRS) also provides some

evidence that the glutamatergic system is potentially involved in depression. Since it is difficult

to assign an unequivocal resonance peak to glutamate alone at a lower field when within the

NMR spectrum, a combination of glutamate, glutamine and GABA, named Glx is typically

measured. Although the Glx is a combined indicator, it is reflective of the level of glutamate.

Research adopting this method has demonstrated that Glx content was reduced in the PFC

regions of MDD patients who were experiencing a depressive episode (Auer et al. 2000; Hasler

et al. 2007). On the contrary, elevated glutamate levels were observed in the occipital region of

MDD patients who were in active depressive episodes or remission, as well as young people with

increased MDD risk (Sanacora et al. 2004; Bhagwagar et al. 2007; Taylor et al. 2011).

Consistent with this finding, increased glutamate metabolites were reported in the frontal cortex

of patients experiencing late life depression as well as post-stroke depression (Glodzik-Sobanska

et al. 2006; Wang et al. 2012). Parallel studies on g-aminobutyric acid (GABA) indicated a drop

of GABA content in the PFC, which may suggest that the dysregulation and imbalance of the

excitatory and inhibitory neurotransmission contribute to a diseased state (Hasler et al. 2007).

23

1.3.4.2 Altered NMDA receptor subunit expressions

Other than the change of glutamate level, some post-mortem approaches for examining MDD

patients or suicidal victims have also addressed the alternate expression of glutamate receptor

subunits and change of receptor function (Murrough et al. 2017).

Several NMDA receptor subunits have been reported altered in MDD either in protein

expression or at the transcription level in various brain regions. NR2A expression was found to

be raised in the lateral amygdala, and increased NR2C subunits were found in the locus

coeruleus (Karolewicz et al. 2005; Karolewicz et al. 2009). Consistent with this result is a study

from suicidal victims which revealed that genes related to NR2B and NR2C were highly

expressed (Chandley et al. 2014). However, in the PFC, decreased expression of NR2A and

NR2B subunits was discovered (Feyissa et al. 2009). Gene expression assay also indicated that

there is an upregulation of NMDA receptors and glutamate related genes in the dorsolateral PFC

in female MDD patients (Gray et al. 2015). Since MDD is a very heterogenous disorder, samples

human subjects have large variations caused by different environmental, social and physiological

states, and the data do display some discrepancies.

1.3.4.3 Ketamine: A recent discovered anti-depressant

Ketamine, a street drug usually referred to as "K", was developed in 1962 as a general anesthetic

for initiating and maintaining anesthesia. Ketamine is a potent non-selective NMDA allosteric

inhibitor and has therefore been recognized as an anti-depressant since 2000. A randomized

double-blinded and placebo-controlled trial involving seven patients demonstrated that IV

injection of low dose, 0.5 mg/ kg, ketamine can significantly improve depressive symptoms for

72 hours (Berman et al. 2000). A single dose of ketamine has recently been shown to have fast

and persistent anti-depressive effects in both human and animal models of depression. In terms

of chemistry, ketamine has a single stereocenter featuring R or S configuration. Although in a

clinical setting racemic mixtures are often used and two isomers share similar pharmacokinetics

(Nadjat-Haiem 2010), recent studies have shown that the R isomers might have a stronger and

long-lasting therapeutic effect with fewer adverse properties when compared to S isomer as well

as the racemic mixture (Zhang et al. 2014; Yang et al. 2015; Zanos et al. 2016).

24

Once the drug is parentally administered, due to its high lipophilicity, it quickly distributed and

readily gets through the BBB. The binding site of the ketamine resides inside the channel cavity

and it blocks the flux of ions similar to that produced by Mg2+ occlusion. There are few

hypotheses trying to explain the mechanism of action of ketamine (Zanos and Gould 2018).

Since previous studies have shown an imbalance between excitatory and inhibitory

neurotransmission, some people believe ketamine can selectively block NMDA receptor on

GABAergic neurons, resulting in disinhibition of pyramidal neurons and an increase in

glutamatergic transmission. Glutamate will then bind AMPA receptors and increase the release

of brain-derived neurotrophic factor (BDNF) which exerts an anti-depressive effect by

promoting protein synthesis via mechanistic targeting of the rapamycin (mTOR) pathway.

Moreover, it can block the miniature excitatory post-synaptic current (mEPSC) induced by

spontaneous release of glutamate. Since mEPSC plays an important role in the suppression of

protein synthesis via inhibition of eukaryotic elongation factor 2 kinase (eEF-2K), blocking

mEPSC can boost protein translation, including BDNF. Ketamine is also hypothesized to inhibit

the extra-synaptic NMDA receptors which can be activated by low level ambient glutamate.

Blockade of extra-synaptic NMDA receptors may enhance mTORC1, which in turn stimulates

protein synthesis in post-synaptic neurons. Several downstream pathways might also engage,

including AMPA receptor trafficking, BDNF release, phosphorylation of eEF-2K and mTOR

pathways, making the entire process more complex (Zanos and Gould 2018). In addition, A

recently published study revealed a new mechanism that the anti-depressive effect of ketamine

might be mediated by blocking bursting in the lateral habenula (Yang et al. 2018).

Compared to many approved anti-depressants which usually have delayed therapeutic effects,

ketamine possesses a rapid onset of activity, however, its common use as a street drug limits its

utilization in clinical practices. Recently, the US Food and Drug Administration approved the

esketamine nasal spray for indication of treatment-resistant major depression (2019).

Nonetheless, due to the potential of abuse and adverse effects, the esketamine is available only at

a certified doctor’s office and clinic.

1.4 Rationale In 2010, Pei and colleagues reported that the D1-D2 receptor complex is upregulated in post-

mortem MDD patients and this direct protein-protein interaction may be responsible for the

25

pathology of depression (Pei et al. 2010). They subsequently developed an interfering peptide

that is able to disrupt this receptor interaction, decreasing the concentration of D1-D2 complex.

This peptide has demonstrated anti-depressive effects when delivered to the prefrontal cortex

(PFC).

Follow up on this study, in 2014, Brown et al. showed that the intranasal delivery of this

interfering peptide using a Pressurized Olfactory Device (POD) significantly reduced the

immobility time of animals in FST showing another viable route of administration (Brown and

Liu 2014). In addition, work done by a previous graduate student Boychuk, also demonstrated

that the intravenous injection of this peptide can also elicit its anti-depressive effects (Boychuk

2017). The evidence described above suggests that the novel interfering peptide has anti-

depressant potential as a therapeutic.

