Philiastides, Alexandra; (2019) Towards understanding selective neuronal vulnerability: establishing an in-vitro model for strain selection. Doctoral thesis (Ph.D), UCL (University College London).

Preview

Text AP_Thesis Final Submission_2018-2019.pdf Download (3MB) | Preview

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 Me7infected 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. 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 peptidylpropyl 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 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.

Download activity - last month

Export as CSVJSONXML

Download activity - last 12 months

Export as XMLJSONCSV

Downloads by country - last 12 months

Export as JSONXMLCSV

Archive Staff Only

View Item