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View and Expressed As the Number of Mhspb1 Positive Cells Over Total Cells Interrogating the Functional Consequences of Peripheral Neuropathy Associated Mutations in Heat Shock Protein B1 Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Patrick Lawrence Heilman Ohio State University Biochemistry Graduate Program The Ohio State University 2017 Dissertation Committee Dr. Stephen Kolb, Advisor Dr. Juan Alfonzo Dr. Sharon Amacher Dr. Mark Parthun Copyrighted by Patrick Lawrence Heilman 2017 ii ABSTRACT Heat shock protein beta-1 (HSPB1), is a ubiquitously expressed, multifunctional protein chaperone that plays an important role in protein quality control, inflammation, apoptosis, cell growth and cytoskeleton maintenance. Mutations in HSPB1 result in the development of a late-onset, distal hereditary motor neuropathy type II (dHMN) and axonal Charcot-Marie-Tooth disease with sensory involvement (CMT2F). The functional consequences of HSPB1 mutations associated with hereditary neuropathy are unknown. HSPB1 also displays neuroprotective properties in many neuronal disease models, including the motor neuron disease amyotrophic lateral sclerosis (ALS). HSPB1 is upregulated in SOD1-ALS animal models during disease progression, predominately in glial cells. Glial cells are known to contribute to motor neuron loss in ALS through a non-cell autonomous mechanism. Here, I studied the non- cell autonomous role of wild-type and mutant HSPB1 in an astrocyte-motor neuron co-culture model system of ALS. Astrocyte-specific overexpression of wild-type HSPB1 was sufficient to attenuate SOD1(G93A) astrocyte-mediated toxicity in motor neurons, whereas, overexpression of mutHSPB1 failed to ameliorate motor neuron toxicity. Expression of a phosphomimetic HSPB1 mutant in SOD1(G93A) astrocytes also reduced toxicity to motor neurons, i suggesting that phosphorylation may contribute to HSPB1 mediated- neuroprotection. To elucidate a mechanism of HSPB1-mediated neuroprotection, I examined the role of wild-type and mutant HSPB1 in regulating inflammatory gene expression through activation of NF-kB signaling and mRNA decay apthways. Wild-type, but not mutant HSPB1 was able to reduce the activation of NF-kB signaling, a major inflammatory signaling cascade, through an unidentified mechanism. Additionally, I found no evidence of HSPB1’s involvement in regulating AU-rich element mRNA decay. Together, these data suggest that HSPB1 neuroprotection may be mediated in part by its regulation of inflammatory signaling. ii DEDICATION To my parents, Richard and Patricia Heilman iii ACKNOWLEDGMENTS There are numerous people to whom I owe a tremendous amount of thanks and gratitude to. First and foremost, to my Ph.D. advisor, Dr. Stephen Kolb, for his guidance, mentorship and patience. I am forever grateful for everything he has taught me, from how to isolate the hippocampus from a mouse to properly writing scientific literature. He has helped me to mature into the scientist I am today, and his mentorship will serve as an important foundation for the rest of my scientific career. To my committee members, Dr. Juan Alfonzo, Dr. Sharon Amacher and Dr. Mark Parthun. Their support and guidance has taught me to keep an open mind and always look at a problem from multiple angles. A particular thank you goes to Dr. Dan Battle, who spent countless hours helping me to develop and analyze data from my co-immunoprecipitation experiments. This work would not have been possible without the help and support of all the alumni and current members of the Kolb Lab Group. To Samantha Renusch and Christopher Wier, thank you for being amazing friends and colleagues, who were always willing to help with an experiment, to lend an ear, or share a beer. I iv hope that we can maintain our friendship with one another and help one another in the future. A special thanks must also go to our collaborators in Dr. Brian Kaspar’s lab, Dr’s. Kathrin Meyer, SungWon Song, and Carlos Miranda, who were instrumental in teaching me about viral production and how to culture primary neuronal cell lines. Without them, this work would never have been possible. A heart-felt thanks goes out to my family. To my girlfriend, Lauren Stober, who has kept me sane over the past 3 years, and been there to pick me up every time I stumbled. To my brothers, Paul and Raymond, for being supportive of my life choices and putting up with all my scientific rambling. To my parents, Richard and Patricia who have loved and supported me every step of the way, from deciding to major in Biochemistry after high school to the decision to enter a Ph.D. program. No matter how hard things got, they were always there for me, and have taught me that hard work always pays off in the end. I cannot thank you enough for all