Up to date, most of the studies and drug developments regarding MDD concentrate on

monoamine neural transmission, including the dopaminergic system. Nonetheless, the

glutamatergic and cholinergic systems also play a significant role in the pathobiology of MDD

and are gaining recent attention.

In previous studies, our lab has demonstrated that the α7nAChR is able to form a protein

complex with NMDA receptors through protein-protein interactions. Li et al. showed that this

protein-protein interaction is critical in nicotine addiction, since the interfering peptide is able to

block the cue-induced reinstatement of nicotine use (Li et al. 2012). Furthermore, the CNS might

use α7nAChR to regulate the function of NMDA receptors. By conducting electrophysiological

experiments, Li et al. (Li et al. 2013) demonstrated that the activation of α7nAChR is able to

boost NMDA-mediated whole cell current and that the application of the interfering peptide can

intercept this functional enhancement. As the activated α7nAChR can upregulate the function of

NMDA, and MDD pathobiology is directly connected to the hyperactivity of the cholinergic and

glutamatergic systems, these two receptors may have synergistic effects that contribute to the

pathology of MDD.

Since this α7nAChR-NR2A interfering peptide can disrupt the receptor interaction and lower the

activity of NMDA receptor, it might also have anti-depressive effects. Unlike other current MDD

medications, the interfering peptide is designed to specifically disrupt the interaction between

α7nAChR and NMDA receptors, allowing for specific targeting without other side effects.

26

1.5 Hypothesis Based on the previous discovery, we made the following hypotheses:

1. Delivery of the interfering peptide will specifically interrupt the protein-protein interaction

between α7nAChR and NMDA receptors in various brain regions.

2. Since the α7nAChR and NMDA are both involved in the depression abnormality,

disruption of the interaction will illustrate an anti-depressive-like effect and alleviate the

disease phenotypes.

3. The interfering peptide will not alter the locomotor activity which may cause false positive

result in FST screening test.

This is the first time that this interfering peptide is introduced as an anti-depressive agent.

Therefore, its detailed pharmacology is unclear. Although we think even a small dosage is able

to elicit an anti-depressive-like effect, we will set the initial dosage to 4 nmol, the maximum

dosage we can deliver to the animal’s brain.

27

Materials and methods

2.1 Animals Male Sprague-Dawley rats, weighing between 200-225 g, were purchased from Charles River

Laboratory (Wilmington, MA). After arrival, they were allowed to acclimate to the environment

for a week before any further procedures occurred. The animals were housed in a controlled

environment where the temperature was maintained between 20-23 °C with a 12-hour day-night

cycle (7 AM-7 PM). They were housed in pairs initially and then single housed after surgery.

The animal protocol has been approved by the Animal Care Committee at Centre for Addiction

and Mental Health (Toronto, Canada) and all experiments proceeded with corresponding

approved guidelines.

2.2 Drugs

2.2.1 TAT peptide

The TAT-peptide was chemically synthesized and purchased from Gen Script (New Jersey,

USA) with > 95% purity. The sequence of the peptide is YGRKKRRQRRRR. The TAT peptide

was diluted with filtered saline with a final concentration of 10 mM. The stock solution was

aliquoted and stored in -80 °C.

Since peptide and other large molecules need active transport mechanism to cross the cell

membrane, the peptide cannot easily get transported into the cell (Koren et al. 2011). The TAT

peptide falls into a class of peptide called cell-penetrating peptide (CPP) and this peptide is

originally derived from human immunodeficiency virus (HIV). The function of this peptide is

used to overcome the lipophilic cell membrane bilayers and facilitate the internalization of small

or large molecules (Koren et al. 2011; Bechara and Sagan 2013). Fusion of this peptide to

another has been shown to allow the penetration of the conjugated peptide into cells and across

the BBB.

2.2.2 TAT-a7-peptide

The fusion peptide, TAT-alpha7-peptide, was chemically synthesized and purchased from

Biomatik (Cambridge, ON) with > 90% purity. The sequence of the peptide is

YGRKKRRQRRRRLNWCAWFLRM with a TAT peptide sequence in front. The TAT-alpha7-

28

peptide is diluted with filtered saline with a final concentration of 10 mM. The stock solution

was aliquoted and stored in -80 °C.

2.2.3 Imipramine

Imipramine (Sigma-Aldrichs) as a TCA was used as a classic anti-depressant for positive control.

The drug is weighted and diluted using filtered saline on the day of the injection, and a final

diluted concentration of 10 mg/ mL.

2.3 Drug administration

2.3.1 Surgical implantation of cannula

A cannula (HRS Scientific) was implanted into the right lateral ventricle by a stereotaxic

surgery. On the day of surgery, the rat was anaesthetized with 5% (v/v) isoflurane (Baxter

Corporation) in the induction chamber before being placed onto the stereotaxic frame;

anaesthesia was maintained by 2% (v/v) isoflurane. Carprofen (Pfizer) (5 mg/ kg) as an analgesia

was diluted with saline and subcutaneously injected to the incision site.

The following flat skull coordinates were used to locate the right cerebral ventricle: 1.0 mm

posterior to the Bregma, 1.4 mm lateral to the midline and 3.6 mm ventral to the surface of the

skull. The cannula was slowly lowered into place and secured with three stainless steel screws

and dental cement, and a dummy (HRS Scientific) was inserted into the canula to prevent

clogging. After surgical procedures, the rats were given 1 mL of saline subcutaneously to prevent

dehydration and allowed to recover for a week. During the recovery, wet mash and extra water

were supplied to facilitate the recovery process.

2.3.2 Intracerebroventricular (ICV) injection

Two different peptide drugs, TAT and TAT-α7-peptide, or saline were administered via ICV

injection through the implanted cannula. The concentration of each drug was 1 nmol/ µL and

different volumes of the drug were injected under two differing dose regimens. One regimen

consists of three injections: immediately after the pre-test of forced swim test, and five hours and

one hour before the behavioural tests (Figure 2-1). The second regimen was a single injection

one hour prior to the swim tests (Figure 2-2).

29

During the injection, the rat was covered with a towel and the dummy canula was taken out. The

injector was inserted by the guidance of the implanted canula. All peptides were injected by a 10

µL syringe (Hamilton) in two minutes. The maximum volume of injection was 4 µL as a volume

greater than 4 µL might cause brain damage. Once the injection was complete, the injector

stayed in place for another two minutes to ensure the successful delivery of the peptide. Then,

the injector was removed, and the dummy was put back to its original state. After injection, the

animals were closely monitored for abnormal behaviours for four hours with one-hour interval.

