Single molecule study of C9ORF72 repeat RNA metabolisms in ALS/FTD

Johns Hopkins University, Baltimore MD

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Abstract

Amyotrophic lateral sclerosis (ALS), an adult-onset motor neuron degenerative disease, is increasingly recognized to have clinical, pathological and genetic overlaps with Frontotemporal dementia (FTD). The expansion of GGGGCC repeats in the first intron of C9ORF72 is the most common genetic cause of both ALS and FTD. The disease has been predominantly attributed to the RNA repeats induced toxicity, including RNA granules that sequester essential RNA binding proteins (RBP), and the toxic poly-dipeptides produced through repeat-associated non-AUG (RAN) translation. Additionally, there is bi-directional transcription which leads to formation of sense (GGGGCC)n and antisense (CCCCGG)n RNA foci in nuclei, as well as accumulation of five different DPRs (poly-GA, poly-GR, poly-PA, poly-PR and poly-GP) translated from both strands in C9ORF72- ALS/FTD patients. Understanding the repeat RNA metabolism is important for uncovering disease mechanisms and identifying potential therapeutic targets. Combining the cutting-edge single molecule imaging approach with biochemical and high-throughput sequencing assays, work from previous period systematically elucidated the sense repeat RNA processing pathways and identified novel neurotoxicity mechanisms. We now propose to apply similar technologies and platforms to determine the metabolism of antisense repeat RNA and elucidate the relationships between sense and antisense RNA repeats. Recently, the clinical trial of antisense oligonucleotide (ASO) targeting the sense repeat-containing RNA showed no neuroprotection, and even indicated a faster decline in C9ORF72-ALS patients. While there could be various scenarios, one plausible hypothesis is that the antisense repeat RNA also contributes to the disease pathogenesis, therefore targeting sense repeats is not sufficient. Furthermore, the sense and antisense strands might affect each other’s metabolisms and toxicity. Therefore, understanding the RNA metabolism of antisense repeats and how sense and antisense RNAs influence each other will be crucial for developing effective therapeutic strategies. In this proposal, we will explore the relationships between sense and antisense repeat RNAs, and assess how knocking down one strand by ASO affects the RNA metabolism of the other strand. We will identify the antisense (CCCCGG)n repeat RNA-associated proteins from intact cells, and determine its influence on cellular RNA metabolism. Finally, we aim to identify genetic modifiers that can reduce the accumulation of both sense and antisense repeat RNAs, thereby eliminating all possible gain-of-toxicity species from the repeat expansion. Overall, this proposal tackles critical biological questions directly relevant to disease mechanisms that are largely understudied in C9ORF72-ALS/FTD. The cutting-edge technologies provide opportunities to address the mechanistic problems that are intractable by other techniques, potentially leading to new discoveries. The findings from this study will provide valuable insights into the disease mechanism and therapy development.