Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.721673
Title: Establishing a neural progenitor cell model of Huntington's disease
Author: Smith, Edward John
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2017
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Abstract:
Huntington's disease (HD) is caused by the expansion of a CAG repeat in the huntingtin (HTT) gene The R6/2 transgenic mouse model exhibits a rapid onset of Huntington's disease-like phenotypes including neurodegeneration and aggregation of mutant huntingtin protein (mHTT). Neural progenitor cells (NPCs) are a pool of cells with stem cell-like properties and are responsible for self-renewal and differentiation into the cells of the central nervous system and mature brain. In this thesis, NPC lines were established from cells extracted from foetal R6/2 and wildtype mouse embryos and cultured in optimised culture media. NPCs were successfully maintained in a mitotic state as monolayer cultures for multiple passages without effects to karyotype or CAG repeat length. Cultures were differentiated by removal of growth factors, into mixed neurons and glia populations that expressed proteins indicative of mature cell types; neurons showed evidence of synaptophysin expression at junctions between cell neurites, suggesting synaptic functionality and formation of rudimentary neural networks. After differentiation, mHTT aggregation was detectable using immunohistochemistry from 14 days of differentiation in 5% of R6/2 cell nuclei, rising to 20% by 28 days, recapitulating an HD-like phenotype found in vivo. Detection of detergent insoluble mHTT-aggregated protein was also validated via western blotting. Super high resolution cell imaging showed aggregation of mHTT is also present in the cytoplasm. High-content imaging analysis system was performed using the Operetta system to explore morphological differences between WT and R6/2 cultures, as well as within the subset of cells with detectable aggregation. R6/2 nuclei were found to be larger than those of WT cells. Novel compounds known to affect protein aggregation were applied to the cell lines to assess their potential use in screening for approaches to modulation mHTT aggregation. The cells developed in this thesis are a novel and useful complement to the R6/2 mouse; phenotypes observed in vivo can be interrogated at the molecular level in terms of how mHTT protein misfolding and aggregation occur and how this affects cellular function.
Supervisor: Watt, Fiona Mary ; Bates, Gillian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.721673  DOI: Not available
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