Abstract
Hexanucleotide repeat expansion in C9orf72 is the most common pathogenic mutation in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite the lack of an ATG start codon, the repeat expansion is translated in all reading frames into dipeptide repeat (DPR) proteins, which form insoluble, ubiquitinated, p62-positive aggregates that are most abundant in the cerebral cortex and cerebellum. To specifically analyze DPR toxicity and aggregation, we expressed DPR proteins from synthetic genes containing a start codon but lacking extensive GGGGCC repeats. Poly-Gly-Ala (GA) formed p62-positive cytoplasmic aggregates, inhibited dendritic arborization and induced apoptosis in primary neurons. Quantitative mass spectrometry analysis to identify poly-GA co-aggregating proteins revealed a significant enrichment of proteins of the ubiquitin–proteasome system. Among the other interacting proteins, we identified the transport factor Unc119, which has been previously linked to neuromuscular and axonal function, as a poly-GA co-aggregating protein. Strikingly, the levels of soluble Unc119 are strongly reduced upon poly-GA expression in neurons, suggesting a loss of function mechanism. Similar to poly-GA expression, Unc119 knockdown inhibits dendritic branching and causes neurotoxicity. Unc119 overexpression partially rescues poly-GA toxicity suggesting that poly-GA expression causes Unc119 loss of function. In C9orf72 patients, Unc119 is detectable in 9.5 % of GA inclusions in the frontal cortex, but only in 1.6 % of GA inclusions in the cerebellum, an area largely spared of neurodegeneration. A fraction of neurons with Unc119 inclusions shows loss of cytosolic staining. Poly-GA-induced Unc119 loss of function may thereby contribute to selective vulnerability of neurons with DPR protein inclusions in the pathogenesis of C9orf72 FTLD/ALS.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-014-1329-4) contains supplementary material, which is available to authorized users.
Highlights
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are severe neurodegenerative diseases with no effective treatment
Changing the original hexanucleotide repeat sequence, but maintaining the dipeptide repeat (DPR) protein sequence, allowed us to focus on protein toxicity rather than GGGGCC or CCCCGG RNA toxicity
Using quantitative analysis of the poly-GGGGCC repeats. PolyGly-Ala (GA) interactome, we identified a novel co-aggregating protein, Unc119, which has been linked to axon maintenance in C. elegans previously [23, 30]
Summary
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are severe neurodegenerative diseases with no effective treatment. Depending on the affected regions, FTLD patients suffer from dementia, behavioral abnormalities, language impairment and personality changes [21]. Both diseases have overlapping clinical, neuropathological and genetic features and are often described as extreme ends of a disease spectrum [22]. A mutation in the non-coding region of the C9orf gene has been identified as the most common genetic cause of both ALS and FTLD [12, 20, 41]. Mutation carriers have a GGGGCC hexanucleotide repeat expansion either in the first intron or the promoter region, depending on the isoform of the C9orf transcript [5]. C9orf patients exhibit clinical symptoms similar to other FTLD or ALS subtypes, but suffer from an unusually high incidence of psychosis [13]
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