Determining the Mechanism of RAN Translation in C9orf72-Associated ALS/FTD

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Abstract The most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a G4C2 hexanucleotide repeat expansion in the first intron of the C9orf72 gene. This repeat undergoes a non-canonical form of protein translation initiation known as repeat associated non-AUG (RAN) translation (RANT). RANT initiates from non-AUG codons and in C9orf72-associated ALS/FTD (C9ALS/FTD) produces dipeptide repeat proteins (DPRs) that aggregate within inclusions in patient brains. DPR expression separate from potentially toxic repeat-containing RNA species is sufficient to induce cell death and reduce cell viability in many model systems, including primary mammalian neurons. As such, RANT is thought to contribute to disease pathogenesis. Despite this central importance, the exact endogenous repeat-containing transcripts from which DPRs are translated remains unknown. The repeat is located within the first intron and typically, introns are spliced from pre-mRNA as RNA lariats and rapidly degraded. In C9ALS/FTD, repeat RNAs are observed in the cytoplasm and undergo translation. Initial studies suggested that RANT could occur from a linear mRNA in which the repeat-containing intron is retained or via internal ribosomal entry from a C9orf72 lariat that fails to undergo degradation. However, our group recently identified a third potential source template for C9RANT: repeat triggered transcriptional initiation within the first intron itself that generates capped repeat containing mRNAs. This proposal will determine the RNA templates that undergo C9RANT in patient neurons and whether targeting pathways which influence their production and stability are capable of modulating RANT production and suppressing toxicity in C9ALS/FTD patient-derived iNeurons. Using patient-derived neurons, I will determine the molecular nature of repeat containing C9orf72 transcripts that undergo RANT using polysome profiling, qRT-PCR, and hybridization chain reaction coupled with confocal microscopy and biochemical assays. In parallel, I will develop antisense oligonucleotides (ASOs) that selectively target this novel C9orf72 transcript and determine the impact of modulating C9orf72 splicing and RNA species production on C9RANT generation and repeat toxicity. The training described in this proposal will prepare me to work at the interface between cellular and molecular neurobiology and its application towards patient-oriented therapeutics.