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.

Keywords: C9orf72; amyotrophic lateral sclerosis; cell biology; dipeptide repeat proteins; human; importin beta; karyopherins; mouse; neuroscience; nucleocytoplasmic transport.

Figure 1.. R-DPRs bind importin β and inhibit nuclear import.

(A) Schematic of Rango FRET sensor, consisting of the importin β-binding domain (IBB) of importin α1 (KPNA2), flanked by CyPET (donor) and YPet (acceptor). (B–C) Rango spectral profile (B) and FRET ratio (C) demonstrating increase in FRET by adding hydrolysis-deficient Ran-Q69L-GTP to importin β-bound Rango (representative of three experiments). (D) Change in Rango FRET ratio induced by adding DPRs (10-mers) to importin β-bound Rango (representative of five experiments, data in C-D fit to non-linear model with one binding site). (E) GFP-trap co-immunoprecipitation of importin β by Rango in the presence of GR10 and PR10. (F) Quantification of Rango-bound importin β in (E), normalized to Rango and expressed as a fraction of untreated lysate (mean ± SD, three technical replicates, legend as in D). (G) Diagram of permeabilized cell nuclear import assay, which was adapted and validated for primary neurons (Figure 1—figure supplement 1). (H) Longitudinal wide-field images of Rango import in permeabilized mouse primary cortical neurons. Scale bar = 10 µm. (I–J) Nuclear to cytoplasmic (N/C) ratio of Rango import in (H), calculated by automated high content analysis. GR and PR graphs are separated for clarity; the control values are identical. All data are normalized to cells lacking energy/lysate and expressed as percent untreated controls (mean ± SEM of n = 4 biological replicates, 189 ± 125 cells per data point). (K) Steady state N/C ratio of Rango in primary neurons fixed after 2 hr (mean ± SEM of n = 7 biological replicates, 409 ± 202 cells per data point, *p<0.05, **p<0.01, ****p<0.001 vs. untreated cells, one-way ANOVA with Dunnett’s post hoc test). (L) IC50 of R-DPRs for inhibition of nuclear import of designated cargoes, from (K) and Figure 1—figure supplement 2. 95% confidence intervals are shown (n = 3–6 biological replicates/condition, 409 ± 202 cells/ replicate for neurons, 1290 ± 305 cells/replicate for HeLa).>20 denotes conditions for which the IC50 was not reached up to the highest dose tested (20 µM). See source file for raw data and exact p values.

Figure 1—figure supplement 1.. Neuron permeabilized cell assay validation.

(A) Mouse primary cortical neurons were permeabilized with hypotonic buffer + BSA cushion, and incubated for 2 hr with 200 nM Rango and 70 kD Texas Red dextran in the indicated conditions. Scale bar = 5 µm. (B) The automated method for N/C ratio determination calculates the mean intensity at a defined distance inside and outside the nuclear rim (inset; nuclear boundaries determined by Hoechst signal). Representative raw data are shown for conditions in (A), from a single well of a 96-well plate. (C) N/C ratio of Rango import in neurons treated with 100 µM GP10, GA10, PA10, or importazole (IPZ). All data are normalized to cells lacking energy/lysate and expressed as percent untreated controls (mean ± SEM of n = 4 biological replicates, 278 ± 238 cells per data point). See source file for raw data.

Figure 1—figure supplement 2.. Extended HeLa nuclear import data.

Diagram of cargo import mechanisms and HeLa nuclear import data in the presence of increasing concentrations of DPR 10- and 20-mers, at steady state (2 hr for Rango (A–C), 4 hr for NLS-GFP (D–F), and 2 hr for M9 (G–I)). These data correspond to those summarized in the table in Figure 1L. Mean ± SEM is shown, n ≥ 3 biological replicates for R-DPRs, n ≥ 2 biological replicates for GP, GA, and PA. 1209 ± 305 cells per replicate, *p<0.05, **p<0.01, ***p<0.001 vs. untreated cells, one-way ANOVA with Dunnett’s post-hoc test. See source file for raw data and exact p values.

Figure 2.. R-DPRs bind importin β in the bead halo assay.

(A) Confocal images of AF488-labeled C9orf72 DPRs added to neutravidin beads coated with biotinylated ‘bait’ proteins, in binding buffer or in the presence of 1 mg/ml neuron lysate (at right). FITC-dextran = negative control (-), Rango sensor = positive control (+). Scale bar = 4 µm. B Rim vs. background ratio in binding buffer (see Figure 2—figure supplement 1 for quantification method). (C–D) Rim vs. background ratio for GR10 (C) and PR10 (D) in 1 mg/ml neuron lysate. In B–D, mean ± SEM is shown for n = 20 beads (5 intensity profiles/bead). **p<0.01, ***p<0.001 vs. control beads by two-way ANOVA with Tukey post-hoc test. See source file for raw data and exact p values.

