IDENTIFICATION OF NEUROPROTECTIVE GENES AGAINST ALPHA- SYNUCLEIN TOXICITY USING A CAENORHABDITIS ELEGANS PARKINSON DISEASE MODEL by SHUSEI HAMAMICHI A DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biological Sciences in the Graduate School of The University of Alabama TUSCALOOSA, ALABAMA 2009 Copyright Shusei Hamamichi 2009 ALL RIGHTS RESERVED ABSTRACT Recent functional analyses of nine gene products linked to familial forms of Parkinson disease (PD) have revealed several cellular mechanisms that are associated with PD pathogenesis. For example, α-synuclein ( α-syn), a primary component of Lewy bodies found in both familial and idiopathic forms of PD, has been shown to cause defects in proteasomal and lysosomal protein degradation machineries and induce mitochondrial/oxidative stress. These findings are further supported by the fact that additional gene products are involved in the same pathways. While these studies have been invaluable to elucidate the etiology of this disease, it has been reported that monogenic forms of PD only account for 5-10% of all PD cases, indicating that multiple genetic susceptibility factors and intrinsic metabolic changes associated with aging may play a significant role. Here we report the use of an organism, Caenorhabditis elegans , to model two central PD pathological features to rapidly identify genetic components that modify α-syn misfolding in body wall muscles and neurodegeneration in DA neurons. We determined that proteins that function in lysosomal protein degradation, signal transduction, vesicle trafficking, and glycolysis, when knocked down by RNAi, enhanced α-syn misfolding. Furthermore, these components, when overexpressed, rescued DA neurons from α-syn-induced neurodegeneration, and several of them have been validated using mammalian system. Taken together, this study represents a novel set of gene products that are putative genetic susceptibility loci and potential therapeutic targets for PD. ii LIST OF ABBREVIATIONS AND SYMBOLS 6-OHDA 6-Hydroxydopamine AD Alzheimer disease ADE Anterior deirid neuron bp Base pair °C Celsius cAMP Cyclic adenosine monophosphate cDNA Complementary DNA CEP Cephalic neuron cGMP Cyclic guanosine monophosphate COR C-terminal of Roc D2 Dopamine 2 D3 Dopamine 3 DA Dopamine DEPC Diethylpyrocarbonate DNA Deoxyribonucleic acid DOG 2-Deoxyglucose dsRNA Double-stranded RNA E1 Ubiquitin-activating enzyme E2 Ubiquitin-conjugating enzyme iii E3 Ubiquitin ligase ER Endoplasmic reticulum ERAD Endoplasmic reticulum-associated degradation FRAP Fluorescence recovery after photobleaching GAL4 Galactose metabolism 4 GFP Green fluorescent protein GO Gene ontology HD Huntington disease HMG-CoA 3-Hydroxy-3-methyl-glutaryl-Coenzyme A hr Hour IPTG Isopropyl β-D-thiogalactoside kDa Kilodalton KOG Eukaryotic orthologous group L3 Larval stage 3 L4 Larval stage 4 LB Luria-Bertani L-DOPA L-3,4-Dihydroxyphenylalanine MPP+ 1-Methyl-4-phenylpyridinium MPTP 1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine mRNA messenger RNA µg Microgram iv µl Microliter miRNA MicroRNA mg Milligram ml Milliliter mM Millimolar MAPK Mitogen-activated protein kinase MAPKK Mitogen-activated protein kinase kinase MAPKKK Mitogen-activated protein kinase kinase kinase n/a Not applicable NGM Nematode growth medium PARK Parkinson disease gene PCR Polymerase chain reaction PD Parkinson disease PDE Posterior