High throughput screen of 100,000 small molecules in C9orf72 ALS neurons missing files
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Epithelial Cell
SUBMITTER: 
Hans Voshol
LAB HEAD: Hans Voshol
PROVIDER: PXD034097 | Pride | 2025-03-07
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| PXP0486_G4C_3_13_BQE1_2018_0245.raw | Raw | |||
| PXP0486_G4C_3_7_BQE1_2018_0238.raw | Raw | |||
| id_placeholder.xlsx | Xlsx |
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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: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:Nuclear RNA foci are often associated with mis-regulation of RNA processing in repeat expansion diseases. Here we report transcriptomic analysis of flies carrying expanded G4C2 repeats, the most common genetic cause of FTD/ALS. By obtaining an average of 32 million reads per library, we show that the presence of numerous nuclear G4C2 repeat RNA foci does not cause extensive changes in RNA processing in this model organism. Comparision of the transcriptomic profile of flies expressing 5R or 160R from two different chromosomes
2015-09-11 | E-GEOD-72771 | biostudies-arrayexpress
Project description:Nuclear RNA foci are often associated with mis-regulation of RNA processing in repeat expansion diseases. Here we report transcriptomic analysis of flies carrying expanded G4C2 repeats, the most common genetic cause of FTD/ALS. By obtaining an average of 32 million reads per library, we show that the presence of numerous nuclear G4C2 repeat RNA foci does not cause extensive changes in RNA processing in this model organism.
2015-09-11 | GSE72771 | GEO
Project description:An abnormal expansion of a G4C2 hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we developed a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform curbed the expression of the G4C2 repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci, reduced the production of a dipeptide repeat protein, and reversed transcriptional deficits. This high-fidelity system possessed improved transcriptome-wide specificity compared to its native form and mediated targeting in motor neuron-like cells derived from a patient with ALS. These results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.
2024-11-28 | GSE251822 | GEO
Project description:An abnormal expansion of a G4C2 hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we developed a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform curbed the expression of the G4C2 repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci, reduced the production of a dipeptide repeat protein, and reversed transcriptional deficits. This high-fidelity system possessed improved transcriptome-wide specificity compared to its native form and mediated targeting in motor neuron-like cells derived from a patient with ALS. These results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.
2024-11-28 | GSE250633 | GEO
Project description:Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease with available treatments only marginally slowing progression or improving survival. A hexanucleotide repeat expansion mutation in the C9ORF72 gene is the most commonly known genetic cause of both sporadic and familial cases of ALS and frontotemporal dementia (FTD). The C9ORF72 expansion mutation produces five dipeptide repeat proteins (DPRs), and while the mechanistic determinants of DPR-mediated neurotoxicity remain incompletely understood, evidence suggests that disruption of nucleocytoplasmic transport and increased DNA damage contributes to pathology. Therefore, characterizing these disturbances and determining the relative contribution of different DPRs is needed to facilitate the development of novel therapeutics for C9ALS/FTD. To this end, we generated a series of nucleocytoplasmic transport "biosensors", composed of the green fluorescent protein (GFP), fused to different classes of nuclear localization signals (NLSs) and nuclear export signals (NESs). Using these biosensors in conjunction with automated microscopy, we investigated the role of the three most neurotoxic DPRs (PR, GR, and GA) on seven nuclear import and two export pathways. In addition to other DPRs, we found that PR had pronounced inhibitory effects on the classical nuclear export pathway and several nuclear import pathways. To identify compounds capable of counteracting the effects of PR on nucleocytoplasmic transport, we developed a nucleocytoplasmic transport assay and screened several commercially available compound libraries, totaling 2714 compounds. In addition to restoring nucleocytoplasmic transport efficiencies, hits from the screen also counteract the cytotoxic effects of PR. Selected hits were subsequently tested for their ability to rescue another C9ALS/FTD phenotype-persistent DNA double strand breakage. Overall, we found that DPRs disrupt multiple nucleocytoplasmic transport pathways and we identified small molecules that counteract these effects-resulting in increased viability of PR-expressing cells and decreased DNA damage markers in patient-derived motor neurons. Several HDAC inhibitors were validated as hits, supporting previous studies that show that HDAC inhibitors confer therapeutic effects in neurodegenerative models.
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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.
2021-03-20 | GSE151826 | GEO
Project description:Mutations in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis (ALS). Both toxic gain of function and loss of function pathogenic mechanisms have been proposed. Accruing evidence from mouse knockout studies point to a role for C9ORF72 as a regulator of immune function. To provide further insight into its cellular function, we performed a genome-wide synthetic lethal CRISPR screen in human myeloid cells lacking C9ORF72. We discovered a strong synthetic lethal genetic interaction between C9ORF72 and FIS1, which encodes a mitochondrial membrane protein involved in mitochondrial fission and mitophagy. Mass spectrometry experiments revealed that in C9ORF72 knockout cells, FIS1 strongly bound to a class of immune regulators that activate the receptor for advanced glycation end (RAGE) products and trigger inflammatory cascades. These findings present a novel genetic interactor for C9ORF72 and suggest a compensatory role for FIS1 in suppressing inflammatory signaling in the absence of C9ORF72.
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