Figure 2—figure supplement 1.. Quantification method for bead halo assay.

Examples of line intensity profiles for control versus PR10 beads (Fiji), with rim vs. background levels indicated.

Figure 2—figure supplement 2.. Free importin β competes for R-DPR binding to importin β-coated beads.

(A) Confocal images of AF488-labeled GR10 and PR10 added to neutravidin beads coated with biotinylated proteins as indicated, in assay buffer or in the presence of increasing concentrations of free (unlabeled) importin β. Scale bar = 4 µm. (B–C) Rim vs. background ratio in binding buffer for GR10 (B) and PR10 (C) ± free importin β. In B-C, mean ± SEM is shown for n = 20 beads (5 intensity profiles/bead). ***p<0.001 (vs. no importin β) by one-way ANOVA with Tukey post-hoc test. See source file for raw data and exact p values.

Figure 3.. PR and GR accelerate passive nuclear influx.

(A) Confocal time-lapse imaging of GFP nuclear influx in permeabilized HeLa cells following ≥ 30 min. incubation with buffer (untreated), 20 µM GR20, 20 µM PR20, or 0.8 mg/ml wheat germ agglutinin (WGA, positive control). Scale bar = 10 µm. (B–C) Nuclear GFP (B) and 40 kD dextran (C) intensity normalized to background fluorescence, expressed vs. time 0 (no influx = 1). GR and PR are separated for clarity; the control values are identical. All experiments included lysate and energy. See Figure 3—figure supplement 1 for validation of assay conditions and non-R-DPR testing, and Figure 3—figure supplement 2 for binding studies with FG-domains which contribute to the NPC selectivity barrier. Data are mean ± SEM for n = 3–6 biological replicates/condition (20–30 cells/replicate). *p<0.05, **p<0.01, ***p<0.001 vs. untreated cells at 5 min by one-way ANOVA with Dunnett’s post hoc test. See source file for raw data and exact p values.

Figure 3—figure supplement 1.. Validation of passive nuclear influx assay.

(A) Confocal images of permeabilized HeLa cells incubated with Texas Red-labeled dextrans of the indicated molecular weight for 15 min. Scale bar = 10 µm. (B) Time lapse imaging of dextran nuclear influx from 0 to 5 min. Nuclear intensity is normalized to time 0 for each cell (1 = no influx). Mean ± SEM is shown for n = 3 biological replicates (20–30 cells/replicate/condition). (C) Time lapse imaging of GFP nuclear influx, with or without lysate/energy, to verify passive transport of this 27 kD, non-NLS-containing protein. A subset of cells were pre-incubated with 0.8 mg/ml WGA as a positive control for impediment to transport. n = 1 (20–30 cells/condition). (D–E) Nuclear GFP (D) and 40 kD dextran (E) intensity for cells preincubated with 100 µM GP10, GA10, or PA10. All experiments included lysate and energy. Mean ± SEM is shown for n = 3 biological replicates (20–30 cells/replicate/condition, ns = no significant difference vs. untreated controls).

Figure 3—figure supplement 2.. R-DPRs show modest binding to FG-domains in the bead halo assay, which can be augmented by importin β.

(A) Confocal images of AF488-labeled DPRs added to glutathione beads coated with yeast FG- and GLFG-domain GST-fusion proteins, in binding buffer with or without added unlabeled importin β. FITC-dextran = negative control (-), full length AF647-importin β = positive control (+). Scale bar = 4 µm. (B) Intensity profiles (rim vs. background) across all beads tested, including the Nup116 F→ A mutant which is used to define the background/non-specific binding level as indicated by the horizontal dashed lines. Correspondence between yeast and human Nups is given in the inset. Mean ± SEM is shown, for n = 20 beads (5 intensity profiles/bead). *p<0.05, **p<0.01, ***p<0.001 vs. Nup116 F→A by two-way ANOVA with Tukey post-hoc test (*denotes GR statistics, # denotes PR statistics, red arrows denote augmentation of binding by importin β (p<0.001)). See source file for raw data and exact p values.

Figure 4.. R-DPR-induced aggregates recruit NCT proteins.

