Investigating the reversibility and tissue specificity of mitochondrial disorders Marina Bartsakoulia, MSc Thesis submitted to Newcastle University in candidature for the degree of Doctor of Philosophy Wellcome Trust Centre for Mitochondrial Research Institute of Genetic Medicine, Faculty of Medical Sciences October, 2016 Abstract Mitochondrial disorders comprise a large group of heterogeneous disorders which are characterized by impairments in the cellular energy production. One of the great challenges of mitochondrial disease is the variety of clinical features present in patients. Mitochondrial disorders affect more than one organ leading to complex multisystem dysfunctions. Tissues, in which the metabolic demand is higher, such as skeletal muscle, neurons, liver or heart are typically affected. Mutations in both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) often lead to mitochondrial disorders. Although mtDNA encodes key proteins for the normal function of the mitochondrial respiratory chain enzymes, the vast majority of the essential components and proteins needed for the maintenance and replication of the mitochondrial DNA are encoded by the nDNA. Exome sequencing in combination with bioinformatics tools has proven really effective in determining novel alterations in the genomic sequence. One aim of this thesis was to evaluate novel mutations from affected patients with combined respiratory deficiencies. As a result, mutations in C12orf65 and in the novel disease gene MiD49, associated with mitochondrial disorders, are thoroughly presented. Vitamin supplements, pharmacological agents and exercise therapy are common strategies used in patients suffering from mitochondrial disorders. It has been shown that in cell lines of patients suffering from two rare reversible infantile mitochondrial diseases (reversible infantile respiratory chain deficiency and reversible infantile hepatopathy due to TRMU deficiency) supplementation of L-cysteine resulted in an improvement in most respiratory chain complexes activities. During my PhD I studied and proved that L-cysteine supplementation was also beneficial in cells from patients suffering from common forms of mitochondrial disorders such as MELAS and MERRF as the supplementation resulted in improved mitochondrial respiratory chain function. Finally, direct conversion of fibroblasts to neuronal progenitor cells was used to model mitochondrial disorders and study the tissue specificity. This project was very challenging due to the complex characteristics of mitochondrial biology. In summary, this thesis reveals the description of novel genes and mutations associated with combined mitochondrial deficiencies. Furthermore, we detected a positive effect of L-cysteine on a subset of mitochondrial disorders, which can be the base of further therapy development. ii Acknowledgements Firstly, I would like to thank and express my deepest appreciation to my supervisor Prof. Rita Horvath, who believed in me and continually supported me during my PhD. Her guidance helped me throughout the experimental and writing up stages of my project. She has always been patient and encouraging. In addition, I would like to thank Dr Veronika Boczonadi for her help, support and encouragement, Dr Aurora-Gomez Duran for her insightful comments and finally Dr Juliane Mueller for her support and contribution to my research. My sincere thanks also goes to my assessors Prof. Hanns Lochmuller and Prof. David Elliott for their helpful comments and their hard questions which widened my research from various perspectives. I would also like to thank all the lab members for the last three years I spent in the lab. They have always been supportive, helpful and created an enjoyable working environment. The completion of my PhD would not have been possible without the continuous support of my friends. They always helped me during the hard times, encouraged and supported me. Last but not least, I would like to thank my family: my parents and my brother. Without their love, support and encouragement I would not have been able to complete my thesis. They have always been standing by me and supporting every decision I made in life. iii Introduction .......................................................................................................... 1 Mitochondrion .................................................................................................................. 1 Origin and Structure .................................................................................................. 1 Function .................................................................................................................... 3 Electron Transport Chain and Oxidative Phosphorylation (OXPHOS) ................... 3 Reactive Oxygen Species (ROS) .............................................................................. 6 Mitochondrial Genetics .................................................................................................... 7 Mitochondrial Genome ............................................................................................. 7 Mitochondrial DNA structure ................................................................................... 9 Mitochondrial DNA inheritance - Heteroplasmy ................................................... 10 Mitochondrial DNA maintenance, replication and transcription ............................ 11 Mitochondrial translation ........................................................................................ 12 Mitochondrial Disorders ................................................................................................ 15 Mitochondrial encoded mutations .......................................................................... 16 Nuclear encoded mutations ..................................................................................... 21 Treatment of mitochondrial disorders ..................................................................... 28 Mitochondrial Dynamics ............................................................................................... 32 Mitochondrial Fusion .............................................................................................. 32 Mitochondrial Fission ............................................................................................. 34 Aims ..................................................................................................................... 38 Overview ........................................................................................................................ 38 Materials and Methods ....................................................................................... 39 Cell Culture .................................................................................................................... 39 Fibroblasts and myoblasts maintenance ................................................................. 39 iv Cell sub culturing .................................................................................................... 39 Cell counting ........................................................................................................... 40 Preservation of the cells .......................................................................................... 40 Mycoplasma detection ............................................................................................ 41 L-cysteine and N-acetyl cysteine (NAC) supplementation .................................... 41 Purifications ................................................................................................................... 41 DNA purification from cultured cells ..................................................................... 41 DNA purification from whole blood ...................................................................... 42 RNA purification from cultured cells ..................................................................... 43 RNA purification from whole blood ....................................................................... 44 DNA extraction for agarose gel .............................................................................. 44 Total protein extraction from frozen muscle tissue ................................................ 45 Quantifications ............................................................................................................... 45 Protein quantification .............................................................................................. 45 Heteroplasmy measurement .................................................................................... 47 Copy number quantification ................................................................................... 48 Levels of protein expression .......................................................................................... 51 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) ........ 51 Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) ............................. 52 Polymerase Chain Reaction (PCR) ................................................................................ 58 Reverse Transcription PCR ............................................................................................ 59 Agarose Gel Electrophoresis .......................................................................................... 60 Sanger Sequencing ........................................................................................................