Age-dependent hippocampal network dysfunction in a mouse model of alpha-synucleinopathy Clare Tweedy Thesis submitted for the degree of Doctor of Philosophy at Newcastle University September 2018 Supervisors: Dr Fiona LeBeau, Dr Gavin Clowry, Dr Amy Reeve, Dr John-Paul Taylor Industrial Supervisors: Dr Fiona Randall and Dr Peter Atkinson ii Abstract Aggregation of the protein alpha-synuclein (ASYN) is a key pathological feature of the alpha-synucleinopathies, a group of diseases including Lewy body dementia and Parkinson’s disease. A common symptom of alpha-synucleinopathy is cognitive dysfunction, and impairment in hippocampal gamma-frequency oscillations may underlie some of the cognitive deficits associated with ASYN pathology. The Thy-1 A30P mouse model overexpresses human mutant ASYN, with mice developing hippocampal spatial memory impairment by 12 months (Freichel et al. 2007). The aim of this thesis was to explore age-related hippocampal network changes in 2-4 month (A30P2+) mice and 10-13 month (A30P10+) mice to assess the effect of overexpression of mutant ASYN on hippocampal network activity in vitro. Using acute brain slice preparations of isolated hippocampi, A30P2+ mouse slices were found to exhibit excitatory/inhibitory network changes in region CA3 in the form of increased spontaneous sharp wave amplitude, increased frequency and amplitude of inhibitory postsynaptic potentials, and increased power of kainate-induced gamma-frequency oscillations. Immunohistochemistry revealed an increase in the density of parvalbumin- positive interneurons alongside a decrease in calbindin-positive interneurons. This change was accompanied by a more depolarised resting membrane potential in A30P2+ mouse CA3 pyramidal cells, and a sensitivity to interictal discharges in response to either kainate receptor agonism or GABAA receptor antagonism. With ageing, levels of excitability in A30P10+ mice were comparable to WT10+ mice. A30P10+ mice instead exhibited an impairment in cholinergic-induced, but not kainate- induced or spontaneous, gamma-frequency network oscillations. While mitochondrial dysfunction was not detectable with COX/SDH histochemistry until 15+ months in A30P mice, A30P10+ mice did show increased immunoreactivity for Iba1+ microglia. An environment of inflammation and excitotoxicity may be present in older A30P mice as a result of early network hyperexcitability, and this thesis explores early network changes in A30P mice and the wider dysfunction that follows. iii iv Acknowledgements I would like to start by extending my gratitude to my supervisory team at Newcastle University for all their support and guidance over the years: Dr Fiona LeBeau, Dr Gavin Clowry, Dr Amy Reeve, and Dr John-Paul Taylor. A particular thank you must go to Fiona for being so supportive of me and for the good company during my time here. I have developed both as a person and as a scientist over the past four years, and to all of you I am grateful. Thank you to Gavin for all your help with immunohistochemistry, Amy for your help with the mitochondrial experiments, and John-Paul for your help with understanding DLB! A huge thank you to my industrial supervisors Dr Fiona Randall and Dr Peter Atkinson, for making an industrial placement as part of my PhD a successful venture! I also extend my gratitude to Eisai and the Medical Research Council for funding the MRC Industrial CASE Studentship. Without the funding and support this research really would not have been possible. I learned so much during my time in the US, and thank you in particular to Fiona for taking me under your wing while I was there! I learned some amazing scientific techniques, and also made some lasting friendships. Thank you to everyone at Eisai’s AiM Institute and the other interns including Iain for making my experience a memorable one. I would also like to acknowledge the guidance and contributions of the members of my progress panel for keeping me on track: Prof. Mark Cunningham, Dr Elizabeta Mukaetova-Ladinska, and Dr Sasha Gartside. Getting some perspective and learning how to defend my experimental choices was incredibly helpful! All the work presented in this thesis is my own, with the exception of assistance from undergraduate and Masters students that I have supervised and worked with over the years. Megan Ingham, Keir Cox, Elizaveta Olkhova, and Joshua Curry assisted with data collection for a number of electrophysiological experiments detailed throughout this thesis. Nathan Kindred and Christopher Williams assisted with data collection for electrophysiological experiments detailed in Chapter 4 of this thesis, as well as assisting in the immunohistochemical experiments detailed throughout. Pippa Gleave assisted with immunohistochemical data collection for