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Chapter 1 - Introduction and Overview AN ANALYSIS OF APOPTOTIC AND AUTOPHAGIC GENES IN NOVEL DROSOPHILA MODELS OF PARKINSON DISEASE by © Peter Githure M'Angale A Thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology Memorial University of Newfoundland January 2017 St. John's Newfoundland and Labrador Abstract Parkinson disease (PD) is the most common neurodegenerative movement disorder in humans and is strongly associated with the selective loss of dopaminergic (DA) neurons. The clinical features include motor dysfunction, resting tremor and in some cases non- motor features such as autonomic, cognitive and psychiatric disorders. The neuropathologic hallmarks are Lewy bodies and Lewy neurites in surviving neurons which are composed of proteinaceous inclusions comprised of α-synuclein, ubiquitin and other proteins. Pathological mechanisms implicated in PD include aberrant protein aggregation, mitochondrial dysfunction, oxidative stress, and failed cellular processes such as apoptosis, autophagy, proteasomal pathway, and several cellular stressors. To analyse the implication and contribution of some of these cellular processes, we altered the expression of the; classical apoptotic genes namely Buffy and Debcl; autophagy genes Atg6 and Pi3K59F; the antiapoptotic transmembrane Bax inhibitor-1 containing motif (TMBIM) family Bax inhibitor-1, Lifeguard and GHITM; mitochondrial genes such as HtrA2, MICU1, porin/VDAC, Pink1 and parkin; and an increased disease risk gene pyridoxal kinase in DA neurons and in the developing eye. We show that the altered expression of key genes can either be detrimental or beneficial to the health of DA neurons as determined by lifespan and locomotor function, in addition to supportive results obtained from biometric analysis of phenotypes from the neuron rich eye. We found that the overexpression of Buffy, the sole pro-survival Bcl-2 homologue in Drosophila could suppress the loss of function-induced phenotypes. ii Acknowledgements I am forever grateful to Dr. Brian E. Staveley for seeing the potential in me and granting me the opportunity to pursue graduate studies, first as a master’s student and secondly as a doctorate student. He has been a solid rock during my period here, his generosity and guidance have been invaluable. I am also grateful to the members of my supervisory committee, Dr. Dawn Marshall and Dr. Kapil Tahlan, for your support and input for my thesis. Additionally, I would like to thank past and current members of the Staveley lab, your generosity and bail outs during times of ageing or climbing crises was much appreciated. Heartfelt gratitude to my wife Margaret Nduta and sons Michael Mugambi, Tamwa Karega and Imani Gitonga for their steadfast belief in me, even when the urge to demand I come back home was highest, they still sacrificed their needs and granted me the strength that kept me going. I would not have made it this far without their unwavering and overwhelming love, kindness and support. To my mom who is definitely going to ululate when this is over. To my sisters and friends, thank you for your love and kindness. iii Dedication This thesis is dedicated to my late father, for whom this would have been a proud moment, a story to tell his clique about the achievements of his son. I know he has a big smile right now and may God rest his soul in eternal peace. iv Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iii Dedication .......................................................................................................................... iv Table of Contents ................................................................................................................ v List of Figures .................................................................................................................. xiv List of Appendices .......................................................................................................... xvii List of Symbols and Abbreviations ................................................................................ xviii Chapter 1 - Introduction and Overview .............................................................................. 1 Parkinson Disease ............................................................................................................ 1 Genetics of Parkinson disease ...................................................................................... 1 Drosophila models of Parkinson disease ..................................................................... 3 The α-synuclein model ......................................................................................................... 4 The Parkin model ................................................................................................................. 6 The Pink1 model................................................................................................................... 8 The HtrA2 model ................................................................................................................ 10 Mitochondrial dysfunction in Parkinson disease ........................................................... 12 Mitochondrial electron transport chain complexes in PD .......................................... 12 Mitochondrial permeability transition pore (mPTP) and PD ..................................... 15 Role of PD-linked genes in mitochondrial dysfunction ............................................. 16 Molecular mechanisms of Parkinson disease................................................................. 20 Neuronal apoptosis ..................................................................................................... 20 Extrinsic death pathway ..................................................................................................... 21 Intrinsic death pathway ..................................................................................................... 23 The role of apoptosis in PD ................................................................................................ 27 Endosomal-lysosomal system .................................................................................... 28 Autophagy .......................................................................................................................... 29 Mitophagy .......................................................................................................................... 32 The ubiquitin-proteasome system .............................................................................. 33 Oxidative stress .......................................................................................................... 35 v Dopamine synthesis ........................................................................................................... 36 Mitochondrial ROS generation .......................................................................................... 38 Endoplasmic reticulum stress and ROS in PD ............................................................... 39 Unfolded protein response pathway ........................................................................... 40 ER ROS production ................................................................................................... 43 ER stress link to PD ................................................................................................... 44 ER-Mitochondrial interaction and calcium homeostasis ........................................... 45 ER-Golgi axis and Golgi stress in PD ........................................................................ 48 Goals of study ................................................................................................................ 48 References ...................................................................................................................... 50 Co-Authorship Statement .................................................................................................. 63 Chapter 2 - The Bcl-2 homologue Buffy rescues α-synuclein-induced Parkinson disease- like phenotypes in Drosophila .......................................................................................... 64 Abstract .......................................................................................................................... 65 Background .................................................................................................................... 66 Materials and Methods ................................................................................................... 69 Drosophila media and culture .................................................................................... 69 Drosophila stocks and derivative lines....................................................................... 69 Ageing assay .............................................................................................................. 70 Climbing assay ........................................................................................................... 70 Scanning electron microscopy of the drosophila eye ................................................. 70 Results ...........................................................................................................................
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