The C9ORF72 repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This G4C2 repeat expansion has been shown to generate toxic RNA molecules that sequester proteins which disrupts their function. These RNAs can also be translated into dipeptide repeats (DPRs) via a non-canonical pathway and accumulate. Both G4C2 RNAs and DPRs have been shown to be toxic when they accumulate in neurons and glia. We and others have recently shown that nucleocytoplasmic trafficking is disrupted in C9ORF72 fly models and induced pluripotent stem cell (iPSC) neurons due to the presence of these molecules. Importantly, genetically enhancing nuclear protein import or reducing nuclear export is neuroprotective in G4C2-expressing flies. In this R21 research proposal, we will employ various fly models of G4C2 RNA and DPR based toxicity and test multiple compounds to pharmacologically modulate nucleocytoplasmic trafficking using inhibitors of the nuclear exporter, exportin-1. Compounds that rescue neural degeneration and motor phenotypes in fly models of C9ORF72 ALS/FTD will then be tested for efficacy in C9ORF72 iPSC motor neurons. We will first investigate C9ORF72 ALS/FTD related pathology including G4C2 RNA foci and DPR accumulation. Next we will assess if these compounds protect against known susceptibility to extracellular stressors and survival employing longitudinal imaging analysis. Finally, we will determine how these compounds affect nucleocytoplasmic trafficking deficits in C9ORF72 ALS motor neurons. Taken together, these studies will determine if pharmacologically modulating the nucleocytoplasmic trafficking pathway is a viable therapeutic strategy to treat neurodegenerative diseases related to the C9ORF72 repeat expansion. Furthermore, we will have identified the neuroprotective mechanism of action for these novel compounds.
Recent work has implicated nuclear pore dysfunction and nucleocytoplasmic trafficking in the pathogenesis of amyotrophic lateral sclerosis caused by the C9ORF72 repeat expansion. Preliminary evidence suggests that genetic or pharmacological inhibition of inhibiting nuclear export is neuroprotective in C9ORF72 models. This proposal will employ multiple C9ORF72 Drosophila models and induced pluripotent stem cell motor neurons to investigate whether therapeutic modulation of nuclear export rescues C9ORF72 mediated neural injury and evaluate the mechanism of protection.
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