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

ABSTRACT: 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, but not stop codon-interrupted "RNA-only" repeats in Drosophila caused adult-onset neurodegeneration. Thus, expanded repeats promoted neurodegeneration through dipeptide repeat proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence revealed that 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.

SUBMITTER: Mizielinska S

PROVIDER: S-EPMC4944841 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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

Mizielinska Sarah S Grönke Sebastian S Niccoli Teresa T Ridler Charlotte E CE Clayton Emma L EL Devoy Anny A Moens Thomas T Norona Frances E FE Woollacott Ione O C IOC Pietrzyk Julian J Cleverley Karen K Nicoll Andrew J AJ Pickering-Brown Stuart S Dols Jacqueline J Cabecinha Melissa M Hendrich Oliver O Fratta Pietro P Fratta Pietro P Fisher Elizabeth M C EMC Partridge Linda L Isaacs Adrian M AM

Science (New York, N.Y.) 20140807 6201

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, but not stop codon-interrupted "RNA-only" rep ...[more]

PMID: 25103406

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Project description:GGGGCC repeat expansions of C9orf72 represent the most common genetic variant of amyotrophic lateral sclerosis and frontotemporal degeneration, but the mechanism of pathogenesis is unclear. Recent reports have suggested that the transcribed repeat might form toxic RNA foci that sequester various RNA processing proteins. Consensus as to the identity of the binding partners is missing and whole neuronal proteome investigation is needed. Using RNA fluorescence in situ hybridization we first identified nuclear and cytoplasmic RNA foci in peripheral and central nervous system biosamples from patients with amyotrophic lateral sclerosis with a repeat expansion of C9orf72 (C9orf72+), but not from those patients without a repeat expansion of C9orf72 (C9orf72-) or control subjects. Moreover, in the cases examined, the distribution of foci-positive neurons correlated with the clinical phenotype (t-test P < 0.05). As expected, RNA foci are ablated by RNase treatment. Interestingly, we identified foci in fibroblasts from an asymptomatic C9orf72+ carrier. We next performed pulldown assays, with GGGGCC5, in conjunction with mass spectrometry analysis, to identify candidate binding partners of the GGGGCC repeat expansion. Proteins containing RNA recognition motifs and involved in splicing, messenger RNA nuclear export and/or translation were significantly enriched. Immunohistochemistry in central nervous system tissue from C9orf72+ patients with amyotrophic lateral sclerosis demonstrated co-localization of RNA foci with SRSF2, hnRNP H1/F, ALYREF and hnRNP A1 in cerebellar granule cells and with SRSF2, hnRNP H1/F and ALYREF in motor neurons, the primary target of pathology in amyotrophic lateral sclerosis. Direct binding of proteins to GGGGCC repeat RNA was confirmed in vitro by ultraviolet-crosslinking assays. Co-localization was only detected in a small proportion of RNA foci, suggesting dynamic sequestration rather than irreversible binding. Additional immunohistochemistry demonstrated that neurons with and without RNA foci were equally likely to show nuclear depletion of TDP-43 (χ(2) P = 0.75) or poly-GA dipeptide repeat protein inclusions (χ(2) P = 0.46). Our findings suggest two non-exclusive pathogenic mechanisms: (i) functional depletion of RNA-processing proteins resulting in disruption of messenger RNA splicing; and (ii) licensing of expanded C9orf72 pre-messenger RNA for nuclear export by inappropriate association with messenger RNA export adaptor protein(s) leading to cytoplasmic repeat associated non-ATG translation and formation of potentially toxic dipeptide repeat protein.

Dataset Information

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

Publications

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

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