Towards Understanding the Mechanism of Clioquinol Neurotoxicity by Jamuna Chhetri BPharm (Hons), Kathmandu University, Nepal MPharmSc, University of Tasmania, Australia Submitted in fulfilment of the requirements for the Doctor of Philosophy Division of Pharmacy, School of Medicine, Faculty of Health University of Tasmania, Hobart October 2016 Declaration of Originality This thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledged in the thesis, and to the best of my knowledge and belief no material previously published or written by another person except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright. Jamuna Chhetri 4 October 2016 i Authority of Access This thesis is not to be made available for loan or copying for two years following the date this statement was signed. following that time, the thesis may be made available for loan and limited copying and communication in accordance with the Copyright Act 1968. Jamuna Chhetri 4 October 2016 ii Statement of Ethical Conduct The research associated with this thesis abides by the international and Australian codes on human and animal experimentation, the guidelines by the Australian Government's Office of the Gene Technology Regulator and the rulings of the Safety, Ethics and Institutional Biosafety Committees of the University. All animal experiments were approved by the Animal Ethics Committee (AEC), University of Tasmania, Australia (Animal Ethics approval numbers: A12817 and A0015097). iii Statement Regarding Published Work Contained in Thesis The publishers of the papers comprising Chapters 1 and 2 hold the copyright for that content, and access to the material should be sought from the respective journals. The remaining non published content of the thesis may be made available for loan and limited copying and communication in accordance with the Copyright Act 1968. iv List of Publications and Statement of Co-authorship The following people and institutions contributed to the publication of the work undertaken as part of this thesis: Candidate: Jamuna Chhetri1 (JC) Author 1: Nuri Gueven1 (NG) Author 2: Glenn Jacobson1 (GJ) Author 3: Anna E. King2 (AEK) 1Pharmacy, School of Medicine, Faculty of Health, University of Tasmania, Hobart, Tasmania, Australia 2Wicking Dementia Research and Education Centre, Faculty of Health, University of Tasmania, Hobart, Tasmania, Australia Author details and their roles: Peer-reviewed journal publications Paper 1, Chhetri J, Jacobson G, Gueven N. Zebrafish-on the move towards ophthalmological research. Eye 2014;7(10):19. (IF: 2.213) Located in chapter 1 Candidate was the primary author and with NG and GJ contributed to the idea, its formalisation and refinement. NG and GJ assisted in editing the manuscript. v Paper 2, Chhetri J, Gueven N. Targeting mitochondrial function to protect against vision loss. Expert Opin Ther Targets 2016:1-16. (IF: 4.79) Located in chapter 1 Candidate was the primary author and with NG contributed to the idea and its formalisation and refinement. NG also assisted in evaluating and editing the manuscript. Paper 3, Alzheimer’s Disease and NQO1: Is there a Link? (Accepted for Publication: Current Alzheimer Research) (IF: 3.58) Located in chapter 1 and chapter 2 Candidate was the primary author and with NG and AEK contributed to the idea, its formalisation and refinement. NG and AEK also assisted in evaluating and editing the manuscript. Conference abstracts (Oral) (Presenting author is underlined) 1. Chhetri J, Gasperini R, Jacobson G, Foa L, Gueven N. Mechanism of Clioquinol Toxicity-relevance to neurodegenerative diseases. APSA-ASCEPT (29 Nov-2 Dec 2015), Hobart, Australia. Conference abstracts (Poster) (Presenting author is underlined) 1. Chhetri J, Jacobson G, Gueven N. Development of a zebrafish model to detect mitochondrial dysfunction-induced vision loss. University of Tasmania Graduate Research conference (Aug 2013, Hobart, Australia). 2. Chhetri J, Jacobson G, Gueven N. Detoxification of clioquinol by NQO1. University of Tasmania Higher Degree Research (HDR) conference (Jul 2014, Hobart, Australia). vi 3. Chhetri J, Dilek J, Jacobson G, Gasperini R, Foa L, Gueven N. Clioquinol induces mitochondrial dysfunction by inhibiting NQO1. AussieMit conference (Dec 1-3 2014, Perth, Western Australia). 4. Chhetri J, Gasperini R, Jacobson G, Foa L, Gueven N. Mechanism of Clioquinol Toxicity-relevance to neurodegenerative diseases. APSA-ASCEPT (29 Nov-Dec 2015, Hobart, Australia). 