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Genetic Analyses of Signal Transduction Pathways

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Genetic Analyses of Signal Transduction Pathways GENETIC ANALYSES OF SIGNAL TRANSDUCTION PATHWAYS INVOLVED IN NEUROMUSCULAR EXCITABILITY AND NEURODEGENERATION IN CAENORHABTIDIS ELEGANS by BWARENABA KAUTU GUY A. CALDWELL, COMMITTEE CHAIR KIM A. CALDWELL JANIS M. O’DONNELL STEVAN MARCUS KATRINA RAMONELL ANDREW WEST 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 2012 Copyright Bwarenaba Kautu 2012 ALL RIGHTS RESERVED ABSTRACT Signal transduction pathways regulate many cellular and molecular aspects of the brain including neurotransmission and cell survival. Defects in neuronal signaling can lead to a variety of cognitive and affective disorders such as epilepsy and Parkinson’s disease. Here, I use a genetically tractable organism, Caenorhabditis elegans (C. elegans), as a model system to study the impact of two canonical signaling pathways on neuronal activity and survival. Using molecular and genetic tools, pharmacological assays, and microscopy techniques, I showed that the canonical Rac GTPase pathway regulates neuronal synchrony in the GABAergic neurons of C. elegans. In our experiments we observed that Rac GTPase mutants exhibited behavioral responses to a GABAA receptor antagonist, pentylenetetrazole. These mutants also exhibited hypersensitivities to an acetylcholinesterase inhibitor, aldicarb, suggesting deficiencies in GABA transmission. Knockdown of selected cytoskeletal genes in Rac hypomorph mutants revealed synergistic interactions, particularly between the dynein motor complex and some members of the canonical Rac-signaling pathway. Examination of the nerve cords of C. elegans revealed that these genetic factors function to regulate vesicle transport in the GABAergic neurons of C. elegans. In my second project, I characterized the role of the heterotrimeric G protein Gαq in the context of neuronal survival, using a C. elegans model of Parkinson’s disease. In this work, we found that activation of Gαq (EGL-30) can significantly protect the dopaminergic neurons against a human Parkinson’s gene, α-synuclein (α-syn). Interestingly, inactivation of ii downstream effectors of Gαq exacerbated the loss of dopaminergic neurons in the presence of α- syn suggesting that these factors likey function in a common pathway with Gαq to provide protection for the dopaminergic neurons against α-syn-induced toxicity. These data suggest that activation of Gαq signaling pathway can offer protection to the dopaminergic neurons and could be a potential therapeutic target for neurodegenerative diseases like Parkinson’s disease. Taken together, my work showed that Rac GTPase signaling pathway controls neuromuscular excitability in C. elegans and Gαq signaling modulates protection of the dopaminergic neurons. iii DEDICATION This dissertation is dedicated to two people. First and foremost, to my grandmother Kaarite Titiba who not only raised me in the absence of my biological parents, but also made a significant impact on my personal life and goals. Second, I dedicate this writing to my aunt (adoptive mother) Tekarawarawa Tematang who accepted me (an orphan looking for a home) to be a part of her family, and provided me with care and education. iv LIST OF ABBREVIATIONS AND SYMBOLS 6OHDA 6-hydroxydopamine α Alpha Ach Acetylcholine ADE Anterior deirid neuron β Beta bp Base pair 0C Degrees Celsius Ca2+ Calcium ion cDNA Complementary DNA CEP Cephalic neuron dsRNA double-stranded RNA D2 Dopamine 2 receptor DA Dopamine DAG Diacylglyerol DNA Deoxyribonucleic acid ε Epsilon EGL Egg-laying defective GABA GAMA aminobutyric acid GAP GTPase activating protein GDP Guanosine-5'-diphosphate v GEF Guanine nucleotide exchange factor GTP Guanosine-5'-triphosphate GFP Green Fluorescent Protein H4 Neuroglioma cell line kDa Kilodalton MAPK Mitogen-activated protein kinase MAPKK Mitogen-activated protein kinase kinase MAPKKK Mitogen-activated protein kinase kinase kinase MPP+ 1-Methyl-4-phenylpyridinium MPTP 1-Methyl-4-phenyl-1, 2, 5, 6-tetrahydropyridine μg Microgram μM Micromolar mg Milligram ml Milliliter mM Millimolar mRNA messenger RNA miRNA microRNA n sample size number NMDA N-methyl-D-aspartic acid NMJ Neuromuscular junction p Probability of null hypothesis PARK Parkinson’s disease gene PD Parkinson’s disease vi PDE Posterior deirid neuron PTZ Pentylenetetrazole RGS Regulator of G protein signaling RNA Ribonucleic acid RNAi RNA interference RT-PCR Reverse transcription polymerase chain reaction SD Standard deviation SEM Standard error of the mean Unc Uncoordinated movement UPR Unfolded protein response UPS Upiquitin proteasome system VA Valproic acid WT Wildtype Proteins/genes α-syn Alpha synuclein CAT-1 C. elegans vesicular monoamine