Comparing the Effects of Α5gabaa Receptor Negative Allosteric Modulators on Inhibitory Currents in Hippocampal Neurons

Comparing the Effects of α5GABAA Receptor Negative Allosteric Modulators on Inhibitory Currents in Hippocampal Neurons

Marc Anthony Manzo

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

Department of Physiology

University of Toronto

Comparing the effects of α5GABAA receptor negative allosteric modulators on inhibitory currents in hippocampal

neurons

Marc Anthony Manzo

Master of Science

Department of Physiology

University of Toronto

2020 Abstract

Overactivity of α5 subunit-containing GABAA (α5GABAA) receptors contributes to cognitive deficits in many neurological disorders. Negative allosteric modulators that

preferentially inhibit α5GABAA receptors (α5-NAMs) have been developed to treat such

deficits. α5-NAMs have been primarily studied using recombinant GABAA receptors expressed in non-neuronal cells. Surprisingly, although the native neuronal environment

influences GABAA receptor pharmacology, no study has directly compared α5-NAM

effects on GABAA receptors expressed in primary neurons. This comparison would aid in the development and selection of more effective compounds for clinical trials. Thus, the current study was undertaken to compare the effects of five α5-NAMs on the function of

GABAA receptors in cultured mouse hippocampal neurons, using whole-cell voltage-

clamp recordings. While α5-NAMs similarly inhibited GABAA receptor-mediated currents; the most efficacious concentrations varied 100-fold. Given that maximal efficacy is similar

among α5-NAMs, factors such as potency, selectivity, and toxicity should be emphasized in the development and selection of α5-NAMs for clinical trials.

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Acknowledgments

Over the last two years, I have grown tremendously as both a person and scientist.

This growth was a consequence of the talented people I worked with and learned from on a daily basis. I want to thank my supervisor Dr. Beverley Orser for her constant encouragement and mentorship. The dedication and passion you have for your work, along with the positive impact you have made in the community, will always inspire me. I would also like to thank the members of my advisory committee, Dr. Bonin and Dr.

Matthews, for their time and support. Thank you for pushing me to think critically about my work and excel as a researcher.

Thank you, Dr. Dian-Shi Wang, for being one of the best teachers I have ever had.

I will cherish the lessons you have taught me, and always remember, “there are no shortcuts in life.” Thank you to Dr. Lilia Kaustov for always going out of your way to support me and for all those delicious desserts you made for the lab. To Dr. Ali A.

Ghavanini and Dr. Woosuk Chung, thank you both for being such great friends and mentors. It was a privilege to learn from experts like yourselves. I want to thank Shahin

Khodaei, Arsène Pinguelo, Winston Li and Leo Liu for being the best lab mates I could have asked for. I learned so much from each of you and I am grateful for the friendships we have made. I would also like to thank the other outstanding lab members I had to the opportunity to work with: Raza Syed, Allison Chown and Sina Kiani. It was a privilege to work with all of you and I thank you for making my time in the lab one that I will treasure forever.

Finally, I want to thank my family. Your unconditional love and support have helped me to step outside of my comfort zone and accomplish my goals.

Thank you for making me a better person.

These two years have been the most memorable and influential years of my life, and I owe that to all the amazing people who have been a part of it.

List of Contributions

Marc Anthony Manzo produced all the data presented in this thesis, except for the results shown in Figure 4.5b. These data were obtained with the help of a previous MSc candidate, Winston Wenhuan Li.

The material presented in chapter 4 was prepared in collaboration with Drs. Dian- Shi Wang, Mariana Popa, John Atack and Beverley Orser, as the work was submitted for publication.

The work of Marc Manzo was supported by an Ontario Graduate Scholarship, a Frederick Banting and Charles Best Canada Graduate Scholarship-master’s awarded from the Canadian Institutes of Health Research and a Kirk Weber Research Award in Anesthesia from the Department of Anesthesia, Sunnybrook Health Sciences Centre.

Table of Contents

Acknowledgments iv

List of Contributions vi

Table of Contents vii

List of Tables x

List of Figures xi

List of Abbreviations xii

Chapter 1: Thesis Overview 1

1.1 Rationale and goal 1

1.2 Specific Aims 4

1.3 Thesis structure 7

Chapter 2: General Introduction 10

2.1 GABA and GABAA receptors 10

2.1.1 GABA: synthesis, release, transport, and metabolism 10

2.1.2 Overview of GABA receptors 12

2.1.3 GABAB receptors 13

2.1.4 GABAA receptors 14

2.1.5 GABAA receptor-mediated inhibition 15

2.1.6 Subunit composition of GABAA receptors 19

2.1.7 Synaptic GABAA receptors 20

2.1.8 Extrasynaptic GABAA receptors 22

2.1.9 GABAA receptor trafficking and phosphorylation 24

2.1.10 GABAA receptor pharmacology and benzodiazepines 26

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2.2 α5GABAA receptors 27

2.2.1 α5GABAA receptor expression 27

2.2.2 α5GABAA receptor properties and function 28

2.3 α5GABAA receptor related disorders 31

2.3.1 Perioperative neurocognitive disorder 31

2.3.2 Traumatic brain injury 32

2.3.3 Schizophrenia 33

2.3.4 Down syndrome 34

2.3.5 Autism spectrum disorder 35

2.3.6 Stroke 36

2.3.7 Alzheimer’s disease 37

2.3.8 Major depressive disorder 38

2.4 α5GABAA receptor negative allosteric modulators (α5-NAMs) 39

2.4.1 Overview of α5-NAM pharmacology 39

2.4.2 RO4938581 41

2.4.3 Basmisanil (RG1662) 42

2.4.4 α5IA 43

2.4.5 MRK-016 44

2.4.6 PWZ-029 45

2.4.7 L-655,708 46

2.4.8 ONO-8590580 47

2.4.9 S44819 47

2.4.10 XLi-093 48

2.5 Summary 49

Chapter 3: General materials and methods 51

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3.1 Study approval 51

3.2 Electrophysiological recordings in cell culture 51

3.2.1 Preparation of primary cell cultures 51

3.2.2 Whole-cell voltage clamp recordings in cell culture 52

3.3 Selection and preparation of α5-NAMs 54

3.4 Data and statistical analyses 57

Chapter 4: Inhibition of tonic but not synaptic current by α5GABAA receptor negative allosteric modulators in hippocampal neurons 58

4.1 Introduction 58

4.2 Methods 60

4.2.1 Primary hippocampal neuronal culture 60

4.2.2 Electrophysiology 60

4.3 Results 60

4.3.1 α5-NAMs inhibit the tonic current 60

4.3.2 All α5-NAMs have similar efficacy at inhibiting the tonic current 64

4.3.3 α5-NAMs have no effect on peak or steady-state current evoked by a saturating concentration of GABA 66

4.3.4 α5-NAMs do not inhibit miniature inhibitory postsynaptic currents 69

4.3.5 α5-NAMs alone and DMSO do not modify GABAA receptor function 72

4.4 Discussion 74

Chapter 5: General Discussion 80

5.1 Summary 80

5.2 Future directions 81

5.3 Conclusions 83

Chapter 6: References 84

List of Tables

Table 4.1 α5-NAMs do not modify peak or steady-state current evoked by a saturating concentration of GABA (1 mM) 68

Table 4.2 α5-NAMs do not modify miniature inhibitory postsynaptic currents 71

List of Figures

Figure 1.1 α5-NAMs reduce the tonic current and improve cognition 6

Figure 3.1 Structures of five α5-NAMs evaluated in the present study 56

Figure 4.1 Tonic current is inhibited by α5-NAMs 62

Figure 4.2 All α5-NAMs similarly inhibit the tonic current at their most efficacious concentrations 65

Figure 4.3 Peak and steady-state current evoked by a saturating concentration of GABA are not modulated by basmisanil 67

Figure 4.4 Basmisanil does not affect miniature inhibitory postsynaptic currents 70

Figure 4.5 α5-NAMs alone and DMSO do not modify GABAA receptor function 73

List of Abbreviations

α5 GABAA α5 subunit-containing γ-aminobutyric acid type A (receptors)

α5 NAM negative allosteric modulator of α5 GABAA receptors

α5 PAM α5 positive allosteric modulator of α5 GABAA receptors

ANOVA Analysis of variance

APV (2R)-amino-5-phosphonovaleric acid

ATP Adenosine triphosphate

CA1 Cornu Ammonis area 1

CA3 Cornu Ammonis area 3 cAMP Cyclic adenosine monophosphate

CNQX 6-Cyano-7-nitroquinoxaline-2,3-dione

CNS Central nervous system

CSDS Chronic social defeat stress

EGTA Ethylene glycol-bis (2-aminoethylether)-N, N, N’, N’-tetra acetic acid

GABA γ-Aminobutyric acid

GABAA γ-Aminobutyric acid type A (receptors)

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GABAB γ-Aminobutyric acid type B (receptors)

GABARAP GABAA receptor-associated protein

GAD Glutamate decarboxylase

GAT γ-Aminobutyric acid transporter

GDP Guanosine diphosphate

GIRK G protein-coupled inwardly-rectifying potassium channels

GPCR G protein-coupled receptor

GTP Guanosine triphosphate

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

IL-1β Interleukin-1 beta

IPSC Inhibitory postsynaptic current

LTP Long-term potentiation mIPSC Miniature inhibitory postsynaptic current mRNA Messenger ribonucleic acid

NMDA N-methyl-d-aspartic acid

PKA Protein kinase A

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PKC Protein kinase C

PND Perioperative neurocognitive disorder

TEA Triethylammonium

TTX Tetrodotoxin

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Chapter 1: Thesis Overview

1.1 Rationale and goal

The γ-aminobutyric acid type A (GABAA) receptor is the major inhibitory receptor system in the mammalian brain. The primary function of GABAA receptors is to reduce neuronal excitability through the influx of anions. The GABAA receptor family contains at least 19 different protein subunits that assemble as heteropentamers (Farrant and Nusser

2005). This collection of subunits gives rise to many different subtypes of GABAA receptors that differ in their kinetics, pharmacological properties, and anatomical distributions. GABAA receptors mediate two distinct forms of inhibition: synaptic and tonic.

Synaptic inhibition is mediated by postsynaptic GABAA receptors that are activated by high transient concentrations of GABA. In contrast, tonic inhibition is generated predominantly by extrasynaptic GABAA receptors that are exposed to ambient levels of

GABA (Farrant and Nusser 2005).

A subtype of GABAA receptors that contain the α5 subunit (α5GABAA) have emerged as a potential therapeutic target to treat cognitive dysfunction (Rudolph and Mohler 2014;

Martin et al. 2009). These receptors are preferentially located in the hippocampus where they regulate learning and memory (Collinson et al. 2002; Sur et al. 1999). α5GABAA receptors are expressed primarily, but not exclusively, in the extrasynaptic regions of neurons where they are major contributors to the tonic current (Caraiscos et al. 2004;

Brunig et al. 2002). However, α5GABAA receptor expression has also been detected in the synaptic regions of neurons, albeit to a lesser extent (Serwanski et al. 2006).

The role α5GABAA receptors have in regulating cognition likely stems from their contribution to the tonic current. Tonic current has been shown to regulate neuronal excitability and hippocampal-dependent processes such as long-term potentiation (Bonin et al. 2007; Martin et al. 2010). Specifically, increased function of α5GABAA receptors has been shown to contribute to cognitive deficits in many neurological disorders including

Down syndrome, traumatic brain injury, and perioperative neurocognitive disorder

(Rudolph and Mohler 2014; Khodaei et al. 2019; Zurek et al. 2014). This rise in α5GABAA receptor function increases tonic inhibition and impairs synaptic plasticity. Furthermore, using genetic or pharmacological approaches to inhibit α5GABAA receptor activity has shown to improve cognitive performance in preclinical models of disease (Martinez-Cue et al. 2013).

This notion of reducing GABAA receptor function to improve cognition has been long established. Bicuculline, a non-selective competitive antagonist of GABAA receptors, has also been shown to improve cognitive performance (Brioni and McGaugh 1988); however, such non-selective GABAA receptor blockade evokes anxiety and has proconvulsive effects, making it unsuitable for clinical use (Dorow et al. 1983; Horowski and Dorrow

2002). Thus, specifically targeting α5GABAA receptors instead of all GABAA receptor subtypes is necessary to treat cognitive dysfunction and avoid unwanted side-effects.

Collectively, these findings motivated studies of compounds that selectively reduce the function of α5GABAA receptors.

A class of drug known as α5GABAA receptor negative allosteric modulators (α5-

NAMs) are currently under development. These drugs bind to GABAA receptors at one or more allosteric sites and reduce GABA-induced currents (Maramai et al. 2019; Sigel and

Ernst 2018). This reduction in GABAA receptor function increases neuronal excitability and improves cognition (Collinson et al. 2002; Atack et al. 2006; Bonin et al. 2007).

Currently, there have been at least eight α5-NAMs developed, including several that have been studied in clinical trials (Maramai et al. 2019). Importantly, despite the success of

α5-NAMs in preclinical studies, none of the compounds have progressed to clinical practice. Therefore, additional research is needed to provide novel insights to guide the development and selection of more effective α5-NAMs for future clinical trials.

To date, studies have investigated several properties of α5-NAMs including their selectivity, potency, and efficacy. Selectivity is the ability of a drug to bind its intended receptor while limiting interactions at off-target sites (Kawasaki and Freire 2011). α5-NAM selectivity can be achieved through preferential binding affinity and/or efficacy for the

α5GABAA receptor. Potency is the concentration of a drug required to elicit a given effect and is often measured as the EC50 (the half maximal effective concentration) (Neubig et al. 2003). A more potent drug will have a lower the EC50. Lastly, efficacy is the capacity of a drug to evoke a certain effect (Neubig et al. 2003). In the case of α5-NAMs, efficacy is the ability to reduce GABA-mediated current.

Most studies that examined and compared the selectivity, potency and efficacy of α5-

NAMs have utilized GABAA receptors recombinantly expressed in heterologous systems

(Sieghart and Savic 2018). Typically, a single subtype of mouse or human GABAA

receptor is transiently transfected into non-neuronal cells such as Xenopus laevis oocytes, human embryonic kidney (HEK) 293 cells or L(tk-) mouse fibroblast cells. The strength of this experimental approach is that it allows a direct comparison of drug effects on receptor subtypes. However, the approach also has drawbacks as non-neuronal cells and neurons differ in terms of several factors that modulate the physiological and pharmacological properties of GABAA receptors. These factors include key intracellular scaffolding proteins such as gephyrin and radixin, and cell-signaling pathways including kinases and phosphatases that regulate receptor phosphorylation (Moss et al. 1992;

Jacob 2019). Also, a single neuron expresses multiple different subtypes of GABAA receptors in contrast to the expression of single subtypes in heterologous model systems.

Thus, the effect of α5-NAMs on native GABAA receptors in neurons may differ from those expressed in recombinant systems.