5. Chhetri J, Farooq M, Dilek J, Jacobson G, Gasperini R, Foa L, Gueven N. NQO1- dependent toxicity of clioquinol. United Mitochondrial Disease Foundation, Mitochondrial Medicine (15-18th of June 2016, Seattle, WA, USA, presented by N Gueven). We the undersigned agree with the above stated “proportion of work undertaken” for each of the above published (or submitted) peer-reviewed manuscripts contributing to this thesis: Signed: A/Prof Nuri Gueven Prof Ben Canny Supervisor Head of School School of Medicine School of Medicine University of Tasmania University of Tasmania Date: 22/9/2016 vii Acknowledgements This PhD thesis would have never been possible if I was not given an opportunity in the first place. Therefore, first and foremost I would like to thank the University of Tasmania, in particular, Professor Gregory Peterson, Deputy Dean (Research), in believing my potential and providing me with an opportunity to pursue PhD study at the University of Tasmania. I am very much grateful to my primary supervisor, A/Professor Nuri Gueven who introduced me to research in a real sense and nurtured my enthusiasm for research. I appreciate his time, immense support and expert suggestions for my PhD study. He has always been a tremendous mentor who has always encouraged my research and allowed me to grow as an independent researcher. I am so much impressed with his immense knowledge of the subject matter, problem-solving skills, and networking skills. All in all, it has been a great honour to be his PhD student. I would also like to express my sincere gratitude to my co-supervisors A/Professor Lisa Foa, Dr Robert Gasperini and Dr Glenn Jacobson for their support and insightful comments on my research project. In addition, I am thankful to Lisa for letting me use the zebrafish facility at Medical Science Precinct (MSP) for the in-vivo experiments. My special thanks go to Dr Peter Traill, Anthony Whitty, Melissa Aubrey of Pharmacy and Dr David Steele, Sarah Kane of Medical Science Precinct (MSP) for the efficient management of logistics to facilitate the smooth running of my day-to-day research work. I could not stay without thanking Mr Steven Weston, Ms Narelle Phillips, Dr Ellen Bennett from MSP for so willingly sharing their histology expertise with me whenever I required them. I would also like viii to thank A/Professor Noel Davies, Central Science Laboratory, University of Tasmania, for his help with mass spectrometric analysis, Petr Smejkal and Aliaa Shallan for their help with 3D printing, Adrian Thompson for providing me with zebrafish eggs for the experiments, Jem Dilek for the initial drug screening and Mohammed Farooq for assisting me with some of the in-vitro studies. I would also like to express my sincere thanks to Emeritus Professor Stuart Mclean for providing me insightful feedback on my write-up, Dr Rosanne Guijt, Dr Rahul Patel and Dr Syed Tabish R. Zaidi for their support and suggestions on my research project and future career. I am thankful to my colleagues in Pharmacy for their companionship, emotional support and of course for the wonderful time we spent together. Last but not the least, I am very much indebted to my family, who has always been a source of inspiration to me and has always stood by me in all my pursuits. They are the reason why I am here today and I happily dedicate this thesis to them. ix List of Abbreviations Acronym Definition 8-HQ 8-hydroxyquinoline Acetyl-CoA acetyl-coenzyme A AD Alzheimer’s disease ADOA autosomal dominant optic atrophy ADP adenosine diphosphate AEC animal ethics committee AEG advance glycation end products ALDH4 aldehyde dehydrogenase 4 AMD age-related macular degeneration ANOVA analysis of variance ANT adenine nucleotide transporter APA acetaminophen APP amyloid precursor protein APS ammonium persulphate ARE antioxidant response element ATP adenosine triphosphate AVMA American Veterinary Medical Association Aβ amyloid beta BSA bovine albumin serum CAG cytosine adenine guanine Cat catalase CM-H2DCFDA chloromethyl 2',7'-dichlorodihydrofluorescein diacetate x CoQ coenzyme ubiquinone Cu Copper Cu/ZnSOD copper/zinc superoxide dismutase Cul 3 cullin 3 Cys cysteine Cytc cytochrome c DAB 3, 3’-diaminobenzidine DCPIP dicholorophenolindophenol DCs double cones Dic dicoumarol DLG dorsal lateral geniculate nucleus DMD digital micromirror device DMEM dulbecco’s modified eagle’s medium DMSO dimethylsulfoxide DNA deoxyribonucleic acid DR diabetic retinopathy DTT dithiothreitol E2 ubiquitin-conjugation enzyme ECL enhanced chemiluminescence EDTA ethylenediaminetetraacetic acid EGTA ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid EMA European Medicines Agency ER escape response ETC electron transport chain FADH2 flavin adenine dinucleotide reduced xi Fe iron Fe-S iron-sulphur centres FMN flavin mononucleotide GAPDH glyceraldehyde-3-phosphate dehydrogenase GLS-2 glutaminase 2 GPT glutamate