2.3.3 Intraperitoneal (IP) injection

Imipramine was also used as a positive control with a dose of 20 mg/ kg and under the same dose

regimen as described above. The drug was administered by intraperitoneal injection into the

abdominal cavity. The animals that received IP injections were well-handled and therefore, no

anaesthesia procedure was required.

2.4 Behavioural Tests

2.4.1 Body weight measurement

Since body weight can be a factor that influences the results of FST, each animal’s body weight

was measured before FST. To measure body weight, an empty cage was used as tare weight, and

an animal was placed into the cage. After the balance reached a steady reading, the body weight

of animal was recorded.

2.4.2 Forced swim test

A complete FST is divided into two parts, pre-test and swim test, and there is a 24-hour time

interval between the two tests. In both tests, an acrylic cylinder 60 cm high by 20 cm in diameter

was filled with water at a temperature of 23-25 °C. The water was filled to the top of the

cylinder, approximately 40 cm, so that the rats were unable to escape from the cylinder, while

their tails were unable to touch the bottom of the cylinder.

In the pre-test, the rats were forced to swim in the cylinder for 15 minutes after which they were

hand-dried by towels and placed under a heat lamp for at least 15 minutes. Afterwards, according

to dose regimen, they received an injection of the drug. 24 hours later, the rats were forced to

30

Figure 2-1. FST setup and three-dose regimen.

The behavioural paradigm is shown on the top of the axis and the dose regimen is shown at the bottom. The FST was divided into two test sessions, pre-test and swim test. According to the groups, the animals received the drug three times between a 24-hour time window, immediately after pre-test, five hours before and one hour before the swim test.

31

Figure 2-2. FST setup and single-dose regimen.

The behavioural paradigm is shown on the top of the axis and the dose regimen is shown at the bottom. The FST was divided into two test sessions, pre-test and swim test. According to the groups, the animals received the drug once between a 24-hour time window, one hour before the swim test.

32

swim for 5 minutes in the swim test. The tests were carried out in the same room as the pre-test,

were video recorded, and immobility time was documented.

2.4.3 Locomotor test

The locomotor test was employed to rule out any false-positive results of the anti-depressive

effect. After the swim test, the rats were allowed to recover for a week, and all animals were kept

in the same treatment groups as FST. Before the locomotor test, the peptides (TAT, TAT-α7-

peptide and saline) were injected ICV following the same dose regimen as the FST. Each animal

was placed in a 20 cm high, 20 cm wide and 30 cm long cage. The locomotor activities were

measured by the interruption of the horizontal beams which transect the bottom of the cage. The

recording lasted for one hour with 5-minute intervals.

2.5 Biochemical analyses

2.5.1 Tissue harvesting and sample preparation

Animals were sacrificed one week after the locomotor test. One hour prior to sacrifice, the rats

received the same amount of either TAT or TAT-α7-peptide, and saline. The rats were

anaesthetized by 5% isoflurane in the chamber and then decapitation was performed. Three

different brain regions including the prefrontal cortex (PFC), hippocampus, and striatum were

quickly harvested, placed on ice and stored in a -80 °C freezer for further biochemical analysis.

All samples were homogenized in RIPA buffer supplemented with a protease inhibitor cocktail.

The protein concentration in each sample was quantified by bicinchoninic acid (BCA) assay.

After the equalization of protein concentrations, samples were boiled 10 minutes in Laemmli

buffer (Bio-rad Laboratory) and subjected to Western blot analysis.

2.5.2 Gel electrophoresis and Western blotting

Total protein samples (50-120 µg) were loaded and separated by a 7.5 % SDS-PAGE gel. The

proteins were then transferred onto a nitrocellulose membrane. The membrane was blocked

using 5% non-fat skim milk in TBST (Tris-buffered saline with Tween-20) at room temperature

for 1 hour.

The blots were incubated overnight at 4 °C with the following primary antibodies: anti-

nAChRα7 (1:1000, Santa Cruz Biotechnology), anti-NR2A (1:1000, Novus Biologicals) and

33

anti-α-Tubulin (1:20000). The next day, the blots were incubated with the species-specific

Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000) for two hours at room

temperature and subsequently illuminated using ECL enhanced reagents (GE Healthcare). The

blot images were collected using Bio-rad ChemiDoc MP imaging system (Bio-rad Laboratory).

2.5.3 Co-immunoprecipitation

The 500 µg of solubilized protein from three brain regions was mixed with protein-A/G

conjugated agarose beads (30 µL, Santa Cruz Biotechnology) and 2 µg of either control IgG

(Millipore) or anti-nAChRα7 antibody (Santa Cruz Biotechnology). The mixture was incubated

overnight at 4 °C. The next day, the beads were washed three times with phosphate-buffered

saline with Tween-20 (PBST) and boiled for 10 minutes in Laemmli buffer.

The samples were separated using SDS-PAGE and then transferred onto a nitrocellulose

membrane. The membrane was blocked using 5% non-fat skim milk in TBST and incubated with

the following antibodies: anti-nAChRα7 (1:1000, Abcam) and anti-NR2A (1:1000, Novus

Biologicals). The following day, the blots were incubated with the species-specific Horseradish

peroxidase (HRP)-conjugated secondary antibodies which specifically bind to the light chain of

the primary antibody (1:5000, Abcam). Incubation lasted for two hours at room temperature and

were subsequently illuminated using ECL enhanced reagents (GE Healthcare). The blot images

were collected using Bio-rad ChemiDoc MP imaging system (Bio-rad Laboratory).

2.6 Statistical analyses The densitometry analysis of the Western blot was performed by ImageLab (Bio-Rad

Laboratory) and videotaped behavioural tests were analyzed manually.

Results are presented as the mean ± standard error of the mean (SEM) and dots on the bar

display each data point. For group comparison, student's t-test was performed to determine the

statistical significance between the two groups. One-way or two-way ANOVA (Graphpad Prism)

followed by post hoc Tukey test for multiple testing was used for multiple group comparisons in

all other experiments.

34

2.7 Experimental design

2.7.1 Anti-depressive effect of TAT-α7-peptide

The anti-depressive effect of the TAT-α7-peptide was assessed by FST. For the first three-dose

regimens the animals were randomly divided into three treatment groups: TAT (ICV, n = 12)

TAT-α7-peptide (ICV, n = 14) and imipramine (IP, n = 7).

For the single injection regimen, the animals were randomly assigned to four treatment groups:

Saline (ICV, n = 6), TAT (ICV, n = 7), TAT-α7-peptide (ICV, n = 7), and imipramine (IP, n =

9).

