C9orf72 frontotemporal dementia and amyotrophic lateral sclerosis: investigating repeat pathology in cell culture models and human post-mortem brain

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

C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins

An expanded GGGGCC repeat in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. A fundamental question is whether toxicity is driven by the repeat RNA itself and/or by dipeptide repeat proteins generated by repeat-associated, non-ATG translation. To address this question we developed in vitro and in vivo models to dissect repeat RNA and dipeptide repeat protein toxicity. Expression of pure repeats in Drosophila caused adult-onset neurodegeneration attributable to poly-(glycine-arginine) proteins. Thus, expanded repeats promoted neurodegeneration through neurotoxic proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence showed both poly-(glycine-arginine) and poly-(proline-arginine) proteins caused neurodegeneration. These findings are consistent with a dual toxicity mechanism, whereby both arginine-rich proteins and repeat RNA contribute to C9orf72-mediated neurodegeneration

C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci.

An expanded GGGGCC repeat in a non-coding region of the C9orf72 gene is a common cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis. Non-coding repeat expansions may cause disease by reducing the expression level of the gene they reside in, by producing toxic aggregates of repeat RNA termed RNA foci, or by producing toxic proteins generated by repeat-associated non-ATG translation. We present the first definitive report of C9orf72 repeat sense and antisense RNA foci using a series of C9FTLD cases, and neurodegenerative disease and normal controls. A sensitive and specific fluorescence in situ hybridisation protocol was combined with protein immunostaining to show that both sense and antisense foci were frequent, specific to C9FTLD, and present in neurons of the frontal cortex, hippocampus and cerebellum. High-resolution imaging also allowed accurate analyses of foci number and subcellular localisation. RNA foci were most abundant in the frontal cortex, where 51 % of neurons contained foci. RNA foci also occurred in astrocytes, microglia and oligodendrocytes but to a lesser degree than in neurons. RNA foci were observed in both TDP-43- and p62-inclusion bearing neurons, but not at a greater frequency than expected by chance. RNA foci abundance in the frontal cortex showed a significant inverse correlation with age at onset of disease. These data establish that sense and antisense C9orf72 repeat RNA foci are a consistent and specific feature of C9FTLD, providing new insight into the pathogenesis of C9FTLD

Temporal control of a dendritogenesis-linked gene via REST-dependent regulation of nuclear factor I occupancy

How the timing of gene expression is controlled during neuronal development is largely unknown. Here we describe a temporal mechanism of gene regulation in differentiating postmitotic neurons involving delayed promoter site occupancy by nuclear factor I and the control of its initial onset by the trans-repressor REST