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Phe-tRNA aminoacylation is disrupted by C4G2 repeat RNA C9orf72mutation associated with ALS and FTD

ABSTRACT: Expanded hexanucleotide GGGGCC repeat mutation in the C9orf72 gene is the main genetic cause of neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense transcripts of the repeats form nuclear RNA foci and bind various RNA binding proteins. Sequestration of these proteins is predicted to be cytotoxic. In this study we identified proteins that bind to antisense repeat RNA with RNA pull-down assay in combination with mass spectrometry. The interactions were confirmed in human brain lysates. Among interacting proteins is phenylalanine-tRNA synthetase (FARS). We confirmed interaction with FARS in C9orf72 mutation-positive patient-derived cells with RNA fluorescent in situ hybridization in combination with immunocytochemistry and with RNA-protein proximity ligation assay. We predict this interaction impacts the function of FARS as we observed decreased levels of charged Phe-tRNA in C9orf72 mutation-positive patient-derived cells compared to control. This study shows that antisense RNA interaction with FARS could influence protein synthesis and reveals so far unknown connection between RNA repeats and aminoacil tRNA synthetases in C9orf72 mutation.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Xiaoke Yin

LAB HEAD: Manuel Mayr

PROVIDER: PXD024527 | Pride | 2023-09-18

REPOSITORIES: Pride

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			Action	DRS
	B_orange_-05.mzid.gz	Mzid
	B_orange_-05.mzid_B_orange_-05.MGF	Mzid
	B_orange_-05.raw	Raw
	B_orange_-06.mzid.gz	Mzid
	B_orange_-06.mzid_B_orange_-06.MGF	Mzid

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Phenylalanine-tRNA aminoacylation is compromised by ALS/FTD-associated C9orf72 C4G2 repeat RNA

Project description:The expanded hexanucleotide GGGGCC repeat mutation in the C9orf72 gene is the main genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Under one disease mechanism, sense and antisense transcripts of the repeat are predicted to bind various RNA-binding proteins, compromise their function and cause cytotoxicity. Focusing on cytoplasmic interaction of the CCCCGG antisense repeat RNA this study identifies phenylalanine-tRNA synthetase (FARS) subunit alpha (FARSA) as the main interactor. The aminoacylation of tRNAPhe by FARS is inhibited by antisense RNA, leading to decreased levels of charged tRNAPhe. Remarkably, this is associated with global reduction of phenylalanine incorporation in the proteome and significant decrease in expression of phenylalanine-rich proteins in cellular models and patient tissues. In conclusion, this research reveals functional inhibition of FARSA in the presence of antisense RNA repeats. Compromised aminoacylation of tRNA could lead to impairments in protein synthesis and further contribute to C9orf72 mutation-associated pathology.

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Project description:Noncoding expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Here we report transgenic mice carrying a bacterial artificial chromosome (BAC) containing the full human C9orf72 gene with either a normal allele (15 repeats) or disease-associated expansion (~100â??1,000 repeats; C9-BACexp). C9-BACexp mice displayed pathologic features seen in C9orf72 expansion patients, including widespread RNA foci and repeat-associated non-ATG (RAN) translated dipeptides, which were suppressed by antisense oligonucleotides targeting human C9orf72. Nucleolin distribution was altered, supporting that either C9orf72 transcripts or RAN dipeptides promote nucleolar dysfunction. Despite early and widespread production of RNA foci and RAN dipeptides in C9-BACexp mice, behavioral abnormalities and neurodegeneration were not observed even at advanced ages, supporting the hypothesis that RNA foci and RAN dipeptides occur presymptomatically and are not sufficient to drive neurodegeneration in mice at levels seen in patients. To compare the RNA Seq profiles from the cortex and spinal cord of transgenic mice expressing unexpanded human C9orf72 (F08, n=4), expanded human C9orf72 (F112, n=3/4), and nontransgenic controls (n=4).

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Project description:N6-methyladenosine (m6A) is the most prevalent internal mRNA modification and regulates RNA metabolism. Repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). Here, we showed that m6A is downregulated in C9ORF72-ALS/FTD patient-derived iPSC-differentiated neurons and postmortem brain tissues. The global m6A hypomethylation leads to transcriptome-wide mRNA stabilization and upregulated gene expression, especially ones involved in synaptic activity and neuronal functions. The m6A modification in the C9ORF72 intron sequence preceding the expanded repeats enhances RNA degradation via the nuclear reader YTHDC1. The m6A reduction leads to increased accumulation of repeat RNA and poly-dipeptides. Moreover, the antisense RNA can also be regulated by m6A. Elevating m6A level significantly reduces both sense and antisense repeat RNA and poly-dipeptides, rescues global mRNA homeostasis, and improves survival of C9ORF72-ALS/FTD patient neurons.

