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.

SUMMARY GGGGCC repeat expansions in C9ORF72 are the most common genetic cause of both ALS and FTD. To uncover underlying pathogenic mechanisms, we found DNA damage was greater, in an age-dependent manner, in motor neurons differentiated from iPSCs of multiple C9ORF72 patients than control neurons. Ectopic expression of the dipeptide repeat (DPR) protein (GR)80 in iPSC-derived control neurons increased DNA damage, suggesting poly(GR) contributes to DNA damage in aged C9ORF72 neurons. Oxidative stress was also increased in C9ORF72 neurons in an age-dependent manner. Pharmacological or genetic reduction of oxidative stress partially rescued DNA damage in C9ORF72 neurons and control neurons expressing (GR)80 or (GR)80-induced cellular toxicity in flies. Moreover, interactome analysis revealed that (GR)80 preferentially bound to mitochondrial ribosomal proteins and caused mitochondrial dysfunction. Thus, poly(GR) in C9ORF72 neurons compromises mitochondrial function and causes DNA damage in part by increasing oxidative stress, revealing another pathogenic mechanism in C9ORF72-related ALS and FTD.

SUMMARY Dipeptide repeat (DPR) proteins are toxic in various models of FTD/ALS with GGGGCC (G4C2) repeat expansion. However, it is unclear whether nuclear G4C2 RNA foci also induce neurotoxicity. Here, we describe a novel Drosophila model expressing 160 G4C2 repeats (160R) flanked by human intronic and exonic sequences. Spliced intronic 160R formed nuclear G4C2 sense RNA foci in glia and neurons about 10 times more abundantly than in human neurons; however, they had little effect on global RNA processing and neuronal survival. In contrast, highly toxic 36R in the context of poly(A)+ mRNA were exported to the cytoplasm where DPR proteins were produced at >100-fold higher level than that in 160R flies. Moreover, the modest toxicity of intronic 160R expressed at higher temperature correlated with increased DPR production but not RNA foci. Thus, nuclear RNA foci are neutral intermediates or possibly neuroprotective through preventing G4C2 RNA export and subsequent DPR production.

C9ORF72 repeat expansion is the most common genetic mutation in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Abnormal dipeptide repeat proteins (DPRs) generated from repeat-associated non-AUG (RAN) translation of repeat-containing RNAs are thought to be pathogenic; however, the mechanisms are unknown. Here we report that (GR)80 and (PR)80 are toxic in neuronal and non-neuronal cells in Drosophila. In contrast to reported shorter poly(GR) forms, (GR)80 is mostly localized throughout the cytosol without detectable accumulation in the nucleolus, accompanied by suppression of Notch signaling and cell loss in the wing. Some Notch target genes are also downregulated in brains and iPSC-derived cortical neurons of C9ORF72 patients. Increased Notch expression largely suppressed (GR)80-induced cell loss in the wing. When co-expressed in Drosophila, HeLa cells, or human neurons, (GA)80 recruited (GR)80 into cytoplasmic inclusions, partially decreasing the toxicity of (GR)80 and restoring Notch signaling in Drosophila. Thus, different DPRs have opposing roles in cell loss and we identify the Notch pathway as one of the receptor signaling pathways that might be compromised in C9ORF72 FTD/ALS.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1448-6) contains supplementary material, which is available to authorized users.

BackgroundTDP-43 is an evolutionarily conserved RNA-binding protein implicated in the pathogenesis of frontotemporal dementia (FTD), sporadic and familial amyotrophic lateral sclerosis (ALS), and possibly other neurodegenerative diseases. In diseased neurons, TDP-43 is depleted in the nucleus, suggesting a loss-of-function pathogenic mechanism. However, the normal function of TDP-43 in postmitotic neurons is largely unknown.ResultsHere we demonstrate that overexpression of Drosophila TDP-43 (dTDP-43) in vivo significantly increases dendritic branching of sensory neurons in Drosophila larvae. Loss of dTDP-43 function, either in a genetic null mutant or through RNAi knockdown, decreased dendritic branching. Further genetic analysis demonstrated a cell-autonomous role for dTDP-43 in dendrite formation. Moreover, human TDP-43 (hTDP-43) promoted dendritic branching in Drosophila neurons, and this function was attenuated by mutations associated with ALS.ConclusionThese findings reveal an essential role for TDP-43 in dendritic structural integrity, supporting the notion that loss of normal TDP-43 function in diseased neurons may compromise neuronal connectivity before neuronal cell loss in FTD and ALS.