2.7.2 Dose response of TAT-α7-peptide

In order to investigate the pharmacological and anti-depressive effect of the TAT-α7-peptide,

three different doses and TATs were tested. The animals were randomly divided into four

treatment groups: TAT (ICV, n = 8), TAT-α7-peptide (ICV, 1 nmol, n = 8), TAT-α7-peptide

(ICV, 2 nmol, n = 8), TAT-α7-peptide (ICV, 4 nmol, n = 8).

35

Results

3.1 TAT-α7-peptide showed an anti-depressive effect in FST To test whether the peptide has an anti-depressive effect, we delivered the peptide 3 times with

maximum dose (4 nmol). The experimental setup and dose regimens were shown in the Figure 2-

1. To assess the anti-depressive effect of the peptide, the time of immobility for each treatment

group: TAT (ICV, 4 nmol, n = 12), TAT-α7-peptide (ICV, 4 nmol, n = 14) and imipramine (IP,

20 mg/ kg, n = 7), was counted.

As shown in the Figure 3-1, the positive control imipramine, a TCA, showed an anti-depressive

effect with a significantly lower immobility time in FST when compared to the TAT control (p <

0.05); this effect is through a different mechanism of action than the interfering peptide.

Compared to the TAT control, the TAT-α7-peptide was able to significantly reduce the

immobility time in FST demonstrating a promising anti-depressive effect of the interfering

peptide (p < 0.001). Surprisingly, the average immobility time of TAT-α7-peptide is even lower

than that of imipramine. Although there is no statistical significance between the two groups (p >

0.05), it might indicate the TAT-α7-peptide may have a greater efficacy than traditional TCA.

3.2 High dosage of TAT-α7-peptide may alter the locomotor activity

As discussed previously, the FST can be easily affected by stimulants or sedatives, resulting in a

false-negative or false-positive outcome. To rule out the factor of hyperactivity, a locomotor test

was performed.

The rats that received three doses of TAT-α7-peptide showed an increase amount of locomotor

activity when compared to both the TAT control and the classic anti-depressant, imipramine

(Figure 3-2). There was no statistical significance between each treatment group (p > 0.05) even

including two outliers in the data. However, the increasing trend of the locomotor activity may

still suggest that the high dosage and/or multiple injections may cause hyperactivity in animal as

well as the biased FST results.

36

Figure 3-1. Immobility time after three ICV drug injections.

Experimental animal was placed in a water-filled cylinder for a 5-minute session, 24 hours after the pre-test. The immobility time of animals that received three injections (4 nmol of TAT or TAT-α7-peptide ICV, and 20 mg/kg imipramine IP) in the FST. The drug was injected immediately after pre-test, 5 hours before actual swim test and 1 hour before actual swim test. FST was performed 1 hour after the last injection and the test was video-recorded. Immobility time for each animal was counted manually. Data were presented as mean ± SEM and were analyzed using one-way ANOVA followed by a post-hoc Tukey test (ns – no statistical significance, *p < 0.05, ***p < 0.001).

TAT

TAT-α7-peptid

e

Imipramine

0

100

200

300Im

mob

ility

Tim

e (s) ��

�

ns

37

Figure 3-2. Locomotor activity after three ICV drug injections.

A. Locomotor activity of animals that received three injections (4 nmol of TAT or TAT-α7-peptide ICV, and 20 mg/kg imipramine IP). The drug was injected 24 hours, 5 hours and 1 hour before the locomotor test. The locomotor activity was measured after 1 hour of the last injection. The locomotor activity was measured by total beam breaks in a 1-hour time interval. Data were presented as mean ± SEM and were analyzed using one-way ANOVA followed by a post-hoc Tukey test (ns – no statistical significance).

B. Locomotor activity of animals when two outliers in the TAT-α7-peptide group were removed (Outlier is defined as the data value is more than three standard error of the mean). Data were presented as mean ± SEM and were analyzed using one-way ANOVA followed by a post-hoc Tukey test (ns – no statistical significance).

9 12 9

TAT

TAT-α7-peptide

Imipramine

0

1000

2000

3000

4000

Tota

l Bea

m B

reak

s (1 h

our)

ns

TAT

TAT-α7-peptide

Imipramine

0

500

1000

1500

2000

Tota

l Bea

m B

reak

s (1 h

our)

ns

A B

38

3.3 Single injection of TAT-α7-peptide can still exert an anti-depressive effect

Since high dosage and multiple injections lead to a potential increasing trend in locomotor

activity, we decided to reduce the number of injections to a single dose (the dose regimen is

shown in the Figure 2-2). The initial dosage was set to 4 nmol in 4 µL which is the maximum

volume that can be delivered into rat brain in order to achieve maximum therapeutic effect.

As seen in Figure 3-3, even a single injection of the interfering peptide can still demonstrate an

anti-depressive effect in FST. The immobility time of the rats receiving TAT-α7-peptide was

significantly lower compared to saline or TAT groups.

FST can be affected by other factors, such as body weight when performing the behavioural test.

Therefore, the body weights of the animals were recorded. The body weights did not have any

statistical significance across all four treatment groups (Figure 3-4).

3.4 Single injection of TAT-α7-peptide does not alter locomotor activity

The three injections of the interfering peptide illustrated an increasing trend in locomotor

activity. Nevertheless, single injection of the peptide was also able to eliminate potential

hyperactivity caused by the interfering peptide in rats (Figure 3-5). There is no statistical

significance between animals that were received saline, TAT and TAT-α7-peptide.

3.5 Dose response of the TAT-α7-peptide anti-depressive effect This is the first time that TAT-α7-peptide was examined in relation to depressive disorders, and

its pharmacology still needs investigation. Here, we tested the anti-depressive effect of different

dosages starting from a maximum 4 nmol dose (Figure 3-6). All three dosages demonstrated a

tendency to lower immobility time. However, only the 4 nmol dosage had statistical significance

(p < 0.05) when compared with saline and TAT groups.

To show the relationship between the immobility time and the peptide dosage, a dose response

curve was constructed (Figure 3-7), and the impact of the animals’ weight was excluded (Figure

3-8).

39

Figure 3-3. Immobility time after single ICV drug injection.

Experimental animal was placed in a water-filled cylinder for a 5-minute session, 24 hours after the pre-test. The immobility time of animals that received a single injection (4 nmol of TAT or TAT-α7-peptide ICV, and 20 mg/kg imipramine IP) in the FST. FST was performed 1 hour after the last injection and the test was video-recorded. Immobility time for each animal was counted manually. Data were presented as mean ± SEM and were analyzed using one-way ANOVA followed by a post-hoc Tukey test (ns – no statistical significance, **p < 0.01, ***p < 0.001).

