The Cellular Phenotype of the Neurodegenerative Disease Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay Teisha Y Bradshaw A thesis submitted to Queen Mary, University of London, for the degree of Doctor of Philosophy September 2014 Centre for Endocrinology William Harvey Research Institute Barts and the London School of Medicine and Dentistry Queen Mary University of London 1 Abstract _____________________________________________________________________________ Abstract Autosomal recessive spastic ataxia of Charlevoix Saguenay (ARSACS) is an early onset neurodegenerative disorder resulting from mutations in the SACS gene that encodes the protein sacsin. Sacsin is a 520kDa multi-domain protein localised at the cytosolic face of the outer mitochondrial membrane with suggested roles in proteostasis and most recently in the regulation of mitochondrial morphology. An excessively interconnected mitochondrial network was observed as a consequence of reduced levels of sacsin protein following SACS knockdown in neuroblastoma cells as well as in an ARSACS patient carrying the common Quebec homozygous SACS mutation 8844delT. Moreover, it was suggested that sacsin has a role in mitochondrial fission as it was found to interact with mitochondrial fission protein Dynamin related protein 1 (Drp1). The aim of this thesis was to explore sacsin’s role in the regulation of mitochondrial morphology and dynamics in non-Quebec ARSACS patients and sacsin knockdown fibroblasts. This study shows that loss of sacsin function promotes a more interconnected mitochondrial network in non-Quebec ARSACS patients and in sacsin knockdown fibroblasts. Moreover, recruitment of the essential mitochondrial fission protein Drp1 to the mitochondria was significantly reduced in ARSACS patient cells and in sacsin knockdown fibroblasts. This reduced recruitment of Drp1 to mitochondria also occurred when cells were treated to induce mitochondrial fission. Furthermore, both the size and intensity of Drp1 foci localised to the mitochondria were significantly reduced in both sacsin knockdown and patient fibroblasts. Finally, reduced ATP production, decreased respiratory capacity of mitochondria and an increase in mitochondrial reactive oxygen species demonstrated impaired mitochondrial function in ARSACS patient and sacsin knockdown fibroblasts. These results suggest a role for sacsin in the stabilisation or recruitment of cytoplasmic Drp1 to prospective sites of mitochondrial fission similar to that observed by other mitochondrial fission accessory proteins. 2 Acknowledgements _____________________________________________________________________________ Acknowledgements I would first like to take this opportunity to thank the Ataxia Charlevoix-Saguenay foundation and Queen Mary, University of London for funding this work. I would like to express my appreciation and gratitude to my supervisors Professor Paul Chapple and Dr Greg Michael. Their guidance, support and their fervent attention to detail have truly been invaluable. I would also like to express my sincere thanks to members of the Chapple group past and present, Dr David Parfitt, Dr Esmeralda Vermeulen, Emma Duncan and Dr Suran Nethisinghe for their encouragement and friendship. It has been great to be a part of Team Sacsin! I have been privileged to work amongst such world renowned academics in the Centre for Endocrinology at Barts and the London School of Medicine and Dentistry. Their passion for research and innovative methodologies has been truly inspirational. In particular I would like to thank Dr Eirini Meimaridou and Dr Harshini Katugampola for stimulating good discussions, for being there when I needed to troubleshoot, for their helpful advice and for cake and coffee. Thank you to my family and friends for their tremendous support and love, getting through these years would not have been possible without you. I dedicate this thesis to my parents, Ishmael and Ruthlyn Bradshaw who have inspired me to follow my dreams. Last but not least, thanks to God, who makes all things possible. 3 Statement of Originality _______________________________________________________________________ Statement of Originality I, Teisha Yo-Stella Bradshaw, confirm that the research included within this thesis is my own work or that where it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. Previously published material is also acknowledged below. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. Signature: Date: 30th September 2014 4 Table of Contents _______________________________________________________________________ Table of Contents Abstract ......................................................................................................................................... 2 Acknowledgements ....................................................................................................................... 3 Statement of Originality ................................................................................................................ 4 Table of Figures ............................................................................................................................. 9 Table of Tables ............................................................................................................................ 12 List of Abbreviations ................................................................................................................... 14 Chapter 1 ..................................................................................................................................... 18 Introduction ................................................................................................................................ 18 1.1 Cerebellum and Cerebellar Dysfunction ..................................................................... 19 1.1.1 X-Linked Ataxia .................................................................................................... 20 1.1.2 Autosomal Dominant Ataxia ............................................................................... 20 1.1.3 Autosomal Recessive Cerebellar Ataxia .............................................................. 25 1.1.4 Prevalent Forms of Autosomal Recessive Cerebellar Ataxia .............................. 28 1.2 Autosomal Recessive Spastic Ataxia of Charlevoix Saguenay and Sacsin ................... 29 1.2.1 Autosomal Recessive Spastic Ataxia of Charlevoix Saguenay ............................. 29 1.2.2 ARSACS Clinical Features ..................................................................................... 30 1.2.3 Molecular Genetics ............................................................................................. 32 1.2.4 Sacsin................................................................................................................... 43 1.2.5 Sacsin Function ................................................................................................... 51 1.3 Mitochondria Biogenesis, Dynamics and Disease ....................................................... 53 1.3.1 Mitochondrial Dynamics ..................................................................................... 54 1.3.2 Mitochondrial Biogenesis.................................................................................... 62 1.3.3 Mitophagy ........................................................................................................... 65 1.3.4 Mitochondrial Dysfunction in Neurodegenerative Disorders ............................. 65 1.4 Aims and objectives .................................................................................................... 70 Chapter 2 ..................................................................................................................................... 73 Methods and Materials ............................................................................................................... 73 2.1 Cell Culture .................................................................................................................. 74 2.1.1 Culture of SH-SY5Y Cells ...................................................................................... 74 2.1.2 Culture of ARSACS Patient and Control Fibroblasts ............................................ 74 2.1.3 Maintenance of Cells in Culture .......................................................................... 74 5 Table of Contents _______________________________________________________________________ 2.1.4 Freezing Cells ...................................................................................................... 75 2.1.5 Cell Counting ....................................................................................................... 76 2.1.6 Mycoplasma Testing ..........................................................................................