High throughput screen of 100,000 small molecules in C9orf72 ALS neurons

ABSTRACT: 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

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Hans Voshol  

LAB HEAD: Hans Voshol

PROVIDER: PXD034016 | Pride | 2025-03-07

REPOSITORIES: pride