Disease Mechanisms in Mitochondrial Maintenance Disorders Kamil Sebastian Sitarz MSc Thesis submitted to Newcastle University in candidature for the Degree of Doctor of Philosophy Institute of Genetic Medicine Faculty of Medical Sciences Newcastle University September 2012 This thesis is dedicated to my beloved wife Agnieszka and our dearest daughters Zuzia and Gabrysia for their constant support and unconditional love. Abstract OPA1 and MFN2 are two critical mitochondrial membrane proteins required for mitochondrial fusion. OPA1 mutations account for approximately 60% of cases of autosomal-dominant optic atrophy (DOA) and up to 20% of mutational carriers develop a more severe multi-systemic neurological phenotype (DOA+) in addition to visual failure. MFN2 mutations result in Charcot-Marie-Tooth disease type 2A (CMT-2A) and in a subgroup of patients, the peripheral neuropathy is complicated by optic atrophy, highlighting a degree of phenotypic overlap with DOA+. POLG1 encodes the catalytic subunit of DNA polymerase gamma (POLG) and POLG1- related diseases are clinically highly heterogeneous, ranging from early-onset Alpers- Huttenlocher syndrome to late-onset isolated chronic progressive external ophthalmoplegia. OPA1, MFN2 and POLG1 mutations all result in disturbed mitochondrial DNA (mtDNA) maintenance, with both quantitative (depletion) and qualitative (point mutations and deletions) mtDNA abnormalities having been identified in patient tissue samples. In this PhD project, the disease mechanisms underpinning these nuclear mitochondrial disorders have been studied further. OPA1 and MFN2 mutations were found to result in significant mtDNA proliferation as a likely compensatory mechanism to impaired mitochondrial oxidative phosphorylation. Using a previously-validated repopulation assay, mtDNA replication in cultured POLG1-mutant fibroblasts was severely depressed following a period of ethidium bromide-induced mtDNA depletion. A similar observation was made with OPA1-mutant fibroblasts, but this effect was not as marked as for POLG1-mutant fibroblasts. Significant reorganisation of the mitochondrial network was also apparent for both groups of mutant fibroblasts. Although neuromyelitis optica (NMO) shares some clinical features with DOA, genetic variations within OPA1 are not associated with the risk of developing NMO. Finally, research into DOA and other mitochondrial optic neuropathies have been severely restricted by the lack of the access to retinal ganglion cells (RGCs), precluding direct studies to be performed on the cell type which is preferentially affected in this group of disorders. To circumvent this limitation, human induced pluripotent stem cell (hiPSC) lines have been generated from patient-derived i fibroblasts harbouring confirmed OPA1 mutations. The future differentiation of these induced pluripotent stem cell lines into RGCs will hopefully provide a powerful and versatile tool for disease modelling and the development of targeted therapeutic strategies. ii Acknowledgements First and foremost, I owe my sincerest gratitude to my supervisors, Prof. Patrick Chinnery and Dr Rita Horvath for providing me with the opportunity to undertake this PhD project. I am grateful for their thought-provoking guidance, inspiration and continued support over the past three years. It was the greatest pleasure to be a part of their group. I would like to address my special thanks to Dr Patrick Yu-Wai-Man, who although not being my official supervisor, has provided me with the inspiring suggestions, continual encouragement and invaluable support throughout the entire course of my PhD. I am grateful for all our constructive discussions and the helpful feedback on the drafts of this thesis. I would like to express my appreciation to all the collaborators I had the pleasure to work with. I owe my gratitude to the members of Prof Majlinda Lako’s Stem Cell Group for their continuous willingness to help. At this point, I would like to thank especially Dr Katarzyna Tilgner, for her excellent assistance with the stem cell culture, her patience, and most importantly, for her friendship. My acknowledgements also extend to Dr David Samuels for offering me support with the statistical analyses, and all the members of the Mitochondrial Diagnostic Service (Newcastle upon Tyne) for providing me with the patient tissue samples. A special mention is also deserved by all my fellow colleagues from the PFC group for making the working environment so friendly. I owe many thanks to Drs Angela Pyle and Gavin Hudson, who have been a great support during my PhD. I would also like to thank Phillippa “Pip” Carling and Gerald “Gerry” Pfeffer, for all these great chats we have had over the much-needed cups of coffee. I would like to extend my deepest gratitude to my parents for all their love, constant understanding and endless support in every situation of my life. Lastly, and most importantly, I would like to express my love and appreciation to my wonderful wife, Agnieszka. You gave me the strength to face all the difficulties and you were always by my side during ups and downs. I am so lucky to have you as my best friend, my soul-mate and my wife. iii Author’s Declaration This thesis is submitted for the degree of Doctor of Philosophy in Newcastle University. The research detailed inside was performed in the Mitochondrial Research Group within Institute of Genetic Medicine under the supervision and guidance of Prof. Patrick Chinnery and Dr Rita Horvath, between September 2009 and August 2012. I hereby declare that none of the material presented in this thesis has been previously submitted by me for a degree or any other qualification at this or any other University. Furthermore, it is my own independent work, unless stated otherwise. This copy has been supplied in the understanding that it is copyright material and that no quotation from the thesis may be made without appropriate acknowledgement. Kamil S. Sitarz iv List of Contents Abstract ......................................................................................................................... i Acknowledgements ...................................................................................................... iii Author’s Declaration .................................................................................................. iv List of Contents ............................................................................................................ v Table of Contents ........................................................................................................ vi List of Figures ............................................................................................................. xi List of Tables ........................................................................................................... xviii List of Publications ................................................................................................... xxi Abbreviations ........................................................................................................... xxii Chapter 1 General Introduction .................................................................................. 1 Chapter 2 Research Aims ......................................................................................... 77 Chapter 3 Materials and Methods ............................................................................. 79 Chapter 4 Disruption of Mitochondrial Fusion and MtDNA Maintenance ........... 103 Chapter 5 Investigations of MFN2-Positive Muscle Biopsy .................................. 117 Chapter 6 Neuromyelitis Optica and Genetic Variations within OPA1 ................. 131 Chapter 7 MtDNA Repopulation Assay and Mitochondrial Network Analysis .... 142 Chapter 8 Generation of hiPSCs from OPA1-Mutant Human Fibroblasts ............. 204 Chapter 9 General Discussion ................................................................................ 244 Appendices .............................................................................................................. 252 Bibliography ........................................................................................................... 279 Publications ............................................................................................................. 311 v Table of Contents Chapter 1 General Introduction ............................................................................... 1 1.1 THE MITOCHONDRION ............................................................................................................... 5 1.1.1 Origin ................................................................................................................................... 5 1.1.2 Structure............................................................................................................................... 5 1.2 RESPIRATORY CHAIN ................................................................................................................. 7 1.2.1 Complex I ............................................................................................................................ 8 1.2.2 Complex II ........................................................................................................................... 9 1.2.3 Complex III .......................................................................................................................... 9 1.2.4 Complex IV ......................................................................................................................