Balendra, Rubika; (2017) Molecular mechanisms and therapeutic strategies in C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia. Doctoral thesis (Ph.D), UCL (University College London).

Abstract

Background A hexanucleotide expansion in C9orf72 is a common cause of the fatal neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia. We have found evidence in a Drosophila model that neurotoxicity is mediated by dipeptide repeat (DPR) proteins generated by repeat-associated non-ATG translation. Here we aimed to evaluate in models of amyotrophic lateral sclerosis and frontotemporal dementia caused by the C9orf72 mutation (C9FTD/ALS) whether DPR proteins cause nucleolar dysfunction and whether small molecules that bind C9orf72 G-quadruplex repeat RNA reduce disease phenotypes. Methods We assessed nucleolar function in Drosophila models. Nucleolar volume was measured with immunofluorescence and confocal microscopy, using automated image analysis. Human induced pluripotent stem cells (iPSCs) from patients with C9orf72-ALS and from healthy controls were taken through neural induction and patterning to derive spinal motor neuron populations. Disease phenotypes were measured with fluorescence in-situ hybridisation for the typical RNA foci seen in C9orf72-ALS patients and an immunoassay for DPRs. Small molecules binding to C9orf72-repeat RNA were applied to the human iPSC-derived spinal motor neurons and fed to C9orf72 Drosophila to evaluate rescue of disease phenotypes. Findings The DPR poly-GR led to increased nucleolar volumes in Drosophila brain. The DPR poly-GA also led to increased nucleolar volume, but to a much lesser extent. Small molecules binding to G-quadruplex GGGGCC RNA showed efficacy in C9orf72 patient-derived neurons and in C9orf72 Drosophila. Interpretation Emerging evidence suggests that nucleolar dysfunction is a key mechanism in C9FTD/ALS. We have shown that DPR proteins lead to nucleolar dysfunction in C9orf72 Drosophila and in C9FTLD patient frontal cortex. The high prevalence of C9orf72-ALS makes use of targeted therapies a compelling strategy. Evidence for amelioration of C9orf72 phenotypes in patient-derived neurons and in Drosophila suggests that targeting G-quadruplex GGGGCC repeat RNA is a potential strategy for treating disease, which requires further optimisation and validation.

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