Chapter 3. I am grateful to the assistance you all provided in the data collection process. I must also extend my gratitude to Karen, for keeping our labs running from the technical side, and always being happy to help me out when I needed it! v A PhD can be an incredibly stressful experience, and so I would like to acknowledge the friendship and support provided by the members of the office and lab over the years who I have come to call good friends. A particular thank you must go to Felix Chan, for being the greatest friend one could wish for, even though you’re now across the pond! Myrto: for your constant support, friendship, and supply of baklava. Jean, Anderson, Tamara, Ashan, Beth, Mark, Nelson: I will miss lunch club. Thank you for many laughs and making the office a fun place to work in. Even when you all judge me for my unhealthy consumption of a certain carbonated beverage, you’ve all been amazing in keeping me sane and motivated and I can’t thank you all enough! To Grace for the endless support and friendship, and to those who were once a part of the office and lab but are now spread across the country (and even the world): Yingdi, John, Gemma, Joe, Chris T, Claire. To Abby, my Boston tour guide and now long-distance friend – thank you for all your support during the writing process. My family have been incredibly supportive of me during the PhD process: from dealing with my mood swings after a long day of experimenting or thesis writing, to being unsure why I am so excited about finding statistical significance but being excited for me all the same! Mum and Dad: I did it! I am indebted to you for your constant support, love, and advice. As much as I know you’ve been counting down the years (!) till I finally get a job, hearing how proud you are of me certainly kept me going. Thank you for also taking me back in home to write my thesis after my trip to Boston when I returned homeless and disorientated! Having my cat Pebbles to assist me with the writing process really brightened my days. A special thank you also to my sister Carol, Rob, Faye, and my niece Charlotte: I think I had more fun playing with your toys to destress from thesis writing than you did! And I can’t wait to do the same with my future nephew to destress from whichever jobs I end up in! To my Grandma for all your support, and also my late Nana and Grandad who I know would have loved to hear all about my research and be at my graduation. Thank you all. The only person that remains left to thank is Adam, for your endless support and reassurance. I couldn’t have done this without you. Thank you. vi Conference Presentations Work from this thesis was presented at the following conferences: British Neuroscience Association (Edinburgh, UK) April 2015 Poster: “The role of NMDA receptors and synaptic plasticity in the induction and build- up of hippocampal gamma-frequency oscillations in vitro”. Federation of European Neuroscience Societies (Copenhagen, Denmark) July 2016 Poster: “Hippocampal gamma-frequency oscillations and hyperexcitability in a mouse model of alpha-synucleinopathy” North East Postgraduate Conference (Newcastle, UK) November 2016 Poster and Oral Presentation: “Hippocampal gamma-frequency oscillations and hyperexcitability in a mouse model of alpha-synucleinopathy” 13th International Conference on Alzheimer’s & Parkinson’s Diseases (Vienna, Austria) March 2017 Poster: “Impaired hippocampal gamma-frequency oscillations and hyperexcitability in a mouse model of alpha-synucleinopathy” Society for Neuroscience (Washington D.C., USA) November 2017 Posters: “Early hippocampal network excitability in a mouse model of alpha- synucleinopathy” and “Impaired hippocampal gamma-frequency oscillations and mitochondrial dysfunction in a mouse model of alpha-synucleinopathy” Federation of European Neuroscience Societies (Berlin, Germany) July 2018 Poster: “Hippocampal hyperexcitability precedes gamma-frequency oscillation impairment in a mouse model of alpha-synucleinopathy”. vii Publications Work expanded on in Chapter 5 has been published with shared first authorship: Robson E, Tweedy C, Manzanza N, Taylor JP, Atkinson P, Randall F, Reeve A, Clowry GJ, LeBeau FEN. Impaired Fast Network Oscillations and Mitochondrial Dysfunction in a Mouse Model of Alpha-synucleinopathy (A30P). Neuroscience (2018) 377: 161- 173. DOI: 10.1016/j.neuroscience.2018.02.032. viii Table of Contents Abstract ........................................................................................................................ iii Acknowledgements ....................................................................................................... v Conference Presentations ..........................................................................................