Tiago Fleming de Oliveira Outeiro YEAST AS A MODEL ORGANISM TO STUDY DISEASES OF PROTEIN MISFOLDING Instituto de Ciências Biomédicas de Abel Salazar Universidade do Porto Porto 2004 Tiago Fleming de Oliveira Outeiro YEAST AS A MODEL ORGANISM TO STUDY DISEASES OF PROTEIN MISFOLDING Dissertação de candidatura ao Grau de Doutor em Ciências Biomédicas submetida ao Instituto de Ciências Biomédicas de Abel Salazar. Orientadora - Professora Susan Lindquist Co-orientadora - Professora Maria João Saraiva Para os devidos efeitos, e de acordo com o disposto no n° 2 do Artigo 8 do Decreto-Lei n° 388/70, o autor desta dissertação declara que interveio na concepção e execução do trabalho experimental, na interpretação e discussão dos resultados e na preparação dos manuscriptos publicados e em vias de publicação. Na presente dissertação incluem-se os resultados das seguintes publicações: Outeiro, TF, Lindquist, S, (2003) Yeast cells provide insight into alpha-synuclein biology and pathobiology, Science, 302:1772-5. Willingham S, Outeiro TF, DeVit MJ, Lindquist SL, Muchowski PJ (2003) Yeast genes that enhance the toxicity of a mutant huntingtin fragment or alpha- synuclein, Science, 302:1769-72. Outeiro, TF, et al., Drug screening yields new insight into neurodegeneration, Nature, in press. Table of Contents TABLE OF CONTENTS ACKNOWLEDGEMENTS XIII ABSTRACT XVII RESUMO XIX OVERVIEW XXIII ORGANIZATION OF THE THESIS XXV ABBREVIATIONS XXVII CHAPTER 1. GENERAL INTRODUCTION 3 1.1 PROTEIN FOLDING AND MISFOLDING 3 1.2 CELLULAR QUALITY CONTROL MECHANISMS 6 1.2.1 Molecular Chaperones 7 1.2.2 Ubiquitin-Proteasome Pathway 8 1.3 PROTEIN MISFOLDING DISEASES 10 1.3.1 Parkinson's Disease 15 1.3.1.1 Genetics of PD 18 1.3.1.1.1 PARK1 19 1.3.1.1.2 PARK2 20 1.3.1.1.3 PARK5 22 1.3.1.1.4 PARK7 23 1.3.1.2 a-Synuclein 23 1.3.1.2.1 aSyn Structure and Interacting Partners 24 1.3.1.2.2 Putative Functions of aSyn 27 1.3.1.2.3 aSyn Mutations and PD 30 1.3.1.2.4 Animal Models of PD based on Expression of aSyn 32 1.3.1.2.5 aSyn Oligomers: Zooming in on the Toxic Species 35 1.3.1.2.6 Selective Neuronal Degeneration in PD: aSyn and Dopamine 37 1.3.2 Polyglutamine Diseases 38 1.3.2.1 Huntington's Disease 40 1.3.2.1.1.1 Genetics of HD 42 1.3.2.1.2 Model Organisms for Studying HD and Other PolyQ Diseases 43 1.4 YEAST AS A "SIMPLE" MODEL ORGANISM FOR STUDYING HUMAN DISEASES 47 1.4.1 The "Awesome Power" of Yeast Genetics 48 1.4.2 Modeling Protein Aggregation Diseases in Yeast 50 1.4.2.1 Friedreich's Ataxia 51 1.4.2.2 Amyotrophic Lateral Sclerosis (ALS) 52 1.4.3 Functional genomics approaches to studying protein aggregation in yeast... 53 1.4.4 Limitations of Yeast to Study Neurodegenerative Disorders 56 1.4.5 Yeast and Drug Screens 56 - ix - Table of Contents CHAPTER 2. YEAST CELLS PROVIDE INSIGHT INTO ALPHA-SYNUCLEIN BIOLOGY AND PATHOBIOLOGY 61 ABSTRACT 61 2.1 INTRODUCTION 62 2.2 MATERIALS AND METHODS 63 2.2.1 Plasmid constructions 63 2.2.2 Yeast Strains and Genetic Procedures 63 2.2.3 Spotting Experiments 65 2.2.4 Plasmid Maintenance Studies 65 2.2.5 SDS-PAGE and Immunoblottings 65 2.2.6 Microscopy. 66 2.3 RESULTS 68 2.4 CONCLUSIONS 85 CHAPTER 3. YEAST GENES THAT ENHANCE THE TOXICITY OF A MUTANT HUNTINGTIN FRAGMENT OR a-SYNUCLEIN 89 ABSTRACT 89 3.1 INTRODUCTION 90 3.2 MATERIALS AND METHODS 92 3.2.1 Yeast screening methods 92 3.2.2 Identification of yeast gene deletion strains that are synthetically sick or lethal with HD53Q or aSyn 93 3.2.3 Analysis of viability in yeast gene deletion strains transformed with HD53Q or a- synuclein 93 3.2.4 Sedimentation analysis of Rnqlp 94 3.3 RESULTS 95 3.4 CONCLUSIONS 107 CHAPTER 4. DRUG SCREENING USING YEAST MODELS OF NEURODEGENERATIVE DISEASE 111 ABSTRACT m 4.1 INTRODUCTION 112 4.1.1 Yeast Model for a-Synucleinopathies 115 4.1.2 Yeast Model for Huntington's Disease (HD) 116 4.2 MATERIALS AND METHODS 119 4.2.1 One-Step Gene Deletions 119 4.2.2 Yeast Strains, Manipulations and Media 120 4.2.3 Drug Library 121 4.2.4 Screening Strategy. 121 4.2.5 Screening Procedures 122 4.3 RESULTS 124 4.3.1 aSyn Screen 124 4.3.2 Huntingtin Screen 127 4.4 DISCUSSION 129 4.5 DRUG SCREENING YIELDS NEW APPROACHES TO NEURODEGENERATION 131 4.5.1 Introduction 131 4.5.2 Discussion 144 Table of Contents CHAPTER 5. BIOLOGICAL EFFECTS OF ALPHA-SYNUCLEIN EXPRESSION IN YEAST 147 ABSTRACT 147 5.1 INTRODUCTION 148 5.2 MATERIALS AND METHODS 151 5.2.1 Plasmid Construction 151 5.2.2 Yeast Strains and Manipulations 151 5.2.3 Growth Rates and Survivorship Assays 152 5.2.4 Growth Curves 153 5.2.5 Protein Preparation, Antibodies, and SDS-PAGE and Immunoblottings 154 5.2.6 GFP Imaging 154 5.2.7 Biochemical Assays and Immunofluorescence 154 5.2.8 ROS Detection 155 5.3.1 Increased Expression of aSyn is Toxic to Yeast. 