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The Discovery of Novel Gsk3 Substrates and Their Role in the Brain

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The Discovery of Novel Gsk3 Substrates and Their Role in the Brain THE DISCOVERY OF NOVEL GSK3 SUBSTRATES AND THEIR ROLE IN THE BRAIN James Robinson A DISSERTATION IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY St. Vincent’s Clinical School Facility of Medicine The University of New South Wales May 2015 ORIGINALITY STATEMENT ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed ……………………………………………… Date ………………………………………………… ii |Page Abstract Bipolar Disorder (BD) is a debilitating disease that dramatically impairs people’s lives and severe cases can lead to exclusion from society and suicide. There are no clear genetic or environmental causes, and current treatments suffer from limiting side effects. Therefore, alternative intervention strategies are urgently required. Our approach is to determine mechanisms of action of current drug therapies, in the hope that they will lead to discovery of next generation therapeutic targets. Lithium has been the mainstay treatment for BD for over 50 years, although its mechanism of action is not yet clear. A major target of lithium (and other mood stabilizers) is Glycogen Synthase Kinase-3 (GSK-3), a Ser/Thr protein kinase that is dysregulated in BD. Pathogenic targets of GSK3 could become novel therapeutic targets for improved treatment of BD, although these are not yet known. Identifying these targets is the primary goal of this project. Our lab used a combination of bioinformatics and phosphoproteomics to discover 45 novel substrates of GSK3 involved in vesicular trafficking events. Here, I focus on two promising trafficking proteins; the lipid kinase phosphatidylinositol 4-kinase II alpha (PI4KII) and the AP-2 kinase adaptin associated kinase-1 (AAK1). PI4KIIα regulates cell-surface expression of AMPA receptors in neurons and is therefore likely to affect neurotransmission in the brain. Phosphorylation regulates this process by promoting binding of PI4KIIα to Adaptin of the AP-3 complex for trafficking to the lysosome to be degraded. Depletion of PI4KIIα in neurons of Drosophila increased their locomotor activity, consistent with hyperactivity exhibited by BD patients in their manic phase, and this was prevented with lithium treatment. Separately, I demonstrate that AAK1 regulates trafficking of the AP-2 complex to recycling endosomes in cells. Surprisingly, depletion of AAK1 increases autophagy flux, implicating it as a potential target in autophagy-related Parkinson’s disease (PD). Accordingly, depletion of AAK1 in Drosophila neurons increased their susceptibility to PD and autophagy-related death. This project identifies two novel substrates of GSK3 that are linked to debilitating neurological disorders. This provides a valuable basis for future research investigating the therapeutic potential of these and other novel GSK3 substrates discovered in our lab. iii |Page Acknowledgements I would like to thank my primary supervisor and mentor Dr. Adam Cole for taking me on as a student as part of the Neurosignalling and Mood Disorders group at the Garvan Institute of Medical Research. Thank you for all your support and guidance throughout my PhD. Thank you for you endless help on daily tasks, refining experimental design, reviewing presentations and thesis drafts and the continual encouragement needed to make me a better scientist. Your office door was always open for educative discussions and I appreciate all the time you made available for me. I had a great PhD experience and I have learnt so much. I would like to thank Hovik Farghaian, a key member of the Neurosignalling and Mood Disorders group, and my very good friend. Your support, expertise and friendship where invaluable throughout my time at the Garvan Institute. Thank you for being such a great friend. I would also like to thank my associate supervisors Prof. Herbert Herzog and Dr. Greg Neely for all your support and wisdom during laboratory meetings and presentations and for your critical evaluation of my work. Thank you to all members, past and present, of the Functional Genomic group for all your collaboration and support over the years. Thank you to our collaborators Dr Vladimir Sytnyk and Iryna Leshchyns’ka at The University of New South Wales (UNSW) for their hippocampal neuron work and to Dr William Hughes for his help and support with microscopy. I would like to thank my family and friends for their endless support and expressing an interest in my work. Special thanks to my beautiful wife Jessica Robinson for all your love, support and understanding during this time. I couldn’t have asked for a better partner in life. Without their combined efforts, the completion of this project would not have been possible. iv |Page Contents ABSTRACT III ACKNOWLEDGEMENTS IV LIST OF TABLES VIII LIST OF FIGURES IX ABBREVIATIONS XII CHAPTER 1 – INTRODUCTION AND LITERATURE REVIEW 1 1.1 CHARACTERISATION OF GSK3 ............................................................................................................... 1 1.1.1 GSK3 GENES ........................................................................................................................................ 1 1.1.2 GSK3 PROTEINS ................................................................................................................................... 2 1.1.3 GSK3 EXPRESSION ............................................................................................................................... 3 1.1.4 GSK3 KINASE ACTIVITY ....................................................................................................................... 3 1.2 SIGNALLING PATHWAYS REGULATING GSK3 ACTIVITY ............................................................................... 6 1.2.1 THE GROWTH FACTOR SIGNALLING PATHWAY ..................................................................................... 6 1.2.2 THE WNT PATHWAY ............................................................................................................................. 8 1.2.3 THE HEDGEHOG PATHWAY ................................................................................................................ 10 1.2.4 THE NOTCH PATHWAY ....................................................................................................................... 12 1.3 PHYSIOLOGY OF GSK3 MUTANT MICE ............................................................................................... 14 1.3.1 GSK3 MUTANT MICE ........................................................................................................................ 14 1.3.2 GSK3 MUTANT MICE ....................................................................................................................... 15 1.4 GSK3 SUBSTRATES .............................................................................................................................. 16 1.4.1 GSK3 AND ITS SUBSTRATES INVOLVED IN APOPTOSIS ....................................................................... 16 1.4.1.1 The role of GSK3 in pro-apoptotic signalling ................................................................................ 16 1.4.1.2 The role of GSK3 in cell survival signalling .................................................................................. 19 1.4.1.3 Summary: GSK3 is a key regulator of cell apoptosis ..................................................................... 21 1.4.2 THE ROLE OF GSK3 IN THE REGULATION OF IMMUNE RESPONSES ...................................................... 21 1.4.2.1 Innate immunity ............................................................................................................................. 21 1.4.2.2 Adaptive immunity ......................................................................................................................... 22 1.4.2.3 Immune transcription factors .......................................................................................................... 23 1.4.3 GSK3’S ROLE IN STEM CELL PROLIFERATION VS. DIFFERENTIATION .................................................. 24 1.4.3.1 Signalling pathways in stem cells .................................................................................................. 24 1.4.3.2 GSK3 mediates the regulation of gene expression downstream of transcription factors ............... 27 1.4.3.3 Summary: GSK3 activity is important in the regulation of cell-fate .............................................. 28 1.4.4 GSK3 REGULATION OF NEUROGENESIS .............................................................................................. 29 1.4.4.1 GSK3 is a critical regulator of neurogenesis downstream of signalling pathways ......................... 29 1.4.4.2 Summary: GSK3’s dynamic control of neurogenesis .................................................................... 31 1.4.5 GSK3 AND NEURONAL MORPHOLOGY ...............................................................................................
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