The Involvement of Anti-Oxidative Response and Mitochondrial Dynamics in the Pathogenesis of Friedreich's Ataxia: Relevance T
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The Involvement of Anti-Oxidative Response and Mitochondrial Dynamics in the Pathogenesis of Friedreich’s Ataxia: Relevance to the Development of Future Therapeutics Shannon Chiang B. Med. Sci. Grad. Dip. Sc. Doctor of Philosophy (Medicine) Discipline of Pathology Faculty of Medicine and Health The University of Sydney 2019 A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy in the Faculty of Medicine and Health at the University of Sydney ACKNOWLEDGEMENTS I would like to first thank my supervisor, Prof. Des Richardson, for you have given me this extraordinary opportunity to pursue my interest in scientific research, allowing me to be part of your laboratory, and to fulfil my PhD studies. Thank you for believing in me. I am grateful that you have accepted me. Your knowledge and insight have helped me over the years, and I appreciate all the efforts and time you have spent to guide me through this journey. Thank you for your many hours of proofreading and writing assistance in preparing the articles and thesis that have been the backbone of this contribution. To my associate supervisor and friend, Dr. Michael Huang, thank you very much for all the help, support, and guidance you have given me. You are a wonderful mentor and you have taught me many valuable lessons. Thank you for being so patient, caring, and supportive. I am grateful to have been your student, and I enjoy our partnership. As you have once said, ‘every moment is an opportunity to learn something new’, I will continue to do so. Thank you to Prof. Clare Hawkins, Dr. Bronwyn Brown, and Dr. Amy Anzovino, for your tremendous help in bringing such a fantastic paper into fruition during my PhD candidature. To the mentors from Vet Histology, ACMM, and Bosch Institute of the University of Sydney, thank you, Elaine Chew, for your great help with the histological studies. Thank you Naveena Gokoolparsadh, Dr. Hongwei Liu, Dr. Gerald Shami, Dr. Melanie Laird, Dr. Donna Lai and Dr. Sheng Hua, for your patience and guidance, and for trusting me to work in your facilities with your precious equipment. I am grateful for the learning experience over the years. i To the present and past members of the Molecular Pharmacology and Pathology Program, I am fortunate to have been a part of such a diverse, hardworking, and friendly group of brilliant minded people. Thank you, Dr. Danuta Kalinowski, for your joyful presence and continuous encouragements. You are knowledgeable and have been a very helpful and kind mentor to me. Thank you, Dr. Sumit Sahni, for your insight and advice along the way. I will always cherish the time we shared with your family at the conference in Tokyo Japan. Thank you, Dr. Patric Jansson, for being so understanding and supportive all the time. You might not realise this, but your mentality inspires hope for me. Thank you, Dr. Dong-Hun Bae, for being so kind and helpful. I greatly appreciate the insight you have given me when it came to the writing of the thesis. Thank you, Dr. Angelica Merlot, for your kindness and for reassuring my confidence. The conference in Portugal had been a fabulous time, and I am grateful for the experience we shared in this trip. To my fellow PhD friends (some have now received their well-earned title), I want to particularly thank Dr. Sharleen Menezes, for your positive nature, intelligence, and support has had a huge influence on me. You are a wonderful friend and you have been inspirational. Thank you to, Dr. Kevin Park, for your companionship and motivation. You encourage me all the time and you make me believe in myself. Thank you, Jasmina Paluncic, for the spirited friendship that we have together. You help me challenge myself, yet at the same time, you guide me and make me stronger. Thank you to, Leyla Fouani, for being such a kind friend, and for sharing uplifting conversations during times of stress. Thank you, Sukriti Krishan and Lionel Leck, for being such amazing and supportive friends. Thank you for ceaselessly helping each other out towards the completion of our papers and theses, and especially the support for each other with drinks and games. And yes, you too Rachal Poon, thank you for bringing laugher and joy and Cooper into this stressful ii environment that desperately needs the occasional bliss. My PhD was made enjoyable and possible because of each of you. I am thankful for the experience and memories we shared, both within and outside of our research lives. Most important of all, I would like to thank my family for their unconditional love and continuing support. In particular, thank you to my Mum for always believing in me and my abilities, for encouraging me to pursue my interests, and for raising me into who I