To date, no study has directly compared the pharmacological properties of different

α5-NAMs on native GABAA receptors in neurons, including the tonic current which is the primary target of α5-NAMs. Thus, the goal of the current study was to compare the effects of several different α5-NAMs on the function of GABAA receptors expressed in cultured mouse hippocampal neurons. Specifically, we examined the ability of the α5-

NAMs to modulate GABA-induced currents, including the tonic current.

1.2 Specific Aims

Aim 1: To characterize and compare the effects of each α5-NAM on the tonic current in

primary hippocampal neurons evoked by a low ambient concentration of GABA.

Aim 2: To compare the effects of α5-NAMs on currents evoked by a prolonged application

of a saturating concentration of GABA.

Aim 3: To compare the effects of each α5-NAM on spontaneous miniature inhibitory

postsynaptic currents.

Figure 1.1. α5-NAMs reduce the tonic current and improve cognition. α5-NAMs target extrasynaptic α5GABAA receptors and decrease their activity. Inhibition of α5GABAA receptors by α5-NAMs reduces the tonic current. The subsequent increase in neuronal excitability enhances long-term potentiation and improves cognition.

1.3 Thesis structure

An overview of the organization of the thesis is provided below. In Chapter 2, I provide an overview of the function, expression, pharmacology, and trafficking of GABAA receptors. Next, I focus on the properties of α5GABAA receptors and describe their subtype-specific expression patterns. Afterwards, I discuss the involvement of α5GABAA receptors in neuroplasticity and cognition. Following this, I discuss the role of α5GABAA receptors in the pathophysiology of several neurological and psychiatric disorders such as Alzheimer’s disease and schizophrenia. Chapter 2 then provides an in-depth review of several α5-NAMs that have been studied in preclinical trials, and a summary of several early clinical studies investigating the therapeutic potential of α5-NAMs.

In Chapter 3, I describe the methodologies used in the study. This information is provided in detail to facilitate the reproducibility of all experiments. The methods used in my studies include the preparation of cultures of hippocampal neurons isolated from fetal mice and whole-cell voltage clamp techniques.

In Chapter 4, I have included the contents of a manuscript, which I prepared as the first co-author entitled, “Inhibition of tonic but not synaptic current by α5GABAA receptor negative allosteric modulators in hippocampal neurons.” This study addresses the three aims outlined in the overview. To address Aim1, I characterize and compare the effects of α5-NAMs on the tonic current. Tonic current in the hippocampus has been shown to be largely mediated by α5GABAA receptors and functions to regulate the excitability of neurons (Caraiscos et al. 2004; Bonin et al. 2007). Here, I compared the inhibitory effect

of each α5-NAM on the tonic current using their most effective concentration. This comparison revealed that all α5-NAMs caused a similar reduction of the tonic current.

Notably, the most efficacious concentrations varied up to 100-fold among the α5-NAMs tested in this study. This is the first report to demonstrate that these five α5-NAMs similarly inhibit the tonic current generated by native GABAA receptors in cultured mouse hippocampal neurons.

Studies utilizing recombinantly expressed GABAA receptors have demonstrated that despite the relative selectivity of α5-NAMs for α5GABAA receptors, these drugs can modulate other subtypes of GABAA receptors at higher concentrations. Therefore, we studied α5-NAM modulation of currents generated by a heterogeneous population of receptors. Specifically, aim 2 of my thesis compares the effects of α5-NAMs on currents evoked by a saturating concentration of GABA. The application of a high concentration of

GABA activates both low-affinity synaptic GABAA receptors and higher-affinity extrasynaptic GABAA receptors. The results of this experiment showed that α5-NAMs do not modulate GABAA receptors activated by prolonged, saturating concentrations of

GABA suggesting a selectivity for current evoked by a low concentration of agonist.

Aim 3 was to compare the effects of each α5-NAM on spontaneous miniature inhibitory postsynaptic currents (mIPSCs). mIPSCs are generated by postsynaptic

GABAA receptors that are transiently activated by the vesicular release of GABA. Here, I recorded miniature inhibitory postsynaptic currents in the absence and presence of each

α5-NAM. The results showed that α5-NAMs did not influence the amplitude, frequency,

or kinetics of mIPSCs. These findings suggest that α5-NAMs do not modulate currents generated by postsynaptic GABAA receptors.

Lastly, I studied whether α5-NAMs, which are designed as allosteric modulators, evoke current on their own. I investigated the effects of each α5-NAM on GABAA receptor function in the absence of GABA. α5-NAMs did not induce current. Next, the effects of the vehicle, DMSO, was also tested on GABAA receptor function. The results showed that the concentration of DMSO used to dilute the study drugs (≤ 0.2% DMSO) did not influence GABAA receptor-mediated current when applied alone.

To conclude, in Chapter 4 I discuss the results considering current literature and highlight the significance of the findings and their relevance for future studies.

Finally, in Chapter 5, I summarize the main findings of my thesis and expand on the discussion presented in Chapter 4. In this section, I provide future directions outlining key experiments and other factors that should be considered to enhance the implications of my findings.

Chapter 2: General Introduction

2.1 GABA and GABAA receptors 2.1.1 GABA: synthesis, release, transport, and metabolism

GABA is the principle inhibitory neurotransmitter of the central nervous system. As such, it plays a crucial role in regulating neuronal activity and synaptic plasticity (Farrant and Nusser 2005). Structurally, GABA is an amino acid that exists as a zwitterion, with no net electrical charge under physiological conditions (pH = 7.4). This is an important feature of the GABA molecule as it allows it to traverse the synapse without interference from electrostatic forces (Mody et al. 1994).

GABA is synthesized from the excitatory neurotransmitter glutamate. This is accomplished through a decarboxylation reaction catalyzed by glutamic acid decarboxylase (GAD). The GAD enzyme has two major isoforms that are encoded by separate genes. Each isoform requires the presence of their activating cofactor, pyridoxal phosphate (PLP) to function. The GAD isoforms differ in their subcellular location and molecular weights of either 65 or 67 kilodaltons (GAD65 and GAD67, respectively)

(Erlander and Tobin 1991; Soghomonian and Martin 1998).

GAD67 is ubiquitously expressed throughout the intracellular environment of neurons

(Gonzales et al. 1991). Distinctly, GAD67 is found mostly in the active form as it is saturated by PLP. These conditions allow GAD67 to produce constant levels of GABA

that are required for cellular metabolic processes including the tricarboxylic acid cycle. In contrast, GAD65 is preferentially located in synaptic terminals where it synthesizes GABA for vesicular release (Erlander and Tobin 1991; Battaglioli et al. 2003; Walls et al. 2010).

To facilitate this process, GAD65 forms a protein complex with the vesicular GABA transporter (VGAT) to produce GABA-containing vesicles (Jin et al. 2003). Most GAD65 exists in its inactive form and is activated by PLP when GABAergic transmission occurs

(Battaglioli et al. 2003).

The vesicular release of GABA is stimulated by the arrival of action potentials that depolarize the axon terminal. This depolarization results in the activation of voltage-gated calcium channels, driving the influx of calcium. This increase in calcium triggers the fusion of vesicles to the plasma membrane and the exocytosis of GABA (Jin et al. 2003).

Typically, the vesicular release of GABA produces synaptic concentrations in the millimolar range (Farrant and Nusser 2005). However, the high concentrations of GABA are rapidly cleared from the cleft through both diffusion and reuptake by neurons and glia

(Barberis et al. 2004; Scimemi 2014). The diffusion of GABA into the extracellular space

(i.e. GABA spillover) is one source of ambient GABA that stimulates extrasynaptic GABAA receptors to produce tonic inhibition (Farrant and Nusser 2005; Glykys and Mody 2007).

An additional source of ambient GABA concentrations are the reverse transport of GABA transporters (GATs) and release from bestrophin1 channels in astrocytes (Heja et al.

2009; Pandit et al. 2015). The concentration of GABA in the extracellular space of the hippocampus was determined using microdialysis techniques and was found to range from 0.2 - 0.8 μM (Lerma et al. 1986; Tossman et al. 1986).

GABA must first be taken up into cells before it is broken down, due to the intracellular localization of its degrading enzyme. As mentioned earlier, the cellular reuptake of GABA occurs via GATs that are expressed in both neurons and astrocytes. In total, there are 4 isoforms of GATs (GAT1-4) that exist, which each function by coupling the transport of

GABA to the inward driving force of Na+ and Cl- ions (Scimemi 2014; Zhou and Danbolt

2013). The GAT isoforms differ in their anatomical distribution, whereby GAT2 and GAT4 are mostly expressed outside the brain in regions such as the liver and kidney (Zhou and

Danbolt 2013). On the other hand, GAT1 and GAT3 are expressed inside the brain, with the former being preferentially expressed in the synaptic regions of neurons and the latter in astrocytic processes (Conti et al. 2004).

In the cellular environment, GABA undergoes enzymatic degradation by GABA transaminase (Rowley et al. 2012). GABA is broken down into succinic semialdehyde, a substrate of the tricyclic acid cycle, which can enter the glutamine cycle to reproduce glutamate and GABA (Bak et al. 2006).

2.1.2 Overview of GABA receptors.

As the major inhibitory neurotransmitter of the mammalian CNS, GABA mediates its inhibitory effects by activating GABA receptors in the postsynaptic membrane (Farrant and Nusser 2005). GABA receptors are classified into two major receptor subclasses:

GABAA and GABAB (Hill and Bowery 1981). GABAA receptors are ionotropic receptors that produce rapid inhibitory currents, whereas GABAB receptors are metabotropic receptors that produce slow, prolonged inhibitory effects in response to GABA (Bowery

and Smart 2006). I will provide a description of both GABA receptor classes, with an expanded overview of GABAA receptors.

2.1.3 GABAB receptors

The metabotropic GABAB receptor exists as a heterodimeric structure comprised of a GABAB1 and GABAB2 subunit (Bowery and Smart 2006). The extracellular N-terminal domain of the GABAB1 subunit is responsible for the binding of GABA, whereas the

GABAB2 is involved in the activation of intracellular G-proteins which are typically of the

Gi/o family (Bettler et al. 2004). These G-protein coupled receptors modulate the activity of other ion channels, as well as enzymes, to produce its inhibitory effect.

Binding of GABA to the GABAB receptor leads to the activation of the associated G- protein through the binding of guanosine triphosphate (GTP). G-proteins are heterotrimeric proteins that belong to a larger family of GTPases. When activated by the

GABAB receptor, G-proteins dissociate into a Gαi/o and Gβi/o subunit (Padgett and

Slesinger 2010). The Gαi/o protein subunit inhibits adenylate cyclase, an enzyme that produces cyclic adenylyl monophosphate (cAMP) from adenosine triphosphate (ATP)

(Federman et al. 1992). Contrarily, Gβi/o alters the activity of ion channels to hyperpolarize cells. Neuronal hyperpolarization via Gβi/o is achieved through the inhibition of voltage- gated calcium channels and the activation of potassium channels that belong to the family of G-protein gated inward rectifying (GIRK) channels (Padgett and Slesinger 2010).

2.1.4 GABAA receptors

GABAA receptors are categorized as part of the cysteine-loop superfamily which is composed of structurally similar ligand-gated ion channels including the glycine receptor, nicotinic acetylcholine receptor (nAchRs), 5-hydroxytryptamine type 3 (5-HT3) receptor, and zinc-activated receptor (Sigel and Steinmann 2012). GABAA receptors are homo- or heteropentameric structures that form an ion channel permeable to Cl- and bicarbonate

- (HCO3 ).

Each GABAA receptor subunit shares a similar structural configuration. Subunits are comprised of a large hydrophilic extracellular N-terminal domain that contains the Cys- loop and is trailed by four transmembrane segments (M1-M4) (Sigel and Steinmann 2012;

Olsen and Sieghart 2009). The transmembrane domain possesses the intracellular loop which is located between the M3 and M4 sequences and can be modulated by phosphorylation. The M2 transmembrane segment forms the channel pore. Following the transmembrane domain is the hydrophilic extracellular C-terminal domain, completing the subunit.

The extracellular N-terminal domain is in the joining of adjacent subunits to form a receptor, as well as creating the ligand-binding sites for GABA and allosteric modulators

(Sigel and Steinmann 2012). Although all subunits share a similar topological organization, they each possess distinct expression patterns throughout the brain, along with unique biophysical and pharmacological properties (Olsen and Sieghart 2009).

2.1.5 GABAA receptor-mediated inhibition

GABAA receptor-mediated current is produced by the movement of ions across the cell membrane, which is dictated by the opening or closing of the channel pore, a process referred to as “gating”. Electrophysiological recordings are frequently employed to measure the function and properties of these receptors.

The direction of current is defined as the movement of positive charge. Therefore, an inward current can be achieved through either the influx of cations or the efflux of anions.

In both cases, the inward current depolarizes the cell. The opposite is true for outward current which hyperpolarizes the cell (Hille 2001). Overall, there are two major factors that determine how much current passes through an ion channel: 1) the channel conductance and 2) the membrane potential. The relationship between current, conductance and membrane potential are summarized by Ohm’s law:

I = gV

where I represents current, g is conductance and V is the membrane potential. Thus, if the membrane potential is held constant, an increase in conductance will produce a larger current. Whether an ion flows into or out of the cell is dependent on its chemical gradient and the membrane potential of the cell. Ultimately, the driving force on a particular ion is described as the difference between the equilibrium (or reversal) potential

(E) of that ion and the membrane potential (Vm) of the cell (Hille 2001). As a result, the equation from Ohm’s law can be adjusted to account for an ion’s driving force:

I = g (Vm - E)

Here, the equilibrium potential (E) is determined by an ion’s concentration gradient across the cell membrane. When the difference between the equilibrium potential of an ion and the membrane potential are zero, the driving force will also be zero, resulting in no net movement of ions across the cell membrane (I = 0) (Hille 2001). Furthermore, the equilibrium potential for a single ion can be calculated using the Nernst equation, which takes into consideration the ion’s intracellular and extracellular concentrations.

RT [x] extracellular E = ln x zF [x] intracellular

Ex represents the equilibrium potential for ion X, R is the thermodynamic gas constant,

T is temperature, z is the valence of the ion (e.g., -1 for Cl and +1 for Na), F is Faraday’s constant, and X denotes the ion of interest. Under circumstances where the valence is a negative value, such as with chloride, then the [x] extracellular and [x] intracellular are interchanged to satisfy the logarithmic function.

However, a neuron is permeable to many different ions, especially Na+, K+ and Cl-.

Therefore, the Goldman equation can be used to calculate a cell’s equilibrium potential with respect to several ions. Using the Goldman equation, a cell’s equilibrium potential is determined by considering each ion’s concentration and relative permeability, denoted as pion:

+ + − 푅푇 푝k[K ]extra + 푝Na[Na ]extra + 푝Cl[Cl ]intra 푉m = ln ( + + − ) 퐹 푝k[K ]intra + 푝Na[Na ]intra + 푝Cl[Cl ]extra

- - Since GABAA receptors are permeable to the anions Cl and HCO3 , the equilibrium potential of GABA is determined by each of these ions (Bormann 1988). However, the

- - permeability of Cl is up to five times greater than that of HCO3 , and thus, will have a much stronger impact on EGABA (Fatima-Shad and Barry 1993). As a result, the cellular

- mechanisms that maintain the Cl gradient are critical in establishing EGABA.

