Partial depletion of SRSF1 rescued the C9ORF72-repeat neurodegeneration-associated locomotor deficits in G4C2x36 Drosophila lines
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PROVIDER: PRJNA576478 | ENA |
REPOSITORIES: ENA
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Project description:In this study we investigated the role of SRSF1 in mitigating locomotor defect in C9ORF72-ALS/FTD Drosophila lines. We analysed the transcriptomes of Drosophila heads from the same lines: (i) control flies expressing 3 G4C2 repeats and a luciferase-RNAi control (G4C2x3 + C-RNAi); (ii) C9ORF72-ALS/FTD model expressing 36 G4C2 repeats and the RNAi control (G4C2x36 + C-RNAi); (iii) C9ORF72-ALS/FTD-neuroprotected flies expressing 36 G4C2 repeats and the disrupted SRSF1 allele (G4C2x36 + ΔSRSF1). RNA samples were profiled using Drosophila 3' IVT gene expression Affymetrix microarrays prior to qRT-PCR validation of the SRSF1 depletion in neuroprotected flies. This study is part of a larger study that investigates the mitigating role of SRSF1 at whole-genome level to detect transcriptomal alterations and neuronal hyperexcitability in C9ORF72-linked amyotrophic lateral sclerosis.
2021-07-21 | GSE138592 | GEO
Project description:Loss of motor neurons in amyotrophic lateral sclerosis (ALS) leads to relentless paralysis and death usually within a few years from symptom onset. Thousands of RNA molecules with roles in multiple cellular pathways are compromised in disease challenging the identification of alterations causing pathogenesis over downstream changes consequent to neurodegeneration. We recently showed that partial depletion of SR-rich splicing factor SRSF1 inhibits the nuclear export of pathological C9ORF72-repeat transcripts and subsequent translation of dipeptide-repeat proteins in patient-derived neurons and Drosophila, providing in turn a promising strategy of neuroprotection for the most common form of ALS. While the roles of SRSF1 remain poorly characterised in neurons, its therapeutic manipulation offers a rare opportunity to identify disease-modifying gene expression changes. Here, we report diseased and neuroprotected transcriptomes from human-derived neurons and Drosophila heads. Strikingly, while the depletion of SRSF1 has limited effects on genome-wide expression, splicing and nuclear export of RNAs, reversal of 90 disease-modifying transcripts were identified over 2,000 RNA changes in human disease. Functional validation further demonstrated that conserved potassium intermediate/small conductance calcium-activated KCNN channel inhibitors mitigate C9ORF72-ALS mediated hyperexcitability and death of human motor neurons as well as Drosophila motor deficits. Our data support efficacy and safety of SRSF1 manipulation as a therapeutic approach leading to rescue of multiple biological processes without disrupting neuronal transcriptomes. In addition to a potential new therapeutic target for pharmacological inhibition, we also identified in vitro and in vivo disease-modifying gene expression signatures to guide the development of new biomarkers and novel therapeutic approaches in C9ORF72-ALS/FTD.
2021-07-19 | GSE139900 | GEO
Project description:Mus musculus C9orf72, C9orf72, member of C9orf72-SMCR8 complex [Source:MGI Symbol;Acc:MGI:1920455], is differentially expressed in 129 experiment(s);
| ENSMUSG00000028300 | ExpressionAtlas
Project description:Mus musculus C9orf72, C9orf72, member of C9orf72-SMCR8 complex [Source:MGI Symbol;Acc:MGI:1920455], is expressed in 40 baseline experiment(s);
| ENSMUSG00000028300 | ExpressionAtlas
Project description:Background. An intronic G4C2 repeat expansion in the C9orf72 gene is the major known cause for Amyotrophic Lateral Sclerosis. There is evidence forthree possible disease mechanisms: a pathological gain of function of nuclear repeat RNA foci, repeat associated noncanonical (RAN) translation into toxic dipeptide repeat (DPR) polyproteins as well as C9orf72 haploinsufficiency. Which of these defects need to be restored to prevent or halt disease progression remains a fundamental question for the development of ALS therapeutics. Methods. Here we developed a high content imaging assay for G4C2 RNA foci detection in C9orf72 patient iPS derived neurons miniaturised to the 1536well format. 