Hexanucleotide repeat translation in ALS and Frontotemporal Dementia The most common genetic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal dementia (FTD) is an intronic GGGGCC (G4C2) hexanucleotide repeat expansion in the gene C9orf72 (C9FTD/ALS). Despite its position within an intron, the C9orf72 repeat triggers synthesis of dipeptide repeat proteins (DPRs) via a process known as Repeat Associated Non-AUG (RAN) Translation. Studies by our group and others over the past decade have defined key mechanistic parameters that regulate RAN translational initiation and identified selective modulators of RAN translation that suppress disease relevant phenotypes in model systems. In this renewal application, we will address two key questions. 1) What mRNA template(s) are used for C9RAN translation endogenously? Some data suggests that repeat-containing lariats are stabilized and translated. We propose an alternative model where aberrant transcription initiation within the intron itself generates linear 5’ M7G capped mRNA species that robustly support RAN translation. 2) What impact does repeat RNA structure have on C9RAN translational initiation and elongation? Our preliminary studies suggest that both repeat RNA structure dynamics and ribosomal quality control (RQC) pathways act as critical modulators of RAN translation by eliciting ribosomal stalling and altering translational initiation and elongation rates. Our central hypothesis is that GC-rich repeats generate aberrant mRNA species whose RNA structure directly influences their capacity for translation and neurotoxicity. Our goals are to determine the relative contributions of different potential endogenous mRNA species to C9RAN translation and the impact of repeat RNA structure on RAN translational efficiency and RQC engagement. Our central premise is that a detailed understanding of C9RAN translation will both uncover potential therapeutic targets for repeat expansion disorders including C9 FTD/ALS and reveal novel biological insights into aberrant translation events in neurons. In sum, this work will rigorously explore the mechanisms underlying C9 RAN translation, enhance our understanding of protein translational dynamics in neurons and repeat expansion disorder pathogenesis while simultaneously providing a rational path towards therapeutic development in ALS and FTD.