αα-Synuclein-Sy-Synucleinnuclein phosphorylationphosphorylation andand relatedrelated kinaseskinases inin ParkinsonParkinson’s diseasedisease Jin-XiaJin-Xia ZhouZhou A thesis submitted in fulfillment of the requirement of the degree of Doctor of Philosophy School of Medical Sciences, Faculty of Medicine and Neuroscience Research Australia November 2013 I PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Zhou First name: Jin-Xia Other name/s: I Abbreviation for degree as given in the University calendar: PhD School: School of Medical Sciences Faculty: Medicine lation and related kinases in Parkinson's disease Abstract 350 words maximum: (PLEASE TYPE) ' Parkinson's disease (PO) is the most common neurodegenerative movement disorder pathologically identified by degeneration of the nigrostriatal system and the presence of Lcwy bodies (LBs) and neurites. structuTal pathologies largely made from insoluble a-synuclein phosphorylated at serine 129 (S 129P). Several kinases have been suggested to facilitate a-synuciein phosphorylation in PD, but without significant human data the changes that precipitate such pathology remain conjecture. The major aims of this pr~ject were to assess the dynamic changes of a -synuclein phosphorylation and related kinases in the progression of PD and in animal models of PD. and to determine whether Tenuigenin (TEN), a Chinese medicinal herb, can prevent cc-synucleln-induc.?d toxicity in a cell model. The levels of non-phosphorylated a-synuclein decreased over the course ofPD, becoming increasingly phosphorylated and insoluble. There was a dramatic increase in phosphorylated a-synuclein that preceded LB formation. Importantly, three a-synuc!ein-relatec ki nases [polo-like kinase 2 {PLK2), lcuc.:inc- rich repeat kinase 2 (LRRK2l and cyclin G-~tssoc i ated kinase (GAK)] were found to be involved at different times in the evolution of LB formation in P.O. A naly.~is of a subacute \IIPTP model of PO neurodegeneration revealed similar increases in S 129P a-synuclein and PLK2/3 levels occurred immediately after the toxic insult, revealing this in 1•ivo model as suitable to assess this molecular mechanism. Assessment of a-sy11uclein over-expressing cell models of PO cytotoxicity 1 revealed increased S l29P a-synuclein but no increase in PLK2/3 levels, however treatment of these cells with the Chinese herbal ~ extract TEN was effective in reducing the increased S 129P a-synuclein and cell toxicity in association with reducing levels of PLK3. 1 This suggests that TEN may be an effective treatment for S129P' a-synuclein induced cytotoxicity as observed in PD. I In summary, these studies indicate that an increase in S 129P a-synuclein occurs early in response to cell damage in both PD and toxin­ induced PD models and that the levels of PLK2/3 increase in concert. Human data suggests that different kinases play a precipitating role in LB formation and compaction. Importantly TEN treatment appears to reduce PLK3 levels and ameliorateS 129P a-synuclein toxicity, identifying this herbal extract as a potential therapeutic drug for this PO relevant mechanism. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or Its agents the right to archive and to make available my thesis or dissertation in whole or 111 parr in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). -s.- \ ls-- ~ • i I ~ • • "• • • • • • • • • • • + • • • " • • • • • • • • •' •• • • ~ • "•' • "• t •• 1 • •• • •• • • • Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Reqllests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional Circumstances and re utre the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS COPYRIGHT STATEMENT 'I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. 1 also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this Is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed clfi((l(~') Date •• • •• • •••••• • • • ' ••• •• •••• 0 •• • ••••• •••• •• •••••••• 4 ••••• ~ •••••••••••••••• • ••••• AUTHENTICITY STATEMENT 'I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.' I i'vv'(r•'e-v'2J~ Signed ................. ~- ..... ' .................................................... Q'-¥1 II / )lOt) Date AcknowledgmentsAcknowledgments Foremost, I would like to express the deepest appreciation to my primary supervisor Professor Glenda Halliday. Thank you so much for the guidance and continuous support of my PhD study. Your extreme enthusiasm on science has inspired me to continue on this field and your patience and immense knowledge on neuroscience make this project special. You are the first-class supervisor and I feel so lucky to be your PhD student. I would like also thank my co-supervisor Dr. Yue Huang for all the help, support and encouragement for my studying and living during the PhD period. You have taught me scientific thinking and problem solving skills. And also your patience and consideration made my life easier in Sydney. I feel like you are not only a supervisor but also a friend. My sincere thanks also go to my conjoint-supervisor Professor Xiao Min Wang from China Capital Medical University, Beijing, China. Thank you for all your generous support, encouragement and consideration for my PhD project. Your excellent organization and management have provided me a valuable and fruitful exchange experience during my PhD study. Without your support, this project would not have gone so well. Thank you to all my colleagues in Halliday group in Neuroscience Research Australia. You all have made my working environment exciting, motivating and fun. A special thanks to Ms Heidi Cartwright, Ms Heather McCann, Ms Shelley Forrest, Ms Danielle Small, Ms Germaine Chua and Ms Amanda Gysbers for technical assistance. This thesis would not have been possible without your help. III Also thank you to all the colleagues in the Xiao Min Group in China Capital Medical University. Your friendly and easy going characters made the laboratory environment so comfortable and fun. A special thanks to Ms Hao Bo Zhang and Dr. E Lv for the experimental cooperation. Your selfless contribution has made this project more comprehensive. I would also like to thank all my friends at Neuroscience Research Australia. Your pleasant company has colored my life during my PhD. A special thanks to Ms Bonnie Lam, Ms Puika Yeung, Ms Jac Kee Low, Dr. Claire Stevens, Dr. Andy Liang and Dr. Jie Zhang. Thank you for leaving me such beautiful memories. The friendship from you all is a treasure to me. Very special and sincere thanks go to all my family. Thank you all for supporting me spiritually throughout my life. Without you, I could not have gone so far. Words can not express my gratitude to you. Finally, thanks to the Australian Government, University of New South Wales (UNSW) and Neuroscience Research Australia (NeuRA) for supporting me financially with the International Postgraduate Research Scholarship (IRPS) and NeuRA PhD Scholarship. It has been a great honor receiving these scholarships, and I will continue research in the future. The PhD journey was very challenging for me and I wish to thank a lot of other people. Although I can not state all the names here, I have a deep appreciation for everyone in my heart. Thank you. IV AbstractAbstract Parkinson’s disease (PD) is the most common neurodegenerative movement disorder pathologically identified by degeneration of the nigrostriatal system and the presence of Lewy bodies (LBs) and neurites, structural pathologies largely made from insoluble α- synuclein phosphorylated at serine 129 (S129P). Several kinases have been suggested to facilitate α-synuclein phosphorylation in PD, but without significant human data the changes that precipitate such pathology remain conjecture. The major aims of this project were to assess the dynamic changes of α-synuclein phosphorylation and related kinases in the progression of PD and in animal models of PD, and to determine whether Tenuigenin (TEN), a Chinese medicinal herb, can prevent α-synuclein-induced toxicity in a cell model. The levels of non-phosphorylated α-synuclein decreased over the course of PD, becoming increasingly phosphorylated and insoluble. There was a dramatic increase in phosphorylated α-synuclein that preceded LB formation. Importantly, three α-synuclein- related kinases [polo-like kinase 2 (PLK2), leucine-rich repeat kinase 2 (LRRK2) and cyclin G-associated kinase (GAK)] were found to be involved at different times in the evolution of LB formation in PD.