GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport

Abstract

GGGGCC (G4C2) repeat expansion in a noncoding region of C9ORF72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)1,2. The basis for pathogenesis is unknown. To capture 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 (GFP) to detect repeat-associated non-AUG (RAN) translation. These transgenic animals show dosage-dependent, repeat length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins as observed in patients. 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 iPSC-derived neurons from C9ORF72 patients. 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.

Timing and significance of pathological features inC9orf72expansion-associated frontotemporal dementia

“…Whether these neurons represent an early C9orf72-related pathological transition stage or a consequence of surgery, local tissue hypoxia, or epilepsy remains unclear. If these neurons reflect C9orf72 disease, nuclear TDP-43 depletion could have resulted from diminished TDP-43 expression or restricted access to the nucleus due to nucleocytoplasmic transport impairment (Freibaum et al, 2015;Jovicic et al, 2015;Zhang et al, 2015). Further work is needed to determine whether loss of nuclear TDP-43 in the absence of a TDP-43 inclusion relates to particular C9orf72-specific inclusions, as suggested in a recent study (Cooper-Knock et al, 2015).…”

Nucleocytoplasmic transport defect in a North American patient with ALS8

“…Disruption of the Ran GTPase‐mediated nucleocytoplasmic transport has been of interest since it was found to be disrupted in SOD1 and C9orf72 ALS 11, 12, 13, 20. In the ALS8 patient cells we found abnormal Ran distribution with increased cytoplasmic localization.…”

“…An SOD1 transgenic mouse model of ALS displayed defects in importin transport proteins in the anterior horn, indicative of a nucleocytoplasmic transport defect in ALS 11. Disruption of nucleocytoplasmic transport system has also been described in familial ALS due to C9orf72 repeat expansion 12, 13. More specifically, Ras‐related nuclear protein (Ran) GTPase is mislocalized to the cytoplasm in C9orf72 cells 13.…”

Nucleocytoplasmic transport inC9orf72-mediated ALS/FTD

“…[15][16][17] Cytoplasmic mislocalization and aggregation of nuclear proteins in ALS/FTD Protein aggregation is a pathological hallmark of neurodegenerative diseases including ALS/FTD, Alzheimer's, Parkinson's, and Huntington's diseases. 18 Cytoplasmic mislocalization and aggregation of TDP-43 (TAR DNA-binding protein of 43 k D ) is observed in >95% of ALS cases and »45% of FTD.…”