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PIAS1 and Huntingtin in Protein Homeostasis

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PIAS1 and Huntingtin in Protein Homeostasis UNIVERSITY OF CALIFORNIA, IRVINE Finding balance in Huntington's disease: PIAS1 and huntingtin in protein homeostasis DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biological Sciences by Joseph Ochaba Dissertation Committee: Professor Leslie Thompson, Chair Assistant Professor Kim Green Professor Peter Kaiser Professor Marcelo Wood 2016 Portions of Chapter 1 © 2014 Proceedings of the National Academy of Sciences of the United States of America Portions of Chapter 2 © 2013 Elsevier Inc. Portions of Chapters 3 © 2016 Elsevier Inc. All other materials © 2016 Joseph Ochaba DEDICATION This dissertation is dedicated to my wife, Kelsey whose unyielding love, support, and encouragement have enriched my soul and inspired me to pursue and complete this research. I also dedicate this body of work to my parents, Andrea and David. I hope that this achievement will complete the dream that you had for me all those years ago when you chose to give me the best education you could. ii TABLE OF CONTENTS Page LIST OF FIGURES AND TABLES iv LIST OF ABBREVIATIONS vii ACKNOWLEDGMENTS viii CURRICULUM VITAE x ABSTRACT OF THE DISSERTATION xvi INTRODUCTION 1 A. Huntington’s disease: From protein to pathogenesis 1 B. Protein clearance pathways in Huntington’s disease 5 C. The role of SUMOylation: A post-translational modification with 8 disease relevance D. PIAS1: An SUMO-HTT E3 ligase and Transcriptional repressor 13 E. Inflammation and Huntington’s disease 16 CHAPTER 1: A potential function for the huntingtin protein as a scaffold 23 for selective autophagy CHAPTER 2: SUMO-2 and PIAS1 modulate insoluble mutant huntingtin 67 protein accumulation CHAPTER 3: PIAS1 regulates mutant huntingtin accumulation and 115 Huntington's disease-associated phenotypes in vivo CHAPTER 4: The role of SUMO-interaction motifs in huntingtin and 169 a -synuclein accumulation and aggregation DISSERTATION CONCLUDING REMARKS 193 REFERENCES 197 iii LIST OF FIGURES AND TABLES Page 1 The SUMOylation pathway 9 1.1 Loss of HTT function in Drosophila larvae or conditional knockout 51 of HTT in the mouse CNS inhibits autophagy 1.2 p62, lipofuscin and ubiquitin accumulate in Htt knockout mouse 52 brain 1.3 HTT copurifies with proteins required for regulation of autophagy 53 1.4 Alignment of the N-terminal domain of HTTs with yeast Atg23s 55 1.5 Alignment of the central domain of HTTs with yeast Vac8s 56 1.6 Alignment of the C-terminal domain of HTTs with yeast Atg11s 58 1.7 The C-terminal domain of HTT is toxic in primary cortical neurons 60 1.8 HTT contains a conserved WXXL domain at W3037 61 1.9 xLIRs in human Huntingtin 62 1.10 WXXLs in human Huntingtin 63 1.11 xLIRs and WXXL domains in Saccharomyces cerevisiae Atg11 64 1.12 The C-terminal domain of HTT interacts with GABARAPL1 65 and LC3B 1.13 Theoretical model of HTT as a scaffold protein required for 66 selective autophagy Table 2.1 qPCR primers 98 2.1 Illustration showing modulation of PIAS1 alters levels of insoluble mHTT 99 accumulation. 2.2 K6 and K9 are primary sites for HTTex1p in vitro SUMOylation 100 2.3 SUMO-1 and SUMO-2 are differentially expressed in HD mice 101 versus control 2.4 mRNA Expression of the SUMO Enzymes 102 2.5 Establishment of SUMOylation Assay 104 2.6 HTT is modified by both SUMO-1 and SUMO-2 106 2.7 PIAS1 is a SUMO E3 ligase for HTT 108 iv Page 2.8 A 586aa fragment of HTT is SUMO modified downstream of exon 1 109 2.9 SUMO-2 causes mutant HTT to accumulate 111 2.10 SUMO-2 proteins accumulate in HD brain 113 3.1 Silencing efficacy of artificial miRNAs targeting mouse Pias1 148 expression 3.2 PIAS1 levels are modulated in R6/2 mouse model following 149 viral-mediated knockdown or overexpression 3.3 Soluble/Insoluble fractionation represents Cytoplasmic/Nuclear 151 fractions, respectively 3.4 Insoluble/nuclear PIAS1 levels are elevated in R6/2 HD 152 mouse model 3.5 Control miSAFE expression is equivalent to Vehicle injected 153 R6/2 mice and does not influence HTT levels or alter behavioral outcomes 3.6 Real-time PCR of PIAS1 and human HTT transgene expression 154 in R6/2 mice 3.7 Effects of PIAS1 modulation on behavior in R6/2 mice 155 3.8 PIAS1 modulation has no effect on mouse weight 156 3.9 PIAS1 knockdown rescue on Rotarod task disappears at 9 weeks 157 3.10 Effect of PIAS1 modulation on overall survival and physiological 158 assessment of R6/2 mice 3.11 PIAS1 modulates insoluble protein accumulation in R6/2 mice 159 3.12 PIAS1 knockdown reduces mHTT inclusions in HD mouse 160 striatum 3.13 PIAS1 modulation alters levels of synaptophysin in the striatum 161 of R6/2 mice 3.14 miPIAS1.3 expression corresponds to altered levels of 162 synaptophysin in