96’200 small molecules were screened for RNA foci modulation, with counterscreens for cell toxicity and general gene expression inhibition. Hits were validated in relevant cell models from different C9orf72 donors and selected for further characterization of their cellular and molecular mode of action. Results. Among the validated hits we identified analogues of known modulators of SF3B1, a major component of the U2 snRNP spliceosomal subunit. Structure activity relationship and genome wide splice analysis confirmed that compounds to clear nuclear G4C2 RNA foci by targeting SF3B1. A combination of genetically engineered G4C2 reporter systems and RNA-protein interactome studies reveals that pharmacological SF3B1 modulation promotes recruitment of SRSF1 to nuclear G4C2 RNA, mobilizing it from RNA foci into nucleocytoplasmic export. Thisleadsto increased RAN translation and, ultimately, enhanced DPR cell toxicity. Preventing SRSF1 phosphorylation by small molecule inhibition of SR protein kinase (SRPK) conversely results in a build up of nuclear RNA foci, antagonistic to SF3B1 modulation. Conclusion. Together, our data provide a new pharmacological entry point with publically available, antagonistic tool compoundsfor modulating G4C2 repeat expansion pathology in C9orf72 ALS models. This will facilitate the dissection of C9orf72 pathobiology by uncoupling RNA foci from RAN translation. Based on our data we propose that therapeutic RNA foci elimination strategies warrant caution due to a potential storage function, counteracting translation into toxic dipeptide repeat polyproteins. Instead, our data support modulation of nuclear export via SRSF1 or SR protein kinases as possible targets for future pharmacological drug discovery
2025-03-07 | PXD034016 | Pride
Project description:Background. An intronic G4C2 repeat expansion in the C9orf72 gene is the major known cause for Amyotrophic Lateral Sclerosis. There is evidence forthree possible disease mechanisms: a pathological gain of function of nuclear repeat RNA foci, repeat associated noncanonical (RAN) translation into toxic dipeptide repeat (DPR) polyproteins as well as C9orf72 haploinsufficiency. Which of these defects need to be restored to prevent or halt disease progression remains a fundamental question for the development of ALS therapeutics. Methods. Here we developed a high content imaging assay for G4C2 RNA foci detection in C9orf72 patient iPS derived neurons miniaturised to the 1536well format. 96’200 small molecules were screened for RNA foci modulation, with counterscreens for cell toxicity and general gene expression inhibition. Hits were validated in relevant cell models from different C9orf72 donors and selected for further characterization of their cellular and molecular mode of action. Results. Among the validated hits we identified analogues of known modulators of SF3B1, a major component of the U2 snRNP spliceosomal subunit. Structure activity relationship and genome wide splice analysis confirmed that compounds to clear nuclear G4C2 RNA foci by targeting SF3B1. A combination of genetically engineered G4C2 reporter systems and RNA-protein interactome studies reveals that pharmacological SF3B1 modulation promotes recruitment of SRSF1 to nuclear G4C2 RNA, mobilizing it from RNA foci into nucleocytoplasmic export. Thisleadsto increased RAN translation and, ultimately, enhanced DPR cell toxicity. Preventing SRSF1 phosphorylation by small molecule inhibition of SR protein kinase (SRPK) conversely results in a build up of nuclear RNA foci, antagonistic to SF3B1 modulation. Conclusion. Together, our data provide a new pharmacological entry point with publically available, antagonistic tool compoundsfor modulating G4C2 repeat expansion pathology in C9orf72 ALS models. This will facilitate the dissection of C9orf72 pathobiology by uncoupling RNA foci from RAN translation. Based on our data we propose that therapeutic RNA foci elimination strategies warrant caution due to a potential storage function, counteracting translation into toxic dipeptide repeat polyproteins. Instead, our data support modulation of nuclear export via SRSF1 or SR protein kinases as possible targets for future pharmacological drug discovery
2025-03-07 | PXD034093 | Pride
Project description:Background. An intronic G4C2 repeat expansion in the C9orf72 gene is the major known cause for Amyotrophic Lateral Sclerosis. There is evidence forthree possible disease mechanisms: a pathological gain of function of nuclear repeat RNA foci, repeat associated noncanonical (RAN) translation into toxic dipeptide repeat (DPR) polyproteins as well as C9orf72 haploinsufficiency. Which of these defects need to be restored to prevent or halt disease progression remains a fundamental question for the development of ALS therapeutics. Methods. Here we developed a high content imaging assay for G4C2 RNA foci detection in C9orf72 patient iPS derived neurons miniaturised to the 1536well format. 