Saline

TAT

TAT-α7-peptid

e

Imipramine

0

100

200

300Im

mob

ility

Tim

e (s)

ns ��

��

40

Figure 3-4. Weight of the animals when performing FST.

Body weight of animal was measured individually before FST. Data were presented as mean ± SEM and were analyzed using one-way ANOVA followed by a post-hoc Tukey test (ns – no statistical significance).

6 7 7 7

Saline

TAT

TAT-α7-peptid

e

Imipramine

0

100

200

300

400

500Bo

dy W

eigh

t (g)

ns

41

Figure 3-5. Locomotor activity after single ICV drug injection.

Locomotor activity of animals that received a single injection (4 nmol of TAT or TAT-α7-peptide ICV, and saline ICV). The drug was injected 1 hour before the locomotor test. The locomotor activity was measured by total beam breaks in a 1-hour time interval. Data were presented as mean ± SEM and were analyzed using one-way ANOVA followed by a post-hoc Tukey test (ns – no statistical significance).

6 7 7

Saline TAT TAT-α7-peptide0

1000

2000

3000

Tota

l Bea

m B

reak

s (1 h

our)

ns

42

Figure 3-6. Immobility time of different dosage of interfering peptide.

Experimental animal was placed in a water-filled cylinder for a 5-minute session, 24 hours after the pre-test. The immobility time of animals that received a single injection (4 nmol, 2 nmol and 1 nmol of TAT or TAT-α7-peptide ICV, and saline ICV) in the FST. FST was performed 1 hour after the last injection and the test was video-recorded. Immobility time was counted manually. Data were presented as mean ± SEM and were analyzed using one-way ANOVA followed by a post-hoc Tukey test (*p < 0.05).

8 8 8 8 8

Saline

TAT

TAT-α7-peptid

e 1 nmol

TAT-α7-peptid

e 2 nmol

TAT-α7-peptid

e 4 nmol0

100

200

300Im

mob

ility

Tim

e (s)

�

�

43

Figure 3-7. Dose response curve of the interfering peptide.

Experimental animal was placed in a water-filled cylinder for a 5-minute session, 24 hours after the pre-test. The immobility time of animals that received a single injection (4 nmol, 2 nmol and 1 nmol of TAT or TAT-α7-peptide ICV, and saline ICV) in the FST. FST was performed 1 hour after the last injection and the test was video-recorded. Immobility time was counted manually. Data were presented as mean ± SEM.

TAT

TAT-α7-peptid

e 1 nmol

TAT-α7-peptid

e 2 nmol

TAT-α7-peptid

e 4 nmol0

50

100

150

200Im

mob

ility

Tim

e (s)

44

Figure 3-8. Weight of the animals when performing FST for dose responsiveness.

Body weight of animal was measured individually before FST. Data were presented as mean ± SEM and were analyzed using one-way ANOVA followed by a post-hoc Tukey test (ns – no statistical significance).

8 8 8 8 8

Saline

TAT

TAT-α7-peptid

e 1 nmol

TAT-α7-peptid

e 2 nmol

TAT-α7-peptid

e 4 nmol0

100

200

300

400

500Bo

dy W

eigh

t (g)

ns

45

3.6 Disruption of α7nAChR-NR2A interaction in different brain regions

Since the peptide is designed to interfere with the direct protein-protein interaction between

α7nAChR and NR2A, a co-immunoprecipitation experiment is necessary to demonstrate the

disruption of the receptor coupling. After finishing the behavioural tests, the animals were

sacrificed. We collected three brain regions, hippocampus, PFC and striatum, that are thought to

be critical in the underlying pathobiology of MDD and examine the effectiveness of the

interfering peptide.

3.6.1 Disruption of α7nAChR-NR2A interaction in hippocampus by TAT-α7-peptide

In hippocampus, the densitometry analysis revealed that the protein-protein interaction was

significantly lower in the animals that received TAT-α7-peptide compared to that of TAT. A

representative immunoblot image of NR2A immunoprecipitated by anti-α7nAChR antibody and

associated densitometric analysis are shown in the Figure 3-9.

3.6.2 TAT-α7-peptide does not change α7nAChR-NR2A interaction in PFC

We also compared the protein interaction in the PFC. Unlike the hippocampus, the α7nAChR-

NR2A interaction was not changed by administering the interfering peptide. A representative

immunoblot image of NR2A immunoprecipitated by anti-α7nAChR antibody and associated

densitometric analysis are shown in the Figure 3-10.

3.6.3 TAT-α7-peptide does not change α7nAChR-NR2A interaction in PFC

We also compared the protein interaction in the PFC. Similar to the PFC, the α7nAChR-NR2A

interaction was not changed by administering the interfering peptide. A representative

immunoblot image of NR2A immunoprecipitated by anti-α7nAChR antibody and associated

densitometric analysis are shown in the Figure 3-11.

46

Figure 3-9. TAT-α7-peptide is able to decrease the α7nAChR-NR2A interaction in rat

hippocampus.

A. Representative blot image of co-immunoprecipitation which shows that TAT-α7-peptide, but not TAT, is able to decrease the α7nAChR-NR2A interaction in rat hippocampus. Tissue extract was used as positive control and IgG isotype was used to rule out the non-specific interaction between the antibody and protein of interest.

B. Densitometric analysis of the level of NR2A co-immunoprecipitated by α7nAChR antibody in hippocampal lysate of rats injected with TAT, or TAT-α7-peptide. The level of co-immunoprecipitated NR2A (NR2A Co-IP) was normalized after being divided by the level of precipitated α7nAChR (α7nAChR IP). Results for each sample are presented as the percentage of TAT group. *p < 0.05 as compared to TAT group, n = 7, Student’s t-test was performed to examine the statistical significance. Data were shown as mean ± SEM.

47

Figure 3-10. TAT-α7-peptide does not change the level of α7nAChR-NR2A interaction in

rat PFC.

A. Representative blot image of co-immunoprecipitation which shows that the levels of α7nAChR-NR2A interaction in rat prefrontal cortex are not different between rats injected with TAT and TAT-α7-peptide. Tissue extract was used as positive control and IgG isotype was used to rule out the non-specific interaction between the antibody and protein of interest.

B. Densitometric analysis of the level of NR2A co-immunoprecipitated by α7nAChR antibody in lysate of rat prefrontal cortex injected with TAT, or TAT-α7-peptide. The level of co-immunoprecipitated NR2A (NR2A Co-IP) was normalized after being divided by the level of precipitated α7nAChR (α7nAChR IP). Results for each sample are presented as the percentage of TAT group. n = 7, Student’s t-test was performed to examine the statistical significance (ns – no statistical significance). Data were shown as mean ± SEM.