the effort and sacrifice. v VITA 2011 ....................................................... B.S. Biochemistry, Duquesne University of the Holy Spirit 2011-Present .......................................... Graduate Research/Teaching Associate, The Ohio State Biochemistry Program, The Ohio State University PUBLICATIONS Heilman, P.L., Song, S.W., Miranda, C., Meyer, K., Wier, C.G., Knapp, A.R., Kaspar, B.K., Kolb, S.J. Hereditary neuropathy-associated HSPB1 mutations disrupt non-cell autonomous protection of motor neurons. Experimental Neurology 2017; 297:101-109 vi FIELDS OF STUDY Major Field: Biochemistry vii TABLE OF CONTENTS ABSTRACT ............................................................................................................ i DEDICATION ........................................................................................................ iii ACKNOWLEDGMENTS ....................................................................................... iv VITA...................................................................................................................... vi TABLE OF CONTENTS ...................................................................................... viii LIST OF TABLES ................................................................................................. xii LIST OF FIGURES .............................................................................................. xiii CHAPTER 1: INTRODUCTION ............................................................................. 1 1.1 HEAT SHOCK PROTEINS .......................................................................... 1 1.1.1 THE HEAT SHOCK PROTEIN FAMILY ................................................ 1 1.2 HEAT SHOCK PROTEIN BETA 1 (HSPB1) ................................................ 7 1.2.1 STRUCTURE AND GENE REGULATION ............................................ 7 1.2.2 HSPB1 PHOSPHORYLATION ........................................................... 11 1.2.3 HSPB1 OLIGOMERIZATION .............................................................. 11 1.2.4 DIFFERENTIAL REGULATION AND CELLULAR LOCALIZATION ... 13 1.3 CELLULAR FUNCTIONS OF HSPB1 ....................................................... 14 1.3.1 MOLECULAR CHAPERONE .............................................................. 15 1.3.2 REGULATION OF CELL SIGNALING/APOPTOSIS .......................... 19 1.3.3 REGULATION OF THE CYTOSKELETON......................................... 23 1.4 HSPB1 AND THE NERVOUS SYSTEM .................................................... 25 1.4.1 EXPRESSION OF HSPB1 IN THE NERVOUS SYSTEM ................... 25 1.4.2 HSPB1 AND NEURONAL INJURY ..................................................... 26 1.4.3 HSPB1 AND ALS ................................................................................ 27 1.4.4 HSPB1 AND PARKINSON’S DISEASE .............................................. 28 1.4.5 HSPB1 and ALZHEIMER’S DISEASE ............................................ 29 1.5 MUTATIONS IN HSPB1 RESULT IN MOTOR NEURON DISEASE ......... 30 1.5.1 CLINICAL DIAGNOSIS OF HSPB1-ASSOCIATED CMT2/dHMNII .... 30 viii 1.5.2 POTENTIAL MECHANISMS OF HSPB1-LINKED CMT2 AND dHMNII ..................................................................................................................... 34 CHAPTER 2: NON-CELL AUTONOMOUS NEUROPROTECTION OF HSPB1 IS DISRUPTED BY MUTATIONS THAT RESULT IN MOTOR NEUROPATHY ..... 39 2.1 INTRODUCTION ....................................................................................... 39 2.2 EXPERIMENTAL METHODS .................................................................... 42 2.2.1 MICE ................................................................................................... 42 2.2.2 HUMAN TISSUE SAMPLES ............................................................... 42 2.2.3 ES MOTOR NEURON DIFFERENTIATION ....................................... 43 2.2.4 MOUSE NPC ISOLATION AND ASTROCYTE DIFFERENTIATION .. 43 2.2.5 CONVERSION OF SKIN FIBROBLASTS TO iNPCs AND DIFFERENTIATION INTO ASTROCYTES .................................................. 44 2.2.6 LENTIVIRAL PRODUCTION .............................................................. 45 2.2.7 ASTROCYTE-MOTOR NEURON CO-CULTURE ASSAY .................. 47 2.2.8 ASTROCYTE VIABILITY ASSAY ....................................................... 48 2.2.9 WESTERN BLOTTING ....................................................................... 48 2.2.10 IMMUNOFLUORESCENCE .............................................................. 49 2.2.11 HSPB1 ELISA ................................................................................... 50 2.2.12 PROTEIN SECRETION ASSAY ......................................................
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