Novel single molecule imaging approaches reveal structure-function alterations to the nuclear pore complex in early C9ORF72-associated TDP-43 proteinopathy

Nucleocytoplasmic transport through the nuclear pore complex is essential for the maintenance of cellular homeostasis by regulating the movement of molecules between the nucleus and the cytoplasm. This process becomes dysfunctional in many diseases but has been particularly implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) linked to the C9ORF72 mutation and associated aberrantly produced polypeptides. To directly study nucleocytoplasmic transport in intact non-genetically modified cells we have developed two new approaches for single molecule tracking through nuclear pore subdomains and super-resolved imaging of nuclear pore structural organisation. Using these techniques we have examined the early impact of the neurotoxic C9ORF72 polypeptide poly(glycine-arginine) on nuclear pore transport dynamics and structure. We find that soluble poly(glycine-arginine) peptides can disrupt molecular flow of passive cargo predominantly during nuclear export which is associated with altered structural organisation and molecular interaction of nuclear basket and central nuclear pore complex domains. These changes converge with perturbed nucleocytoplasmic homeostasis of the key ALS/FTD pathological protein TDP-43 and begin to explain this initiating step in disease.