A network analysis based proteomic and transcriptomic investigation into HIV-Tat induced neuronal dysfunction and the neuroprotective effect of lithium By Tariq Ahmad Ganief University of Cape Town GNFTAR001 Dissertation submitted for the degree of Doctor of Philosophy In the Department of Integrative Biomedical Sciences University of Cape Town October 2015 Supervisor: Professor Jonathan M. Blackburn 1 The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town Declaration I, Tariq Ahmad Ganief, declare that this thesis is my own work (except where acknowledgements indicate otherwise). Neither the whole work nor part thereof has been, is being, or is to be submitted for any degree or examination at any other university. I empower the University of Cape Town to reproduce for the purposes of research either the whole or any part of the contents of this thesis, in any manner whatsoever. Signature of candidate: Tariq Ganief Signed on the 28 day of October, 2015 2 | P a g e Acknowledgements During the years of my PhD, I have received a tremendous amount of support both personally and professionally; without which, this road would have proved significantly more arduous. While words are an abysmal return for all your friendship and counsel, please do accept them, even if only as an acknowledgement in gratitude. Shaun, through all the debates and discussions in the years we have worked together I have come to appreciate your single-minded determination to achieve. Through your determination and help, I myself have learned and achieved. Your help and friendship has been invaluable. Nelson, despite being a ‘recent’ addition to our lab, you quickly proved your value and became indispensable to the success of our group. Your knowledge and advice has and will always be appreciated. To my supervisor and mentor, Professor Jonathan Blackburn; your brilliant mind, positive attitude, simple solutions countering seemingly incalculable complexity, unwavering support, patience and guidance have been a source of inspiration and motivation throughout this PhD. You have undoubtedly shaped me as a scientist and I truly and sincerely appreciate all the opportunities you have given me. I am proud to have been your student. Lastly and most importantly; to my dear my parents, Sofwaan and Mariam Ganief; I thank you for the years of quiet sacrifice, continued support and pride in spite of difficulties. I thank you for raising me with a sense of wonder, perseverance and determination; traits which have been paramount to the completion of this PhD. i Table of Contents Acknowledgements ........................................................................................ i Abbreviations ............................................................................................... vii Abstract ......................................................................................................... 1 Chapter 1 General introduction ..................................................................... 3 HIV-Associated Dementia ..................................................................................... 3 HIV Life cycle ......................................................................................................... 4 HIV in the brain ..................................................................................................... 7 Innate antiviral response and inflammation ......................................................... 9 Neuroinflammation and neurodegeneration ..................................................... 12 Possible origins of disease .................................................................................. 14 ARV therapy and HIV Dementia .......................................................................... 14 Neuroinflammation in HIV infection ................................................................... 15 Neurotoxic viral proteins .................................................................................... 16 HIV Transactivator of transcription ..................................................................... 17 HIV and protein translation ................................................................................. 20 HIV and the cytoskeleton .................................................................................... 22 Glycogen Synthase Kinase in HAND .................................................................... 23 Lithium ................................................................................................................ 24 Teasing apart the Differences ............................................................................. 26 Assaying complex biology ................................................................................... 26 Two-dimensional gel electrophoresis ................................................................. 27 A new ‘proteomics’ ............................................................................................. 28 Mass spectrometry ............................................................................................. 29 Mass spectrometry-based proteomics ............................................................... 32 Limitations of MS ................................................................................................ 35 Decreasing biological complexity for MS analysis .............................................. 36 Chromatographic particle/bead size ................................................................... 37 Bottom-up proteomics data processing ............................................................. 38 Aims of This Thesis .................................................................................................. 45 Chapter 2 Establishing a model for HIV-Tat induced neuronal apoptosis ...... 46 ii | P a g e Introduction ............................................................................................................ 46 Materials and Methods ........................................................................................... 48 Cell culture .......................................................................................................... 48 Subculture ........................................................................................................... 48 MTT assay ............................................................................................................ 48 Results and Discussion ............................................................................................ 49 Remnants of apoptotic cells produce formazan ................................................. 50 Reduced, monomeric HIV-Tat is essential for the induction of apoptosis ......... 51 Chapter 3 Quantitative proteomic analysis of HIV-1 Tat-induced damage in SH-SY5Y neuroblastoma cells ................................................................... 56 Abstract ................................................................................................................... 56 Introduction ............................................................................................................ 57 Materials and Methods ........................................................................................... 59 Cell culture and SILAC labelling ........................................................................... 59 Treatment with recombinant HIV-1 Tat CLADE-B: .............................................. 59 Sample preparation ............................................................................................. 60 Mass spectrometry ............................................................................................. 61 Data processing and bioinformatic analysis ....................................................... 62 Functional enrichment analysis .......................................................................... 62 Results ..................................................................................................................... 63 Monomeric HIV-Tat is required to induced apoptosis in SH-SY5Y cells ............. 63 Proteome dynamics ............................................................................................ 64 Discussion ................................................................................................................ 68 HIV clade-B Tat as an HIV dementia causative agent ......................................... 68 Bioinformatic identification of dysregulated proteins upon HIV-Tat treatment 68 HIV-Tat alters protein translation and cytoskeletal regulation .......................... 72 Cytoskeletal dysregulation features prominently in highly differentially regulated dataset ................................................................................................ 73 HIV-Tat affects key systems seemingly unrelated to protein translation or cytoskeletal regulation ........................................................................................ 75 Chapter 4 Extensive pathway analysis of SILAC labelled proteomic and transcriptomic data from HIV-Tat treated SY-SY5Y neuroblastoma cells ...... 77 iii | P a g e Abstract 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