Assessing the Excitability Changes of DRG Neurons in Models of Diabetic Neuropathy Katerina Kaloyanova Kaloyanova A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy The University of Sheffield Faculty of Science Department of Biomedical Science April / 2021 ACKNOWLEDGEMENTS I wish to express my deepest gratitude to my supervisor Dr. Mohammed Nassar. From the day we met, he has shown unwavering belief in me and my potential. He always prioritised my health and mental wellbeing and made me believe in myself, even during my most difficult times of self-doubt. Since day one, he has treated me as an equal colleague and listened and considered my ideas and questions. Despite his extremely busy schedule, he always found time to help with any difficulties I had, both personal and professional. Because of his never- ending patience and support, I never felt alone in my challenges throughout the last four years. Dr. Nassar’s extensive scientific knowledge and professionalism made this work what it is, but his kindness, enthusiasm and profound belief in my abilities made me the scientist I am today. I feel truly lucky and blessed to have had the best supervisor one could have asked for. Thank you! You made me a better scientist and person and for that I will be eternally grateful. I am extremely grateful to my advisors Dr. Liz Seward and Prof. Walter Marcotti for their encouragement as well as expert guidance and constructive remarks, which helped refine and advance this research. I would also like to extend my sincerest thanks to Zainab Mohammed for training and helping me in the first months of this degree and trusting me with continuing some of her own work. I would also like to thank Nadya and Jiahao, whose contribution to the data analysis for Chapter 5 was instrumental to the results presented here. During this degree, I was fortunate to meet some exceptional people: Eve, Judit and Mohamed, who were always around to lend a listening ear, for a laugh or just a chat. With you around, this experience was enjoyable despite the difficulties. My special thanks are extended to Mohamed – thank you for checking on me and keeping me company during the long hours of calcium imaging experiments, for your endless encouraging and comforting words and for supporting me through my worries – big or small. I wish to also thank my closest friends Elisabetta, Beth and Ainhoa who have been by my side since we all started our first science degrees together. Thank you all for your understanding and positive vibes. Ainhoa, we went through this hand in hand and you understood my struggles better than anyone. Thank you for the numerous coffees, chats, advice and your sincere friendship. My sincere appreciation goes to Patrick Wood, for all his love and continuing support. Thank you for encouraging me to be ambitious and embark on this journey. Thank you for helping me practice all my talks and presentations, for listening to my venting, for making me laugh so hard I cry and for always taking care of me when I could not do it myself. You are my home and family here. Finally, I would like to extend my deepest gratitude to my loving family in Bulgaria. Thank you for encouraging your only child to begin her education in a country so far away from you 8 years ago. Thank you for all your sacrifices, all your phone calls, all the postcards and parcels you have sent. Thank you for reminding me of how far I have come in times when I felt that I could not continue. Your tremendous belief and trust empower me to achieve things I never believed I could. This work is fully dedicated to you – my parents. I love you both. ABSTRACT Diabetic neuropathy (DN) is the most common type of neuropathy, with 25-50% of patients experiencing pain at any stage of its progression. However, the understanding of the pathophysiology of DN is still incomplete. No disease-modifying treatments have yet been developed and pain is only treated symptomatically with drugs targeting the central nervous system (CNS). These have limited efficacy and can produce serious side effects. Research into novel analgesics is turning away from the CNS and now focusing on the peripheral nervous system (PNS) and more specifically the pain-sensing neurons (nociceptors) to produce better-targeted and safer treatments. However, to this end, a suitable medium-to-high throughput screening tool is needed, able to discriminate nociceptors from non-nociceptors in a heterogeneous cell population, such as the dorsal root ganglia (DRG). Previously, our lab showed a link between the veratridine (VTD)-induced oscillatory (OS) and slow decay (SD) calcium response profiles with nociceptors and non-nociceptors, respectively. Here, we validated the VTD-response profiles as broad functional markers of