48

Figure 3-11. TAT-a7-peptide does not change the level of a7nAChR-NR2A interaction in

rat striatum.

A. Representative blot image of co-immunoprecipitation which shows that the levels of α7nAChR-NR2A interaction in rat prefrontal cortex are not different between rats injected with TAT and TAT-α7-peptide. Tissue extract was used as positive control and IgG isotype was used to rule out the non-specific interaction between the antibody and protein of interest.

B. Densitometric analysis of the level of NR2A co-immunoprecipitated by α7nAChR antibody in striatal lysate of rats injected with TAT, or TAT-α7-peptide. The level of co-immunoprecipitated NR2A (NR2A Co-IP) was normalized after being divided by the level of precipitated α7nAChR (α7nAChR IP). Results for each sample are presented as the percentage of TAT group. n = 4, Student’s t-test was performed to examine the statistical significance (ns – no statistical significance). Data were shown as mean ± SEM.

49

3.7 Expressions of α7nAChR and NR2A in different brain regions

To test whether the anti-depressive effect is mediated by the interruption of the direct protein-

protein interaction between α7nAChR and NR2A, or by changes to protein expression, Western

blot experiments were carried out in three different brain regions.

3.7.1 Expression of α7nAChR and NR2A were not changed in rat hippocampus

Protein expression was tested in the same animals that received the treatment and behavioural

test. The representative western blot image and corresponding densitometric analysis (Figure 3-

12) showed that the expressions of α7nAChR and NR2A was not altered in rat hippocampus.

3.7.2 Expression of α7nAChR and NR2A were not changed in rat PFC


tests. The representative western blot image and corresponding densitometric analysis (Figure 3-

13) showed that the expressions of α7nAChR and NR2A was not altered in rat PFC.

3.7.3 Expression of α7nAChR and NR2A were not changed in rat striatum


tests. The representative western blot image and corresponding densitometric analysis (Figure 3-

14) showed that the expressions of α7nAChR and NR2A was not altered in rat striatum.

50

Figure 3-12. TAT-α7-peptide does not alter the expression of α7nAChR and NR2A in the

rat hippocampus.

A. Representative Western blot image shows no difference in expression levels of α7nAChR and NR2A in lysate of rat prefrontal cortex injected with TAT, or TAT-α7-peptide. α-Tubulin was used as a loading control.

B. Densitometric analysis of the expression levels of α7nAChR in hippocampal lysate of rats injected with TAT, or TAT-α7-peptide. The level of α7nAChR was normalized after being divided by the level of α-Tubulin. Results for each sample are presented as the percentage of the TAT samples. n = 7, Student’s t-test was performed to examine the statistical significance (ns – no statistical significance). Data were shown as mean ± SEM.

C. Densitometric analysis of the expression levels of NR2A in hippocampal lysate of rats injected with TAT, or TAT-α7-peptide. The level of NR2A was normalized after being divided by the level of α-Tubulin. Results for each sample are presented as the percentage of the TAT samples. n = 7, Student’s t-test was performed to examine the statistical significance. Data were shown as mean ± SEM.

51

Figure 3-13. TAT-α7-peptide does not alter the expression of α7nAChR and NR2A in the

rat PFC.

A. Representative Western blot image shows no difference in expression levels of α7nAChR and NR2A in lysate of rat prefrontal cortex injected with TAT, or TAT-α7-peptide. α-Tubulin was used as a loading control.

B. Densitometric analysis of the expression levels of α7nAChR in lysate of rat prefrontal cortex injected with TAT, or TAT-α7-peptide. The level of α7nAChR was normalized after being divided by the level of α-Tubulin. Results for each sample are presented as the percentage of the TAT samples. n = 7, Student’s t-test was performed to examine the statistical significance (ns – no statistical significance). Data were shown as mean ± SEM.

C. Densitometric analysis of the expression levels of NR2A in lysate of rat prefrontal cortex injected with TAT, or TAT-α7-peptide. The level of NR2A was normalized after being divided by the level of α-Tubulin. Results for each sample are presented as the percentage of the TAT samples. n = 7, Student’s t-test was performed to examine the statistical significance. Data were shown as mean ± SEM.

52

Figure 3-14. TAT- α7-peptide does not alter the expression of α7nAChR and NR2A in the

rat striatum.

A. Representative Western blot image shows no difference in expression levels of α7nAChR and NR2A in striatal lysate of rats injected with TAT, or TAT-α7-peptide. α-Tubulin was used as a loading control.

B. Densitometric analysis of the expression levels of α7nAChR in striatal lysate of rats injected with TAT, or TAT-α7-peptide. The level of α7nAChR was normalized after being divided by the level of α-Tubulin. Results for each sample are presented as the percentage of the TAT samples. n = 4, Student’s t-test was performed to examine the statistical significance. Data were shown as mean ± SEM.

C. Densitometric analysis of the expression levels of NR2A in striatal lysate of rats injected with TAT, or TAT-α7-peptide. The level of NR2A was normalized after being divided by the level of α-Tubulin. Results for each sample are presented as the percentage of the TAT samples. n = 4, Student’s t-test was performed to examine the statistical significance. Data was shown as mean ± SEM.

53

Discussion

4.1 Overall findings The main goals of this project were to examine whether the interruption of two critical

membrane receptors, α7nAChR and NMDA receptor, could produce an anti-depressive effect

and further to understand the pharmacology of the peptide drug.

In this study, by conducting FST in rats, we have revealed that the TAT-α7-peptide, a peptide

designed to disrupt direct protein-protein interaction between α7nAChR and NMDA receptor

subunit NR2A, has a promising acute anti-depressive effect even with a single injection. Hence,

the collected data suggests that this protein interaction could be a new therapeutic target, and that

this peptide intervention might also be a novel treatment for MDD.

Interestingly, we have found that multiple injections of high dose of peptide have better

performance than the classical tricyclic anti-depressant, imipramine. Nonetheless, this can also

lead to overdose and overdose of the peptide might cause an enhancement of motor activity in

animals and confound the anti-depressive outcome.

In the previous study, this α7nAChR and NMDA receptor coupling was only observed in the

hippocampus. In my study, I also demonstrated this direct protein interaction also exists in other

brain regions such as PFC and striatum. The Western blot experiments have shown that the

interfering peptide does not alter the receptor expression in the brain, and co-

immunoprecipitation have illustrated that the direct protein-protein interaction between

α7nAChR and NMDA was indeed disrupted in the hippocampus but not in PFC and striatum.

