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Towards Understanding Selective Neuronal Vulnerability: Establishing an In-Vitro Model for Strain Selection

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Towards Understanding Selective Neuronal Vulnerability: Establishing an In-Vitro Model for Strain Selection Towards understanding selective neuronal vulnerability: Establishing an in-vitro model for strain selection A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy from University College London Alexandra Philiastides MRC Prion Unit at UCL Institute of Prion Diseases University College London 2018 1 Declaration I, Alexandra Philiastides, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Dedication This thesis is dedicated to the memory of my dearest grandfather Andreas Philiastides and to my beloved grandparents Michalis and Theano Themistokleous. 3 Acknowledgments Firstly, I would like to express my sincere gratitude to my PhD supervisor Dr. Peter- Christian Kloehn, for the continuous support, scientific input, encouragement and patience throughout my PhD. I would like to thank my previous supervisor Dr. Sarah Lloyd, for her great support, guidance and kind advice during the first 18 months of my PhD. A special thank you to Dr. Craig Brown, who introduced me to cell culture and molecular cloning. It was a real privilege for me to share of his exceptional scientific knowledge which was pivotal for the accomplishment of the work presented in this thesis. I would also like to thank Dr. Juan Ribes, for his continuous support, help with data analysis, experimental planning and protocols and for his encouragement when the PhD seemed like an unending struggle. I would like to express my special appreciation and thanks to my secondary supervisor Prof. Parmjit Jat, for his valuable advice on my research as well as on my career. I am also grateful to my departmental graduate tutor Prof. Elizabeth Fisher, for her tremendous support and advice during the difficult times of my PhD. Many thanks are due to Dr. Graham Jackson, for his insightful comments and valuable suggestions which helped me during the write up of this thesis. This work would not have been possible without the invaluable scientific input and patience of Daniel Yip, Christian Schmidt and Parvin Ahmed who carried out the automated Scrapie Cell assays and devoted their time to help me collect data for my PhD thesis. I am grateful to Nunu Arora, Mark Batchelor, Susan Joiner, Jessica Sells, Adam Wenborn, Dr. Emmanuelle Vire, Emma Quarterman and Gary Adamson for the scientific input, encouragement and help throughout the past four years. I am thankful to my friends Luke Dabin, Laura Pulford, Justin Tosh, Lucy Sheytanova, Nora Thoeng, Matt Rickman, Dr. Karen Cleverley, Billy West, Xun Yu Choong, Julia Ravey, Madeleine Reilly and Shaheen Akhtar, with whom I have shared many happy moments and who were always encouraging throughout my PhD. I would also like to thank my colleagues with whom I have shared the PhD office, for the support and the happy memories which I treasure so much: Lucianne Dobson, James Miller, Charlotte Ridler, Prasanth Sivakumar, David Thomas, Grace O’Regan, Karolin Koriath, Caroline 4 Casey, Angelos Armen, Rachele Saccon, Daniel Wright, Francesca De Giorgio, Rhia Ghosh, Marta Benedekova, Claudia Cannavo, Heather Whittaker, Thanos Dimitriadis, Ines Whitworth, Bernie Simone and Emma Jones. I am grateful to the people in the IT and administration office who were always friendly and happy to help: Ryan Peter, Samantha MacLeod, Amir Mayahi, David Ruegg, Alison Church and Oke Avwenagha. This work would not have been possible without the funding provided by John Collinge through the Medical Research Council. I am thankful and grateful beyond words to my best friend Ruchi Kumari for her immense support and love, and for being the most amazing, caring, genuine and loving friend I could ever ask for. Thank you for the love, the patience, the endless deep conversations, the daily coffee breaks and for believing in me and reminding me that things will always be ok in the end. I would not have been able to go through this without you. To my dearest parents, Antony and Stalo, and to my precious brothers Andreas and Michalis, thank you for the continuous support, the endless love and for helping me overcome situations when continuing seemed impossible. To my amazing fiancé, Charlie, thank you for the love, support, encouragement and never-ending patience. 