Identification and validation of hits modulating C9ORF72-dipeptide repeat proteins level in vitro and vivo models

The intronic hexanucleotide repeat expansion, GGGGCC (or G4C2), in the C9ORF72 gene is the most common genetic cause of Amyotrophic Lateral Sclerosis. When it is expanded above 30 and up to hundreds repeats, the G4C2 repeat expansion is transcribed in aberrant RNAs that fold in G-quadruplex structures and generate RNA foci within motoneurons. Additionally, aberrant transcripts also shuttle in the cytoplasm and can be translated via the unconventional repeat-associated non-ATG translation (RANT) into five toxic dipeptide repeat (DPR) proteins that induce neurodegeneration. However, so far, no effective pharmacological approach, to reduce the pathological load of DPRs is currently available. Here, we developed and performed a high-throughput drug-screening fluorescent assay in HEK293T cells to identify positively and negatively modulators of RANT. We selected and characterized 3 hits by assessing whether they affected DPRs level at transcriptional, translational or post-translational level. Finally, we tested the efficacy of one hit in vivo, using Drosophila flies carrying the G4C2X36 repeats and evaluating the hit’s capacity to rescue the climbing ability and increase life span of mutant flies.