Cation-chloride co-transporters (CCCs) located within the plasma membrane play a major role in regulating intracellular chloride levels (Kaila et al. 2014). In neurons, the two major CCCs are the sodium-potassium chloride co-transporter (NKCC1) which transports chloride into the cell and the potassium-chloride co-transporter (KCC2) which moves chloride out of the cell. The expression levels of each transporter change over the course of development and in certain neurological disorders, which significantly impact GABAA receptor function (Kaila et al. 2014).

In early development, NKCC1 is expressed at much higher levels in comparison to

KCC2 (Kaila et al. 2014). NKCC1 transports 2 Cl- into the cell along with 1 Na+ and 1 K+, resulting in a relatively high intracellular concentration of Cl-. This gradient produces a

- - positive equilibrium potential for Cl , and thus, when GABAA receptors are activated, Cl flows out of the cell resulting in depolarization (Ben-Ari et al. 2012). This excitatory effect of GABA plays an important role throughout development as it stimulates intracellular pathways that produce neurotrophic effects in neurons (Ben-Ari et al. 2012).

In the later stages of neurodevelopment, KCC2 expression rises and NKCC1 expression is reduced (Kaila et al. 2014). KCC2 functions to extrude 1 Cl- from the cell, co-transporting it with 1 K+ (Ben-Ari et al. 2012). By increasing the levels of KCC2 relative

to NKCC1, the intracellular Cl- concentrations are reduced in comparison to the extracellular environment (Kaila et al. 2014). This change in the Cl- gradient results in a more negative equilibrium potential for Cl-, such that when the cells membrane is at a slightly more positive membrane potential, GABAA receptor activation will lead to the

- influx of Cl and cell hyperpolarization (Ben-Ari et al. 2012). However, GABAA receptors may also exert their inhibitory effects through shunting inhibition (Kaila et al. 2014). For example, if EGABA is equal to the resting membrane potential of the cell (typically -65 to -

70 mV in hippocampal neurons), then there will be little to no driving force on Cl-. Instead, the opening of the GABAA receptor channels will increase conductance/decrease membrane resistance and short-circuit any excitatory input the cell receives (Kaila et al.

2014).

- In periods of prolonged or repetitive activation of GABAA receptors, the influx of Cl can accumulate and exceed the rate of Cl- removal by KCC2 (Lambert and Grover 1995).

- - Due to the collapse of the Cl gradient in such cases, the equilibrium potential of HCO3

(EHCO3- = -12 mV) which is much more positive than ECl- becomes the primary determinant of EGABA (Isomura et al. 2003; Staley et al. 1995). Thus, in these cases, receptor activation causes an initial hyperpolarization followed by a depolarizing response (Lambert and

Grover 1995). Additionally, in disease states such as epilepsy and chronic pain,

GABAergic neurotransmission can produce depolarizing responses (Kaila et al. 2014).

Under these conditions, KCC2 function and/or expression is downregulated resulting in a more positive EGABA. Therefore, CCCs have an important role in maintaining normal neurophysiological functioning. For the purposes of my thesis, I will be focusing on

GABAA receptor function in healthy mature neurons where receptor activation produces a hyperpolarizing response.

Now that the general function of GABAA receptors has been reviewed, it is important to understand how the unique subunit composition of GABAA receptor subtypes influences their function and anatomical distribution.

2.1.6 Subunit composition of GABAA receptors

GABAA receptors exist as heteropentamers that are assembled from a repertoire of subunits (α1−6, β1−3, γ1−3, δ, ε, θ, π, and ρ1−3) (Farrant and Nusser 2005). Although this array of subunits could give rise to over 150,000 theoretical subunit combinations, only a small number have been identified to exist in humans (Olsen and Sieghart 2008).

In addition, subunits vary in their regional expression patterns throughout the brain. The

α1, β1, β2, β3, and γ2 subunits are found to be ubiquitously expressed throughout the brain, whereas the α2, α3, α4, α5, α6, and γ1 have specific patterns of distribution (Olsen and Sieghart 2009). The most abundant GABAA receptor isoform contains 2α, 2β and a

γ subunit, which are joined in a counter-clockwise fashion in the following sequence

γ−β−α−β−α (Farrant and Nusser 2005). Other receptor isoforms may include a γ1, γ3 or

δ subunit in place of the γ2 subunit, although they are not as highly expressed. Other uncommon receptor isoforms include the dimeric αβ or homomeric ρ receptor isoforms

(Mortensen and Smart 2006). Whereas αβ receptors are expressed throughout the brain, the ρ receptor subtype, formerly designated as a GABAC receptor, is localized in the retina. The ρ GABA receptor is still considered to be a part of the GABAA receptor family due to sequence and structure homology (Boue-Grabot et al. 1998; Olsen and Sieghart

2008). Collectively, the GABAA receptor population is a heterogenous one, where the subunit composition determines the physiological properties and subcellular distribution or receptors.

2.1.7 Synaptic GABAA receptors

The synaptic GABAA receptors population mediates phasic inhibition. This current is generated by the transient activation of postsynaptic GABAA receptors through the vesicular release of GABA (Farrant and Nusser 2005). Such release can produce millimolar concentrations of GABA in the synaptic cleft that get cleared from the synapse in under a millisecond (Farrant and Nusser 2005). Here, the brief activation of GABAA receptors generates rapid inhibitory postsynaptic currents (IPSCs). The time-course of an IPSC can be influenced by a number of factors. These include the number and organization of GABAA receptors in the postsynaptic membrane, as well as the structure of the synaptic cleft.

Synaptic GABAA receptors are predominantly expressed in the synaptic regions of neurons, although these subtypes can also be detected in extrasynaptic regions to a lesser extent (Farrant and Nusser 2005). GABAA receptors transition through 3 main conformational states that give rise to the IPSC, which are: 1) the closed state, 2) the open state (GABA-bound, channel open), and 3) the desensitized state (GABA-bound, channel closed). The rate and extent of these conformational changes are largely dependent on the receptor’s biophysical properties which are determined by the subunit composition.

Microscopy coupled with immunolabelling techniques have revealed that the majority of synaptic GABAA receptors contain an α1, α2, or α3 subunit (Farrant and Nusser 2005).

The kinetic and pharmacological properties of these receptor subtypes were studied using electrophysiological techniques. With this approach, properties such as potency, desensitization and conductance were determined. These studies have revealed that synaptic GABAA receptors have a relatively low affinity for GABA and rapidly desensitize to prolonged GABA exposures to reduce the overall inhibitory drive on the cell. They also have a higher channel conductance in comparison to extrasynaptic receptors (Bai et al.

2001).

The α subunit of a receptor is critical in determining the receptor’s potency for GABA, given that the GABA binding site is located at the α/β subunit interface. The potency of a receptor is typically indicated by the EC50 value which is the GABA concentration that elicits half of the maximal GABA response for that receptor subtype. Of the conventional synaptic GABAA receptors, the α3 subtype was shown to have the highest EC50 (lowest potency) and the α1 the lowest (highest potency) (Bohme et al. 2004). The EC50 value ranges from approximately 1 to 50 μM for these receptors (Bohme et al. 2004; Karim et al. 2013). However, this relatively low GABA affinity for synaptic GABAA receptors in comparison to extrasynaptic receptors is appropriate considering the saturating GABA concentrations they are exposed to (>1 mM).

Another property of the receptor that is attributed to the α subunit is the channel kinetics. Channel activation, or transition from the closed to open state, is shown to be fastest in α1 subunit-containing GABAA receptors, and slowest in α3 subunit-containing

receptors (Picton and Fisher 2007). Desensitization, which is the transition between the ligand-bound open state to the ligand-bound closed state, also differs among the α subunits. In general, the rate of desensitization is faster in the α1-3 synaptic GABAA receptors compared to the extrasynaptic receptors, which typically contain an α5 or δ subunit (Farrant and Nusser 2005). Lastly, deactivation occurs when GABA dissociates from the receptor, thereby moving the receptor into the closed state. Overall, the fastest to slowest deactivating synaptic GABAA receptor subtypes are the α1-, α2- and α3- subtype, respectively (Picton and Fisher 2007).

2.1.8 Extrasynaptic GABAA receptors

Extrasynaptic GABAA receptors are located outside the synapse where they are exposed to ambient levels of GABA. Such low concentrations of GABA generate a persistent baseline current known as the tonic current (Farrant and Nusser 2005). The amplitude of this current can be quite small, and due to its persistent nature, requires a

GABAA receptor antagonist to reveal (Lee and Maguire 2014). Application of bicuculline, a GABAA receptor competitive antagonist, will result in a shift in the holding current. The magnitude of this shift represents the amplitude of the tonic current (Bright and Smart

2013). Tonic inhibition occurs in several regions of the brain including the cortex, hypothalamus and hippocampus.

As for any GABAA receptor, the subunit composition of extrasynaptic GABAA receptors is a key determinant of their expression and functional properties. Receptors that contain an α4, α5, α6, or δ subunit are the predominant extrasynaptic receptor subtypes that generate the tonic current (Farrant and Nusser 2005). For example, α4δ

GABAA receptors are major contributors to tonic current in the thalamus, whereas α6δ

GABAA receptors drive tonic current in the cerebellum (Glykys and Mody 2007; Chandra et al. 2006). Moreover, tonic inhibition in hippocampal neurons has been shown to be largely mediated by α5 subunit-containing GABAA receptors, although other receptors such as the δ subtype also contribute (Caraiscos et al. 2004; Glykys et al. 2008).

Extrasynaptic receptors are activated by ambient concentrations of GABA in the extracellular space that can range from 0.2 – 0.8 μM, depending on the synaptic conditions (Lerma et al. 1986; Tossman et al. 1986; Bright and Smart 2013). Factors that influence ambient GABA levels include synaptic-mediated release, reverse activity of

GABA transporters, and the astrocytic release of GABA through bestrophin-1 channels

(Glykys and Mody 2007; Pandit et al. 2015). In comparison to synaptic GABAA receptors, the extrasynaptic subtypes have a much higher affinity for GABA, extending from the nanomolar to low micromolar range (Karim et al. 2013). This property enables these receptors to have a high sensitivity for the low, ambient GABA levels in the extracellular space (Karim et al. 2013). Secondly, their slow rate of desensitization enables them to produce long-lasting inhibitory currents as they are continually activated by such low concentrations of GABA (Bianchi and Macdonald 2002). Lastly, their slow rate of deactivation further increases their inhibitory effect on cells (Banks and Pearce 2000).

Collectively, a high affinity and slow rate of desensitization/deactivation make extrasynaptic receptors well-suited to generate the tonic current.

2.1.9 GABAA receptor trafficking and phosphorylation

Synaptic and extrasynaptic GABAA receptors are not restrained within the plasma membrane as they can diffuse laterally within it and also cycle between the cytoplasm and neuronal surface (Vithlani et al. 2011). These trafficking processes determine the number of receptors in the synaptic and extrasynaptic regions of neurons, and thus, the extent of phasic and tonic inhibition, respectively. Both synaptic and extrasynaptic receptors are trafficked to the plasma membrane in a similar manner. GABAA receptors are trafficked to the extrasynaptic regions of neurons where they can either remain or diffuse into synaptic regions (Bogdanov et al. 2006; Thomas et al. 2005).

GABAA receptor trafficking originates in the endoplasmic reticulum, where subunits are assembled into a receptor. Following this, GABAA receptors are transported in vesicles and inserted into the cell’s membrane. This is a complex process that is executed by several accessory proteins. For example, the GABAA receptor associated protein

(GABARAP) is involved in the trafficking of receptors from the endoplasmic reticulum to the neuronal membrane. Increasing GABARAP expression has shown to enhance the surface expression of γ2 subunit-containing receptors (Wang et al. 1999; Leil et al. 2004).

Conversely, GABAA receptors can be removed from the plasma membrane via clathrin- mediated endocytosis. This process is regulated by the clathrin adaptor protein 2 (AP2) which directly interacts with the receptor’s subunits and aids in the assembly of clathrin- coated pits (Kittler et al. 2000).

Two major intracellular scaffolding proteins have been implicated in the clustering of

GABAA receptors at synaptic and extrasynaptic sites. The first is gephyrin, a scaffolding

protein involved in the synaptic clustering of GABAA receptors. Eliminating the expression of gephyrin resulted in drastic reductions in the clustering of synaptic GABAA receptors

(Kneussel et al. 1999). Radixin is the other major intracellular scaffolding protein that has been shown to stabilize GABAA receptors in extrasynaptic regions, especially those containing the α5 subunit (Loebrich et al. 2006). Studies that depleted the expression of radixin found an increased number of α5 subunit-containing GABAA receptors in the synaptic regions of neurons along with an increase in synaptic-mediated inhibition. This is due to the diffusion of receptors from the extrasynaptic to synaptic region (Hausrat et al. 2015).

Lastly, GABAA receptor trafficking and function can also be altered directly by phosphorylation. For example, GABAA receptor phosphorylation by protein kinase A or C

(PKA and PKC, respectively) have shown to modulate the trafficking of receptors to the plasma membrane as well as their channel conductance (Vithlani and Moss 2009). Using recombinantly expressed GABAA receptors in human embryonic kidney (HEK293) cells,

PKA phosphorylation had different effects on channel conductance depending on the β subunit expressed. Specifically, phosphorylation of β1-containing receptors decreased

GABAA receptor function, whereas β3 subunit-containing GABAA receptors were potentiated in (McDonald et al. 1998; Moss et al. 1992). In contrast, β2 subunit-containing receptors were not modulated by PKA in HEK293 cells. Furthermore, kinase modulation of GABAA receptors is also dependent on the heterologous system utilized. For example,

Calcium/Calmodulin-dependent Kinase II did not influence β3 subunit-containing receptors expressed in HEK293 cells, whereas it potentiated the same receptors when expressed in a neuroblastoma-glioma hybrid cell line (Houston and Smart 2006). This

latter point emphasizes the importance of studying receptor function in native environments given the importance of intracellular signaling pathways and their modulation of receptor function.

2.1.10 GABAA receptor pharmacology and benzodiazepines

Many pharmacological agents have been developed to modulate GABAA receptor function, with benzodiazepines being one of the most widely studied and used in clinical practice (Sigel and Ernst 2018). Benzodiazepines, which are positive allosteric modulators (PAMs) of GABAA receptors, function by potentiating GABA-induced currents to increase neuronal inhibition. The potentiating effect of benzodiazepines is achieved by increasing the receptor’s affinity for GABA which results in an increase in the frequency of channel opening (Sigel and Ernst 2018). Overall, benzodiazepines induce a number of behavioural effects including anxiolysis, sedation, and cognition-impairing effects (Curran

1986, 1991).

