C9orf72 arginine-rich dipeptide repeat proteins disrupt karyopherin-mediated nuclear import

Abstract

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases, including ALS caused by a C9orf72 hexanucleotide repeat expansion. However, the mechanism(s) remain unclear. Karyopherins, including importin β and its cargo adaptors, have been shown to co-precipitate with the C9orf72 arginine-containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline arginine (PR), and are protective in genetic modifier screens. Here, we show that R-DPRs interact with importin β, disrupt its cargo loading, and inhibit nuclear import of importin β, importin α/β, and transportin cargoes in permeabilized mouse neurons and HeLa cells, in a manner that can be rescued by RNA. Although R-DPRs induce widespread protein aggregation in this in vitro system, transport disruption is not due to nucleocytoplasmic transport protein sequestration, nor blockade of the phenylalanine-glycine (FG)-rich nuclear pore complex. Our results support a model in which R-DPRs interfere with cargo loading on karyopherins.

Multiple pathways of toxicity induced byC9orf72dipeptide repeat aggregates and G4C2RNA in a cellular model

“…Interestingly, other DPRs have also been reported to inhibit additional aspects of nucleocytoplasmic transport. Poly-PR can directly obstruct the central channel of the nuclear pore by binding to the FG domains of nuclear pore proteins (Shi et al, 2017), and R-DPRs can disrupt cargo loading onto karyopherins (Hayes et al, 2020).…”

“…mRNA containing the pathological G4C2 expansion has been shown to form stable Gquadruplexes in the nucleus that retain RNA binding proteins and induce splicing defects (Cooper-Knock et al, 2014;Donnelly et al, 2013;Gitler & Tsuiji, 2016;Haeusler et al, 2014;Sareen et al, 2013;Simon-Sanchez et al, 2012;Xu et al, 2013). R-DPRs have been additionally shown to interact with membrane-free phase separated compartments, such as the nucleolus, causing nucleolar stress and dysfunction of nucleolar quality control, as well as impairment of nucleocytoplasmic trafficking (Frottin et al, 2019;Hayes et al, 2020;Kwon et al, 2014;Lee et al, 2016;Mizielinska et al, 2017;Shi et al, 2017;Tao et al, 2015;Vanneste et al, 2019;White et al, 2019). Additionally, cytoplasmic poly-GA aggregates associate extensively with proteasomes, interfere with proteasome activity (Guo et al, 2018), and have been found to colocalize with p62, ubiquitin and several components of the proteasome machinery in patient brain (Al-Sarraj et al, 2011;Guo et al, 2018;May et al, 2014;.…”

“…Particularly, R-DPRs have also been shown to directly bind and interfere with cargo loading onto importin at the nuclear pore (Hayes et al, 2020). While cytoplasmic poly-GA aggregates had no apparent effect on the localization of the nuclear pore complex ( Figure 1B), we found that cells with cytoplasmic GA65-GFP aggregates also frequently contained aggregate foci of importins α1 (KPNA2), α3 (KPNA4) and β1 (KPNB1), not co-localizing with the poly-GA inclusions (Figure 4-figure supplement 1).…”

Matrin 3 in neuromuscular disease: physiology and pathophysiology

“…Instead, MATR3(Ser85Cys) is markedly more resistant to detergent extraction and preferentially accumulates within the insoluble fraction of transfected cells ( 51 ). Even MATR3(WT) becomes markedly insoluble under conditions of thermal or proteotoxic stress, suggesting that the Ser85Cys mutation may lower the threshold for this behavior, rather than introducing novel properties per se ( 95 , 96 ). Furthermore, in MATR3 variants that are unable to bind RNA and undergo LLPS, the Ser85Cys mutation dramatically affects the internal dynamics of MATR3 within liquid-like droplets.…”

Nup62 is recruited to pathological condensates and promotes TDP-43 insolubility in C9orf72 and sporadic ALS/FTLD.

“…Expression of codon-optimized sequences encoding DPRs cause excitotoxicity in neurons, alters nucleolar and mitochondrial functions, disrupts SG dynamics, causes ribosomal disfunction and are cytotoxic [49][50][51][52][53][54][55][56][57][58] . However, perhaps the most highlighted cellular biology disrupted in C9-ALS/FTLD is the nucleocytoplasmic transport pathway [59][60][61][62][63][64][65][66] .…”