Drosophila screen connects nuclear transport genes to DPR pathology in c9ALS/FTD

Abstract

Hexanucleotide repeat expansions in C9orf72 are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) (c9ALS/FTD). Unconventional translation of these repeats produces dipeptide repeat proteins (DPRs) that may cause neurodegeneration. We performed a modifier screen in Drosophila and discovered a critical role for importins and exportins, Ran-GTP cycle regulators, nuclear pore components, and arginine methylases in mediating DPR toxicity. These findings provide evidence for an important role for nucleocytoplasmic transport in the pathogenic mechanism of c9ALS/FTD.

Figure 1. Genes implicated in nuclear transport are potent modifiers of PR toxicity in Drosophila.

(a) Expression of a PR25 construct in the fly eye induces a moderate degenerative eye phenotype compared to the eyes of non-affected PA-expressing flies or severely affected flies expressing two copies of the PR25 transgene. (b) Setup of RNAi screen for PR25 modifiers. (c) Knockdown of four karyopherins (Trn, Fs(2)Ket, Kap-alpha3 and Ranbp11) strongly enhanced PR toxicity. (d) Knockdown of RanGTP cycle regulators Rangap and Rcc1 both enhance eye degeneration. (e) Knockdown of some nuclear pore complex components (Nup50, Nup107 and Nup154) suppressed degeneration others (Nup44A, Nup62, Nup93-1) enhanced the phenotype. (a,e) show males, (c,d) females.

Figure 2. Transportin-1 and arginine methylation are directly implicated in DPR models and C9 patients.

(a) Trn knockdown potently enhanced the PR-induced degenerative eye phenotype. Females are shown. (b) Computational conformational docking predictions predict that PR can fit the transportin-1 binding pocket. Positive arginine side chains of PR (red) interact with the negative side chains (blue) of the binding pocket. (c) Elav is mislocalized to the cytoplasm in PR expressing flies. Mislocalization is exacerbated upon Trn RNAi knockdown. Arrowheads indicate cytoplasmic staining. Scale bar indicates 5 μm. (d) hnRNPA3 is mislocalized in c9FTD cases (arrowheads) but not in disease-negative controls. Picture shows dentate gyrus. Scale bar indicates 10 μm. (e) Art1 knockdown enhances the PR-induced eye phenotype. Males are shown. (f) PR colocalizes with PRMT1 upon cotransfection in HeLa cells, as determined by super resolution microscopy (SIM). (g) GR staining associates with (arrowheads) and partially colocalizes with (arrow) ASYM24 staining in transfected neuroblastoma cells, as determined by SIM. Scale bars indicate 10 μm. (h) Immunostaining with ASYM24 detects methylated pathological aggregates (arrowheads) in dentate gyrus of c9FTD patient samples but not in controls. Scale bar indicates 10 μm.