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A GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the G4C2 RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats identified RanGAP (Drosophila ortholog of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9ORF72 ALS patient-derived induced pluripotent stem cells (iPSNs), and in C9ORF72 patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9ORF72 iPSNs, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD amenable to pharmacotherapeutic intervention.

Abstract. Ran is a nuclear Ras-like GTPase that is required for the bidirectional transport of proteins and ribonucleoproteins across the nuclear pore complex (NPC). A key regulator of the Ran GTP/GDP cycle is the 70:kD Ran-GTPase-activating protein RanGAP1. Here, we report the identification and localization of a novel form of RanGAP1. Using peptide sequence analysis and specific mAbs, RanGAP1 was found to be modified by conjugation to a ubiquitin-like protein.Immunoblot analysis and immunolocalization by light and EM demonstrated that the 70-kD unmodified form of RanGAP1 is exclusively cytoplasmic, whereas the 90-kD modified form of RanGAP1 is associated with the cytoplasmic fibers of the NPC. The modified form of RanGAP1 also appeared to associate with the mitotic spindle apparatus during mitosis. These findings have specific implications for Ran function and broad implications for protein regulation by ubiquitin-like modifications. Moreover, the variety and function of ubiquitin-like protein modifications in the cell may be more diverse than previously realized.T RANSPORT of macromolecules across the nuclear envelope occurs bidirectionally through nuclear pore complexes (NPCs) 1, large supramolecular assemblies that span both membranes of the nuclear envelope (Rout and Wente, 1994). Whereas small ions and metabolites can passively diffuse through the NPC, most proteins and ribonucleoproteins are transported across the NPC by a signal-and energy-dependent mechanism. Dissection of the events culminating in nuclear import has been aided by the development of a permeabilized cell system that has made possible the identification of soluble cytosolic factors required for nuclear import (Adam et al., 1990), and more recently by the development of solution binding assays that use recombinant transport factors and nucleoporins .Using the permeabilized cell assay in conjunction with biochemical fractionation of cytosolic extracts, four factors required for nuclear import have been purified and characterized (Moore and