am. Thank you to my sister, Cordelia, for being my best friend and a caring sister, for supporting and looking after me every day and night, meals included. Thank you to my lovely girlfriend, Steph, for being the most understanding and considerate person that I have ever known, for loving me and being there for me all the time. Thank you to Pom Pom for being by my side, and on my lap and bed whenever I’m home, and for the unconditional love she perfuses me in support of my work, for more walks. Last but not least, thank you to my Dad, for you have been the one who had sparked my interest in science. From the moment I asked you about gravity as a child to the day you told me about the mysteries of the universe, you have ironically also warned me about pursuing research for it is not an easy adventure. And now, well, it is research, but it’s not physics. You have unknowingly inspired me over the years and helped me find my path. I am extremely grateful for the unending love and support and encouragement from my family. I love you all very much. This thesis is dedicated to my family. iii STATEMENT OF ORIGINALITY This dissertation is written and submitted in accordance with the regulations for the degree of Doctor of Philosophy at the University of Sydney. I certify, to the best of my knowledge, that previously published work and materials has been appropriately acknowledged in the text. This thesis has not been submitted for any degree or other purposes. I certify that the intellectual content of this thesis is the product of my own work and that all the assistance received in preparing this thesis and sources have been acknowledged. Shannon Chiang iv AUTHORSHIP ATTRIBUTION STATEMENT This thesis contains materials published in: ❖ Chiang, S., Kovacevic, Z., Sahni, S., Lane, D.J., Merlot, A.M., Kalinowski, D.S., Huang, M.L., Richardson, D.R. Frataxin and the Molecular Mechanism of Mitochondrial Iron- loading in Friedreich’s Ataxia. Clinical Science, 2016. 130(11):853-70. ❖ Anzovino, A.*, Chiang, S.*, Brown, B.E., Hawkins, C.L., Richardson, D.R., Huang, M.L. Molecular Alterations in a Mouse Cardiac Model of Friedreich’s Ataxia: An Impaired Nrf2 Response Mediated via Up-Regulation of Keap1 and Activation of the Gsk3β Axis. American Journal of Pathology, 2017. 187(12):2858-2875. *Equal first author. ❖ Chiang, S., Kalinowski, D.S., Jansson, P.J., Richardson, D.R., Huang, M.L. Mitochondrial Dysfunction in the Neuro-degenerative and Cardio-degenerative Disease, Friedreich’s Ataxia. Neurochemistry International, 2017. pii: S0197-0186(17)30371-6. ❖ Huang, M.L., Chiang, S., Kalinowski, D.S., Bae, D.H., Sahni, S., Richardson, D.R. The Role of the Antioxidant Response in Mitochondrial Dysfunction in Degenerative Diseases: Cross-Talk between Antioxidant Defense, Autophagy, and Apoptosis. Oxidative Medicine Cell Longevity, 2019. 2019:6392763. Published materials in these articles are presented in Chapter 1, Chapter 2, and Chapter 3. Where there is joint first-authorship, I was a principal contributor, and was involved in designing and performing experiments, statistical analysis of data for the studies (Western blotting, co- immunoprecipitation, histology, PCR, transmission electron microscopy, animal treatments, in vivo work), and I also contributed in figure preparations and writing of manuscripts. As an equal v first-author, Dr. Anzovino was involved in the intellectual design and planning of studies, performing particular experiments (Fig. 3.2; Fig. 3.3A, C; Fig. 3.5; and Fig. 3.7), statistical analysis, and writing of the manuscript that partially contributes to the thesis in Chapter 3. Dr. Huang was also involved in designing and performing certain experiments in Chapter 3 and Chapter 4, and in writing parts of the manuscripts. Permission to include these works was granted by both Dr. Anzovino and Dr. Huang. In addition to the above, in cases where I am not the corresponding author of a published item, permission to include the published material has been granted by the corresponding authors. Shannon Chiang, 21st May 2019 As supervisors for the candidature upon which this thesis is based, I can confirm that the authorship attribution statements above are correct. Des Richardson, 21st May 2019 Michael L. Huang, 21st May 2019 vi ABSTRACT Friedreich’s ataxia (FA) is the most common autosomal recessive ataxia, and patients of the disease are severely afflicted with progressive neuro- and cardio-degeneration. The main cause of FA is due to the deficient expression of the mitochondrial protein, frataxin, and its deficiency has been well reported to be associated with oxidative stress and losses in energy metabolism. The major aim of this thesis was to elucidate the molecular mechanisms involved in the dysregulated anti-oxidative response in frataxin deficiency, which is responsible for the exacerbation of oxidative stress in FA.