C9orf72 arginine-rich dipeptide repeat proteins disrupt importin β-mediated nuclear import

Abstract

15Disruption of nucleocytoplasmic transport (NCT), including mislocalization of the importin b 16 cargo, TDP-43, is a hallmark of amyotrophic lateral sclerosis (ALS), including ALS caused by a 17 hexanucleotide repeat expansion in C9orf72. However, the mechanism(s) remain unclear. 18Importin b and its cargo adaptors have been shown to co-precipitate with the C9orf72-arginine-19 containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline 20 arginine (PR), and are protective in genetic modifier screens. Here, we show that R-DPRs 21 interact with importin b, disrupt its cargo loading, and inhibit nuclear import in permeabilized 22 mouse neurons and HeLa cells, in a manner that can be rescued by RNA. Although R-DPRs 23 induce widespread protein aggregation in this in vitro system, transport disruption is not due to 24 NCT protein sequestration, nor blockade of the phenylalanine-glycine (FG)-rich nuclear pore 25 complex. Our results support a model in which R-DPRs interfere with nuclear transport 26 receptors in the vicinity of the nuclear envelope. 27 101 importin β and compete with the IBB. Synthetic GP10, GA10, and PA10 peptides did not affect 102 6Rango FRET even at high concentrations ( Figure 1D). However, we observed a dose-103 dependent increase in FRET with low-nanomolar PR10 and GR10, indicating these DPRs are 104 capable of binding to importin β and displacing the sensor. To further validate these 105 observations, we used GFP nanobody-coated beads to bind Rango and probe for co-106 immunoprecipitation of importin β in the presence of increasing concentrations of GR10 and 107 PR10 ( Figure 1E-F). Again, we observed the dose-dependent displacement of importin β from 108 the sensor at low nanomolar concentrations, confirming that Rango release was responsible for 109 the increases in FRET. 110To test the functional consequence of R-DPR-importin β interactions for nuclear import, 111we performed the permeabilized cell assay (Adam et al., 1990), in which the plasma membrane 112 of cultured cells is selectively permeabilized, leaving the nuclear membrane intact as verified by 113 nuclear exclusion of 70 kD dextran ( Figure 1G). Fluorescent transport cargo is then added, with 114 energy regeneration mix and cell lysate to provide a source of importins and Ran for nuclear 115 import, which is measured by increasing nuclear fluorescence. Traditionally, this method uses 116 digitonin for permeabilization; however, when attempted with primary mouse cortical neurons, 117 we repeatedly found that even minimal concentrations of digitonin opened both the plasma and 118 nuclear membranes. Since the nuclear envelope is devoid of the digitonin target cholesterol 119 (Colbeau et al., 1971;Adam et al., 1990), we reasoned that its rupture in permeabilized 120 neuronal cells was caused by mechanical perturbation upon removal of cytoplasmic proteins. 121Therefore, we developed a new protocol involving hypotonic cell opening in the presence of a 122 high concentration of BSA as a cushion, which faci...

Nuclear Import Receptors Directly Bind to Arginine-Rich Dipeptide Repeat Proteins and Suppress Their Pathological Interactions

“…Importantly, precipitation of importins by R-rich DPRs also happens under physiological conditions (i.e., in the presence of numerous other cellular poly-GR/PR interactors), as seen in precipitation assays with HeLa cytosol. In line with our findings, precipitation of importins by Rrich DPRs from cell lysates has been reported previously (Boeynaems et al, 2017;Hayes et al, 2019). Importantly, however, we now demonstrate that this precipitation might be due to a direct effect of R-rich DPRs on importin solubility that can be reconstituted in vitro and could happen in the absence of aggregationprone RBP cargoes bound to importins.…”

“…Binding of R-rich DPRs to TNPO1 was determined to be of relatively high affinity (k D in the low nanomolar range), suggesting this interaction is likely to happen in cells. Our data are in line with recent work showing direct binding of poly-PR and poly-GR to Impb in vitro using FRET sensors and bead Halo assays (Hayes et al, 2019), but no other NTRs were analyzed in this study. Here, we find that multiple importins have the capacity to directly bind to R-rich DPRs.…”