Abstract
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.
Highlights
Expanded GGGGCC repeats in intron 1 of the C9orf72 gene represent the most common cause of familial amyotrophic lateral sclerosis (ALS) and familial frontotemporal degeneration (DeJesus-Hernandez et al, 2011; Renton et al, 2011), though how this genetic change results in neuronal injury is not yet understood
We examined the relationship between RNA foci and characteristic neuropathology of C9orf72 + ALS: first, the loss of nuclear TDP-43 in motor neurons, which is the pathological hallmark of ALS (Neumann et al, 2006) and has been shown to correlate with neuronal loss (Brettschneider et al, 2013); and second, the presence of cytoplasmic aggregates containing dipeptide repeat protein, which are a hallmark of C9orf72 + disease (Ash et al, 2013; Mackenzie et al, 2013; Mori et al, 2013b)
There is an urgent need to understand the mechanisms of neuronal injury in C9orf72 + disease
Summary
Expanded GGGGCC repeats in intron 1 of the C9orf gene represent the most common cause of familial amyotrophic lateral sclerosis (ALS) and familial frontotemporal degeneration (DeJesus-Hernandez et al, 2011; Renton et al, 2011), though how this genetic change results in neuronal injury is not yet understood. A number of studies reported that molecular phenotypes correlated with the presence of RNA foci (Donnelly et al, 2013; Lagier-Tourenne et al, 2013; Lee et al, 2013; Mizielinska et al, 2013; Sareen et al, 2013). One study suggested that foci burden in the frontal cortex positively correlated with disease severity in eight patients with C9orf frontotemporal degeneration (Mizielinska et al, 2013) Two of these reports identified co-localization of RNA foci with various proteins (Donnelly et al, 2013; Sareen et al, 2013) and suggested that pathogenic sequestration might be occurring. Further work to characterize protein binding partners of the RNA foci is required, because many of the studies far are in disagreement as to the most important interactions
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