It has been determined that approximately 75% of all GABAA receptors in the brain contain a high-affinity benzodiazepine binding site, which is situated at the interface of an

α1, α2, α3, or α5 and γ2 subunit (D'Hulst et al. 2009). Substitution of the γ2 subunit with either a γ1 or γ3 subunit produces a low-affinity binding site. Benzodiazepines also have an extremely low binding affinity for the α4 and α6 subunit-containing GABAA receptors, which are often referred to as benzodiazepine-insensitive receptors. The insensitivity of these receptors to benzodiazepines is due to an arginine substitution within the α4 and

α6 subunits (Rudolph and Knoflach 2011; Sigel and Ernst 2018). Additional low-affinity

benzodiazepine binding sites have been located in the transmembrane domain of GABAA receptors and are structurally distinct from the extracellular binding site at the α and γ subunit interface (Sigel and Ernst 2018).

Classic benzodiazepines, such as diazepam, bind to all benzodiazepine receptor sites with similar affinity and efficacy (Sigel and Ernst 2018). However, activating all benzodiazepine receptor subtypes (i.e. α1, 2, 3, and 5) is unfavourable due to the variety of adverse behavioural effects they induce. To address this issue, studies utilized genetic and pharmacological approaches to elucidate the subtype-specific functions of GABAA receptors. These studies revealed that the α1 subunit plays a major role in sedation, the

α2 and α3 subunits in anxiolysis, and the α5 subunit in cognition (Soh and Lynch 2015).

The selective function of α5 subunit-containing GABAA (α5GABAA) receptors in cognition has made them an attractive therapeutic target to treat cognitive dysfunction.

2.2 α5GABAA receptors

2.2.1 α5GABAA receptor expression

The anatomical distribution of α5GABAA receptors support their role in cognition.

α5GABAA receptors are not ubiquitously expressed throughout the brain but instead are highly concentrated within the hippocampus (Glykys and Mody 2007). α5GABAA receptors make up about 5% of all GABAA receptors expressed in the entire brain, but constitute approximately 25% of GABAA receptors in the hippocampus (Rudolph and

Mohler 2014). This finding has been confirmed in humans using α5-selective compounds and PET imaging techniques (Lingford-Hughes et al. 2002). Within the hippocampus, the

α5 subunit is mostly expressed in the dendritic layers of the CA1 and CA3 regions, that is the stratum radiatum and stratum oriens (Sur et al. 1999). α5GABAA receptors are also expressed in other brain regions including the cortex, hypothalamus, and amygdala, albeit to a lesser extent than in the hippocampus (Engin et al. 2018).

The subcellular localization of the α5 subunit is also precise. α5GABAA receptors are largely expressed in the extrasynaptic regions of neurons (Farrant and Nusser 2005).

This is achieved through the binding of receptors to radixin, the intracellular scaffolding protein that clusters receptors in the extrasynaptic regions of neurons (Hausrat et al.

2015). However, electron microscopy revealed that a fraction of α5GABAA receptors are also expressed in the synaptic regions of hippocampal pyramidal cells (Serwanski et al.

2006). This finding is further supported through immunoprecipitation experiments that unveiled a direct interaction between the α5 subunit and gephyrin, the synaptic scaffolding protein (Christie and de Blas 2002). Altogether, although α5GABAA receptors are predominantly expressed extrasynaptically where they generate a tonic current, they are also expressed in synaptic regions where they contribute to phasic inhibitory currents as well (Mohamad and Has 2019).

2.2.2 α5GABAA receptor properties and function

The properties of α5GABAA receptors are key in determining their neurophysiological functions. Rather than being exposed to the high transient concentrations of GABA that occur in the synaptic cleft, α5GABAA receptors are typically faced with constant ambient levels of GABA within the extrasynaptic regions of neurons (Glykys and Mody 2007). The

α5GABAA receptors are well-suited to these conditions due to their relatively high affinity

for GABA and slow kinetics (i.e. desensitization and deactivation) enable them to generate a persistent, low-amplitude tonic current (Farrant and Nusser 2005; Banks and

Pearce 2000).

In hippocampal neurons, α5GABAA receptors have been shown to be major contributors to the tonic current. The genetic removal of the α5 subunit in hippocampal neurons drastically reduced the amplitude of the tonic current without influencing miniature inhibitory postsynaptic currents (Caraiscos et al. 2004). A major function of the tonic current is to regulate the overall excitability of neurons, and thus the firing of action potentials (Bonin et al. 2007). Ultimately, this change in firing rate modulates network synchrony and activity that regulates certain cognitive processes such as learning and memory (Towers et al. 2004; Soh and Lynch 2015).

Electrophysiological studies have noted the contribution of α5GABAA receptors to synaptic currents, which is consistent with their expression in the synaptic regions of neurons (Vargas-Caballero et al. 2010; Zarnowska et al. 2009). Synaptic α5GABAA receptors in hippocampal CA1 pyramidal neurons have been shown to be preferentially activated by dendrite-targeting interneurons and under conditions of increased network activity, which give rise to distinct synaptic events with slower kinetics (Schulz et al. 2018;

Jacob 2019). Nevertheless, other studies found that the genetic removal or pharmacological blockade of α5GABAA receptors had no effect on synaptic current (Bonin et al. 2007; Caraiscos et al. 2004). This suggests that α5GABAA receptor contribution to synaptic events may be highly specific regarding both the cell-type and network conditions. Regardless, the role of α5GABAA receptors in mediating synaptic currents

may also contribute to the synchronization of neuronal networks, and thus should not be overlooked when examining the function of α5GABAA receptors in cognition.

α5GABAA receptors ability to regulate neuronal excitability allows them to influence long-term potentiation, the neural correlate of learning and memory. Reducing the function of α5GABAA receptors lowers the threshold required to evoke long-term potentiation, and has been associated with improved cognitive performance in hippocampal-dependent tasks (Martin et al. 2010).

The notion of reducing GABAA receptor function to improve memory has been long established. Bicuculline, a non-selective competitive antagonist of GABAA receptors, has demonstrated pro-cognitive effects in preclinical models (Brioni and McGaugh 1988).

However, the issue with such non-selective GABAA receptor blockade is that it evokes anxiogenic and proconvulsive effects as well, making it unsuitable for clinical use

(Horowski and Dorrow 2002). Thus, it is imperative that a selective approach is utilized in targeting α5GABAA receptors to treat cognitive dysfunction.

Given that α5GABAA receptors play a fundamental role in cognition, they have been considered to be therapeutic targets for the treatment of cognitive dysfunction associated with a wide range of disorders (Rudolph and Mohler 2014; Jacob 2019). Specifically,

α5GABAA receptor function has been shown to be increased in many disorders and impair cognitive performance, whereby reducing receptor activity improves cognition (Maramai et al. 2019). Consequently, α5GABAA receptor function and expression has been examined in the pathophysiology of many cognitive disorders.

2.3 α5GABAA receptor related disorders

Many neurological and psychiatric disorders produce some degree of cognitive dysfunction in patients. Although the causes and underlying pathology of these disorders are often complex and multifactorial, α5GABAA receptors have been identified as a common contributing factor in several models of humans disease (Jacob 2019;

Mohamad and Has 2019). Below, I provide a brief overview on several disorders, highlighting the involvement of α5GABAA receptors and their potential as a therapeutic target.

2.3.1 Perioperative neurocognitive disorder

A subset of patients who undergo surgery and general anesthesia experience persistent cognitive deficits throughout the postoperative period, and this change in cognition is referred to as perioperative neurocognitive disorder (Mahanna-Gabrielli et al.

2019). Although the exact cellular mechanisms underlying these cognitive deficits are unknown, general anesthesia and inflammation triggered by the surgery itself have been shown to be contributing factors (Moller et al. 1998; Subramaniyan and Terrando 2019;

Mahanna-Gabrielli et al. 2019).

Preclinical studies from our laboratory have demonstrated that a single brief exposure to anesthesia can produce persistent memory deficits (Zurek et al. 2014). These deficits are associated with a sustained increase in α5GABAA receptor function within the hippocampus, measured by an increase in the amplitude of the tonic current (Zurek et al.

2014). Furthermore, reducing the activity of α5GABAA receptors using genetic or pharmacological methods attenuated such anesthetic-induced cognitive deficits.

Inflammation has also been shown to induce cognitive deficits. For example, the proinflammatory cytokine IL-1β produced memory deficits in mice. These memory deficits were associated with enhanced α5GABAA receptor function in the hippocampus (Wang,

Zurek, et al. 2012). The cognitive deficits induced by inflammation could be reversed through either pharmacological blockade or genetic removal of the α5GABAA receptor

(Wang, Zurek, et al. 2012). Collectively, this evidence suggests that reducing α5GABAA receptor activity may be an effective treatment for perioperative neurocognitive disorder.

2.3.2 Traumatic brain injury

Traumatic brain injury can result from sudden collisions or impacts to the head, leading to long-lasting cognitive deficits (Kiraly and Kiraly 2007). Although the causes and severity of the injury may vary substantially, several lines of evidence point to the

α5GABAA receptor as a potential therapeutic target. Firstly, this type of injury is associated with selective cell death within the hippocampus, which may disrupt hippocampal-dependent processes that are modulated by α5GABAA receptor activity

(Royo et al. 2006). Secondly, traumatic brain injury triggers the inflammatory response within the brain which has also been shown to upregulate α5GABAA receptor function and induce cognitive deficits (Wang, Zurek, et al. 2012; Kumar et al. 2015). Finally, a recent study has demonstrated that inhibiting α5GABAA receptors in a rodent model of mild traumatic brain injury enhanced neuroplasticity and improved cognition (Khodaei et al.

2019). Collectively, the data indicate that the cognitive dysfunction in traumatic brain

injury may be due to an increase in α5GABAA receptor activity, impairing hippocampal function. Thus, inhibiting α5GABAA receptors could be an effective therapeutic strategy to treat this disorder.

2.3.3 Schizophrenia

Schizophrenia is most notable for its psychotic symptoms including hallucinations and delusions, although patients also experience cognitive deficits in executive function and memory (Charych et al. 2009). GABAergic dysfunction has been strongly implicated in the pathophysiology of schizophrenia with several lines of evidence indicating α5GABAA receptors as potential therapeutic targets (Rudolph and Mohler 2014). One study compared the genomes of schizophrenic patients to healthy controls (Pocklington et al.

2015). This genome-wide analysis found that patients with schizophrenia tended to have duplications in gene segments associated with GABAergic signaling, most often involving the α5, β3 and δ subunits (Pocklington et al. 2015). Such enhanced expression of α5 could lead to increased α5GABAA receptor activity within the hippocampus and impair cognitive processes. In addition, schizophrenic patients exhibit reduced power in gamma oscillations within cortical regions of the brain, which are important in performing certain cognitive functions (Uhlhaas and Singer 2010). α5GABAA receptors have been shown to play a key role in establishing network synchrony and increasing the power of gamma oscillations within the brain (Papatheodoropoulos and Koniaris 2011; Glykys et al. 2008).

Also, in the phenylcyclidine rat model of schizophrenia, pharmacologic inhibition of

α5GABAA receptors improved cognitive performance in a novel object recognition task

(Redrobe et al. 2012). Lastly, in support of the notion of increased GABAergic

transmission, a clinical study found that treating schizophrenic patients with a benzodiazepine worsened cognitive function, whereas administering a negative allosteric modulator improved cognition in a working memory task (Menzies et al. 2007). Despite there being evidence to support the overactivity of α5GABAA receptors in schizophrenia, other studies have reported reduced GABAergic signaling (Perry et al. 1979). Consistent with a deficit in GABA, the α5GABAA receptor positive allosteric modulator, SH-053-

20FR-CH3, was able to restore dopaminergic neurotransmission within the hippocampus of the methylazoxymethanol acetate (MAM) induced model of schizophrenia in rats (Gill et al. 2011). Thus, whether there is an increase or reduction in α5GABAA receptor activity may depend on the preclinical model used. These findings also emphasize the heterogeneity and complexity of the disorder, as seen in the diversity of patient symptoms and variation in the response to treatment (Brennand et al. 2014).

2.3.4 Down syndrome

Down syndrome is caused by human trisomy 21 and can result in severe intellectual disability (Rudolph and Mohler 2014). This disorder has been heavily investigated using several preclinical models that have been established to test potential pharmacological treatments. To date, the Ts65Dn mouse model is the most widely used and best- characterized model (Rudolph and Mohler 2014). Ts65Dn mice are generated through an extra copy of the mouse chromosome orthologous to human chromosome 21 (Liu et al.

2011). In behavioural studies, these mice demonstrate cognitive deficits in the novel object recognition and Morris Water maze assays. Electrophysiological analysis revealed that Ts65Dn mice also have reduced hippocampal neuroplasticity, indicated by deficits in

long-term potentiation (Siarey et al. 1997). These deficits have been attributed to excessive GABAergic function, characterized by an increase in the number of GABAergic synapses and GABA release (Contestabile et al. 2017). In line with these findings, pharmacological inhibition of α5GABAA receptors attenuated deficits in long-term potentiation and improved cognitive performance (Martinez-Cue et al. 2013). Overall, preclinical evidence suggests that increased GABAergic function contributes to cognitive impairment in Down syndrome, and thus, reducing α5GABAA receptor activity to restore function may be a useful approach.

A clinical trial was initiated to determine whether a compound that selectively inhibits

α5GABAA receptors could improve cognition in patients with Down syndrome

(NCT01436955). However, despite compelling preclinical evidence, the compound did not demonstrate pro-cognitive efficacy in patients. Further research is needed to understand why this compound was ineffective.

2.3.5 Autism spectrum disorder

Autism spectrum disorder is a group of neurodevelopmental disorders characterized by social and cognitive impairments. One hypothesis suggests that these deficits arise from genetic dysfunction and produce an imbalance between excitatory and inhibitory neurotransmission in the brain (Cellot and Cherubini 2014). Genetic studies have revealed that this imbalance may come from the duplication or deletion of a chromosomal segment encoding certain GABAA receptor subunits, including the α5 subunit (Mohamad and Has 2019). For example, a post-mortem brain analysis of autism spectrum disorder patients showed an increase in mRNA levels of α5 (Purcell et al. 2001). In contrast, a

positron emission tomography imaging study found reduced binding of an α5-selective compound, RO-15-4513, in patients with autism compared to healthy controls (Mendez et al. 2013). Further supporting this finding of reduced inhibition, a meta-analysis examining different mouse models of autism spectrum disorder found that the number of parvalbumin-positive GABAergic interneurons was reduced, resulting in network dysfunction (Gogolla et al. 2009). In summary, the evidence supports the notion that

GABAergic signaling is dysregulated in autism spectrum disorder. More importantly, given the heterogeneity in this disorder and the underlying pathophysiology, patient stratification may be a beneficial approach to take in selecting the optimal treatment.

2.3.6 Stroke

Stroke injury may lead to long-lasting cognitive dysfunction in patients, severely reducing their quality of life (das Nair et al. 2016). Preclinical studies have demonstrated that excessive inhibitory neurotransmission is a limiting factor in stroke recovery. In fact, human studies found that increasing cortical excitability through repetitive transcranial magnetic stimulation (rTMS) after stroke may aid in recovery (Wang, Tseng, et al. 2012).