This may suggest the hippocampus is playing a crucial role in the underlying pathology of MDD,

and this protein interaction is particularly important in hippocampus.

4.2 Anti-depressive effect of TAT-α7-peptide The ICV administration of the TAT-α7-peptide showed a robust anti-depressive effect in FST.

Initially, to maximize the effect of the interfering peptide, we decided to use the maximum dose

(4 nmol) that the rat brain can accommodate and repeated the injection three times over a 24-

hour interval. This dose regimen has been shown to have an even better anti-depressive effect

than the classic TCA, imipramine. Nevertheless, repeated injection of high dosage induced a

54

trend of increasing locomotor activity. One of the drawbacks of FST is that it can be easily

influenced by locomotor activity, which makes the conclusion ambiguous. Therefore, we thought

the reason for the enhanced locomotor activity was due to repeated high-dose injection, and to

overcome this issue, the ICV administration was reduced to single injection. Even with the single

injection, the peptide still showed an acute anti-depressive effect.

We have observed that overdose of the peptide may lead to abnormal motor enhancement of the

animals, and the threshold dose of the peptide required for anti-depressive effect still remains

unclear. To closely examine the pharmacology and further understand the anti-depressive

efficacy of the peptide, we tested the anti-depressive effect at different doses. Although all doses

have demonstrated some levels of the anti-depressive ability, only the maximum dose ,4 nmol,

has statistical significance. This may suggest that a certain amount of receptor interactions need

to be disrupted in order to achieve a therapeutic effect. In addition, the drug was injected directly

into the ventricles and was distributed to the entire brain. A variable amount of peptide may be

required to interrupt the interaction at a specific brain region to be effective.

4.3 ICV delivery of the peptide The brain features a ventricular system filled with circulating CSF which serves different

functions including protection of brain from trauma and concussion, supply of nutrients, and

elimination of waste metabolites in the CNS (Di Terlizzi and Platt 2009). The peptide was

administered by ICV injection, a direct way to deliver drug into CNS without crossing the BBB.

More significantly, with the circulation of CSF, the drug can be distributed throughout the brain.

The reason why ICV delivery was chosen is that the ICV injection can ensure the peptide is able

to elicit its maximal effect as there will be no loss to the digestive and circulatory system.

4.4 Animal model of MDD In our experiments, only FST was adopted to investigate the anti-depressive effect of the TAT-

α7-peptide. As discussed before, the FST is only a screening test for anti-depressants which lacks

face and construct validity. In addition, FST is an acute test for anti-depressants. Hence, we do

not know the efficacy of the peptide in the chronic and more realistic MDD models such as

social defeat and LH. Based on the observations of this project and previous studies, we

hypothesize that the administration of TAT-α7-peptide could improve the behaviours of social

55

defeats and reduce the escape failure in LH. However, these issues may require further

experimentation in order to validate.

4.5 Possible mechanism of action

4.5.1 Anti-depressive effect

The anti-depressive effect of the TAT-α7-peptide may be mediated by inhibition of NMDA

receptors. In a previous study, we have found that the interaction between α7nAChR-NR2A can

modulate the function of NMDA receptors. Co-administration of choline with NMDA and

glycine can induce a significantly greater NMDA receptor-mediated whole cell current (Li et al.

2013). In addition, activation of α7nAChR can also facilitate NMDA receptor-dependent LTP as

well as mEPSC (Li et al. 2013). Therefore, by blocking the receptor interaction, activation of

α7nAChR is no longer able to enhance the function of NMDA receptors, which in turn elicits an

anti-depressive effect similar to ketamine. Compared to ketamine, the TAT-α7-peptide has acute

therapeutic effects but, it does not possess the same adverse effects as ketamine.

The downstream molecular signaling pathway and effect of the α7nAChR-NR2A interactions are

extremely complicated. A proteomic study has demonstrated that at least 21 biological pathways

are involved in this receptors crosstalk (Zhang et al. 2016). Therefore, the entire picture of the

underlying mechanism remains unclear, and the mechanism of TAT-α7-peptide still requires

further elucidation.

4.5.2 Elevation of locomotor activity

Locomotor activity is a highly variable test, and hence outliers do exist. In our study, when

animals were administered with a high dose of interfering peptide during a short period of time,

their locomotor activity was observed to be enhanced. Although there is no significant increase

in locomotor activity, whether we included or excluded the outliers, we still need to address this

potential issue of the interfering peptide. As discussed previously, the anti-depressive effect may

be mainly mediated through antagonism of NMDA receptor, and the NMDA receptor

antagonism might also contribute to the observed increased locomotor activity. Previous studies

have shown that administration of NMDA antagonists such as ketamine and MK-801 is able to

increase locomotor activity (Imre et al. 2006; Tran et al. 2016). Therefore, the TAT-α7-peptide

can indeed affect the function of NMDA receptor mediated neurotransmission.

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4.6 Expression of receptors and interruption of receptor interaction in different brain regions

4.6.1 Disruption of α7nAChR-NR2A interaction was only observed in the hippocampus

The co-immunoprecipitation is the most commonly approach to determine direct protein-protein

interactions. We performed the experiments in three critical regions in the brain that are

associated with MDD pathobiology including hippocampus, PFC and striatum (Drevets et al.

2008; Koenigs and Grafman 2009; MacQueen and Frodl 2011).

Although these three brain regions are all involved in the MDD, the peptide can disrupt the

interaction differently. The TAT-α7-peptide can only interrupt the α7nAChR and NR2A

interaction in hippocampus but not in PFC and striatum (Figure 3-9, 3-10 and 3-11). The reason

for this difference may tightly associate with the route of administration. The route of

administration used in our study is ICV injection in which the peptide is directly injected to the

lateral ventricles. In the perspective of the anatomy, the hippocampus resides directly next to the

ventricles. Hence, if high concentration of peptide was delivered to the lateral ventricle, they can

easily penetrate the wall of the ventricles entering hippocampus. Therefore, the most profound

interfering outcome was observed in the hippocampus.

The second closed tissue to the lateral ventricles is the striatum which locates besides the

hippocampus. However, there was not noticeable change of the α7nAChR-NR2A complex. As

we discussed in the introduction, each brain region expresses different nAChRs. For example,

α4β2, α4α5β2 and α7 are expressed in the cortex and four different types are found in

hippocampus which are α4β2, α4α5β2, α3β5 and α7. In striatum, α7nAChR is not a predominant

species and therefore, the α7nAChR-NR2A interaction could be limited in the striatum.

