An unbiased genetic screen in Drosophila expressing G4C2-repeat-containing transcripts (repeats that in human cause pathogenesis in C9orf72-related neurological disease) finds genes that encode components of the nuclear pore and nucleocytoplasmic transport machinery, and reveals that G4C2 expanded-repeat-induced alterations in nucleocytoplasmic transport contribute to C9orf72 pathology and neurodegeneration. The most common cause of the debilitating disease amyotrophic lateral sclerosis (ALS) is a hexanucleotide repeat expansion GGGGCC (G4C2) in the C9orf72 gene. Two studies in this issue use contrasting methods to arrive at a molecular mechanism that may cause a familial form of the disease. Using a candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats (Ke Zhang et al.) or an unbiased genetic screen in Drosophila expressing 8, 28 or 58 G4C2 repeat-containing transcripts (Brian Freibaum et al.), the two groups sought genes that enhance or suppress the disease phenotype. Zhang et al. identify the gene encoding RanGAP, a key regulator of nucleocytoplasmic transport, and Freibaum et al. identifies genes that encode components of the nuclear pore and the nucleocytoplasmic transport machinery. Both papers show deficits in nucleocytoplasmic transport in Drosophila cells expressing G4C2 repeats and in iPSC-derived neurons from ALS patients. Zhang et al. show that these defects can be rescued with antisense oligonucleotides or small molecules targeting the G-quadruplexes. The GGGGCC (G4C2) repeat expansion in a noncoding region of C9orf72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis and frontotemporal dementia1,2. The basis for pathogenesis is unknown. To elucidate the consequences of G4C2 repeat expansion in a tractable genetic system, we generated transgenic fly lines expressing 8, 28 or 58 G4C2-repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein to detect repeat-associated non-AUG (RAN) translation. We show that these transgenic animals display dosage-dependent, repeat-length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins, as observed in patients with C9orf72-related disease. This model was used in a large-scale, unbiased genetic screen, ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC), as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results, we found morphological abnormalities in the architecture of the nuclear envelope in cells expressing expanded G4C2 repeats in vitro and in vivo. Moreover, we identified a substantial defect in RNA export resulting in retention of RNA in the nuclei of Drosophila cells expressing expanded G4C2 repeats and also in mammalian cells, including aged induced pluripotent stem-cell-derived neurons from patients with C9orf72-related disease. These studies show that a primary consequence of G4C2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration.

GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport