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Parkinson's Disease INVESTIGATING AUTOPHAGY DYSFUNCTION INDUCED BY A PARKINSON’S DISEASE-CAUSING MUTATION IN VPS35 by Abir Ashfakur Rahman A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomolecular Sciences Boise State University December 2018 © 2018 Abir Ashfakur Rahman ALL RIGHTS RESERVED BOISE STATE UNIVERSITY GRADUATE COLLEGE DEFENSE COMMITTEE AND FINAL READING APPROVALS of the dissertation submitted by Abir Ashfakur Rahman Dissertation Title: Investigating Autophagy Dysfunction Induced by a Parkinson’s Disease-Causing Mutation in VPS35 Date of Final Oral Examination: 30 November 2018 The following individuals read and discussed the dissertation submitted by student Abir Ashfakur Rahman and they evaluated his presentation and response to questions during the final oral examination. They found that the student passed the final oral examination. Brad E. Morrison, Ph.D. Chair, Supervisory Committee Allan Albig, Ph.D. Member, Supervisory Committee Matthew L. Ferguson, Ph.D. Member, Supervisory Committee Julia Thom Oxford, Ph.D. Member, Supervisory Committee The final reading approval of the dissertation was granted by Brad E. Morrison, Ph.D., Chair of the Supervisory Committee. The dissertation was approved by the Graduate College. DEDICATION I would like to dedicate this dissertation to my mother, Lutfa Begum, and my father, Mujibur Rahman. I hope I have made you proud. iv ACKNOWLEDGEMENTS I would like to thank my thesis advisor, Dr. Brad Morrison, for being the mentor that he has been and guiding me every step of the way to my doctorate degree. I also want to thank my committee members, Dr. Allan Albig, Dr. Matt Ferguson and Dr. Julia Oxford, for providing me with guidance and giving me their valuable time and wisdom. I would like to extend my special gratitude to Beth Gee (Momma Beth) in the Biomolecular Sciences Graduate Programs administration, whose help and care has been priceless in the last four years. The same goes for Raquel Brown, Barb Jibben, Tracy Yarnell, Diane Smith, Cindy Keller-Peck, Shin Pu and Crystal Scott. Being at the Biomolecular Research Center was truly an amazing experience. I am grateful to all the faculty and staff of Department of Biomolecular Sciences, the Biomolecular Research Center and Department of Biological Sciences at Boise State University for their support and for giving me a sense of belonging, and for being the amazing human beings that they are. I would also like to especially acknowledge the Boise State University Health Services Center staff members and the Graduate College for being there when I so desperately needed help. I would like to thank Jennifer Aumeier, LMT, from BSU Health Services, and Dr. Toby Bingham and the amazing staff members at Life Chiropractic, for keeping me functional, when it seemed like my body would break apart. I would like to thank my friend Nabil Rahman for helping me with Adobe Illustrator. I am also thankful to all the friends I have made here at Boise. I cannot name v them all for the sake of space. Last but not least, I could not have done this without my amazing wife, Rebeka Sultana, my awesome sister, Tasnuva Rahman, my mom, Lutfa Begum, my dad, Mujibur Rahman and my auntie, Ismat Ara. I love you all and I hope I have made you proud. vi ABSTRACT Parkinson’s Disease (PD) is an idiopathic disorder with no known cure. With number of cases steadily rising around the world, it is imperative to turn to the underlying cellular and molecular mechanisms of the disease manifestation and neurodegeneration to craft novel modes of therapy. VPS35 is one of the few genes that have identified and definitively linked to familial PD. The particular mutation that has been associated is known to cause dysfunction of a key cellular process known as autophagy. This process is primarily responsible for clearance of unwanted, damaged or misfolded proteins, among other things. Our study reveals an important link between VPS35, PI3K-AKT signaling pathway and autophagy dysfunction that might be a potential mechanism of PD pathogenesis. We also address possible perturbed dopaminergic neurogenesis in the substantia nigra brain region as a second plausible disease mechanism, as well as a target for therapeutic intervention. vii TABLE OF CONTENTS DEDICATION ................................................................................................................... iv ACKNOWLEDGEMENTS .................................................................................................v ABSTRACT ...................................................................................................................... vii LIST OF TABLES ............................................................................................................. xi LIST OF FIGURES .......................................................................................................... xii LIST OF ABBREVIATIONS .......................................................................................... xiii CHAPTER ONE: A REVIEW OF THE CONTRIBUTIONS OF VPS35 MUTATIONS IN PARKINSON’S DISEASE ............................................................................................1 Introduction ..............................................................................................................1 Identified VPS35 Mutations ....................................................................................3 VPS35 as Part of the Retromer Complex and Related Functions ............................4 Proposed Mechanisms of VPS35 in Neuronal Dysfunction ....................................6 Current Model Organisms and Systems for Studying VPS35 .................................8 Cell Culture ..................................................................................................8 Mice ...........................................................................................................10 Rat ..............................................................................................................11 Drosophila Melanogaster ...........................................................................11 Saccharomyces Cerevisiae and Caenorhabditis Elegans ...........................12 Future Research Directions ....................................................................................13 viii CHAPTER TWO: ENHANCED HYALURONAN SIGNALING AND AUTOPHAGY DYSFUNCTION BY VPS35 D620N ................................................................................15 Introduction ............................................................................................................16 PD is a Progressive Neurodegenerative Disease .......................................16 Autophagy Misregulation in PD ................................................................16 The PD-Causing VPS35 D620N Mutation Affects Retromer Function and Blocks Autophagy ......................................................................................17 Autophagy Regulation by Phosphatidylinositol-AKT Signaling ..............19 HA-AKT Signaling ....................................................................................19 Results ....................................................................................................................20 VPS35 D620N Inhibits Autophagy ...........................................................20 VPS35 Knockdown Inhibits Autophagy, Phenocopying the VPS35 D620N Mutation. ....................................................................................................22 VPS35 D620N Misregulates ECM-Receptor Interaction and Pi3k-AKT Pathway Gene Expression..........................................................................23 Hyaluronan Inhibits Autophagy in Differentiated SH-SY5Y Cells ..........26 Altered Hyaluronan-AKT Signaling by VPS35 D620N ............................28 AKT Pathway Activation by VPS35 D620N is Independent of Hyaluronan Production ..................................................................................................29 Misregulated CD44 and RHAMM Expression by VPS35 D620N ............30 Discussion ..............................................................................................................33 Methods..................................................................................................................36 Tissue Culture ............................................................................................36 Western Blotting ........................................................................................37 Lentivirus Production and Transduction ....................................................38 Autophagic Flux Assay ..............................................................................38 ix Hyaluronan Treatment ...............................................................................38 Hyaluronan Quantification.........................................................................39 Differential Gene Expression, Splicing and Gene Set Enrichment Analysis......................................................................................................39 Gaussia Luciferase Protein-Fragment Complementation Assay ...............40 Statistical Analysis and Data Management................................................41 Supplementary Figures ..........................................................................................42 REFERENCES ..................................................................................................................45
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