156 5.3.2 Accumulation of Proteasomal Substrate CPY* but not Sec61-2p nor Cytosolic Substrate 157 5.3.3 aSyn Impairs Intracellular Trafficking 160 5.3.4 aSyn Causes Increased Sensitivity to ER Stress 163 5.3.5 aSyn Causes ER Breakdown 165 5.3.6 aSyn Expression Causes Increased ROS Production and Sensitivity to Oxidative Stress 167 5.4 DISCUSSION 173 CHAPTER 6. GENERAL CONCLUSIONS 179 REFERENCES 187 -xi - Acknowledgements ACKNOWLEDGEMENTS The work presented here was only possible due to a great deal of support and help from many different people. I would therefore like to express my sincere gratitude to all of them and make them feel they all played important roles not only in this work, but more importantly, in my life. I would like to thank Prof. Maria João Saraiva for all her help with my initial work, and for her support throughout my PhD. I have always felt her interest even through the distance. I also need to thank the members of her lab, where I spent some time before coming to the US. Monica definitely helped me a lot with the initial molecular biology! Clara, my good friend, your happiness is contagious, and you are one of my best friends! We can be apart for years, but I know you will always be there for me if I need! It would be unfair not to mention Prof. Pedro Moradas Ferreira. I have always admired him, ever since college, and he has been a good friend, helping me dealing with several issues related to the PhD process at ICBAS. During my PhD I learned a great deal, not only in terms of science, but also as a person. A PhD is a big commitment, and having the opportunity to go abroad enables one to grow and develop as a human being as well. Science is a lot about social interactions, and the numerous collaborations showed me that generosity and friendliness are key ingredients for success. I tried to make the most out of all this time, establishing a network of people who I now know well, - XIII - Acknowledgements and with whom I know I will work in the future, for sure. They are not only collaborators, but more importantly, friends. In this group I have to include: Paul Muchowski for very fruitful collaborations, for sharing materials, and for all his friendship and support. Chris Rochet, another good friend and collaborator, with whom I've worked closely. Cole Haynes and Antony Cooper, for an exciting collaboration that is still ongoing. I've come to interact with some of the greatest experts in the field, and I would like to express my gratitude for making me feel like I was at their level. Their excitement and enthusiasm has been extremely important: Jeff Kelly, Peter Lansbury, Brad Hyman, Anne Young, Patrik Brundin... and so many others... Thank you all! To the members of the Lindquist lab, for putting up with me, for helping me getting started, for their critical thinking, for reading my manuscripts and thesis... and for being good friends! Heather, I admire you for your strong will, for your courage, for being such a hard-worker. And also for being such a good and close friend! Thank you for your advice, for guiding me, and for trusting in me! It was hard to see you leave, even though I feel you'll always be close by! Oliver, you've been a good friend, and I feel like our ways have moved in parallel quite often. It has been great working with you! To the Portuguese "gang" both in Chicago and in Boston, for all the good times we've spent together. I've made several good friends I know I will keep in the future! - xiv - Acknowledgements Rita... we've developed such a special friendship... I have to express my gratitude to you in a special way. To my friends Hélder and Marta, I have to say how much you mean to me! Hélder, we have a future together! Our perseverance will take us far! To Nene and Liliana... I feel like you've always been with me... through the good and the not so good moments... Always near, always present! No matter what happens in our lives, our friendship will always be there! To my parents, sister, grandparents and all my family, I thank their unconditional support.