This increase in excitability may enhance neuroplasticity, allowing existing cortical functions to reallocate to undamaged areas of the brain. Using a murine stroke model, tonic inhibition was found to be increased in the peri-infarct region of the brain (Clarkson et al. 2010). Furthermore, both the genetic removal and/or pharmacological inhibition of

α5GABAA receptors improved functional recovery after stroke in rodents (Clarkson et al.

2010). Thus, reducing tonic inhibition in the peri-infarct zone by targeting α5GABAA receptors may be a valuable therapeutic approach in improving patient outcomes.

A competitive antagonist selective for α5GABAA receptors was tested in a clinical trial with stroke patients (NCT02877615). Although the compound was well-tolerated by subjects, it did not improve patient recovery from stroke. It is unclear why this compound is not effective in humans, given the abundance of preclinical findings that support its therapeutic effects.

2.3.7 Alzheimer’s disease

Alzheimer’s disease is a progressive neurodegenerative disorder, characterized by a gradual decline in memory. There are several lines of evidence that suggest α5GABAA receptors can be targeted to improve cognitive function in this disorder. First, both post- mortem and live imaging studies have demonstrated reduced cholinergic neurotransmission and N-methyl-D-aspartate (NMDA) receptor-mediated signaling in these patients (Lin et al. 2014). Although these findings do not directly involve α5GABAA receptors, the resulting impairments in long-term potentiation may be restored by targeting α5GABAA receptors and increasing excitability. Second, post-mortem studies have also revealed that Alzheimer’s patients have a greater number of astrocytes that are in the reactive state (Steele and Robinson 2012; Jo et al. 2014). These reactive astrocytes can produce GABA, leading to higher ambient GABA levels and increased tonic inhibition.

Thirdly, immunohistochemical analysis of post-mortem brain tissue revealed an increase in α5 subunit expression in the CA1 region of the hippocampus in Alzheimer’s patients

(Kwakowsky et al. 2018).

To further determine the role of α5GABAA receptors in Alzheimer’s disease, a preclinical study examined the effects of reducing α5GABAA receptor function on

neuroplasticity and cognitive function. In the 5xFAD mouse model of Alzheimer’s disease, pharmacologic inhibition of α5GABAA receptors restored deficits in long-term potentiation and improved memory performance in the Y-maze spatial memory task (Wu et al. 2014).

Altogether, an increased inhibitory drive within the hippocampus may contribute to the cognitive impairments in Alzheimer’s disease. Thus, the α5GABAA receptors may provide a viable target to selectively reduce inhibition within the hippocampus and restore cognition.

2.3.8 Major depressive disorder

Major depressive disorder is one of the most common neuropsychiatric disorders and often characterized by negative emotions and anhedonia (Ghoneim and O'Hara 2016).

Although some effective antidepressant treatments exist, they can take weeks to months to have an effect (Duman 2018).

Post-mortem analysis of human brain tissue revealed a significant increase in

α5GABAA receptor protein levels in the cortical brain regions of depressed patients (Xiong et al. 2018). Further examining α5GABAA receptor expression in the chronic social defeat stress mouse model of depression, increased protein levels were detected in the cortex and hippocampus (Xiong et al. 2018). In the same mouse model, pharmacological inhibition of α5GABAA receptors reversed deficits in the forced swim test and female urine sniffing test.

The exact mechanism by which α5GABAA receptors elicit these anti-depressive properties is unclear. However, one potential mechanism is through increasing the power

of high frequency oscillations in brain regions involved in mood regulation. The rapid acting anti-depressant ketamine displays such properties, as it induces high amplitude network oscillations. Similarly, reducing α5GABAA receptor activity increases neuronal excitability and has been shown to augment gamma oscillations within the hippocampus

(Zanos et al. 2017). Thus, in addition to their conventional role in cognition, α5GABAA receptors may also serve as a therapeutic target in mood disorders.

Contrary to the findings reported above, there is evidence that supports a GABAergic deficit hypothesis of major depressive disorder (Luscher et al. 2011). Specifically, positive allosteric modulators (PAMs) selective for α5GABAA receptors have also demonstrated antidepressant-like properties in mice studied using the forced swim test (Prevot et al.

2019). Overall, controversy remains around the use of NAMs and PAMs in the treatment of depression. Nevertheless, an imbalance between excitatory and inhibitory neurotransmission appears to be a central issue.

2.4 α5GABAA receptor negative allosteric modulators (α5-NAMs)

2.4.1 Overview of α5-NAM pharmacology

Given the overwhelming evidence implicating α5GABAA receptors in the pathophysiology of neurological and psychiatric disorders, these receptors have garnered a great deal of attention as therapeutic targets. Their attractiveness stems from their role in regulating cognition and their highly specific distribution pattern (Jacob 2019). These features of the receptor limit the potential for unwanted side-effects. In general, animals with reduced expression or ablation of the α5GABAA receptor have demonstrated improved cognitive performance. Importantly, these animals that lacked the α5GABAA

receptor did not show anxiolytic or proconvulsive behaviours which are often associated with non-selective GABAA receptor blockade (Sieghart and Savic 2018; Dorow et al.

1983).

Compounds that selectively inhibit α5GABAA receptors, known as α5GABAA receptor negative allosteric modulators (α5-NAMs), were developed as cognitive-enhancing drugs

(Sieghart and Savic 2018). α5-NAMs bind to GABAA receptors at the benzodiazepine site which is located at the interface of the α/γ subunits (Atack et al. 2009). Upon binding, these compounds reduce GABA-induced currents, increasing neuronal excitability

(Maramai et al. 2019).

α5-NAMs target α5GABAA receptors through a preferential efficacy and/or affinity

(Sieghart and Savic 2018). An α5-NAM that is efficacy-selective binds to many subtypes of GABAA receptor with similar affinity, but preferentially reduces the function of α5GABAA receptors. Contrarily, the affinity-selective α5-NAMs have a similar efficacy for several receptor subtypes but have a much greater affinity for the α5GABAA receptor. It is important to note that α5-NAMs are relatively selective compounds, and thus, still have effects at other GABAA receptor subtypes, especially at higher concentrations (Sieghart and Savic 2018). Therefore, when examining the pharmacology or behavioural properties of these drugs, it is important to consider the contribution of other GABAA receptor subtypes to the observed effect.

In general, the α5-NAMs developed to date have reliably shown to enhance synaptic plasticity and long-term potentiation, improving cognitive performance (Sieghart and

Savic 2018). Importantly, α5-NAMs do not have anxiogenic or proconvulsive effects at the doses required to achieve their pro-cognitive effects (Maramai et al. 2019).

Next, I will describe several of the most widely studied α5-NAMs, reviewing their progression through preclinical and clinical trials. Additionally, I will review two α5- selective compounds that are not negative allosteric modulators. The first is S44819, which is a competitive antagonist at the α5GABAA receptor. The second compound is Xli-

093, which is an α5-selective benzodiazepine receptor antagonist.

2.4.2 RO4938581

The RO4938581 compound was developed by Hoffman La-Roche and classified as an imidazotriazolobenzodiazepine. This compound is affinity- and efficacy-selective for the α5GABAA receptor. It possesses ≥17-fold binding selectivity for α5GABAA receptors compared to other GABAA receptor subtypes. RO4938581 inhibits GABA-evoked currents at the α5 subtype by -40% in comparison to ± 10% at the α1, α2, and α3 subunit- containing GABAA receptors (Ballard et al. 2009). RO4938581 enhanced long-term potentiation and also reversed cognitive deficits in rats that were induced by diazepam and scopolamine (Ballard et al. 2009). In a preclinical model of Down syndrome (Ts65Dn mouse model), RO4938581 treatment prevented the development of neuroanatomical irregularities and rescued cognitive function (Martinez-Cue et al. 2013). Furthermore, in healthy adult monkeys, this compound improved executive function in an object retrieval task (Ballard et al. 2009). Importantly, RO4938581 did not display anxiogenic or proconvulsive effects. However, further clinical development of this compound was not pursued likely due to the drug-induced increase in activity of its metabolizing enzyme

cytochrome P450 1A2 (Bundgaard et al. 2013). The increased metabolism of RO4938581 after repeated dosing makes it unsuitable for chronic use.

2.4.3 Basmisanil (RG1662)

Basmisanil, an isoxadolepyridine, was produced by Hoffman La Roche (Maramai et al. 2019). Although basmisanil has yet to be described in peer-reviewed studies, some of its properties have been outlined in the US patent 8835425B2, in which it is referred to as R1. Like RO4938581, basmisanil is efficacy- and affinity-selective for the α5GABAA receptor (Maramai et al. 2019). Basmisanil demonstrates an affinity over 100-fold greater for the α5GABAA receptor versus other GABAA receptors. This compound also modulates

GABA-evoked currents by approximately -40% at α5GABAA receptors compared to less than -10% at the other subtypes. Basmisanil’s effect on hippocampal neuroplasticity was investigated using the Ts65Dn mouse model of Down syndrome, which characteristically displays deficits in long-term potentiation and cognition. Hippocampal slices obtained from Ts65Dn mice that were pretreated with basmisanil showed significant improvements in long-term potentiation. These results were consistent with findings in the Morris water maze task, where Ts65Dn mice that received basmisanil performed superior to controls.

Notably, basmisanil did not impair motor performance, nor did it induce anxiogenic or proconvulsive effects (US patent 8835425B2).

Based on these findings, basmisanil progressed into phase 2 clinical trials to determine whether it could improve cognitive function in patients with Down syndrome.

Although the compound was well-tolerated by human subjects, it did not demonstrate pro- cognitive efficacy as measured by the Repeatable Battery for the Assessment of

Neuropsychological Status sub-tests (NCT02024789). This test is commonly used to examine multiple cognitive domains including attention and memory. Basmisanil subsequently entered clinical trials for patients with schizophrenia with the primary outcome being cognitive enhancement. This clinical trial was recently completed but results have yet to be published (www.clinicaltrials.gov, NCT02953639).

2.4.4 α5IA

α5IA was developed by the Merck group and categorized as a triazolopyridazine

(Maramai et al. 2019). Unlike the other α5-NAMs, α5IA is strictly an efficacy-selective compound as it has a similar affinity for all GABAA receptor subtypes (Ki = 0.58–0.88 nM).

α5IA reduces GABA-evoked current by approximately 40% at the α5GABAA receptor, whereas effects at the α1, α2 and α3 subunit-containing receptors are -18, +13 and -7%, respectively (Dawson et al. 2006). In murine hippocampal slices, α5IA significantly enhanced long-term potentiation. This change in neuroplasticity was associated with improved cognitive performance by rats in the delayed-matching-to position version of the

Morris Water maze. Moreover, α5IA did not exhibit anxiogenic or proconvulsive properties and did not impact motor function (Dawson et al. 2006).

α5IA was studied in humans using an ethanol-induced impairment word recall task.

This task was selected because it is hippocampal-dependent (Eichenbaum 2004).

Furthermore, alcohol potentiates the function of GABA and can impair memory function

(Wallner et al. 2003). In this experimental paradigm, subjects were pre-treated with α5IA and consumed alcohol 2 hours later. After consuming the alcohol, subjects were given a list of words to memorize and instructed to recall them 30 minutes later. The results

showed that participants who received α5IA performed significantly better on this task

(Nutt et al. 2007). Despite having pro-cognitive effects in humans, additional preclinical testing indicated that chronic dosing of α5IA resulted in renal toxicity, halting the development of this compound (Atack 2010).

2.4.5 MRK-016

MRK-016 was developed by the Merck group as the sequel to α5IA. MRK-016 is characterized as a pyrazolotriazine and is similar to α5IA in that it is an efficacy-selective compound (Maramai et al. 2019). It binds to all receptor subtypes with similar affinity (Ki

= 0.77 – 1.4 nM) but has preferential efficacy at the α5GABAA receptor of -55% compared to -16, 6, and -9% modulation of GABA-evoked currents at the α1, α2 and α3 GABAA receptor subtypes (Atack et al. 2009). In murine hippocampal slices, MRK-016 significantly enhanced long-term potentiation (Atack et al. 2009). The behavioural effects of MRK-016 were examined in a delayed-matching-to-position version of the Morris Water maze test, where it improved cognitive performance in a dose-dependent manner.

Furthermore, MRK-016 did not have any anxiogenic or proconvulsive effects in rodents

(Atack et al. 2009).

MRK-016 has also demonstrated therapeutic effects in a variety of preclinical disease models. First, MRK-016 was effective in reversing memory deficits induced by lipopolysaccharides in a contextual fear conditioning task (Eimerbrink et al. 2015). Our lab has previously shown that the pro-inflammatory cytokine interleukin-1β increases the surface expression of α5GABAA receptors in the hippocampus, increasing the amplitude of the tonic current (Wang, Zurek, et al. 2012). This evidence suggests that α5GABAA

receptor function may be upregulated in inflammatory states, whereby reducing their function can restore cognition. Second, MRK-016 demonstrated antidepressant-like properties in a preclinical model of depression as measured by the forced swim, tail suspension and sucrose preference test (Fischell et al. 2015; Zanos et al. 2017; Xiong et al. 2018). As described in the above section on major depressive disorder, MRK-016 may achieve these antidepressant-like effects by increasing neural excitability and the power of network oscillations. Such effects have been associated with the antidepressant properties of other medications, such as ketamine (Zanos et al. 2017).

However, there were two main findings that terminated MRK-016’s development. One factor was its variable pharmacokinetic profile in humans. A second, and more crucial factor, was that MRK-016 produced adverse effects in the elderly at relatively low doses including nausea and vomiting, while being well-tolerated by young adults (Atack et al.

2009).

2.4.6 PWZ-029

PWZ-029 was developed by a research team led by Dr. James Cook. This imidazobenzodiazepine is both affinity- and efficacy-selective for the α5GABAA receptor.

PWZ-029 has a binding affinity over 30-fold greater for the α5GABAA receptor compared to the other receptor subtypes and also demonstrates its strongest NAM effect of -17% at the α5 subtype (Savic et al. 2008). However, this compound shows relatively high PAM effects at increasing concentrations at the α1, α2 and α3 subunit-containing GABAA receptors (20, 15, and 45% modulation, respectively) (Sieghart and Savic 2018). In rodent behavioural assays, PWZ-0129 enhanced cognitive performance in a passive, but not

active, avoidance learning task (Savic et al. 2008). This compound also reversed scopolamine-induced cognitive deficits in mice in the novel object recognition test, but not in the Morris Water maze task (Harris et al. 2008). In non-human primates, PWZ-029 improved performance in an object retrieval task, but only in trials where the difficulty was relatively high or in the presence of scopolamine-induced cognitive deficits (Clayton et al.

2015). To date, PWZ-029 along with the other imidazobenzodiazepines have not been tested in clinical trials.

