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Title: C9orf72 frontotemporal dementia and amyotrophic lateral sclerosis : investigating repeat pathology in cell culture models and human post-mortem brain
Author: Ridler, Charlotte Elizabeth
ISNI:       0000 0004 7230 1072
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2016
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A GGGGCC repeat expansion in the first intron of chromosome 9 open reading fame 72 (C9orf72) is the most common known genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The repeats are transcribed into RNA in both sense and antisense orientations, which aggregates to form RNA foci in cells. Additionally, repeat RNA undergoes repeat-associated non-ATG initiated (RAN) translation producing dipeptide repeat (DPR) proteins in all six sense and antisense reading frames. This thesis aims to dissect RNA and DPR protein gain-of-function mechanisms operating in C9orf72 FTD/ALS to study their effects in isolation. To achieve this, three classes of DNA constructs were generated: 1) pure GGGGCC repeats that produce RNA foci and DPR proteins, 2) ‘RNA-only’ constructs designed to preclude RAN translation, and 3) ‘protein-only’ constructs coding for all five DPR proteins that do not produce GGGGCC repeat RNA. Cultured cells transfected with pure and RNA-only repeat constructs exhibited a positive correlation between repeat length and both sense and antisense RNA foci formation. Additionally, DPR protein subcellular localisation was examined in protein-only repeat construct transfected cells and compared to the pathology found in patient brain. A collaborative project expressing these constructs in Drosophila showed that arginine-containing DPR proteins were responsible for C9orf72 repeat toxicity in flies. To determine the relevance of the different DPR proteins to human disease, the frequency and distribution of DPR protein inclusions was studied in C9orf72 FTD patient brain, using a novel automated image analysis protocol. Poly(glycine-arginine) DPR protein inclusion-bearing neurons also exhibited an increased nucleolar volume compared to inclusion-negative neurons, confirming recent speculation implicating nucleolar stress in disease pathogenesis. These studies in cell culture, Drosophila and human post-mortem brain implicate poly(glycine-arginine) as a toxic species within C9orf72 FTD, which may aid in the targeting of future treatments for this condition.
Supervisor: Isaacs, A. M. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available