Elucidation of specific nucleocytoplasmic trafficking pathways that are disrupted in C9ORF72 ALS

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Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases for which no substantially effective treatments exist. The most common known genetic cause of both ALS and FTD is a hexanucleotide repeat expansion (HRE) mutation within the C9ORF72 gene. Transcription and subsequent translation of the HRE sequence produces multiple toxic RNAs and dipeptide repeat proteins (DPRs). Genetic screens in fly and yeast models have revealed that modifiers (enhancers and suppressors) of C9ALS pathology overwhelmingly cluster within nucleocytoplasmic trafficking (NCT) pathways, suggesting that HRE RNAs and/or DPRs confer neurotoxicity by disrupting NCT. However, critical gaps in our understanding of disrupted NCT remain. For example, there are multiple NCT pathways, each utilizing unique sub-cellular localization motifs within protein cargos that are recognized by specific transport proteins. No previous attempts have been made to elucidate the specific NCT pathways that are disrupted in C9ALS nor the specific toxic HRE product(s) that are responsible. To investigate these critical mechanisms thought to underlie neurotoxicity in C9ALS, we have generated “biosensors” designed to interrogate specific NCT pathways. These biosensors are composed of fluorescent proteins fused to unique nuclear localization and export signals allowing them to be recognized by different transport proteins. Using an intersectional approach, we will co- transfect each pathway-specific NCT biosensor with each HRE product to identify NCT pathways that are disrupted in C9ALS and the responsible HRE toxin(s). These experiments will be performed in high-throughput using immortalized cells and an automated image acquisition and analysis platform (high-content imaging). Subsequently, we will determine whether the perturbation of specific NCT pathways is recapitulated in patient- derived induced pluripotent stem cell (iPSC) motor neurons, a more disease relevant cellular model system. Finally, we will carry out a focused RNAi screen of the known genetic modifiers of C9ALS to identify those capable of restoring NCT in human cells. The research team is ideally positioned to carry out these studies by virtue of a vast clinical knowledge of C9ALS, expertise in therapeutic development and proficiency in both high content imaging and iPSC model systems. Furthermore, preliminary findings demonstrate both the feasibility of the approach and have informed the overarching hypothesis that NCT biosensors can be used to reveal specific NCT pathways that are disrupted in C9ALS, the HRE products that cause this disruption, and to identify genetic modifiers of NCT in human cellular model systems. The high-throughput nature of the project could be adapted for therapeutic screening, making it uniquely positioned to accelerate progress toward C9ALS therapies that restore NCT. Knowledge gained under the proposed studies is expected to provide critical insight into the pathobiology of C9ALS and lead to the identification of relevant therapeutic targets.