deirid neuron RING Really interesting new gene RNA Ribonucleic acid RNAi RNA interference Roc Ras of complex ROS Reactive oxygen species rpm Revolutions per minute RT Room temperature (25 °C) v RT-PCR Reverse transcriptase polymerase chain reaction SAGE Serial analysis of gene expression SD Standard deviation SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SNP Single nucleotide polymorphism UPR Unfolded protein response UPS Ubiquitin-proteasome system UTR Untranslated region C. elegans Proteins AGE-1 Aging alteration 1 (phosphoinositide 3-kinase) ATGR-7 Autophagy 7 BAR-1 Beta-catenin/armadillo related 1 CDK-5 Cyclin dependent kinase 5 CED-3 Cell death abnormality 3 (caspase) CLK-1 Clock 1 (demethoxyubiquinone hydroxylase) CMK-1 Calcium/calmodulin-dependent protein kinase 1 CSNK-1 Casein kinase 1 DAF-2 Abnormal dauer formation 2 (insulin receptor) DAF-16 Abnormal dauer formation 16 (forkhead Box 01A) DAT-1 Dopamine transporter 1 vi DJR-1 Oncogene DJ-1 DJR-2 Oncogene DJ-1 DOP-2 Dopamine receptor 2 (D2-like receptor) DPY-1 Dumpy 1 (collagen) DPY-5 Dumpy 5 (collagen) EAT-2 Eating 2 (nicotinic acetylcholine receptor) GPI-1 Glucose-6-phosphate isomerase 1 HRD-1 HRD 1 HRDL-1 HRD-like 1 HSF-1 Heat-shock factor 1 ISP-1 Rieske iron sulphur protein LRK-1 Leucine-rich repeats, Ras-like domain, kinase 1 (LRRK2) MOM-4 More of MS 4 (MAPKKK7) NHR-6 Nuclear hormone receptor family 6 (NURR1) NPR-1 Neuropeptide receptor family 1 OBR-1 Oxysterol binding protein 1 PDR-1 Parkinson’s disease related 1 (parkin) PINK-1 PTEN-induced putative kinase 1 PMK-1 p38 MAP kinase ROL-6 Roller 6 (collagen) SMF-1 Yeast SMF homolog (divalent metal transporter) vii TAG-278 Temporary assigned gene 278 TAP-1 TAK kinase/MOM-4 binding protein TOR-2 Torsin 2 (torsinA) TRX-1 Thioredoxin 1 UNC-32 Uncoordinated 32 (vacuolar proton-translocating ATPase) UNC-51 Uncoordinated 51 (unc-51-like kinase 2) UNC-54 Uncoordinated 54 (myosin class II heavy chain) UNC-75 Uncoordinated 75 (CELF/BrunoL protein) VPS-41 Vacuolar protein sorting 41 YKT-6 Yeast YKT6 homolog (v-SNARE) Mammalian Proteins α-Syn Alpha synuclein ALDOA Aldolase A AMF Autocrine motility factor AMFR Autocrine motility factor receptor Amyloid-β Amyloid beta ASK1 Apoptosis signal-regulating kinase 1 ATG7 Autophagy 7 ATP13A2 ATPase, Type 13A2 BAG5 BCL2-associated athanogene 5 viii CSNK1G3 Casein kinase 1, gamma-3 CHIP C-terminus of HSC70-interacting protein Daxx Death-associated protein 6 DJ-1 Oncogene DJ-1 ERV29 Surfeit 4 FBXW7 F-box and WD40 domain protein 7 GAIP G protein alpha-interacting protein GAPDH Glyceraldehyde-3-phosphate dehydrogenase GBA Glucocerebrosidase GIGYF2 GRB10-interacting GYF protein 2 GIPC GAIP C-terminus-interacting protein GPI Glucose-6-phosphate isomerase HDAC6 Histone deacetylase 6 HTRA2 HTRA serine peptidase 2 HSF1 Heat-shock factor 1 HSP70 Heat-shock protein, 70 kDa HSPC117 Hypothetical protein 117 IgG Immunoglobulin G INSR Insulin receptor LRRK2 Leucine-rich repeat kinase 2 NRB54 Nuclear RNA-binding protein, 54 kDa ix p38 Mitogen-activated protein kinase p38 Pael receptor Parkin-associated endothelin receptor PDE9A Phosphodiesterase 9A PI3K Phosphoinositide 3-kinase PINK1 PTEN-induced putative kinase 1 PLK2 Polo-like kinase 2 PRKN Parkin PSF Polypyrimidine tract-binding protein-associated splicing factor Q82 Polyglutamine 82 containing protein RAB1A Ras-associated protein 1A RAB3A Ras-associated