The interfering effect was not seen in the PFC region either. Though the PFC has been

intensively studied for its role in MDD pathology, we did not notice any alternation of

α7nAChR-NR2A complex in PFC. The PFC is the farthest away from the lateral ventricles,

which means the peptide has to diffuse to the PFC crossing layers of brain. The TAT-α7-peptide

consists of 22 amino acid which leads to the large molecular weight of the molecule. Even with

the aid of CPP, TAT fragment, only finite number of peptide molecules can reach the PFC

57

region. Thus, the effect of the peptide would be limited and to address this issue, the peptide may

be injected directly to PFC.

4.6.2 Receptor expression in different brain regions

To confirm the anti-depressive effects are mediated by interruption of the peptide but not by the

alternation of protein expressions, Western blot experiment for each brain tissue was performed.

Although the statistical test was not able to reach significance level, protein expressions were not

consistent, and we did observe a relatively large variation of protein expression especially for

α7nAChR. The α7nAChR expression in some animals were significantly higher (see Figure 3-

12. A - example blot of hippocampus).

The reasons for this observation are complicated. Firstly, different animal may have different

expression and sensitivity to either the environment or the TAT-α7-peptide. Disruption of the

α7nAChR-NR2A protein complex might lead to alternation of receptor signaling, which affect

the trafficking, signaling, degradation and synthesis of the receptors. In addition, prior to drug

administration and behavioural tests, the animals have gone through surgical procedures for

implantation of canula. Therefore, the drugs used in surgery such as anesthetics and analgesia

and even surgery itself may alter the protein expressions(Flood et al. 2002; Umana et al. 2013).

4.7 TAT-α7-peptide as a peptide drug

4.7.1 Benefits of TAT-α7-peptide

The major benefit of TAT-α7-peptide is its specificity. Unlike other small molecule drugs which

usually have off-target effects and lead to unexpected adverse effects, this peptide can selectively

target the protein-protein interaction between α7nAChR and NMDA receptors. This level of

specificity allows the peptide to elicit its therapeutic effect without compromising other signaling

pathways. More importantly, other than currently approved first-line anti-depressants, SSRIs,

which usually takes weeks to have effect, this peptide has a rapid onset of action and robust anti-

depressive effects when the injected one hour prior to the swim test.

4.7.2 Limitation of the interfering peptide

The predominant limitations of the TAT-α7-peptide as a treatment for MDD are due to the

biochemical nature of the peptide. First of all, the major drawback of the interfering peptide is its

58

route of administration. Most current anti-depressants are delivered orally which is the simplest

and most common route of administration. Oral administration can be done routinely with no

pain or any further procedures. Nevertheless, the interfering peptide is a sequence of amino acids

linked by ordinary amide bond. Therefore, the peptide cannot be administered orally otherwise it

could be broken down by the digestive system. Hence, parenteral routes such as intravenous,

subcutaneous and intracranial must be adopted when using peptide drugs. These routes, when

compared with the oral route, are perceived to be both intrusive and painful. For instance, ICV

injection is clinically considered as an invasive route since it requires surgical implantation of a

cannula for multiple administrations. Furthermore, the peptide has a short half-life in the blood-

stream which is mainly caused by enzymatic cleavage. However, this disadvantage of IV route

can be improved by increasing dosage of the peptide or through other chemical modifications.

Moreover, unlike other current anti-depressants which are typically small molecules, the

interfering peptide is comprised of a number of amino acids with an excessively large molecular

weight; and the large molecular weight makes crossing the blood-brain-barrier (BBB) difficult.

Even though the interfering peptide has a TAT sequence in front assisting with the penetration of

the BBB, the amount of peptide that gets delivered into the brain is limited. Therefore, to achieve

maximum therapeutic effect, the peptide may have to be directly injected into the brain. For

example, the route of administration used in the experiments was ICV injection.

Although some of the intrinsic weaknesses of the peptide drug were not observed in the TAT-α7-

peptide, they are still potential threats which need to be taken into consideration. Due to the

physiochemical property of the peptide, they might aggregate or become water insoluble

(Fosgerau and Hoffmann 2015). In the worst case, the administered peptide has immunogenicity

which may activate the immune system and cause unexpected and problematic outcomes.

4.8 Future directions In this project, we have demonstrated that the TAT-α7-peptide can elicit robust anti-depressive

effects in the FST. However, other than the validity of FST, it also introduced some questions for

further investigation. First, demonstrating the distribution of the peptide within the brain.

Although the peptide was administered by ICV injection and can be spread to the entire brain by

the ventricular system, an imaging experiment would be advised to clearly display the

distribution and localization of the peptide. The localization of peptide may give us a hint as to

59

how it exerts its anti-depressive function and more importantly, understand its potential side

effects - which were not uncovered through behavioural testing.

In addition, we have not tested the time-dependency of the peptide. In our experiment, we only

showed that the interfering peptide, dosed one hour prior to the test, can exert an acute anti-

depressive effect, and we do not know how long this effect will last. This is a critical question

when this peptide drug can be applied in a clinical setting. If the peptide cannot perform its anti-

depressive function over a prolonged period of time, multiple administrations of drug may be

necessary, and this can significantly compromise a patients’ quality of life, especially when the

peptide drug is delivered by ICV injection. Moreover, in our experiments, although all three

different dosages showed some level of anti-depressive effect, only the maximum dosage, 4

nmol, significantly reduced immobility time. Thus, there is still room for improvements of

efficacy.

Another important question is by what means can we improve the route of administration of the

peptide. Currently, we have only tested the peptide in ICV injection. Even though this is the most

effective way to deliver the drug to CNS, it is a very invasive method. Therefore, in the future,

other milder routes of administration and their efficiency should be examined, including IV

injection and IN with certain speciality devices.

Finally, the underlying mechanism of the interfering peptide still remains unclear but may be

mediated by α7nAChR and NMDA receptor modification and their downstream signaling

events. This mechanism of action is worth further investigation and might provide a novel

therapeutic target for developing a new generation of anti-depressant drugs.

60

Conclusion Overall, we have confirmed that the interruption of the α7nAChR and NMDA receptor by the

interfering peptide has a potent anti-depressive effect, which could become a novel treatment of

MDD. In addition, this gives us more understanding towards the function of the physical

interaction between two receptors and their involvement in the behavioural context. However,

there are some unsolved questions remains, and translation of the peptide drug to clinical use still

requires more validated animal models. To gain more understanding of the therapeutic effects

and associated side reactions of the peptide, the exact mechanism of action of this interfering

peptide still needs further sophisticated investigations.

61

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Disruption of the Interaction between a7 Nicotinic Acetylcholine … · 2019. 7. 28. · (CDC) demonstrate that between the years 2009 and 2012, 7.6% of American had experienced depression