(A) Aggregates formed by adding R-DPRs to HEK cell lysate in transport buffer (before and after 15 min centrifugation). Venn diagram indicates number of proteins identified by mass spectrometry analysis of pellets (n = 2 technical replicates). Enriched NCT-related GO terms are shown, with fold change and p value calculated by the DAVID algorithm. Overall top GO terms are shown in Figure 4—figure supplement 1. (B) List of identified NCT-related proteins, in all 4 samples (black), n = 2 GR10 samples (blue), and n = 2 PR10 samples (red). Asterisk denotes samples seen in n = 2 GR10 samples and only n = 1 PR10 sample. (C) Western blots for indicated NCT and Nup proteins in pellet vs. supernatant fractions. RCC1 is marked with an asterisk, as this protein was not identified in the MS results and serves as the negative control. All samples were loaded by volume, see Figure 4—figure supplement 2 for membrane protein stain and additional Western blots of disordered RNA binding proteins. (D) Quantification of blots in (C). Mean ± SD for two technical replicates is shown (TP = TNPO1, β = importin β, α = importin α, RG = RanGAP1, RC = RCC1, Ran = RanGTPase). See source file for raw data.

Figure 4—figure supplement 1.. Overall top GO terms enriched in R-DPR aggregates.

(A-B) Top molecular function GO terms for GR10 and PR10 aggregates according to p value (shown as -log₁₀) (A) and fold change (B). In B, selected GO categories enriched in PR samples are highlighted in red, and GR in blue.

Figure 4—figure supplement 2.. Western blots for selected low complexity-domain (LCD)-containing proteins in R-DPR supernatant vs. pellet fractions.

(A) Western blot for indicated proteins identified by R-DPR aggregate mass spectrometry in supernatant vs. pellet, loaded by volume. A representative post-transfer Faststain (total protein stain) is shown. (B) Quantification of blots in (A). Mean ± SD is shown for two technical replicates. See source file for raw data.

Figure 5.. R-DPR nuclear import blockade does not require aggregates and is rescued by RNA.

(A) Rango N/C ratio in permeabilized HeLa transport reactions with 100 µM GR10 or PR10 and increasing concentrations of total HEK cell RNA +/- RNAse. See Figure 5—figure supplement 1 for attempts to rescue with 1,6-hexanediol and importin β. (B) Schematic of fractionated Rango transport assays, run with aggregates present or absent (supernatant only), followed by addition of RNA to a subset of reactions. See Figure 5—figure supplement 2 for western blots of fractionated samples ± RNA. (C) Rango N/C ratio from fractionated transport assays. (D) Confocal images of fractionated transport assays run in the presence of AF488-labeled R-DPRs and AF647-labeled importin β. Arrows mark R-DPR collection around the nuclear membrane in conditions where transport was inhibited. Acquisition parameters were kept constant for all images (scale bar = 10 µm). (E) Schematic of (1) lysate vs. (2) nuclei R-DPR preincubation assays. (F) Rango N/C ratio from preincubation assays. (G) Working model: R-DPRs block nuclear import by binding to importin β and preventing the formation of the importin α•importin β•NLS cargo complex in the soluble phase of the transport reaction, which can be alleviated by RNA. See Figure 5—figure supplement 3 for DPR/RNA electrophoretic mobility shift assay. For A,C,F mean ± SEM of n ≥ 3 biological replicates are shown (each data point represents 1462 ± 555 cells). *p<0.05, **p<0.01, ***p<0.001 vs. untreated cells by one-way ANOVA with Dunnett’s post-hoc test. See source file for raw data and exact p values.

Figure 5—figure supplement 1.. 1,6-HD and importin β do not rescue nuclear import in the permeabilized cell assay.

(A-B) 1,6-hexanediol (1,6-HD) (A) and WT importin β (B) cause dose-dependent inhibition of Rango import in HeLa cells at baseline (mean ± SEM for n = 2 replicates is shown). (C) No rescue of mild Rango import inhibition (25 µM PR10) was seen for either intervention (n = 1 for 1,6-HD, and n = 2 replicates for importin β, 1622 ± 271 cells/data point). Note that values in C are not background corrected as some fell below the level observed for cells without energy or lysate added. See source file for raw data.

Figure 5—figure supplement 2.. RNA only minimally attenuates R-DPR aggregate formation.

(A) Sypro Ruby-stained protein gel showing effect of total HEK RNA on R-DPR-mediated aggregate formation. HEK lysate pellets were prepared as for mass spec and Western blots in Figure 4, +/- 20 µg total HEK RNA. Minimal reduction of predominantly low molecular weight species was apparent by the protein stain (bracketed in red). (B–C) Western blots for indicated proteins +/- RNA in pellet vs. supernatant fractions (B), and quantifification of band intensity, expressed as percent untreated control (C). All samples were loaded by volume as in the parent figure. Mean ± SD for two technical replicates is shown.

Figure 5—figure supplement 3.. RNA binds R-DPRs in an electrophoretic mobility shift assay.

Electrophoretic mobility shift assay of AF488-labeled DPRs (10mers), +/- total HEK RNA, imaged by UV transillumination to simultaneously visualize the AF488 and SYBR Gold signals. Note the co-migration of AF488 R-DPRs with RNA, as visualized by AF488. No SYBR Gold was added to these lanes.