96’200 small molecules were screened for RNA foci modulation, with counterscreens for cell toxicity and general gene expression inhibition. Hits were validated in relevant cell models from different C9orf72 donors and selected for further characterization of their cellular and molecular mode of action. Results. Among the validated hits we identified analogues of known modulators of SF3B1, a major component of the U2 snRNP spliceosomal subunit. Structure activity relationship and genome wide splice analysis confirmed that compounds to clear nuclear G4C2 RNA foci by targeting SF3B1. A combination of genetically engineered G4C2 reporter systems and RNA-protein interactome studies reveals that pharmacological SF3B1 modulation promotes recruitment of SRSF1 to nuclear G4C2 RNA, mobilizing it from RNA foci into nucleocytoplasmic export. Thisleadsto increased RAN translation and, ultimately, enhanced DPR cell toxicity. Preventing SRSF1 phosphorylation by small molecule inhibition of SR protein kinase (SRPK) conversely results in a build up of nuclear RNA foci, antagonistic to SF3B1 modulation. Conclusion. Together, our data provide a new pharmacological entry point with publically available, antagonistic tool compoundsfor modulating G4C2 repeat expansion pathology in C9orf72 ALS models. This will facilitate the dissection of C9orf72 pathobiology by uncoupling RNA foci from RAN translation. Based on our data we propose that therapeutic RNA foci elimination strategies warrant caution due to a potential storage function, counteracting translation into toxic dipeptide repeat polyproteins. Instead, our data support modulation of nuclear export via SRSF1 or SR protein kinases as possible targets for future pharmacological drug discovery
2025-03-07 | PXD034092 | Pride
Project description:Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) patients with the C9orf72 mutation show predominantly cytoplasmic aggregates of poly-GR and poly-PR proteins that are acutely toxic in various model systems. To identify the molecular mediators of neurotoxicity of poly-GR/PR, we analyzed their interactomes in primary neurons. GFP-(GR)149 and (PR)175-GFP preferentially interacted with RNA-binding proteins, including stress granule-associated and nucleolar proteins, as well as ribosomes. Overexpression of the poly-GR/PR interactors Staufen 1/2 (STAU1/2) and YBX1 led to cytoplasmic aggregation of poly-GR/PR into large stress granule-like inclusions, while the poly-GR/PR interactor nucleophosmin (NPM1) recruited poly-GR into the nucleolus. In addition, poly-PR expression reduced ribosome levels and translation, which is consistent with the widespread reduction of synaptic proteins detected by proteomics. Surprisingly, only GFP-(GR)53, but not GFP-(GR)149, localized to the nucleolus and reduced ribosome levels and translation in neurons, suggesting impaired ribosome biogenesis is driving the acute toxicity commonly observed in vitro. In C9orf72 patient brains, we detected co-aggregation of poly-GR/PR inclusions with ribosomes, but not stress granules. Partial sequestration of ribosomes may chronically impair protein synthesis and contribute to C9orf72 ALS/FTD pathogenesis.
2018-06-27 | PXD008691 | Pride
Project description:C9orf72 repeat expansions cause inherited amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) and result in both loss of C9orf72 protein expression and production of potentially toxic RNA and dipeptide repeat proteins. In addition to ALS/FTD, C9orf72 repeat expansions have been reported in a broad array of neurodegenerative syndromes including Alzheimer’s disease. Here we show that C9orf72 deficiency promotes loss of homeostatic signatures in microglia and transition to an inflammatory state characterized by an enhanced type I IFN signature. Furthermore, C9orf72-depleted microglia trigger age-dependent neuronal defects, in particular enhanced cortical synaptic pruning, leading to altered learning and memory behaviors in mice. Interestingly, C9orf72 deficient microglia also promote enhanced synapse loss and neuronal deficits in a mouse model of amyloid accumulation, while paradoxically improving plaque clearance. These findings suggest that altered microglial function due to decreased C9orf72 expression directly contributes to neurodegeneration in repeat expansion carriers independent of gain of function toxicities.
2022-06-15 | GSE164674 | GEO
Project description:C9orf72 repeat expansions cause inherited amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) and result in both loss of C9orf72 protein expression and production of potentially toxic RNA and dipeptide repeat proteins. In addition to ALS/FTD, C9orf72 repeat expansions have been reported in a broad array of neurodegenerative syndromes including Alzheimer’s disease. Here we show that C9orf72 deficiency promotes loss of homeostatic signatures in microglia and transition to an inflammatory state characterized by an enhanced type I IFN signature. Furthermore, C9orf72-depleted microglia trigger age-dependent neuronal defects, in particular enhanced cortical synaptic pruning, leading to altered learning and memory behaviors in mice. Interestingly, C9orf72 deficient microglia also promote enhanced synapse loss and neuronal deficits in a mouse model of amyloid accumulation, while paradoxically improving plaque clearance. These findings suggest that altered microglial function due to decreased C9orf72 expression directly contributes to neurodegeneration in repeat expansion carriers independent of gain of function toxicities.
2022-06-15 | GSE164675 | GEO