AT THE INTERSECTION OF PROTEOSTASIS AND NEURODEGENERATIVE DISEASE: BYSTANDER MISFOLDING AND IMPAIRED PROTEOME INTEGRITY By MICHAEL PACE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2018 © 2018 Michael Pace To my wife, friends, family, and everyone who supported me throughout this endeavor, as well as to the scientific process and an appreciation of critical thinking, without which I would not have made it this far. ACKNOWLEDGMENTS I wish to thank my mentor, Dr. David Borchelt, for always being available to answer my questions and unravel the scientific minutia of my projects whenever I was unable to do so. I wish to thank my colleagues and collaborators Dr. Guilian Xu, Hilda Brown, Susan Fromholt, Dr. Jada Lewis, John Howard, Dr. Sruti Rayaprolu, Dr. Matthew Hamm, Dr. Kevin Strang, Dr. Jacob Ayers, and Keith Crosby. They provided invaluable support in the form of scientific and technical expertise as well as insightful conversation on a daily basis to push my projects forward. I wish to thank the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida for assistance in processing and analyzing samples during proteomic studies. My committee (consisting of Dr. David Borchelt, Dr. Lucia Notterpek, Dr. Benoit Giasson, Dr. Ron Mandel and Dr. Timothy Garrett) provided invaluable scientific insight throughout the progression of my degree. Lastly, I wish to acknowledge Drs. Dawn Bowers and David Vaillancourt, the co-directors of the training grant that I received, for professional development advice and advancing my opportunities to grow and mature as a scientist. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF OBJECTS ....................................................................................................... 12 LIST OF ABBREVIATIONS ........................................................................................... 13 ABSTRACT ................................................................................................................... 16 CHAPTER 1 INTRODUCTION .................................................................................................... 18 Proteostasis Network Composition and Functionality ............................................. 20 Molecular Chaperones ............................................................................................ 21 The Ubiquitin-Proteasome System ......................................................................... 24 The Autohphagy-Lysosomal System ...................................................................... 28 Effects of Pathologic Proteinopathies on Proteostasis ............................................ 32 Evidence for Compromised Cellular Protein Folding in the Face of Neurodegenerative Proteinopathies .................................................................... 36 2 DIFFERENTIAL INDUCTION OF MUTANT SOD1 MISFOLDING AND AGGREGATION BY TAU AND α-SYNUCLEIN PATHOLOGY ............................... 40 Methods .................................................................................................................. 43 Transgenic Mice ............................................................................................... 43 Breeding Scheme to Generate Mice Co-expressing G85R-SOD1:YFP and Mutant Proteins Associated with Human Proteinopathies ............................. 44 Intramuscular Human αSyn Fibril Injections into Hemizygous M83 and Bigenic M83-G85R-SOD1:YFP Transgenic Mice to Seed αSyn Pathology .. 45 Tissue Processing, Immunohistochemistry and Image Microscopy ................. 45 Fluorescence Quantification and Statistical Analysis ....................................... 47 Preparation of Brain and Spinal Cord Tissues for Immunoblot Analysis .......... 47 Immunoblotting and Western Blot Quantification .............................................. 49 Results .................................................................................................................... 50 Mutant Tau Induces G85R-SOD1:YFP Inclusion Pathology in the Spinal Cord and Brain Stem ..................................................................................... 50 Localization of SOD1 Pathology Relative to Tau Pathology ............................. 51 Lack of G85R-SOD1:YFP Inclusion Pathology Induction in the rTg4510 Model of Cortical and Hippocampal Tauopathy ............................................. 52 5 Paucity of Induced G85R-SOD1:YFP Aggregation in the M83 Model of αSynucleinopathy .......................................................................................... 53 Discussion .............................................................................................................. 54 Conclusions ............................................................................................................ 61 3 CHANGES IN PROTEOME SOLUBILITY INDICATE WIDESPREAD PROTEOSTATIC DISRUPTION IN MOUSE MODELS OF NEURODEGENERATIVE DISEASE ...................................................................... 80 Materials and Methods............................................................................................ 82 Transgenic Animals .......................................................................................... 82 Intramuscular Injection of Preformed α-synuclein Fibrils into M83 Transgenic Mice ............................................................................................ 83 Sequential Protein Detergent Extraction from Mouse Forebrain/Spinal Cord and Preparation of Protein Samples for Proteomics Analysis ....................... 84 LC-MS/MS Analysis ......................................................................................... 85 Calibration, Relative Quantification and Bioinformatics of Proteomics Data ..... 86 Western Blot Validations of LC-MS/MS Data ................................................... 88 Expression and Purification of Recombinant K18 Tau and Assembly of Recombinant K18 Tau Fibrils ........................................................................ 88 N2a Cell Culture and K18-mediated Human Tau Seeding in vitro .................... 89 Results .................................................................................................................... 90 Neurofibrillary Tangle Pathology Perturbs the Solubility of CNS Cytosolic Proteins ......................................................................................................... 90 Validation of LC-MS/MS Data in Tauopathy ..................................................... 94 Differential Effects of Pathological Tau Versus Amyloid-β on Proteome Solubility ........................................................................................................ 97 Analysis of Changes in Proteome Solubility in Mouse Models of Spinal Proteinopathy ................................................................................................ 98 Identification of Proteins in SDS-insoluble Fractions from N2a Cell Lysates Prepared with Tau Aggregates .................................................................... 101 Bioinformatic Analysis of Proteins that Aberrantly Fractionate as SDS- insoluble Reveals Commonly Affected Protein Classes .............................. 103 Discussion ............................................................................................................ 104 4 ASSESSING THE ALLEVIATION OF ABERRANT PROTEOME INSOLUBILITY VIA TRANSIENT INHIBITION OF TRANSLATION WITH CYCLOHEXIMIDE ...... 130 Materials and Methods.......................................................................................... 133 Transgenic Mice ............................................................................................. 133 Cycloheximide Preparation, Injection, and Harvesting Procedures ................ 133 Sequential Detergent Extraction of Spinal Cord Tissue .................................. 134 Liquid Chromatrography Tandem Mass Spectrometry Analysis ..................... 135 Bioinformatics, Statistics, and the Calibration/Relative Quantification of Proteomics Data .......................................................................................... 137 Results .................................................................................................................. 138 Discussion ............................................................................................................ 140 6 5 DISCUSSION ....................................................................................................... 148 LIST OF REFERENCES ............................................................................................. 156 BIOGRAPHICAL SKETCH .......................................................................................... 184 7 LIST OF TABLES Table page 2-1 RNAseq expression data for SOD1 in mouse and human tauopathies .............. 79 3-1 Statistical information for different proteinopathy animal groups analyzed via LC-MS/MS .......................................................................................................