PROJECT SUMMARY/ABSTRACT Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD), the second most common form of dementia, comprise a spectrum of fatal neurodegenerative diseases. An intronic GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the C9orf72 gene, have been linked to ALS and FTD, although clinically two distinct diseases. The C9orf72 HRE is the most common cause of both familial and sporadic ALS accounting for ~40% and ~8% of patients respectively. Overall, about 10% of ALS cases are familial with the remaining 90% being sporadic. The molecular mechanisms underlying disease pathogenesis remain poorly understood. Defects in nucleocytoplasmic transport (NCT) and the nuclear pore complex (NPC) have recently emerged as a prominent pathomechanism underlying multiple neurodegenerative diseases including C9orf72 ALS/FTD, subsets of sporadic ALS, Alzheimer’s Disease, and Huntington’s Disease. However, little is known about the nature of the injury to the NPC and its individual nucleoporin components themselves. Using induced pluripotent stem cell derived spinal neurons (iPSNs) and postmortem human tissue, we have amassed data that loss of the transmembrane nucleoporin POM121 from NPCs initiates a pathological cascade impacting NPC composition, function and downstream cellular survival. Notably, loss of POM121 is mediated by pathologic G4C2 repeat RNA and not dipeptide repeat poly peptides or loss of C9ORF72 protein. Given that POM121 protein is not mislocalized and POM121 RNA metabolism is unaltered, we hypothesized that POM121 and subsequently altered nucleoporin proteins are aberrantly degraded in the early stages of C9orf72 ALS/FTD pathogenesis. Recent work in yeast and non-neuronal mammalian cells has shown that nuclear CHMP7 “activates” ESCRT-III mediated degradation of nuclear pore complexes and nuclear envelope components during nuclear pore surveillance and homeostasis. Our new preliminary data suggests that the loss of POM121 from the nucleoplasm and NPCs is initiated by nuclear accumulation of CHMP7. Mechanistically, increased nuclear CHMP7 appears to be the result of G4C2 repeat RNA mediated impaired nuclear export. Thus, our data strongly implicate a CHMP7 degradative pathway in disease pathogenesis. Intriguingly, knockdown of CHMP7 mitigates NPC injury in C9orf72 iPSNs making CHMP7 an attractive therapeutic target in neurodegeneration. In this proposal we will comprehensively investigate this new pathway including studies to 1) Determine the degradative pathway by which CHMP7 mediates nucleoporin removal from NPCs in iPSNs, 2) Investigate the mechanism by which pathologic G4C2 repeat RNA initiates CHMP7 mediated NPC injury. And finally, 3) using a large battery of individual patient iPSN spinal neuron cell lines, evaluate the ability of CHMP7 antisense oligonucleotides to mitigate C9orf72 ALS/FTD and sporadic ALS mediated alterations in the nuclear pore complex and nucleocytoplasmic transport and downstream sen...