these DRG subpopulations using mice with genetically ablated nociceptors (1.8-DTA). Then, by using voltage-gated sodium channel (VGSC) blockers with different specificities, we demonstrated that the VTD- Ca2+ imaging assay can be used as a drug screening platform for drugs, individually or in novel combinations. The VTD-Ca2+ imaging assay was then applied in combination with nociceptive agonists to investigate the excitability changes in distinct neuronal subpopulations during DN. In db/db mice, during the early metabolic phase, small- to medium-diameter nociceptors showed 1.4 -fold increased sensitivity to CAP and 1.2-fold increased VGSC excitability. In the late, NEU phase, small-diameter nociceptors showed increased sensitivity to CAP (1.6-fold) and increased VGSC excitability (1.4-fold), whereas medium nociceptors show decreased sensitivity to AITC (1.8-fold) . This is the first study to characterise phase- and subpopulation- specific excitability changes in the well-established db/db mouse model. Collectively, these results point to specific subpopulations of DRG neurons affected during the early and late stages of DN. These findings could aid in the better targeting of novel therapies for the treatment of DN and pain. Furthermore, we have demonstrated that the VTD-Ca2+ imaging assay can be applied as a tool for the characterisation of excitability changes in distinct DRG subpopulations during neuropathological conditions such as DN. DECLARATION I declare that the work presented in this thesis is my own original research and has been composed entirely by myself. All supporting literature, resources, and collaborative contributions have been acknowledged clearly. Part of this work has been published prior to submission of this thesis under the title: “An unbiased and efficient assessment of excitability of sensory neurons for analgesic drug discovery” PAIN, 2020 Signature: Katerina Kaloyanova Kaloyanova, April 2021 LIST OF ABBREVIATIONS •OH Superhydroxyl AGE Advanced glycation end products AITC Allyl isothiocyanate AMH Aδ-mechano-heat AP Action potential AR Aldose reductase ATP Adenosine 5’-triphosphate AUC Area under curve CAP Capsaicin CGRP Calcitonin gene-related peptide CIP Channelopathy-associated insensitivity to pain CNS Central nervous system DAG Diacylglycerol DN Diabetic neuropathy DoA Days of age DRG Dorsal root ganglia eGFP Enhanced green fluorescent protein FADH2 Flavin dinucleotide + hydrogen FEPS Familial episodic pain syndrome FFAs Free fatty acids GECI Genetically encoded calcium indicator GlcNac 5-diphosphate-N-acetylglucosamine GLUT Glucose transporter GoF Gain of function GP Glutathione peroxidase H2O2 Hydrogen peroxide HFD High fat diet IB4 Isolectin B4 ID Intermediate decay IEM Inherited erythromelalgia IENF Intraepidermal nerve fibre iPSC Induced pluripotent stem cell IR Insulin resistance KO Knock-out LDL Low density lipoproteins LoF Loss of function LOX1 Oxidized-LDL-receptor 1 LTMR Low-threshold mechanoreceptors NADH Nicotinamide dinucleotide + hydrogen NADPH Reduced nicotinamide adenine dinucleotide phosphate NCV Nerve conduction velocity NF200 Neurofilament 200 NOD Non-obese diabetic OS Oscillatory P2X3 P2X purinoceptor 3 PARP Poly(ADP-ribose) polymerase PC12 Pheochromocytoma cells PDN Painful diabetic neuropathy PKA Protein kinase A PKC Protein kinase C PNS Peripheral nervous system RAGE Receptor for advanced glycation end products RD Rapid decay RMP Resting membrane potential ROS Reactive oxygen species SC Schwann cell scRNA-seq Single-cell PCR and RNA sequencing SD Slow decay SNRI Serotonin and noradrenaline reuptake inhibitors STZ Streptozotocin T1D Type 1 diabetes T1DN Type 1 diabetic neuropathy T2D Type 2 diabetes T2DN Type 2 diabetic neuropathy TCA Tricyclic antidepressants TH Tyrosine hydroxylase TLR4 Toll-like receptor 4 TRP Transient receptor potential-activated TTX Tetrodotoxin TTX-R Tetrodotoxin resistant TTX-S Tetrodotoxin sensitive VGCC Voltage-gated calcium channels VGKC Voltage-gated potassium channels VGSC Voltage-gated sodium channel VTD Veratridine WoA Weeks of age TABLE OF CONTENTS ACKNOWLEDGEMENTS ____________________________________________________________ II ABSTRACT _______________________________________________________________________ III DECLARATION ____________________________________________________________________IV LIST OF ABBREVIATIONS ___________________________________________________________ V CHAPTER 1: INTRODUCTION ________________________________________________________ 1 1.1. THE NERVOUS SYSTEM AND DORSAL ROOT GANGLIA (DRG) NEURONS ____________________________ 2 1.1.1. DRG NEURONS: HETEROGENEITY AND CLASSIFICATION ______________________________________ 3 1.1.2. DRG NEURONS EXCITABILITY _______________________________________________________