Investigation of repeat-associated non-AUG translation and dipeptide repeat proteins in C9orf72-associated ALS/FTD

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A hexanucleotide (G4C2) repeat expansion in the gene C9orf72 is the commonest known cause of both familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (DeJesus-Hernandez et al., 2011; Renton et al., 2011). C9orf72 is expressed in neurons where the repeat expansion is transcribed bi-directionally into aberrant repeat-containing RNA (DeJesus-Hernandez et al., 2011). Both sense and antisense transcripts aggregate into cytoplasmic and intranuclear RNA foci (Mizielinska et al., 2014). Despite the lack of a conventional start codon, these transcripts are translated through repeat-associated non-AUG (RAN) translation (Ash et al., 2013; Green et al., 2017).This produces five dipeptide repeat proteins (DPRs): glycine-alanine (GA), glycine-arginine (GR), proline-arginine (PR), proline-alanine (PA) and glycine-proline (GP) (Mori et al., 2013). The DPRs form neuronal inclusions in widespread brain regions (Ash et al., 2013). Additionally, most patient brains show truncation and translocation of the RNA-binding protein TDP-43 from the nucleus to cytoplasmic aggregates, a phenomenon also common in non-C9orf72 ALS and FTD (Neumann et al., 2006).Both RNA foci and DPRs, particularly the arginine-rich GR and PR, were toxic to multiple model systems (Donnelly et al., 2013; Mizielinska et al., 2014; Lopez-Gonzalez et al., 2016). Recent work in our lab showed that inhibiting RAN-translation in a Drosophila model through inserting periodic stop codons into the repeat region retained mRNA foci, but ameliorated neurodegeneration (Mizielinska et al., 2014; Moens et al., 2018). This suggests that reducing the translation of DPRs may be protective against repeat-mediated disease.Following this work, our lab has collaborated with an industry partner to screen 20,000 small molecules (SMs) to identify those that reduce RAN-translation in HeLa cells overexpressing G4C2 repeats. SMs have a chance of passing the blood-brain barrier and are cheaper to produce compared to antisense oligonucleotides, another therapeutic option currently under development (Schludi and Edbauer, 2017; Donnelly et al., 2013).Techniques usedTissue culture, ELISA, SIMOA, immunocytochemistry, confocal microscopy, Southern blotting, protein synthesis assay (Click-iT AHA), RNA FISH, transfection/transduction