protein 3A RAB8A Ras-associated protein 8A RGS Regulators of G protein signaling SEC22 Secretion deficient 22 SNCA Synuclein, alpha SYVN1 Synoviolin 1 Ub Ubiquitin Ubch7 Ubiquitin-conjugating enzyme 7 Ubch8 Ubiquitin-conjugating enzyme 8 UCHL Ubiquitin C-terminal hydrolase UCHL1 Ubiquitin C-terminal hydrolase 1 x USP10 Ubiquitin-specific protein 10 VMAT2 Vesicular monoamine transporter 2 VPS41 Vacuolar protein sorting 41 XIAP X-linked inhibitor of apoptosis xi ACKNOWLEDGMENTS First and foremost, I would like to thank Drs. Guy and Kim Caldwell, and the former and present members of the Caldwell lab. Among them, I wish to especially recognize highly motivated undergraduate students who undertook the enormous challenge of working in the PD research field. Most notably, I want to thank Renee Rivas, Adam “Deuce” Knight, Susan DeLeon, and Paige Dexter. Without their help and contribution, I guarantee that none of our projects would have worked as smoothly as they did. Furthermore, I want to thank Cody Locke for making science intellectually stimulating. Among the former and present non-undergraduates, I would like to acknowledge Dr. Laura Berkowitz, Michelle Norris, Lindsay Faircloth, and Jenny Schieltz for their assistance in many untold and underappreciated areas of research. I also want to thank the usual Wilhagans gang including Jafa Armagost, Adam “Ace” Harrington, and AJ Burdette for making my life as a graduate student more enjoyable and fulfilling. I wish all of them good luck for their future endeavors. Outside of the Caldwell lab, I would like to thank my graduate committee members, Dr. Janis O’Donnell, Dr. Katrina Ramonell, and Dr. Jianhua Zhang for their continuous support and encouragement. Furthermore, I wish to acknowledge other faculty members and staff of the Department of Biological Sciences including Dr. Martha Powell, Dr. Harriett Smith-Somerville, and many others for their assistance whenever I xii needed it and their enthusiasm toward my research. It has been a pleasure sharing research ideas and data annually, and receiving tremendously kind responses from all of you. I also would like to express my gratitude to research collaborators, Dr. Susan Lindquist (Whitehead Institute/MIT), Dr. David Standaert (UAB), Dr. Ted Dawson (Johns Hopkins University), and Dr. Antonio Miranda Vizuete (Universidad Pablo de Olavide). Thank you so much for allowing me to work on multiple influential research projects. Most notably, I would like to thank Dr. Joshua Kritzer and Dr. Chris Pacheco, both post-docs at the Lindquist lab, for believing in me. Outside of the current research world, I want to thank my parents and my brother who are currently in Japan as well as my former PIs, Dr. Hideo Nishigori (Teikyo University) and Dr. Mike Shipley (Midwestern State University). Furthermore, I wish to recognize all of my friends from all over the world. Thank you for our friendship and understanding of what we wish to achieve in our lives. I am who I am, and I do what I do because of you. Lastly, I would like to acknowledge Sylvester Stallone for making a classic movie, Rocky, the ultimate source of my inspiration while working on my PNAS article, presentation slides used for my post-doc interview at Whitehead Institute, and this current dissertation. I only wanted to go the distance, but I had never thought I would have an opportunity to go to Boston.