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C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD

Project description:Noncoding expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Here we report transgenic mice carrying a bacterial artificial chromosome (BAC) containing the full human C9orf72 gene with either a normal allele (15 repeats) or disease-associated expansion (~100–1,000 repeats; C9-BACexp). C9-BACexp mice displayed pathologic features seen in C9orf72 expansion patients, including widespread RNA foci and repeat-associated non-ATG (RAN) translated dipeptides, which were suppressed by antisense oligonucleotides targeting human C9orf72. Nucleolin distribution was altered, supporting that either C9orf72 transcripts or RAN dipeptides promote nucleolar dysfunction. Despite early and widespread production of RNA foci and RAN dipeptides in C9-BACexp mice, behavioral abnormalities and neurodegeneration were not observed even at advanced ages, supporting the hypothesis that RNA foci and RAN dipeptides occur presymptomatically and are not sufficient to drive neurodegeneration in mice at levels seen in patients.

2016-02-10 | GSE77720 | GEO

C9orf72 Intermediate Repeats are Associated with Corticobasal Degeneration, Increased C9orf72 Expression and Disruption of Autophagy

Project description:Microsatellite repeat expansion disease loci can exhibit pleiotropic clinical and biological effects depending on repeat length. Large expansions in C9orf72 (100s-1000s of units) are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). However, whether intermediate expansions also contribute to neurodegenerative disease is not well understood. Several studies have identified intermediate repeats in Parkinson’s disease patients, but the association was not found in autopsy confirmed cases. We hypothesized that intermediate C9orf72 repeats are a genetic risk factor for corticobasal degeneration (CBD), a neurodegenerative disease that can be clinically similar to Parkinson’s but has distinct tau protein pathology. Indeed, intermediate C9orf72 repeats were significantly enriched in autopsy-proven CBD (n=354 cases, odds ratio=3.59, p-value=0.00024). While large C9orf72 repeat expansions are known to decrease C9orf72 expression, intermediate C9orf72 repeats result in increased C9orf72 expression in human brain tissue and CRISPR/cas9 knockin iPSC derived neural progenitor cells. In contrast to cases of FTD/ALS with large C9orf72 expansions, CBD with intermediate C9orf72 repeats was not associated with pathologic RNA foci or dipeptide repeat protein aggregates. Knock-in cells with intermediate repeats exhibit numerous changes in gene expression pathways relating to vesicle trafficking and autophagy. Additionally, overexpression of C9orf72 without the repeat expansion leads to defects in autophagy under nutrient starvation conditions. These results raise the possibility that therapeutic strategies to reduce C9orf72 expression may be beneficial for the treatment of CBD.

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RNA sequencing of human fibroblasts after SUPT4H1 siRNA treatment

Project description:Asses the transcriptome-wide changes associated with depletion of transcription elongation factor, SUPT4H1, in human fibroblasts. An expanded hexanucleotide repeat in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics (e.g., antisense oligonucleotides) are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor, Spt4, selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Inhibition of SUPT4H1, the human ortholog of Spt4, similarly decreased production of sense and antisense RNA foci as well as DPR proteins in c9ALS patient fibroblasts and neurons from patient-derived induced pluripotent stem cells, consistent with our finding that cerebellar DPR protein levels correlate with SUPT4H1 expression in c9FTD/ALS patients. Therapeutic targeting of a single factor to eliminate pathological features of c9FTD/ALS offers advantages over approaches that require targeting sense and antisense repeats separately.

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Project description:G4C2 repeat expansions within the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeats undergo repeat-associated non-ATG translation to generate toxic dipeptide repeat proteins. Here, we show that insulin/Igf signalling is reduced in fly models of C9orf72 repeat expansion using RNA-sequencing of adult brain. We further demonstrate that activation of insulin/Igf signalling can mitigate multiple neurodegenerative phenotypes in flies expressing either expanded G4C2 repeats or the toxic dipeptide repeat protein poly-GR. Levels of poly-GR are reduced when components of the insulin/Igf signalling pathway are genetically activated in the diseased flies, suggesting a mechanism of rescue. Modulating insulin signalling in mammalian cells also lowers poly-GR levels. Remarkably, systemic injection of insulin improves the survival of flies expressing G4C2 repeats. Overall, our data suggest that modulation of insulin/Igf signalling could be an effective therapeutic approach against C9orf72 ALS/FTD.

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