2.4.7 L-655,708

This imidazobenzodiazepine was one of the earlier α5-NAMs developed by the Merck group. L-655,708 is strictly a binding-selective compound with an affinity 30 to 70-fold greater at the α5GABAA receptor (ki = 1 nM) versus α1, α2 and α3 subunit containing receptors (Atack et al. 2006). L-655,708 elicited similar effects at all receptor subtypes, with an efficacy of -17% at the α5GABAA receptor. In murine hippocampal slices, L-

655,708 enhanced long-term potentiation. L-655,708 also improved cognitive performance in healthy adult rats in the Morris Water maze test and reversed anesthetic- induced memory deficits in mice (Atack et al. 2006; Saab et al. 2010).

Although L-655,708 was not proconvulsive, it possessed anxiogenic-like properties which terminated its development (Navarro et al. 2002). It was postulated that such effects may have resulted from actions at the other GABAA receptor subtypes, since α5 knockout mice and other selective α5-NAMs do not induce anxiogenic-like behaviours

(Collinson et al. 2002; Sieghart and Savic 2018). This finding emphasizes the importance

of selecting the appropriate dose for L-655,708, as higher concentrations compromise its selectivity for the α5GABAA receptor which may elicit unwanted side-effects.

2.4.8 ONO-8590580

This α5-NAM is primarily an efficacy-selective compound, similar to α5IA and MRK-

016. ONO-8590580 has a relatively low binding selectivity for the α5GABAA receptor with only a 3 to 17-fold greater affinity compared to α1, α2, and α3 subunit-containing receptors. It is a functionally selective compound that exerts a modulatory effect of -40% at the α5GABAA receptor while having negligible effects at the remaining subtypes

(Kawaharada et al. 2018). ONO-8590580 significantly enhanced long-term potentiation in rat hippocampal slices and improved cognitive performance in two behavioural assays.

First, in a passive-avoidance task, ONO-8590580 attenuated cognitive deficits in rats that were impaired by the N-methyl-D-aspartate receptor antagonist, MK-801. Second, in an

Alzheimer’s disease model that utilizes both scopolamine- and MK-801-induced deficits,

ONO-8590580 significantly improved cognitive performance in the eight-arm radial maze task (Kawaharada et al. 2018). Importantly, ONO-8590580 did not have any proconvulsive or anxiogenic effects in rodents. These results highlight the therapeutic potential of ONO-8590580, especially in the management of Alzheimer’s disease.

2.4.9 S44819

Compound S44819 is distinct from the other compounds discussed this far because it does not function as a negative allosteric modulator (Sieghart and Savic 2018). Instead,

S44819 behaves as a competitive antagonist, meaning it binds to the orthosteric site of

the GABAA receptor and prevents GABA from binding and activating the receptor

(Etherington et al. 2017). The binding selectivity of this compound is determined by several key amino acid residues located within the α5 subunit (Etherington et al. 2017).

In the CA1 region of mouse hippocampal slices, S44819 inhibited the tonic current without influencing miniature inhibitory postsynaptic currents and enhanced long-term potentiation. In rats, S44819 improved memory performance in the novel object recognition assay and reversed scopolamine-induced deficits in the eight-arm radial maze test (Etherington et al. 2017).

S44819 was studied in humans and shown to increase cortical excitability in healthy adults, as measured through electroencephalographic signals (Darmani et al. 2016).

Consequently, S44819 entered clinical trials to investigate its therapeutic potential in stroke recovery, a disorder characterized by excessive GABAergic function (Clarkson et al. 2010). However, after completing a clinical trial in stroke patients, S44819 was not found to alter the prognosis of the disease (Chabriat et al. 2020). These results dispute the role of inhibitory neurotransmission in human patients recovering from stroke.

2.4.10 XLi-093

XLi-093 is a bivalent imidazobenzodiazepine that was also developed by Dr. James

Cook and colleagues. Unlike the rest of the compounds discussed so far, this compound functions as a benzodiazepine receptor antagonist (Sieghart and Savic 2018). Thus, Xli-

093 strictly prevents the potentiation or inhibition of GABA-induced currents by allosteric modulators acting at the benzodiazepine site. Its selectivity for the α5GABAA receptor is

achieved through its binding affinity which is over 60 times greater for α5GABAA receptors in comparison to the other subtypes (Li et al. 2003). In electrophysiological studies, Xli-

093 had no effect on diazepam’s potentiating effects at α1, α2 and α3-subunit containing receptors, but significantly reduced diazepam’s effects at α5GABAA receptors. Consistent with these findings, XLi-093 mitigated diazepam-induced cognitive deficits in the Morris

Water maze test (Clayton et al. 2015). Despite all this, Xli-093 has yet to undergo further preclinical or clinical testing.

2.5 Summary

α5GABAA receptors influence cognition by generating tonic inhibition and controlling neuronal excitability within the hippocampus (Jacob 2019). Previous research has shown that α5GABAA receptors are hyperactive in many cognitive disorders and are associated with cognitive dysfunction. Thus, α5-NAMs have been developed to selectively target

α5GABAA receptors to treat cognitive deficits (Maramai et al. 2019). Despite showing robust cognitive-enhancing properties in clinical trials, these compounds have yet to reach clinical practice due to toxicity or a lack of efficacy. Therefore, novel insights are needed to improve the development and selection of more effective α5-NAMs for future clinical trials.

To date, α5-NAMs have predominantly been studied in recombinant systems that lack the neuronal environment and native complement of GABAA receptors which may influence α5-NAM pharmacology (Sieghart and Savic 2018). Specifically, increased

α5GABAA receptor function enhances the tonic currents which is associated with cognitive impairment (Mohamad and Has 2019). However, no study has yet to

characterize and compare the effects of α5-NAMs on their primary target, the tonic current. This is especially important for two main reasons: 1) the tonic current is mediated by a heterogenous GABAA receptor population, and 2) α5-NAMs can modulate other

GABAA receptors subtypes besides the α5 subtype.

In summary, by comparing α5-NAM effects on GABAA receptor-mediated currents in primary neurons, we provide novel insights into their pharmacological properties that may guide the development and selection of compounds for future clinical trials.

Chapter 3: General materials and methods

3.1 Study approval

All protocols used in the current study were approved by the Local Animal Care

Committee at the University of Toronto. Experimental procedures were conducted in accordance with the guidelines set out by the Canadian Council on Animal Care. All methods have been described thoroughly to allow for the reproducibility of the findings reported in this thesis.

3.2 Electrophysiological recordings in cell culture 3.2.1 Preparation of primary cell cultures

All experimental procedures were approved by the local Animal Care Committee at the University of Toronto. Hippocampal cell cultures were prepared by dissecting the hippocampi of fetuses from pregnant CD1 mice (Charles River, USA). The pregnant mice were euthanized by cervical dislocation and disinfected with alcohol. Next, using a pair of scissors and forceps, a midline cut was made down the center of the abdomen to remove the fetuses (fetuses were approximately E16-18). Once removed, the fetuses were immediately placed in a culture dish filled with phosphate buffered saline (PBS, Sigma-

Aldrich) that was situated atop an ice-filled dish. A new clean pair of scissors and forceps were then used to extract the fetuses from their encapsulating membranes and decapitate them in a fresh dish of PBS. From here on, a light microscope was used to make incisions through the skulls and brains of the fetuses. Once the fetuses were decapitated, vertical

cuts along the midline of the skull were made to reveal the brain which was then removed and transferred to another separate dish containing PBS. Using a clean pair of fine-tip forceps and scalpel, the hemispheres of each brain were separated. Following the separation of the hemispheres, the hippocampi were carefully dissected and placed into a fresh culture dish containing 1 mL of PBS. In total, approximately 8 hippocampi (4 fetuses) were used in a single dissection.

After the hippocampal tissue was removed, they were cut into pieces using a scalpel.

0.5 mL of trypsin was then added for 25 minutes to digest this tissue. After which, 1 mL of Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich) + 10% Fetal Bovine Serum

(FBS, Life technologies) was added to the culture dish to inactivate the trypsin. Finally, neurobasal medium (Life Technologies) that was supplemented with 2% B27 (Life

Technologies) and 0.5 mM L-glutamate (Life technologies) was added to the dish and mixed together with the hippocampal tissue using a pipette. This dish containing the hippocampal tissue and neurobasal medium was then transferred into to twenty 35-mm culture dishes coated with poly-D-lysine (Sigma-Aldrich). Hippocampal cell cultures were

™ then grown for two weeks in a Forma Series II 3110 Water-Jacketed CO2 Incubator

(Thermo Scientific) that was maintained at 37°C and 5% CO2 with 95% humidified air.

Cultures prepared under these conditions primarily contain hippocampal neurons at a density of about 1 x 106 cells/dish.

3.2.2 Whole-cell voltage clamp recordings in cell culture

Whole-cell voltage clamp recordings were performed in cultured hippocampal neurons at a holding potential of -60 mV using an Axopatch 200B amplifier (Molecular

Devices, Sunnyvale, California, USA) controlled with the pClamp 8.0 software (Molecular

Devices) via a Digidata 1322 interface (Molecular Devices). Patch pipettes were pulled from thin-walled borosilicate glass capillary tubes with an open-tip resistance of 2–4 MΩ.

The intracellular solution (ICS) contained (in mM) 140 CsCl, 10 HEPES, 11 EGTA, 2

MgCl2, 1 CaCl2, 2 TEA, and 4 MgATP (pH 7.3 with CsOH, 285 to 295 mOsm).

Extracellular recording solution contained (in mM) 140 NaCl, 2 CaCl2, 1 MgCl2, 5.4 KCl,

25 HEPES, and 28 glucose (pH 7.4, 320 to 330 mOsm). The extracellular solution (ECS) was applied to neurons by a computer-controlled, multibarrelled perfusion system (SF-

77B, Warner Instruments, Hamden, Connecticut, USA). All electrophysiological recordings were performed at room temperature (22–24 oC).

Ionotropic glutamate receptor blockers 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX,

10 μM) and (2R)-amino-5-phosphonovaleric acid (APV, 20 μM) were added to the extracellular solution. Tetrodotoxin (0.2 μM) was used to block voltage-dependent sodium channels. Tetrodotoxin was purchased from Alomone Labs (Jerusalem, Israel). CNQX,

APV, and bicuculline were obtained from Hello Bio Inc. (Princeton, New Jersey, USA); and GABA from Sigma-Aldrich (Oakville, Ontario, Canada).

The whole-cell configuration was achieved by first lowering the pipette onto the soma of the neuron where a tight giga-ohm seal was formed (>1 GΩ). Next, whole-cell capacitance was compensated before rupturing the membrane under the recording pipette by applying negative pressure. The resulting whole-cell configuration provided electrical access to the intracellular environment as well as allowing the ICS held in the

recording pipette to washout the intracellular contents of the cell. The series resistance was monitored by applying a 10 mV hyperpolarizing voltage step, and currents were recorded at a sampling frequency of 5 kHz, except for miniature inhibitory postsynaptic currents (mIPSCs) which were sampled at a frequency of 10 kHz. The low pass filter frequency was set using an 8-pole Bessel filter to 1 kHz and 2 kHz for tonic current and mIPSC recordings, respectively.

Tonic current was recorded by adding exogenous GABA (0.5 µM) to the extracellular solution, and the competitive GABAA receptor antagonist bicuculline (20 µM) was applied to reveal the amplitude of the tonic current. Bicuculline was selected for the current study because it is a potent GABAA receptor blocker with minimal effects on off-target receptors

(Johnston 2013). The GABA concentration of 0.5 µM was chosen because it mimics the ambient levels of GABA that have been detected in vivo (Bright and Smart 2013). This low concentration of GABA will preferentially activate the high-affinity extrasynaptic

GABAA receptors that generate the tonic current.

3.3 Selection and preparation of α5-NAMs

The five α5-NAMs investigated were: basmisanil, Ono-160, L-655,708, α5IA and

MRK-016 (Fig. 3.1). As discussed in Chapter 2, these α5-NAMs were selected for their recent involvement in clinical trials or having been widely studied in preclinical trials. Ono-

160 is a novel compound that is described in a recent patent (WO2015115673A1) and is reported to have a high affinity for α5GABAA receptors (Ki = 0.9 nM).

Four of the five α5-NAMs: basmisanil, Ono-160, α5IA and MRK-016, were synthesized in-house at the Medicines Discovery Institute (Cardiff University, Cardiff,

Wales). L-655,708 was obtained from Sigma-Aldrich (Oakville, Ontario, Canada). All α5-

NAMs were dissolved in dimethyl sulfoxide (DMSO) to produce a primary stock of 10 mM.

The stock was subsequently diluted in ultrapure water to create a secondary stock of 0.1 mM in 1% DMSO, stored at -20 oC. For the in vitro studies, the secondary stock was further diluted in the extracellular solution to obtain the desired α5-NAM concentration in

≤ 0.1% DMSO.

Fig 3.1. Structures of five α5-NAMs evaluated in the present study. Basmisanil is an isoxazole-pyridine. Ono-160 is Compound 160 described in the patent WO

2015/115673 A1. L-655708 is an imidazobenzodiazepine; whereas α5IA and MRK-106 are a triazolophthalazine and pyrazolotriazine, respectively.

3.4 Data and statistical analyses

Currents were analyzed with pClamp 10 software (Molecular Devices). For the tonic current experiment, only a single α5-NAM concentration was tested on each cell, which was normalized to the bicuculline response of that cell. The effects of each α5-NAM on the tonic current were reported as % inhibition which was calculated as (Iα5NAM/Ibicuculline)

× 100%, where Iα5NAM is the current amplitude of the α5-NAM response and Ibicuculline for that of bicuculline. Miniature inhibitory postsynaptic currents (mIPSCs) were analyzed with MiniAnalysis 6.0.3 (Synaptosoft Inc., Fort Lee, New Jersey, USA). Each file was also manually inspected to reject false events caused by noise and include events that were not automatically detected. mIPSCs were recorded for at least 30 seconds under each experimental condition. All graphs were created with GraphPad Prism 6 (Graphpad

Software Inc., La Jolla, California, USA).

Data are presented as mean ± SEM. The normality of data sets was tested with the

D’Agostino–Pearson omnibus test (n ≥ 8) or the Kolmogorov–Smirnov test (n < 8). For comparing three or more groups, the one-way ANOVA followed by the Tukey’s multiple comparisons test was used. If normality was not met, the Kruskal-Wallis test was used.

A paired Student’s t test was used to compare two groups, and when normality assumptions were not satisfied, the nonparametric Wilcoxon matched-pairs signed rank test was utilized. Cumulative distributions of the amplitude and frequency of mIPSCs were compared using the Kolmogorov–Smirnov test. A two-tailed hypothesis test was used, and statistical significance was set to P < 0.05.

Chapter 4: Inhibition of tonic but not synaptic current by α5GABAA receptor negative allosteric modulators in hippocampal neurons

4 4.1 Introduction

Cognitive deficits are associated with multiple neurological and psychiatric disorders

including Alzheimer’s disease, Down syndrome, schizophrenia, traumatic brain injury and

perioperative neurocognitive disorder (Millan et al. 2012; Rabinowitz and Levin 2014;

Belrose and Noppens 2019). These cognitive deficits often result in poor long-term

outcomes, and impose a tremendous burden on patients, their families, and the

healthcare system. To date, no effective pharmacological treatments have been

developed to substantially mitigate cognitive deficits. Thus, there is a large unmet need

for new drugs that improve cognitive performance.

