Stress Granule Assembly Disrupts Nucleocytoplasmic Transport

Abstract

Defects in nucleocytoplasmic transport have been identified as a key pathogenic event in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) mediated by a GGGGCC hexanucleotide repeat expansion in C9ORF72, the most common genetic cause of ALS/FTD. Furthermore, nucleocytoplasmic transport disruption has also been implicated in other neurodegenerative diseases with protein aggregation, suggesting a shared mechanism by which protein stress disrupts nucleocytoplasmic transport. Here, we show that cellular stress disrupts nucleocytoplasmic transport by localizing critical nucleocytoplasmic transport factors into stress granules, RNA/protein complexes that play a crucial role in ALS pathogenesis. Importantly, inhibiting stress granule assembly, such as by knocking down Ataxin-2, suppresses nucleocytoplasmic transport defects as well as neurodegeneration in C9ORF72-mediated ALS/FTD. Our findings identify a link between stress granule assembly and nucleocytoplasmic transport, two fundamental cellular processes implicated in the pathogenesis of C9ORF72-mediated ALS/FTD and other neurodegenerative diseases.

Keywords: ALS; C9ORF72; nucleocytoplasmic transport; stress granule.

Figure 1. Stressors disrupt nucleocytoplasmic transport

(A) HEK293T cells expressing S-tdTomato (red) stained with Ataxin-2 (green) and DAPI (blue). (B) Stressed HEK293T cells expressing S-tdTomato (red) treated with KPT-350 or DMSO and stained with Ataxin-2 (green) and DAPI (blue). (C) HEK293T cells expressing S-GFP stained with Ataxin-2 (red), GFP (green), and DAPI (blue). N: nuclear; W: whole cell. n numbers in graph. ns: not significant *: p<0.05; **: p<0.01; ****: p<0.0001. Data are represented as mean ± SEM.

Figure 2. Nucleocytoplasmic transport factors are localized to stress granules

(A) Untreated (top row), arsenite- (middle row) or sorbitol- (bottom row) treated HEK293T cells stained with Ran (red), Ataxin-2 (green) and DAPI (blue). Arrows indicate co-localization. (B) Arsenite-treated HEK293T cells stained with transport factors (red), TIA-1 (green) and DAPI (blue). Arrows indicate co-localization.

Figure 3. Nucleocytoplasmic transport factors are constituents of stress granules

(A) Subcellular fractionation of HEK293T cells. P₁₀₀₀: pellet from 1,000 g; P₁₈₀₀₀: pellet from 18,000 g; S: supernatant after 18,000 g. (B) Co-IP of nucleocytoplasmic transport factors with G3BP1-GFP from U-2 OS cells expressing G3BP1-GFP. (C) HEK293T cells expressing MBP (control, top) or MBP-tagged M9M (bottom) were stained with Importin β2 (red), TIA-1 (green), DAPI (blue), and MBP (white). Dashed lines separate transfected versus non-transfected cells. White arrowheads indicate co-localization. Yellow arrowheads indicate TIA-1-positive puncta without Importin β2 co-localization. (D) Co-IP of Importin β2 and G3BP1-GFP with chemically synthesized M9M or control peptide. (E) HEK293T cells expressing GFP-tagged wild type (top) or mutant (bottom) Importin β2 (green) stained with TIA-1 (red) and DAPI (blue). Arrowheads indicate co-localization. W: whole cell, n numbers in graph. ****: p<0.0001. Data are represented as mean ± SEM.

Figure 4. Stress granules mediate the nucleocytoplasmic transport defects caused by arsenite

(A) Experimental design. (B and C) HEK293T cells expressing S-tdTomato (red) (B) or S-GFP (green) (C) were treated with arsenite and GSK or ISRIB and stained with Ataxin-2 (green in B and red in C) and DAPI (blue). (D) Control (left two columns) or G3BP1/2 double knockout (G3BP KO) U-2 OS cells (right two columns) expressing S-tdTomato (red) stained with G3BP (green) and (DAPI). N: nuclear; W: whole cell. (E and F) Subcellular fractionation of control and arsenite-treated HEK293T cells with or without GSK or ISRIB pre-treatment (E) or wild-type control or G3BP KO (KO) U-2 OS cells (F). WCL: whole cell lysate; P₁₈₀₀₀: pellet from 18,000 g; S: supernatant after 18,000 g. n numbers in graph. ns: not significant; *: p<0.05; **: p<0.01; ****: p<0.0001. Data are represented as mean ± SEM.

Figure 5. Dipeptide repeat proteins and cytoplasmic TDP-43 cause nucleocytoplasmic transport defects

(A) HEK293T cells co-expressing S-tdTomato (red) with GFP (left column), GFP-tagged 50 repeats of poly-GR (column 2) or poly-PR (column 3), or cytoplasmic TDP-43 (TDP(cyto)) (right column) stained with DAPI (blue). N: nuclear; W: whole cell. (B) HEK293T cells expressing GFP (top row), GFP-tagged (GR)₅₀ (row 2) or (PR)₅₀ (row 3), or TDP(cyto) (bottom row) were stained with Ran (red), Ataxin-2 (green) and DAPI (blue). GFP expression shown on right (white). Arrowheads indicate co-localization. Number of cells measured (n) for each condition indicated in graph. *: p<0.05; ****: p<0.0001. Data are represented as mean ± SEM.

Figure 6. Stress granules contribute to nucleocytoplasmic transport defects caused by poly-GR, poly-PR or cytoplasmic TDP-43

(A) HEK293T cells co-expressing S-tdTomato (red) and GFP-tagged (GR)₅₀, (PR)₅₀, or cytoplasmic TDP-43 (TDP(cyto)) (green) treated with DMSO, GSK or ISRIB and stained with DAPI (blue). Quantification in B. (C) Wild type control or G3BP1/2 double knockout (G3BP KO) U-2 OS cells co-expressing S-tdTomato (red) and GFP-tagged (GR)₅₀, (PR)₅₀, or TDP(cyto) (green) stained with DAPI (blue). Quantification in D. N: nuclear; W: whole cell. n numbers in graph. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. Data are represented as mean ± SEM.

Figure 7. GSK, ISRIB, and Ataxin-2 ASO suppress nucleocytoplasmic transport defects and neurodegeneration in C9-ALS models

(A) C9-ALS iPSNs treated with DMSO, GSK or ISRIB were stained with Ran (red), MAP2 (green), and DAPI (blue). (B) Control or C9-ALS iPSNs treated with scrambled or Ataxin-2 ASOs were stained with Ran (red), MAP2 (green), and DAPI (blue). Bottom right: ASO-treated C9-ALS iPSNs immunoblotted for Ataxin-2 and β-Actin. (C) Fly salivary glands stained with GFP and DAPI. N: nuclear; W: whole cell. (D) Fly eye degeneration. (E) Flight assay. n numbers in the graph. ns: not significant; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. Data are represented as mean ± SEM.