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Contribution of the Pannexin-1 Channel to Amyloid-Β Induced

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Contribution of the Pannexin-1 Channel to Amyloid-Β Induced Contribution of the Pannexin-1 Channel to Amyloid-β Induced Synaptic Dysfunction By Albert Yeung A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirements of the degree of MASTER OF SCIENCE Department of Pharmacology and Therapeutics University of Manitoba Winnipeg, Canada Copyright © 2018 by Albert Yeung ABSTRACT Alzheimer’s disease is a progressive neurodegenerative disease associated with the buildup of amyloid-β protein. Toxic amyloid-β oligomers (AβOs) are known to impair the function of excitatory synapses of the hippocampus, leading to deficits in synaptic plasticity, loss of synaptic structure, and eventual neuronal death. Pannexin-1 (Panx1) is a membrane channel which has an emerging role in central nervous system physiology and pathophysiology. My thesis investigates the contribution of Panx1 to these AβO induced degenerative events by using fluorescence imaging, immunoblotting, and electrophysiology techniques on in vitro primary neuronal cultures and acute hippocampal slices. Here I demonstrate that the activity of Panx1 channels is facilitated by AβO treatment following the stimulation of NMDA receptors. However, KO of Panx1 failed to ameliorate AβO induced synaptic degeneration or deficits in long-term potentiation. i ACKNOWLEDGMENTS I would like to start by acknowledging my supervisor, Dr. Michael Jackson, who provided me with the opportunity to join his lab and conduct the work contained in this thesis. His expertise and mentorship were invaluable to my growth as a researcher, and in more than one occasion helped guide me through the inevitable challenges of scientific inquiry. I would also like to acknowledge my advisory committee members Dr. Tiina Kauppinen and Dr. Tabrez Siddiqui, not only for their guidance in this project, but also for the collaborative opportunities that they have offered me over these past two years. They have opened my eyes to many amazing aspects of neuroscience research. Thank you to Natalie Lavine, Ying Wang, and Chetan Patil, who provided technical support in this project, including the preparation and maintenance of primary neuronal cultures. I would also like to thank Dr. Satchin Katyal for accommodating my use of the Cytation 5, and to Asha Sinha, Marina Mostafizar, and Haynes Yuan in helping me set up these experiments. I am grateful for the help provided by Benjamin Karimi and Shreya Dhume in performing the mouse behavioural tests. It was truly the combined efforts of everyone mentioned that made this thesis possible. I was lucky to have wonderful lab mates, who made lunch time, coffee break and bench- side (or rig-side) chats something to look forward to. First and foremost, I extend my gratitude to my good friends Shubham Tanwar and Harish Gangadharappa, who made me feel truly welcome when I first joined the lab two years ago. Thank you to Chetan and Prajwal Raghunatha, who have helped me out on numerous occasions in and out of the lab, even to this day. And of course, I would like to thank Natalie, who’s advice on all things biochemistry was only trumped by her infectious laugh. ii It is also important for me to acknowledge some of the people behind the scenes. Karen Donald was immensely helpful in navigating departmental requirements, answering my questions and fulfilling any requests I had. I am thankful for the opportunity to undertake this project through the MD/MSc program which was run by Dr. Kent Hayglass and Kim Ormiston and now Dr. Mark Nachtigal and Allison Birch. Funding through the MD/MSc program was provided by the CIHR. Lastly, I am privileged to have a great supporting cast of family and friends. Behind this accomplishment is the unconditional support that my parents have given me over the years. I am also deeply grateful for my partner Linda Lam, who has been one of my greatest role models in this journey and an unwavering source of love and strength. iii TABLE OF CONTENTS ABSTRACT .............................................................................................................................. i ACKNOWLEDGMENTS ...................................................................................................... ii TABLE OF CONTENTS ...................................................................................................... iv LIST OF TABLES ................................................................................................................. xi LIST OF FIGURES ............................................................................................................... xi LIST OF ABBREVIATIONS ............................................................................................. xiv CHAPTER 1: INTRODUCTION .......................................................................................... 1 1.1 Introduction to Alzheimer’s Disease ......................................................................... 1 1.2 Hippocampal dependent plasticity, learning, and memory .................................... 2 1.2.1 Excitatory synapses and glutamate signalling ....................................................... 3 1.2.2 NMDA receptors and LTP in the hippocampus .................................................... 6 1.2.3 Calcium signalling, protein phosphorylation, and bidirectional plasticity............ 8 1.2.4 Maintenance of LTP requires gene transcription and translation ....................... 10 1.2.5 Synaptic plasticity is associated with dynamic structural changes ..................... 10 1.2.6 The synaptic plasticity and memory hypothesis ................................................. 11 1.2.7 Synaptic plasticity and memory in other regions of the hippocampus ............... 13 1.3 Alzheimer’s disease pathogenesis ............................................................................ 14 iv 1.3.1 AD as a proteinopathy: amyloid-β and hyperphosphorylated tau ....................... 14 1.3.2 Amyloid-β cascade hypothesis ............................................................................ 17 1.3.3 AD mouse models and hippocampal synapse dysfunction ................................. 19 1.3.4 Synaptotoxicity is driven by soluble amyloid-β oligomers ................................. 20 1.3.5 Amyloid-targeted therapies ................................................................................. 20 1.4 Chronic glutamate excitotoxicity model of AD ...................................................... 22 1.4.1 Effects on glutamate release and reuptake .......................................................... 23 1.4.2 Post-synaptic binding of amyloid-β oligomers ................................................... 24 1.4.3 Amyloid-β Oligomers and NMDA receptors ...................................................... 25 1.4.4 Calcium dyshomeostasis in synaptic dysfunction and neurodegeneration ......... 26 1.5 Therapeutic development in AD ............................................................................. 27 1.5.1 NMDA receptor blockade in AD ........................................................................ 28 1.5.2 NMDA receptor secondary currents: a possible contributing mechanism to AD? . ............................................................................................................................. 29 1.6 Introduction to Pannexin-1: Structure and function ............................................ 30 1.6.1 Panx1 Structure ................................................................................................... 31 1.6.2 Panx1 permeability and conformational states.................................................... 33 1.6.3 Panx1 pharmacology ........................................................................................... 36 1.7 Panx1 channel regulation and activation mechanisms ............................................ 37 1.7.1 Direct activation .................................................................................................. 40 v 1.7.2 Receptor-mediated activation .............................................................................. 41 1.7.3 Src Family Kinases.............................................................................................. 41 1.7.4 Calcium signalling............................................................................................... 42 1.8 Panx1 in CNS physiology and pathophysiology ........................................................ 43 1.8.1 Panx1 expression in the CNS .............................................................................. 43 1.8.2 Activity dependent recruitment of Panx1 channels in seizure models................ 44 1.8.3 Panx1 link to NMDA channels and excitotoxicity.............................................. 45 1.8.4 Panx1 role in synaptic plasticity and memory .................................................... 46 1.8.5 Panx1 in Alzheimer’s Disease............................................................................. 47 1.9 Experimental aims and hypothesis ......................................................................... 48 CHAPTER 2: MATERIAL AND METHODS................................................................... 50 2.1 Production of secreted amyloid-β oligomers .......................................................... 50 2.2 Validation of amyloid-β oligomers in 7PA2-CM ................................................... 50 2.3 Experimental animals..............................................................................................
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