the striatum of R6/2 mice v Page 3.15 Iba1+ microglia numbers are decreased by PIAS1 reduction in 163 the R6/2 striatum 3.16 Insoluble NF-kB (p65) levels are restored to NT levels following 164 PIAS1 reduction in the R6/2 striatum 3.17 Soluble inflammatory cytokines are altered in the R6/2 striatum 165 and restored to NT levels following PIAS1 knockdown 3.18 Temporal profile of pro-inflammatory cytokines in detergent 166 soluble fraction of striatal tissue homogenates 3.19 Conserved domains of PIAS1 167 3.20 The PIAS1 SIM domain regulates accumulation of insoluble 168 mHTT and self-turnover 4.1 GPS-SUMO prediction evaluation of Huntingtin (586aa) 188 and a -synculein 4.2 a -synuclein secondary structure from PELE via 189 Biology WorkBench 4.3 Aggregation ‘hot spot’ prediction using AGGRESCAN prediction 190 software 4.4 SIM mutations in 25Q and 137Q, 586 aa fragment of HTT alters 191 cleavage and insoluble accumulation. 4.5 SIM mutation in A53T a -synuclein abrogates insoluble 192 accumulation and confirmation state vi LIST OF ABBREVIATIONS AAV Adeno-associated virus a -synculein Synuclein, alpha (non A4 component of amyloid precursor) BACHD An HD transgenic mouse line expressing a neuropathogenic, full- length human mutant Huntingtin (fl-mhtt) gene carrying 97 mixed CAA-CAG repeat CNS Central nervous system COS A fibroblast-like cell line derived from monkey kidney tissue GFAP Glial fibrillary acidic protein HD Huntington’s disease HeLa A cell type in an immortal cell line used in scientific research HMW High molecular weight HTT Human huntingtin protein IFN g Interferon gamma IBA1 Ionophore calcium-binding adapter molecule 1 IL Interleukin IL1-b Interleukin-1 beta JAK/STAT Janus kinase/ signal transducer and activator of transcription mHTT mutant human huntingtin protein miRNA microRNA mRNA Messenger RNA MSN Medium Spiny Neurons NFKB Nuclear factor kappa-light-chain-enhancer of activated B cells NT Nontransgenic controls for HD mice pcDNA Plasmid Cytomegalovirus promoter DNA PD Parkinson’s disease PIAS1 Protein inhibitor of activated STAT-1 Poly Q A polyglutamine tract or polyQ tract is a portion of a protein consisting of a sequence of several glutamine units PTM Post-translational Modification R6/2 An HD transgenic mouse line for the 5' end of the human HD gene carrying approximately 120 +/- 5 (CAG) repeat expansions SIM SUMO-interaction motif STAT1 Signal transducer and activator of transcription-1 SUMO-1 Small ubiquitin-related modifier 1 SUMO-2/3 Small ubiquitin-related modifier 2/3 TNF a Tumor necrosis factor alpha UPS Ubiquitin-Proteasome System zQ175 The zQ175 knock-in (zQ175 KI) allele encodes the human HTT exon 1 sequence with a ~190 CAG repeat tract, replacing the mouse Htt exon 1 vii ACKNOWLEDGMENTS I am forever grateful for the teachings and support of my mentor, Dr. Leslie Thompson. Her intelligence is rivaled only by her enthusiasm and dedication to the field of Huntington’s disease research. Leslie, I have greatly appreciated your encouragement and mentorship over the years, both of which have driven me as an individual and a scientist. It has been a true honor and a pleasure working alongside of you and learning from you. Your ability to manage and involve yourself in so many different areas of research without missing a step is impressive. I admire your generosity and genuine scientific and personal interest in the HD community. Working in your lab and having access to the resources in the community, has allowed me to grow as an individual and learn as a scientist. The amount I have been able to accomplish in such a short time could only have been done with you as a mentor. Thank you for everything. I would also like to thank, Dr. Joan Steffan for all of her intellectual input and thoughtful discussions throughout the course of my graduate career, all of which were invaluable. Joan, I admire your passion, your work ethic, and your dedication to science. You have taught me so much including experimental design, analysis, and technique. I thank you for encouraging me to think independently and yet always being there when I needed your advice. Lastly, thank you for teaching me that it is (in fact) all about autophagy. I am very fortunate to have had access to two great mentors. Thank you both for always being there for me scientifically and personally and I can only hope that I will make you both proud in the years to come. viii I would like to thank my committee members, Dr. Marcelo Wood, Dr. Peter Kaiser, and Dr. Kim Green, for providing helpful suggestions and critique, all of which have been immensely invaluable to these studies. Thank you to the entire Thompson lab for their technical assistance and support both intellectually and emotionally throughout the years of my graduate work; particularly Dr.
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