Comparing the Effects of α5GABAA Receptor Negative Allosteric Modulators on Inhibitory Currents in Hippocampal Neurons by Marc Anthony Manzo A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Physiology University of Toronto © Copyright by Marc Anthony Manzo 2020 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 ii among α5-NAMs, factors such as potency, selectivity, and toxicity should be emphasized in the development and selection of α5-NAMs for clinical trials. iii 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. iv 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. v 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. vi 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 vii 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 viii 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 ix 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 x 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 xi 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) xii 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 xiii PKC Protein kinase C PND Perioperative neurocognitive disorder TEA Triethylammonium TTX Tetrodotoxin xiv 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.