transporter CAT-2 C. elegans Tyrosine Hydroxylase CED-10 C. elegans Rac GTPase protein DJ-1 Oncogene DJ EGL-8 C. elegans Phospholipase enzyme beta EGL-10 C. elegans RGS protein EGL-30 C. elegans heterotrimeric Gαq protein vii ERK-MAPK Extracellular regulated MAP kinase CDK-5 Cyclin dependent kinase 5 GOA-1 C. elegans heterotrimeric g protein Gαo INA-1 C. elegans integrin alpha receptor protein LIS-1 C. elegans lissencephaly protein LRRK2 Leucine-rich repeat kinase MIG-2 C. elegans Rac/Rho protein PTEN Phosphatase and tensin homolog PKC Protein Kinase C RAB-3 Small Ras associated protein SID-1 Double stranded RNA transporter memberane SNARE Soluble NSF attachment protein receptor SNB-1 Synaptobrevin 1 viii ACKNOWLEDGMENTS I want to first thank my academic advisors Guy Caldwell and Kim Caldwell for giving me the opportunity to learn scientific research in their wonderful lab at this institution. I am also thankful for their mentorship and continual support to me since the beginning of my graduate school. I also want to thank my colleagues and friends in the Caldwell Lab for the relationship we have shared together. In particular, I am indebted to Cody Locke who taught me a lot about how to become a better thinker and researcher. In addition, I am grateful for the assistance of former and current undergraduate students who have helped me with several projects. Kyle Lee and Kalen Berry were the first undergraduates whom I had the priviledge to work with. Matthew Hicks, Chris Gilmartin, and Akeem Borom are the recent undergraduate students who have also contributed to our research work. I also want to take this opportunity to acknowledge our wonderful lab manager, Dr. Laura Berkowitz, who has not only kept the lab safe and organized all the time, but has also been actively involved in mentoring students including myself. I thank The University of Alabama and the Department of Biological Sciences for the golden opportunity they have offered me here, i.e. to receive an education from such great institution. I thank all my graduate committee members, Dr.Stevan Marcus, Dr. Janis O’Donnell, Dr. Katrina Ramonell, Dr. Kim Caldwell, and Dr. Guy Caldwell for guiding me throughout my entire graduate study. ix CONTENTS ABSTRACT ................................................................................................ ii DEDICATION ........................................................................................... iv LIST OF ABBREVIATIONS AND SYMBOLS ........................................v ACKNOWLEDGMENTS ......................................................................... ix LIST OF TABLES ................................................................................... xiv LIST OF FIGURES ...................................................................................xv 1. INTRODUCTION a. Use of C. elegans in the study of synaptic transmission ..........................3 b. Methods for studying synaptic transmission in C. elegans......................4 c. Key synaptic transmission pathways in C. elegans .................................8 d. Modulation of neuromuscular excitability in C. elegans ......................11 e. The role of cytoskeletal proteins in neurotransmission..........................13 f. C. elegans as a model system for studying Epilepsy ..............................15 g. C. elegans as a model system for studying Parkinson’s disease............17 h. Using C. elegans to study genetic factors associated with PD ..............17 i. C. elegans α-syn model for neurodegeneration ......................................20 j. Use of C. elegans to identify modifiers of α-syn toxicity .......................22 k. The role of dopamine signaling pathways in the survival of dopaminergic neurons ................................................................................23 x l. Current studies .......................................................................................24 m. References .............................................................................................27 2. USING A COMPLEMENTARY PHARMACOLOGICAL/ BEHAVIORAL APPROACH TO CHARACTERIZE C. ELEGANS SYNAPTIC TRANSMISSION a. Abstract ................................................................................................36 b. Materials/Methods ................................................................................37 c. Results ...................................................................................................39 d. Discussion .............................................................................................41 e. References .............................................................................................43 3. PHARMACOGENETIC ANALYSIS REVEALS THE POSTDEVELOPMENTAL
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