An attractive receptor target for cognition-enhancing drugs is γ-aminobutyric acid type

A (GABAA) receptors containing the α5 subunit (α5GABAA receptors) (Jacob 2019).

These receptors are highly expressed in the hippocampus, and to a lesser extent the

cortex, where they regulate cognition (Sur et al. 1999). α5GABAA receptors are primarily

located in the extrasynaptic regions of neurons, where they generate a tonic conductance;

and are also expressed in synapses, though at lower levels (Glykys et al. 2008; Brunig et

al. 2002; Serwanski et al. 2006). Increased activity of the α5GABAA receptors is

associated with impaired cognition in several disorders, whereby reducing α5GABAA

receptor function, either through pharmacological or genetic approaches, enhances cognitive performance (Zurek et al. 2014; Khodaei et al. 2019; Martinez-Cue et al. 2013).

α5GABAA receptor negative allosteric modulators (α5-NAMs), have been developed to selectively reduce the function of α5GABAA receptors (Sieghart and Savic 2018;

Maramai et al. 2019). α5-NAMs bind to GABAA receptors at a high-affinity benzodiazepine binding site, located at the interface of an α1, 2, 3 or 5 subunit and a γ1, 2 or 3 subunit and preferentially inhibit α5GABAA receptors (Sigel and Ernst 2018). The resulting reduction in permeability of the cell membrane to anions increases the excitability of neurons and enhances cognition (Bonin et al. 2007; Martin et al. 2010).

To date, more than a dozen α5-NAMs have been developed (Sieghart and Savic

2018; Maramai et al. 2019). The selectivity, potency and efficacy of these drugs have been studied using mouse and human GABAA receptors recombinantly expressed in heterologous systems. The results have shown that α5-NAMs differ in terms of their binding affinity, selectivity, and efficacy for α5 subunit-containing versus α1, α2 and α3 subunit-containing GABAA receptors (Maramai et al. 2019; Sieghart and Savic 2018).

However, no study has directly compared the inhibitory effects of α5-NAMs on the function of native GABAA receptors in neurons, including the tonic current. Primary neurons differ substantially from heterologous systems because they express a heterogeneous GABAA receptor population and contain the native neuronal machinery that regulates receptor pharmacology (Houston and Smart 2006; Brunig et al. 2002;

Nakamura et al. 2015). Thus, the goal of this study was to directly compare the effects of five leading α5-NAMs on inhibitory tonic and synaptic currents generated by native

GABAA receptors in hippocampal neurons. These results might offer valuable insights to inform the development of novel α5-NAMs and guide the selection of drugs for clinical trials.

4.2 Methods 4.2.1 Primary hippocampal neuronal culture

Cultures were prepared as described in Chapter 3.2.1.

4.2.2 Electrophysiology

Whole-cell voltage clamp techniques were used to study the effects of α5-NAMs on

GABAA receptor function. Each α5-NAM (basmisanil, Ono-160, L-655,708, α5IA and

MRK-016) was studied at several concentrations to verify their maximal efficacy in our native system. The concentrations were selected from recombinant studies (L-655,708,

α5IA and MRK-016) (Atack 2010; Atack et al. 2009; Atack et al. 2006) and patents (patent

US8835425B2 for basmisanil and patent WO2015115673A1 for Ono-160) based on reported binding affinity and/or efficacy at the α5GABAA receptor.

4.3 Results 4.3.1 α5-NAMs inhibit the tonic current

We first investigated the effect of the five NAMs (basmisanil, Ono-160, L-655,708,

α5IA and MRK-016) on tonic current. This current is primarily generated by high-affinity extrasynaptic GABAA receptors activated by low, ambient concentrations of GABA

(Farrant and Nusser 2005; Brickley and Mody 2012). Cultured hippocampal neurons were

continuously perfused with a low concentration of GABA (0.5 μM) to mimic the low, ambient GABA concentrations that occur in vivo (Farrant and Nusser 2005; Bright and

Smart 2013). A competitive GABAA receptor antagonist, bicuculline (20 µM), was co- applied with GABA to reveal the total amplitude of the tonic current, which was assessed by measuring the reduction in holding current (Fig. 4.1a). Subsequently, following washout of bicuculline, the α5-NAM was co-applied with GABA to determine its ability to block the tonic current. For the analyses, the decrease in holding current induced by the

α5-NAM was normalized to the decrease caused by bicuculline and results are described as “percent inhibition”.

Basmisanil inhibited the tonic current by 14.9 ± 4.7% (10 nM, n = 7) to 56.4 ± 9.1%

(1 µM, n = 8; Fig. 1a). Increasing the concentration to 10 µM failed to further reduce the tonic current. The effect at 1 µM was significantly greater than that at 10 nM (P = 0.004, n = 7) or 100 nM (P = 0.009, n = 7), which shows a concentration-dependent inhibitory effect of basmisanil.

The remaining four α5-NAMs also inhibited the tonic current at each concentration that was tested (Fig. 4.1b-e). In contrast to basmisanil, all four α5-NAMs similarly inhibited the tonic current across all concentrations (P > 0.05) and did not demonstrate a concentration-dependent effect across the concentration range tested.

Fig 4.1. Tonic current is inhibited by α5-NAMs. (a) Representative traces (left) showing basmisanil’s effect on tonic current at 10 nM and 1 µM in comparison to the effect of bicuculline (20 µM). A single α5-NAM concentration was tested on each cell.

Summarized data for four different concentrations of basmisanil (right), illustrating a concentration-dependent effect. n = 7, 7, 8, 5 (left to right). One-way ANOVA, F(3,23) = 6.4,

P = 0.003. *P < 0.01, Tukey’s multiple comparisons test. (b-e) Summarized data for the remaining four α5-NAMs, which do not inhibit the tonic current in a concentration-

dependent manner. One-way ANOVA for all except MRK-016, where the Kruskal-Wallis test was used. (b) Ono-160, n = 8, 8, 9 (left to right). F(2, 22) = 1.2, P = 0.3. (c) L-655,708, n

= 8, 8, 8, 6 (left to right). F(3, 26) = 0.4, P = 0.7. (d) α5IA, n = 9, 9, 9, 7, F(3, 30) = 2.3, P = 0.1.

(e) MRK-016, n = 10, 8, 10, 7 (left to right), H(3) = 7.7, P = 0.052. Data are mean ± SEM.

4.3.2 All α5-NAMs have similar efficacy at inhibiting the tonic current

We next directly compared the maximal effect of the five α5-NAMs, which was replotted from Fig. 1 and arranged based on the concentration that produced the maximal effect (Fig. 4.2). Ono-160 at 10 nM had a maximal effect of 41.2 ± 7.5% (n = 8). α5IA and

MRK-016 at 100 nM inhibited the current by 56.3 ± 8.4% (n = 9) and 52.8 ± 6.4% (n =

10), respectively; whereas L-655,708 at 200 nM produced a maximal effect of 39.4 ± 6.1%

(n = 8). Basmisanil at 1 µM had an effect of 56.4 ± 9.1% (n = 8). Overall, Ono-160 (10 nM) exhibited the lowest maximal effective concentration, whereas basmisanil (1 µM) required the highest concentration to maximally inhibit the tonic current, at the concentrations tested. Thus, the concentrations of α5-NAMs that produced the maximal inhibitory effect varied by approximately 100-fold. More interestingly, no significant differences were observed for the maximal effect between all five NAMs (H4 = 5.7, P =

0.2, Kruskal-Wallis test), indicating that the maximal efficacies of the five α5-NAMs were similar for inhibition of the tonic current.

Fig 4.2. All α5-NAMs similarly inhibit the tonic current at their most efficacious concentrations. Summarized data replotted from Fig. 2 to compare the maximal inhibitory effect for each drug. n = 8, 9, 10, 8, 8 (left to right). H(4) = 5.7, P = 0.2, Kruskal-Wallis test.

Data are mean ± SEM.

4.3.3 α5-NAMs have no effect on peak or steady-state current evoked by a saturating concentration of GABA

Although α5GABAA receptors are predominantly located extrasynaptically, they are

expressed at lower levels in synaptic regions of neurons (Brunig et al. 2002; Serwanski

et al. 2006). Additionally, high concentrations of α5-NAMs (higher than those necessary

to inhibit α5GABAA receptors) can have positive or negative modulatory effects on the

function of other GABAA receptor subtypes (Sieghart and Savic 2018). Therefore, we next

determined whether α5-NAMs also modulated currents evoked by a saturating

concentration of GABA that activates both low-affinity synaptic and high-affinity

extrasynaptic GABAA receptors.

A saturating concentration of GABA (1 mM) was applied for a prolonged duration of

16 seconds to activate a peak current followed by a steady-state current after a fast

desensitization. Peak current was used as an indicator of maximal GABAA receptor

activation, whereas the magnitude of the steady-state current revealed the activity of

receptors that exist in a non-desensitized state. The effects of α5-NAMs were studied at

the concentrations that produced the maximal reduction in tonic current, as outlined in

Fig. 4.2. Each α5-NAM was pre-applied for 10 seconds before being co-applied with

GABA, allowing us to study the effects on both the peak and steady-state currents. As

shown in Fig. 4.3, basmisanil did not alter the amplitude of the peak or steady-state

current (P > 0.05 for both, n = 8; see also Table 4.1). Similarly, the other α5-NAMs also

had no effects (Table 4.1). Overall, the results show that α5-NAMs do not affect GABAA

receptors activated by a prolonged exposure to a high, saturating concentration of GABA.

Fig 4.3. Peak and steady-state current evoked by a saturating concentration of GABA are not modulated by basmisanil. (a) Representative traces showing the effect of basmisanil on current evoked by GABA (1 mM). (b) Summarized data for the peak and steady-state current. n = 8; P = 0.8 and P = 0.1 for the peak and steady-state current, respectively; Student’s paired t test. Data are mean ± SEM.

Table 4.1. α5-NAMs do not modify peak or steady-state current evoked by a saturating concentration of GABA (1 mM). P > 0.05, Student’s paired t test except for

Ono-160 (steady-state) where the Wilcoxon matched-pairs signed rank test was used.

Sample size also applies to the drug treatment. Data are presented as mean ± S.E.M.

Peak (pA) Steady-state (pA)

Control (n = 8) 5264 ± 950 644 ± 134

Basmisanil (1 μM) 5321 ± 943 693 ± 141

Control (n = 7) 4563 ± 1099 646 ± 226

Ono-160 (10 nM) 4252 ± 1091 640 ± 212

Control (n = 7) 4998 ± 1005 752 ± 186

L-655,708 (200 nM) 4945 ± 1025 746 ± 184

Control (n = 6) 4271 ± 764 567 ±140

α5IA (100 nM) 4286 ± 849 549 ± 136

Control (n = 5) 3514 ± 894 349 ± 98

MRK-016 (100 nM) 3390 ± 799 375 ± 84

4.3.4 α5-NAMs do not inhibit miniature inhibitory postsynaptic currents

The currents reported above were evoked by exogenous GABA and are mediated by both extrasynaptic and synaptic GABAA receptor populations. In contrast, mIPSCs are generated by GABA that is released from presynaptic terminals and mediated by postsynaptic GABAA receptors (Farrant and Nusser 2005). Thus, we further investigated the effects of α5-NAMs on synaptic GABAA receptors by recording mIPSCs, in the absence or presence of each α5-NAM.

Basmisanil had no effect on the amplitude or frequency of mIPSCs (Fig. 4.4; P >

0.05). Similarly, the time course and charge transfer of mIPSCs were also unaffected by basmisanil (Table 2; P > 0.05). Likewise, the four other α5-NAMs did not modulate mIPSCs (Table 4.2). These results indicate that α5-NAMs do not modulate postsynaptic

GABAA receptors.

Fig 4.4. Basmisanil (1 µM) does not affect miniature inhibitory postsynaptic currents

(mIPSCs). (a) Representative traces of mIPSCs in the absence and presence of basmisanil. (b) Cumulative distributions of the amplitude (left) and frequency (right) of mIPSCs show that both were not altered by basmisanil. P = 0.1 and P = 0.7 for the amplitude and frequency, respectively; Kolmogorov−Smirnov test.

Table 4.2. α5-NAMs do not modify miniature inhibitory postsynaptic currents. For all

the parameters, P > 0.05, Student’s paired t test except for α5IA (frequency, rise and

area), basmisanil (frequency), L-655,708 (amplitude and area), and MRK-016 (frequency)

where the Wilcoxon matched-pairs signed rank test was used. Sample size also applies

to the drug treatment. Data are presented as mean ± S.E.M.

Amplitude Frequency Rise time Decay time Area (pA) (Hz) (ms) (ms) (pA.ms)

Control (n = 7) 45.5 ± 4.7 3.7 ± 0.2 5.0 ± 0.3 11.0 ± 1.0 552.6 ± 88.7

Basmisanil (1 μM) 41.7 ± 3.2 4.6 ± 0.7 5.3 ± 0.4 9.9 ± 0.8 468.1 ± 48.2

Control (n = 6) 39.0 ± 3.2 4.0 ± 0.9 5.8 ± 0.6 9.0 ± 1.2 399.0 ± 53.2

Ono-160 (10 nM) 40.2 ± 1.8 4.8 ± 0.8 5.6 ± 0.4 8.4 ± 1.1 399.6 ± 49.3

Control (n = 7) 47.2 ± 6.5 5.3 ± 0.8 5.2 ± 0.3 9.9 ± 0.8 533.0 ± 109.0

L-655,708 (200 nM) 42.8 ± 4.7 4.8 ± 1.0 5.3 ± 0.4 9.2 ± 1.2 446.7 ± 78.2

Control (n = 7) 43.8 ± 2.6 1.6 ± 0.3 5.4 ± 0.3 10.3 ± 0.2 514.3 ± 75.8

α5IA (100 nM) 43.0 ± 2.3 2.2 ± 0.4 5.9 ± 0.2 10.7 ± 1.3 510.2 ± 72.6

Control (n = 8) 40.3 ± 4.0 2.5 ± 0.5 6.7 ± 0.4 13.7 ± 1.5 596.8 ± 69.6

MRK-016 (100 nM) 40.0 ± 3.9 2.8 ± 0.7 6.4 ± 0.5 13.8 ± 1.0 591.9 ± 75.8

4.3.5 α5-NAMs alone and DMSO do not modify GABAA receptor function

As allosteric modulators, α5-NAMs require GABA to first bind and open the GABAA receptor channel to modify the GABA-induced current (Sieghart and Savic 2018). Thus,

α5-NAMs should not evoke any current in the absence of GABA. To confirm whether this was true in primary hippocampal neurons, each α5-NAM was tested at their most efficacious concentration as outlined in aim 1 in the absence of GABA. None of the α5-

NAMs induced any current on their own (Fig. 4.5a), indicating they are allosteric modulators.

