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Profiling nucleo/cytoplasmic distribution of mRNAs identifies a role for the Golgi-to-ER trafficking in C9orf72 toxicity

ABSTRACT: Compelling evidence indicates that defects in nucleocytoplasmic transport contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS). In particular, hexanucleotide (G4C2) repeat expansions in C9orf72, the most common cause of genetic ALS, have a widespread impact on the transport machinery that regulates the nucleocytoplasmic distribution of proteins and RNAs, thereby affecting their functions. We have previously reported that the expression of G4C2 hexanucleotide repeats in cultured human and mouse cells caused a marked accumulation of poly(A) mRNAs in cell nuclei, suggesting that mRNA trafficking is impaired by expanded C9orf72-ALS repeats. To further characterize the process, in this work we set out to systematically identify the specific mRNAs that are altered in their nucleocytoplasmic distribution in the presence of C9orf72-ALS repeats. Results from RNAseq profiling of nuclear and cytoplasmic RNA fractions obtained from cells expressing G4C231 repeats show a significant accumulation of mRNAs in the nuclei of G4C231 cells, further suggesting that nuclear retention of mRNAs is a major effect of C9orf72 expanded repeats expression. Interestingly, pathway analysis shows that mRNAs involved in ER-to-Golgi trafficking are particularly enriched among the identified mRNAs. Most importantly, functional studies in cultured cells and Drosophila indicate that the membrane trafficking route regulated by ARFGAP1, a GTPase-activating protein that associates with Golgi, is specifically affected by expanded C9orf72 repeats, as well as by C9orf72-related dipeptide repeat proteins (C9-DPRs), pointing to ER-to-Golgi vesicle-mediated transport as a target of C9orf72 toxicity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE235448 | GEO | 2023/08/21

REPOSITORIES: GEO

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Project description:G4C2 hexanucleotide repeat expansions in a non-coding region of the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). G4C2 insertion length is variable, and patients can carry up to several thousand repeats. Dipeptide repeat proteins (DPR) translated from G4C2 transcripts are thought to be a main driver of toxicity. Experiments in model organisms with relatively short DPR have shown that arginine-rich DPR are most toxic, while polyGlycine-Alanine (GA) DPRs cause only mild toxicity. However, GA is the most abundant DPR in patient brains, and experimental work in animals has generally relied on the use of low numbers of repeats, with DPRs often tagged for in vivo tracking. Whether repeat length, or tagging, affect the toxicity of GA has not been systematically assessed. Therefore, we generated Drosophila fly lines expressing GA100, GA200 or GA400 specifically in adult neurons. Consistent with previous studies, expression of GA100 and GA200 caused only mild toxicity. In contrast, neuronal expression of GA400 drastically reduced climbing ability and survival of the flies, indicating that long GA DPRs can be highly toxic in vivo. This toxicity could be abolished by tagging GA400. Proteomics analysis of fly brains showed a repeat-length-dependent modulation of the brain proteome, with GA400 causing earlier and stronger changes than shorter GA proteins. PolyGA expression up-regulated proteins involved in ER to Golgi trafficking, and down-regulated proteins involved in insulin signalling. Experimental down-regulation of Tango1, a highly conserved regulator of ER-to Golgi transport, partially rescued GA400 toxicity, suggesting that misregulation of this process contributes to polyGA toxicity. Experimentally increasing insulin signaling also rescued GA toxicity. In summary, our data show that long polyGA proteins can be highly toxic in vivo, and that they may therefore contribute to ALS/FTD pathogenesis in humans patients.

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Profiling nucleo/cytoplasmic distribution of mRNAs identifies a role for the Golgi-to-ER trafficking in C9orf72 toxicity

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