Identification of new pathways modulating C9orf72-derived DPRs expression

The hexanucleotide repeat expansion GGGGCCn (also known as G4C2n) localizes in the first intron of the C9ORF72 gene and is the most common genetic cause of ALS and FTD (C9ALS/FTD). The pathomechanisms proposed for C9ALS/FTD suggest that from sense (G4C2)n- and anti-sense (C4G2)n-containing transcripts originate two different mechanisms of toxicity: i) by the alteration of RNA processing due to binding and sequestration of RNA-binding proteins, thereby leading to impairment of RNA metabolism; and ii) by their unconventional Repeat-associated non AUG (RAN) translation into five different dipeptide-repeats (DPRs). In addition, pathological expansion of (G4C2)n reduces the C9orf72 transcription causing loss of function of the C9ORF72 protein. The toxicity of some of these DPRs has been showed in several cell lines, in iPSC-derived neurons, in Drosophila and in mouse models. An impairment of the ubiquitin-proteasome system (UPS) due to aggregation of toxic proteins is largely demonstrated in neurodegenerative disorders and among the mechanisms of DPR-related toxicity. RAN translation of (G4C2)n-RNAs has been recently shown to require a near-cognate start codon upstream of the repeat in frame +1 and to be triggered by stress conditions in a cap-dependent or cap-independent way. However, the mechanism regulating RAN translation is still largely unknown. Importantly, no small molecules are known to selectively modulate RAN translation, even if antisense oligonucleotides (ASOs) and small molecules binding the r(GGGGCC)n have been proposed as therapeutics for C9ALS/FTD. In addition, no effective pharmacological approach to reduce the pathological load of DPRs is currently available. Here, I developed a high-throughput drug-screening assay to identify small molecules and relative molecular targets that can modulate the DPR level. Among the identified hits, two hits reduced DPRs expression levels triggering the protein clearance system in vitro. Moreover, the screening identified compounds having the same target that increased DPRs expression levels indicating the targeted pathway as a crucial modulator of the translation process of the C9orf72 repeat-containing mRNAs. Conversely, I showed that pharmacological inhibition of the pathway reduced DPRs expression levels in vitro, while in vivo it rescued climbing ability and increased life span of Drosophila flies carrying G4C2X36 repeats. Moreover, genetic ablation of the target reduced DPRs expression levels by decreasing their translation efficiency in vitro and rescued the pathological phenotype in vivo. Together, the results show the identification of new pathways as new drug targets for C9ALS/FTD.