5 Abstract Prion diseases are fatal neurodegenerative diseases that affect humans and animals. Prion strains, conformational variants of misfolded prion proteins, are thought to be associated with distinct clinical and pathological phenotypes. Why prion strains cause damage in particular areas of the brain is poorly understood. Although prions are innocuous to most cell lines, differences in their tropism to mouse-adapted prion strains have been broadly observed. While some cell lines show broad susceptibility to prion strains, others are highly selective, suggesting that susceptibility to a specific prion strain is determined by distinct cellular factors. The neuroblastoma cell line N2aPK1 (PK1) is refractory to the murine prion strain Me7, but highly susceptible to RML. Intracerebral inoculation of mice with Me7 induces hippocampal neuronal loss, whereas RML does not cause degeneration in this brain region. The PME2 subclone, is a PK1-derived subclone with low susceptibility to Me7 and this was used as the parental line to derive highly Me7-susceptible cells. To understand the molecular underpinning of selective neuronal vulnerability and cell tropism of prion strains, respectively, we first undertook a series of successive sub cloning experiments to identify rare PK1 cell clones that are susceptible to Me7. Initially, Me7-susceptible clones were identified at a frequency of only 4x10-3. The percentage of Me7-susceptible cells increased by 6-fold and 20-fold, respectively, and by the third and final round of sub cloning, 63% of cell clones were highly susceptible to Me7. Persistently infected PME2 cell clones deposited disease-associated PrP (PrPd) in perinuclear and extracellular stores. Strikingly, Me7-refractory PK1 cells were found to be highly susceptible to prions derived from homogenates of chronically Me7- infected PME2 cells, suggesting that a single passage in PME2 cells changed the strain properties of brain-adapted Me7. This cell model provides the first evidence for prion strain adaptation in genetically similar cell clones. The identification of genetically similar cell clones that differ in their ability to adapt prion strains lays the foundation for future work to gain insights into the molecular mechanisms that underlie prion strain adaptation. During the second half of my PhD, I worked on a separate project, investigating the role of Fkbp proteins in molecular mechanisms of prion propagation. While the prion protein gene is the major genetic determinant of susceptibility to prion disease, several studies have identified additional modifier genes that also influence susceptibility and modify the disease phenotype. 6 A microarray gene expression study which correlated the level of mRNA expression, in uninfected brains, from 5 inbred lines of mice, with their respective incubation times identified several potential prion modifier genes including Fkbp9. Lower levels of expression of Fkbp9 correlated with shorter incubation times in mice, following prion infection. These findings were validated in vitro where Scrapie Cell Assays (SCA) in Fkbp9 stably knocked down cell lines showed a significant increase prion propagation. The Fkbp9 protein is part of the immunophilin family of proteins which are peptidyl- propyl cis-trans isomerases. Fkbp proteins have been implicated in aspects of neurodegenerative disease, including accelerating α-synuclein fibrillisation and aggregation (primarily Fkbp12 but also Fkbp38, 52 and 65) and inhibiting tau induced tubulin polymerisation (Fkbp52) in vitro. Fkbp52 also reduced Aβ levels in a fly model of Alzheimer’s disease and Fkbp51 was shown to block tau clearance through the proteasome resulting in oligomerisation. The aim of this project was to characterise the functional roles of Fkbp family members in prion propagation. I generated a panel of N2aPK1 cell lines by stable gene silencing of four different Fkbp genes and employed the SCA to test whether Fkbp knock down (KD) influences prion propagation. For each Fkbp gene, four to eight KD cell lines were generated. Three out of four Fkbp4 (Fkbp52) KD cell lines with over 50% KD of mRNA expression levels showed a significant reduction in the number of PrPSc- positive cells, as quantified in the SCA. Additionally, KD of Fkbp8 in PK1 cells led to a significant reduction in the number of PrPSc-positive cells in four out of the five cell lines screened in the SCA. In contrast to these findings, in some cell lines with a significant reduction in mRNA expression levels (>60%) of the target Fkbp gene, there was no corresponding decrease in the number of PrPSc-positive cells. We reasoned that shRNA off-target effects arising when an shRNA downregulates unintended gene targets through partial sequence complementarity, may mask the effect of KD of the gene of interest. To examine whether an independent gene silencing approach for the examined gene targets recapitulates the results of stable gene silencing, siRNAs were used to transiently knock down Fkbp genes in chronically RML-infected PK1 cells (iS7 cells). Surprisingly, none of the siRNAs against the specified Fkbp genes reduced the number of PrPSc-positive iS7 cells. After establishing which Fkbp proteins affect prion propagation in the SCA, we aimed to carry out in vitro studies to understand the molecular mechanisms by which Fkbp proteins influence prion propagation. After optimisation of expression and cloning strategies, I successfully induced the expression of recombinant Fkbp9 and Fkbp52 proteins. The aim was to use the recombinant proteins in cell-free assays to test whether Fkbp proteins affect prion 7 replication and/or modulate the fibrillisation of recombinant PrPC. In vitro assays with recombinant Fkbp proteins were not carried out as the project was terminated shortly after my primary supervisor left the Unit.
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