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Activation of nonsense-mediated mRNA decay protects against C9orf72 dipeptide-repeat neurotoxicity

ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a deleterious neurodegenerative disease without effective treatment options. Recent studies have indicated the involvement of the dysregulation of RNA metabolism in the pathogenesis of ALS. Among the various RNA regulatory machineries, nonsense-mediated mRNA decay (NMD) is a stress responsive cellular surveillance system that degrades selected mRNA substrates to prevent the translation of defective or harmful proteins. Whether this pathway is affected in neurodegenerative diseases is unclear. Here we report that the NMD pathway is inhibited in ALS patients with C9orf72 hexanucleotide repeat expansion (HRE), the most common cause of familial ALS. Bioinformatic analysis of multiple transcriptome profiles revealed significant overlap of upregulated genes in NMD-defective cells with those in the brain tissues, micro-dissected motor neurons, or induced pluripotent stem cell-derived motor neurons from ALS patients carrying C9orf72 HRE, suggesting the suppression of NMD pathway in these patients. In contrast, there are few overlapping upregulated genes in brain tissues from sporadic ALS patients with those in NMD-defective cells. Using Drosophila models expressing various C9orf72 HRE products, we have validated the suppression of the NMD pathway by C9orf72 HRE and identified arginine-rich dipeptide repeats (DPRs) generated from HRE as the main culprits of NMD inhibition. Furthermore, in human SH-SY5Y neuroblastoma cells and in mouse brains, expression of arginine-rich DPRs was sufficient to cause NMD inhibition. Remarkably, expression of UPF1, a core gene in the NMD pathway, efficiently blocked neurotoxicity caused by arginine-rich DPRs in both cellular and Drosophila models. Although not as effective as UPF1, expression of another NMD gene UPF2 also ameliorated the degenerative phenotypes in DPR-expressing flies, indicating neuroprotection by genetically reactivating the NMD pathway. Finally, after validating Tranilast as an NMD-activating drug, we demonstrated its therapeutic potential in cellular and Drosophila models of C9orf72 DPR neurotoxicity. Therefore, our study has revealed the suppression of NMD in C9orf72 ALS and has suggested that the restoration of the NMD pathway by Tranilast, an asthma drug with solid safety record, could be a promising therapeutic strategy for ALS.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE123172 | GEO | 2018/12/01

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.

Project description:Importance: An intronic hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest monogenic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Predicting those who will develop neurodegeneration and its timing will be essential to initiating and assessing preventative therapy. This requires consideration of both compensatory, protective mechanisms and pathogenic events prior to overt neurodegeneration. Objective: To identify biochemical changes in individuals at higher risk of developing ALS or FTD via C9orf72 HRE heterozygosity. Design: Cross sectional observational study. Setting: Tertiary ALS or dementia referral centre. Participants: People with established ALS or FTD, either due to C9orf72 HRE or apparently sporadic cases; asymptomatic first-degree relatives of those with a known C9orf72 HRE; asymptomatic non-carrier controls. Exposure: C9orf72 HRE. Main outcomes: Relative abundance of 30 predefined cerebrospinal fluid biomarkers of ALS and FTD comparing asymptomatic C9orf72 HRE carriers and age-matched non-carriers. Differential abundance of proteins quantified using data independent acquisition mass spectrometry and neurofilament light chain measured by electrochemiluminescent assay. Results: Data for 19 people with sporadic ALS, 10 people with C9orf72 ALS, 14 people with sporadic FTD, 10 people with C9orf72 FTD, 48 asymptomatic C9orf72 HRE carriers and 39 non-carrier controls were analysed. Ubiquitin carboxyl-hydrolase isozyme L1 levels were higher in asymptomatic C9orf72 HRE carriers compared with age-matched non-carriers (log2fold change 0.20, FDR-adjusted p-value = 0.034). Neurofilament light chain levels did not differ significantly between groups. Ubiquitin carboxyl-hydrolase isozyme L1 levels remained elevated after exclusion of those with high neurofilament light chain levels, after adjusting for NFL level and after adjusting for age. Conclusions and relevance: Elevated cerebrospinal fluid ubiquitin carboxyl-hydrolase isozyme L1 levels in C9orf72 hexanucleotide repeat expansion carriers occurs in the absence of elevation in neurofilament light chain, potentially reflecting mechanisms that precede the phase of neurodegeneration characterised by rapid neuronal loss. Such mechanisms may have either compensatory or pathogenic roles. Ubiquitin carboxyl-terminal hydrolase isozyme L1 elevation brings forward the window on the changes associated with the C9orf72 HRE carrier state, with the potential to inform understanding penetrance and approaches to prevention.

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Activation of nonsense-mediated mRNA decay protects against C9orf72 dipeptide-repeat neurotoxicity

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