Dimethyl sulfoxide (DMSO) was the drug vehicle utilized in the current study. DMSO is commonly used as a solvent due to its ability to solubilize a wide variety of compounds that have low aqueous solubility. Although DMSO is a relatively safe solvent, previous research has shown that low concentrations of DMSO (1%) can have neurodegenerative effects (Hanslick et al. 2009). Furthermore, another study demonstrated that 3% DMSO reduced GABA-evoked (5 μM) currents in cultured dorsal root ganglion neurons (Nakahiro et al. 1992). Thus, to determine whether the DMSO concentrations (≤ 0.1%) used in the current study influenced GABAA receptor function, we tested DMSO at 0.2% in the tonic current recordings. The results showed that 0.2% DMSO had no effect on GABAA receptor function (Fig 4.5b). This finding is supported by other studies that found low concentrations of DMSO (≤ 2%) do not affect GABAA receptor-mediated currents (Walters et al. 2000; Sancar and Czajkowski 2011).

Fig 4.5. α5-NAMs alone and DMSO do not modify GABAA receptor function. (a) α5-

NAMs do not evoke any current in the absence of GABA. GABA (5 μM) was applied to each cell as a positive control, n = 14. α5-NAMs were then tested in the absence of GABA.

For each α5-NAM, a one sample t-test against zero was conducted, n = 5, 5, 5, 6, 6 (left to right); P > 0.05 for all. (b) 0.2% DMSO does not modify the tonic current. One sample t-test against zero; n = 5, P = 0.06.

4.4 Discussion

The results reported here show that the five α5-NAMs share a similar maximal efficacy for inhibition of the tonic current (ranging from 39% to 56%). The reduction in current is concentration-dependent for basmisanil, but not for the other four compounds, at the concentrations tested. None of the α5-NAMs modify current evoked by a saturating concentration of GABA, nor do they change the frequency, the amplitude or the time course of mIPSCs.

Given that α5GABAA receptors are major contributors to the tonic current in the hippocampus (Glykys et al. 2008; Caraiscos et al. 2004), it was expected that compounds with greater efficacy for inhibiting recombinant α5GABAA receptors would more effectively inhibit the tonic current. For example, MRK-016 was expected to inhibit the tonic current to a greater extent than L-655,708, as these drugs have maximal inhibitory effect on recombinant α5GABAA receptors of 55% and 17%, respectively (Atack et al. 2006; Atack et al. 2009). However, the α5-NAMs’ maximal inhibitory effects on the tonic current were similar. Two factors may account for the discrepancy between our expectations and the study results. First, inhibition of the tonic current depends on the efficacy and potency of

α5-NAMs for each GABAA receptor subtype. Although tonic current has a large component mediated by α5GABAA receptors, other subtypes could also contribute

(Mortensen and Smart 2006; Lee and Maguire 2014). Furthermore, primary neurons express a heterogeneous complement of native GABAA receptor subtypes, which is in striking contrast to heterologous systems which express a relatively homogeneous receptor population (e.g. α5β3γ2). A positive allosteric action at these receptors could

counter the negative inhibitory effects α5-NAMs have at the α5GABAA receptors. Thus, even if α5-NAMs are relatively selective for α5GABAA receptors, minor effects at other

GABA receptor subtypes, which are abundantly expressed in the hippocampus, could obscure differences in α5-NAM efficacy at α5GABA receptors.

In addition, heterologous systems and primary neurons may differ in terms of the intracellular environment and cell signaling pathways, including factors that are known to regulate GABAA receptor function, such as through phosphorylation (Houston and Smart

2006; Nakamura et al. 2015). Phosphorylation regulates ligand binding at the benzodiazepine site which has been shown to modify sensitivity to the behavioral effects of benzodiazepines (Hodge et al. 1999; Churn et al. 2002). Overall, the heterogeneity of

GABAA receptors underlying the tonic current and the native neuronal environment may both contribute to the convergence in the overall effect size on tonic current, despite distinct α5-NAM efficacy at recombinant α5GABAA receptors.

Notably, four of the five α5-NAMs investigated here did not display a concentration- dependent inhibitory effect on the tonic current at the concentrations tested. This observation may be due to PAM effects at GABAA receptor subtypes that do not contain the α5 subunit. For example, L-655,708 at higher concentrations demonstrated PAM effects on extrasynaptic α4 and α6 subunit-containing recombinant GABAA receptors, which also contribute to tonic current (Ramerstorfer et al. 2010). Such PAM effects may counteract the increasing inhibitory effect of higher concentrations of L-655,708 at

α5GABAA receptors. Conversely, basmisanil is devoid of PAM effects and exhibits over

100-fold selectivity for α5 compared with α1-3 subunits, as described in patent

US8835425B2. The lack of PAM effect for basmisanil may account for its concentration- dependent inhibition of the tonic current. Finally, GABAA receptors express multiple benzodiazepine binding sites (Sigel and Ernst 2018). In addition to the high-affinity binding site, low-affinity sites exist in the transmembrane domains of GABAA receptors, and binding at these sites can exert both potentiating and inhibitory effects of benzodiazepines (Sigel and Ernst 2018; Walters et al. 2000; Baur et al. 2008; Middendorp et al. 2015). At high concentrations, α5-NAMs may bind to these low-affinity sites and potentiate receptor function, offsetting the inhibitory effects generated at the high-affinity site. This action at low- and high-affinity binding sites may account for the lack of concentration-dependent effect of Ono-160, α5IA and MRK-016 since they are not known to have PAM effects on α4 and α6 subunit-containing receptors, as seen with L-655,708.

α5-NAM inhibition of the tonic but not synaptic current supports the relative selectivity of these compounds for α5GABAA receptors. In cultured hippocampal neurons, the tonic current is generated by a heterogeneous receptor population including those containing

α4, α5 and δ subunits as well as αβ dimeric subunits (Brickley and Mody 2012; Mortensen and Smart 2006; Lee and Maguire 2014). Previous studies using genetic and pharmacological approaches suggest that α5GABAA receptors mediate a large portion of the tonic current in hippocampal neurons (Caraiscos et al. 2004; Glykys et al. 2008).

Consistently, all five α5-NAMs in this study inhibited the tonic current.

α5-NAMs did not affect currents evoked by a saturating GABA concentration, a result that further supports their relative selectivity for α5GABAA receptors. GABA-evoked currents have two major components: a rapidly desensitizing peak current and a slowly

decaying steady-state current, each with distinct pharmacological and kinetic properties

(Banks and Pearce 2000; Yeung et al. 2003). Although saturating GABA concentrations activate a heterogeneous receptor population, peak current receives large contributions from synaptic receptors, as indicated by the preferential reduction of peak current by synaptic GABAA receptor blockers, such as gabazine (Yeung et al. 2003; Bai et al. 2001).

The steady-state current results from the transition of peak current receptors into the non- conductive desensitized state. Although extrasynaptic GABAA receptors are primed to contribute to the steady-state current (through their slow-desensitizing properties), synaptic receptors also contribute (Li and Akk 2015). Thus, the potential effects of α5-

NAMs may be masked by the high-conductance synaptic GABAA receptors. Altogether, the lack of α5-NAM effect on the peak and steady-state current supports the relative selectivity of α5-NAMs.

α5-NAMs did not inhibit mIPSCs, consistent with the notion that synaptic GABAA receptors are not affected by these agents. mIPSCs are primarily generated by synaptic

GABAA receptors containing α1-3 subunits, and they are activated by the vesicular release of GABA (Farrant and Nusser 2005). Our findings are consistent with those of other studies using pharmacological and genetic approaches, which did not detect an

α5GABAA receptor contribution to synaptic currents (Bonin et al. 2007; Caraiscos et al.

2004). However, previous studies have also demonstrated that α5GABAA receptors are expressed in synaptic regions on the dendrites of hippocampal pyramidal cells, where they contribute to slowly decaying synaptic currents (Serwanski et al. 2006; Vargas-

Caballero et al. 2010; Hausrat et al. 2015). The receptor heterogeneity underlying mIPSCs together with the limited efficacy of α5-NAMs may contribute to this lack of effect

we observed on mIPSCs, especially in terms of the decay time. Synaptic GABAA receptors, α5GABAA receptors, and extrasynaptic GABAA receptors containing the δ subunit all contribute to mIPSCs, which may mask the potential effect of α5-NAMs on decay time (Sun et al. 2018; Farrant and Nusser 2005; Hausrat et al. 2015).

Our results provide several insights into the selection and development of α5-NAMs for future clinical trials. Given that all α5-NAMs share similar efficacy for inhibiting tonic current in primary neurons, factors other than efficacy are worthy of consideration. First, potency may be just as important as efficacy. For drugs with lower potency, a higher concentration in the brain is required to achieve a desired effect. However, the required high concentrations may be difficult to achieve and may lead to unwanted side-effects.

Basmisanil’s maximal effective concentration for inhibiting the tonic current was greater than the four other compounds, which may suggest a lower potency. A low potency for basmisanil might have contributed to its lack of efficacy for participants with Down syndrome, as the concentration of basmisanil in the brain may have been insufficient to achieve an observable effect. Second, the selectivity for GABAA receptor subtypes is another factor to consider. For example, the development of L-655,708 was terminated because of anxiogenic effects in rodents, which arose from non-selective inhibition of

GABAA receptor subtypes other than the α5GABAA receptors (Atack et al. 2006; Navarro et al. 2002). In addition, toxicity must also be contemplated. A clinical study of α5IA was terminated because of renal toxicity (Atack 2010). Finally, novel approaches might be needed to enhance the efficacy at inhibiting α5GABAA receptors. The newly developed

S44819, a competitive antagonist that is selective for α5GABAA receptors, demonstrated selective effect on tonic current and pro-cognitive effects in rodents (Etherington et al.

2017). Future studies would be of interest to determine whether S44819 is more effective than α5-NAMs at inhibiting tonic current.

In conclusion, the α5-NAMs investigated in this study similarly inhibited the tonic current at their most efficacious concentrations without any effect on synaptic current.

Factors besides efficacy including potency, selectivity, and toxicity, as well as novel approaches to inhibit α5GABAA receptors, are worthy of consideration in the development and selection of more effective α5-NAMs for new indications. Such indications include perioperative neurocognitive disorder, as preclinical work has demonstrated that a single anesthetic exposure can trigger a sustained increase in the function of α5GABAA receptors which may contribute to persistent cognitive deficits. Consequently, reducing the function of α5GABAA receptors alleviates the post-anesthetic cognitive deficits (Zurek et al. 2014). Altogether, despite the challenges faced in recent clinical trials, the development of α5-NAMs show promise and will continue, as the hyperactivity of

α5GABAA receptors is implicated in many devastating neurological and psychiatric disorders.

Chapter 5: General Discussion 5.1 Summary

The main objectives of my thesis were to: 1) characterize and compare the effects of

α5-NAMs on the tonic current generated in primary hippocampal neurons, and 2) to compare their effects on a GABA-evoked (1 mM) current and on mIPSCs. Our study is the first to characterize and compare the effects of α5-NAMs on the tonic current generated in murine hippocampal neurons. Moreover, we demonstrated that the five α5-

NAMs studied here all similarly inhibited the tonic current. To confirm their relative selectivity for extrasynaptic α5GABAA receptors, we next tested the α5-NAMs on currents that are predominantly driven by synaptic GABAA receptor populations. Our findings showed that α5-NAMs did not modulate synaptic currents, suggesting that α5-NAMs’ relative selectivity for α5GABAA receptors is preserved in hippocampal neurons.

Our data provides novel insights for future α5-NAM development. As described earlier, α5-NAMs have yet to be adopted into clinical practice despite promising preclinical results. The current study showed that α5-NAMs similarly inhibit the tonic current which is the primary function of α5GABAA receptors. However, the most efficacious concentration varied up to 100-fold among the α5-NAMs. Therefore, rather than selecting compounds based solely on their efficacy at the α5 receptor subtype, other factors such as potency, bioavailability and toxicity may be of equal importance in selecting a compound for future trials.

5.2 Future directions

In our hippocampal cell culture model, we demonstrate that α5-NAMs preferentially inhibit the tonic current without influencing synaptic GABAA receptor-mediated function.

Although this demonstrates that α5-NAMs possess a relative selectivity for α5GABAA receptors, it does not rule out effects at other extrasynaptic receptor subtypes that also contribute to the tonic current. Thus, comparing α5-NAM effects in cell cultures derived from mice that lack the α5 subunit (α5 -/-) would provide additional insight around α5-

NAM selectivity in primary neurons.

To further support our findings that α5-NAMs do not modulate currents evoked by a saturating concentration of GABA or miniature inhibitory postsynaptic currents, a positive control should be incorporated into the experimental design. This could be accomplished by applying the β-carboline, DMCM, which is an inverse agonist of the benzodiazepine site at all GABAA receptors (Sieghart and Savic 2018). Moreover, it has been shown that the application of DMCM can reduce the amplitude of GABA-evoked currents and accelerate the decay time of mIPSCs (Levi et al. 2015; Tietz et al. 1999).

One of the limitations of the in vitro culture model used in the current study is that it does not account for sex differences in GABAA receptor function. Specifically, expression of δ subunit-containing GABAA receptors in the hippocampus have been shown to be influenced by the ovarian cycle in female mice (Cushman et al. 2014). High levels of the hormone progesterone were found to enhance δ subunit expression and increase tonic inhibition (Wu et al. 2013). Therefore, sex differences in the amplitude of the tonic current

could lead to α5-NAMs being less effective in females due to an increase in the tonic current.

The findings presented in my thesis may provide insight into why some clinical trials have failed. For example, basmisanil and S44819 were both well-tolerated in humans but failed due to a lack of efficacy. One factor that may have contributed to the lack of efficacy in these clinical trials is bioavailability. A compound with low bioavailability may not reach the necessary brain concentration to achieve cognitive-enhancing effects. The results of my thesis showed that although all α5-NAMs similarly inhibited the tonic current, basmisanil had the highest maximally effective concentration. Therefore, the lack of efficacy detected for basmisanil could be related to insufficient drug concentrations in the brain.

Overall, it is important to remember that α5-NAMs have shown pro-cognitive effects in both non-human primates and humans (α5IA reversed an ethanol-induced memory impairment in humans) (Sieghart and Savic 2018). This evidence suggests that α5-NAM efficacy may not be the primary issue in progressing these compounds to clinical use.

Heterogeneity in the symptoms and underlying pathophysiology of disorders, such as schizophrenia, are other factors that could obscure the effects of α5-NAMs (Jacob 2019;

Brennand et al. 2014). Moreover, there are still several indications including Alzheimer’s disease and depression that have not been studied in clinical trials. Ultimately, given the role of α5GABAA receptors in cognition and their multitude of related disorders, α5-NAM clinical testing and development will continue to be refined.

5.3 Conclusions

In conclusion, the five α5-NAMs investigated in the current study similarly inhibited the tonic current while having no effect on currents evoked by a saturating concentration of GABA or mIPSCs. Notably, the concentration that caused the maximum inhibition of the tonic current ranged from 10 nM to 1 μM. This data should inform future clinical trials that selecting compounds based on efficacy may be an ineffective approach. Instead, factors that differ among α5-NAMs such as potency, bioavailability and toxicity should be of greater consideration.

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