Hall, CE;
(2016)
Using VCP mutant hiPSC-derived motor neurons and glia to capture early molecular pathogenic events in MND.
Doctoral thesis , UCL (University College London).
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Abstract
Motor neuron disease (MND) is rapidly progressive and invariably fatal, and with no significantly impactful therapies available to date there is desperate and unmet clinical need. Recent discoveries in the underlying pathology and genetics of MND suggest that altered proteostasis, RNA regulation and glial contribution play key roles in molecular pathogenesis. I have used a combination of human induced pluripotent stem cell (hiPSC) models and functional genomic technologies to characterize early pathogenic events in VCP-related MND. I hypothesize that the early pathogenic events in MND differ during the differentiation of motor neurons and astrocytes; therefore I first generated enriched populations of both motor neurons (MNs) and astrocytes (ACs) from control and VCP mutant patient iPSCs. Next to understand if changes in RNA regulation contribute to pathogenic events in VCP-related MND, I carried out a temporal analysis using RNA sequencing (RNAseq) of control and VCP mutant iPSCs neural derivatives. Additionally, I examined the results of this transcriptional study in light of collaborative live cell imaging experiments, performed across a matched time-course, that have uncovered cell-type specific organellar dysfunction in VCP mutant MNs. This paradigm importantly enabled us to discriminate primary from secondary pathogenic events in VCP-related MND. Further, after the onset of pathogenesis I also used individual nucleotide resolution UV cross linking with immunoprecipitation (iCLIP) to study RNA interactions of the candidate RNA-binding protein TDP43, which forms the pathological hallmark of MND. Together, this provides new insights into the earliest molecular pathogenic events in MND and has highlighted the contribution of both motor neurons and astrocytes. By identifying key disease mechanisms this study aims to guide future therapeutic strategy.
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