McLaughlin, Martha; (2023) Comparing dysfunctional RNA processing in TDP-43 and FUS mutant ALS models. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Amylotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease of motor neurons which causes muscle weakness, paralysis and fatality often within 2-3 years of diagnosis. The causes of ALS are not fully understood, and there is currently no treatment that prolongs life more than 3 months, on average. Mislocalisation of protein TDP-43 is found in the vast majority of ALS cases and mutations in TDP-43 and another RNA binding protein called FUS, which does not cause TDP-43 mislocation, are also causative for ALS. The overall aim of my Thesis was to investigate and develop new methods to compare the effects of FUS and TDP-43 dysfunction on RNA processing, in order to identify overlapping impacts of dysfunction that may reveal shared pathways in ALS pathology. Firstly, I investigated an autoregulatory mechanism of FUS involving intron retention of the FUS mRNA transcript. I found that this intron-retained transcript is detained in the nucleus, where it diffuses away from the site of transcription and has a longer half-life in the prescence of nuclear FUS, which may be due to faster splicing into the fully-spliced FUS transcript. Furthermore, the level of FUS intron retention did not appear to be affected in all of the ALS models examined expressing mutations in TDP-43 or other ALS-linked genes. Secondly, I compared small RNA expression in FUS and TDP-43 mutant mouse models and other ALS models with and without TDP-43 mislocalisation. I found that there was some overlapping impact on small RNA expression from FUS cytoplasmic and TDP-43 RNA binding gain of function mutants, as well as FUS and TDP-43 loss of function models. Finally, I tested a method of assessing changes in RNA turnover in TDP-43 mutants using RNA labelling with 4sU to optimise this method for future experiments in TDP-43 and FUS mutant models. Overall, the experiments described in this Thesis show that there are both distinct and shared impacts of FUS and TDP-43 dyfunction on RNA processing and identified specific disrupted genes that could be future therapeutic targets.

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