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dc.contributor.advisorWishart, Thomas
dc.contributor.advisorSkehel, Paul
dc.contributor.authorKing, Declan James Peter
dc.date.accessioned2019-07-15T12:42:25Z
dc.date.available2019-07-15T12:42:25Z
dc.date.issued2019-06-29
dc.identifier.urihttp://hdl.handle.net/1842/35791
dc.description.abstractThe generation and spread of misfolded protein aggregates is common to all Protein Misfolding Diseases (PMDs) including but not limited to Alzheimer’s, Parkinson’s and Prion disease. Yet despite this common feature, the early mechanisms underpinning the apparent perturbation in protein handling and processing in such conditions remain poorly understood. Research in our lab has previously shown protein misfolding may be seeded and supported in different ways in the brain. Mice homozygous for the P101L mutation in murine PrP (101LL) are capable of supporting protein misfolding and amyloid plaque formation after challenge with pre-formed PrP fibrils and wild type mice of the same genetic background do not, and appear to be capable of curtailing this accumulation of abnormal protein in vivo. Thus, both mouse lines provided ideal tools to investigate early cellular and molecular responses to abnormal PrP fibril challenge. PMDs occur in vivo with a regional dependant progression however; such investigations are more amenable to controlled initiation and visualisation in vitro. Therefore, primary neuronal hippocampal cell cultures were generated from each line (101LL and WT) and were thoroughly assessed at the cellular level. Comparative molecular analysis of these cultures and in vivo hippocampal tissue from both WT and 101LL lines was carried out and transcriptome analysis confirmed a broadly comparable global expression profile at a basal level between in vivo, in vitro and genotype. Interestingly, this suggests that the genotype specific differential protein misfolding responses are therefore likely to be the result of regulatory cascades initiated after inoculation. To examine this at the cellular level, cultures were then challenged with fluorescently labelled recombinant PrP fibrils. Fibrils were directly interacting with PrPC on neuronal surfaces but did not cause neurotoxicity. However, post-synaptic marker PSD-95 was significantly reduced in 101LL cultures suggestive that 101LL neurons were more susceptible to fibril insult than WT. Microglial cells were activated post fibril challenge in both genotypes. This generic response was possibly to limit neuronal damage and promote fibril degradation through phagocytosis. Transcriptome analyses supported these observations and showed an increase in expression of genes associated with phagocytosis was occurring after fibril challenge in both genotypes. Hypertrophic astrocytes and an increase in expression of genes associated with astrocyte differentiation and development was only evident in WT fibril-challenged cultures indicating differential glial responses were occurring between genotypes. Intracellular labelling showed localisation with endosomes and lysosomes in WT cells indicating an endolysosomal pathway was operative however; localisation in 101LL cells was primarily in endosomes indicating an impairment in abnormal protein degradation pathways was occurring in 101LL fibril-challenged cultures. Transcriptomic analysis identified reduced gene expression of lysosomal associated genes Laptm5 and Ctsz in 101LL fibril-challenged cultures, supporting endolysosomal processing was reduced in this genotype. Additional transcriptomic profiles obtained post-fibril challenge showed an increase in gene expression in WT cultures associated with endocytosis, macrophage engulfment, immune and defence response and ER-associated misfolded protein catabolic processes none of which were induced in 101LL fibril-challenged cultures. These analyses suggest a dysfunction of multiple mechanisms may be associated with an inability to clear misfolded protein. These data provide key information on pathways and cellular mechanisms involved in either clearance of misfolded protein or initiation of amyloid formation. Identifying these pathways will provide a number of possible test targets with the potential of impacting diagnosis and/or intervention in PMD’s such as AD, PD and prions.en
dc.language.isoenen
dc.publisherThe University of Edinburghen
dc.relation.hasversionBarron, Rona M., Declan King, Martin Jeffrey, Gillian McGovern, Sonya Agarwal, Andrew C. Gill, and Pedro Piccardo. 2016. 'PrP aggregation can be seeded by pre-formed recombinant PrP amyloid fibrils without the replication of infectious prions', Acta Neuropathologica, 132: 611- 24.en
dc.relation.hasversionJeffrey, Martin, Gillian McGovern, Emily V. Chambers, Declan King, Lorenzo González, Jean C. Manson, Bernardino Ghetti, Pedro Piccardo, and Rona M. Barron. 2012. 'Mechanism of PrP-amyloid formation in mice without transmissible spongiform encephalopathy', Brain pathology (Zurich, Switzerland), 22: 58-66.en
dc.subjectamyloid plaquesen
dc.subjectprotein misfoldingen
dc.subject101LLen
dc.subjectamyloid plaque formationen
dc.subjectPrP fibrilsen
dc.titleInvestigating early cellular and molecular responses to misfolded PrP proteinen
dc.typeThesis or Dissertationen
dc.type.qualificationlevelDoctoralen
dc.type.qualificationnamePhD Doctor of Philosophyen


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