Poly(GR) impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis

Abstract

The major genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a C9orf72 G₄C₂ repeat expansion^1,2. Proposed mechanisms by which the expansion causes c9FTD/ALS include toxicity from repeat-containing RNA and from dipeptide repeat proteins translated from these transcripts. To investigate the contribution of poly(GR) dipeptide repeat proteins to c9FTD/ALS pathogenesis in a mammalian in vivo model, we generated mice that expressed GFP-(GR)₁₀₀ in the brain. GFP-(GR)₁₀₀ mice developed age-dependent neurodegeneration, brain atrophy, and motor and memory deficits through the accumulation of diffuse, cytoplasmic poly(GR). Poly(GR) co-localized with ribosomal subunits and the translation initiation factor eIF3η in GFP-(GR)₁₀₀ mice and, of importance, in c9FTD/ALS patients. Combined with the differential expression of ribosome-associated genes in GFP-(GR)₁₀₀ mice, these findings demonstrate poly(GR)-mediated ribosomal distress. Indeed, poly(GR) inhibited canonical and non-canonical protein translation in HEK293T cells, and also induced the formation of stress granules and delayed their disassembly. These data suggest that poly(GR) contributes to c9FTD/ALS by impairing protein translation and stress granule dynamics, consequently causing chronic cellular stress and preventing cells from mounting an effective stress response. Decreasing poly(GR) and/or interrupting interactions between poly(GR) and ribosomal and stress granule-associated proteins may thus represent potential therapeutic strategies to restore homeostasis.

Fig. 1 |. GFP-(GR)₁₀₀ mice exhibited neurodegeneration and behavioral deficits.

a, Immunohistochemical analysis with anti-GFP or anti-GR antibodies in the cortex of 6-month-old mice expressing GFP (n = 12) or GFP−(GR)₁₀₀ (n = 9). Scale bars, 20 μm. b, Immunohistochemical (left) and quantitative (right) analyses of poly(GR) in c9FTD/ALS patient frontal cortex (n = 8). Scale bar, 20 μm. c, An immunoassay was used to compare poly(GR) levels in GFP−(GR)₁₀₀ (n = 8), (G₄C₂)66 (n = 5) and (G₄C₂)₁₄₉ (n = 5) mice, and c9FTD/ALS patients (n = 5). d-g, Representative images of NeuN-labeled cells in the cortex (d) and hippocampus (f) of 6-month-old GFP (n = 8) or GFP-(GR)₁₀₀ (n = 9) mice (scale bars, 200 μm), and quantification of NeuN-labeled cells in the cortex (e) and hippocampus (g) of GFP mice at 1.5 (n = 10), 3 (n = 8) and 6 (n = 8) months of age, or GFP−(GR)₁₀₀ mice at 1.5 (n = 10), 3 (n = 9) or 6 (n = 9) months of age. h, Day 1 and day 2 results from a 4-day rotarod test used to determine motor deficits of 3- and 6-month-old mice expressing GFP (n = 15 per group) or GFP−(GR)₁₀₀ (n = 12 per group) by evaluating latency to fall from a rotating rod (see also Supplementary Fig. 6d for day 3 and day 4 results). i, Results from the fear-conditioning test used to determine associative learning and memory of 3- and 6-month-old mice expressing GFP (n = 15 per group) or GFP-(GR)₁₀₀ (n = 12 per group) by evaluating the percentage of time frozen in response to a conditioned (cued) stimulus. Data are presented as mean±s.e.m. In c, ***P= 0.0006, **P = 0.0023 and *P = 0.0462, one-way analysis of variance (ANOVA), Tukey’s post hoc analysis. In e, ****P< 0.0001 and ***P = 0.0008, two-way ANOVA, Tukey’s post hoc analysis. In g, ****P<0.0001, ***P= 0.0004, *P = 0.0165 and ^#P = 0.0343, two-way ANOVA, Tukey’s post hoc analysis. In h, ****P<0.0001, ***P= 0.0001, **P= 0.0020, ^###P = 0.0003 and ^##P= 0.0021, two-way ANOVA, Tukey’s post hoc analysis. In i, **P = 0.0043 and *P = 0.0286, two-way ANOVA, Tukey’s post hoc analysis.

Fig. 2 |. Poly(GR) proteins co-localized with ribosomal proteins in GFP–(GR)₁₀₀ mice and c9FTD/ALS patients.

a, Double-immunofluorescence staining for GFP–(GR)₁₀₀ and ribosomal proteins (S6, L21 or S25) or the translation initiation factor eIF3η in the cortex of 6-month-old GFP–(GR)₁₀₀ mice (n = 5). b, Double-immunofluorescence staining for poly(GR) and S6 in the cortex of 6-month-old (G₄C₂)₁₄₉-expressing mice (n = 3). c, Double-immunofluorescence staining for poly(GR) and S6, L21 or eIF3η in c9FTD/ALS patient frontal cortex (n = 3). d, Double-immunofluorescence staining for poly(GA) and S6 in the cortex of 6-month-old GFP–(GA)₅₀-expressing mice (top) (n = 3) or c9FTD/ALS patients (bottom) (n = 3). e, Double-immunofluorescence staining for GFP–(GR)₁₀₀ and the endoplasmic reticulum marker KDEL (top) or the mitochondrial marker Tom20 (bottom) in the cortex of 6-month-old GFP–(GR)₁₀₀ mice (n = 5). All scale bars, 5 μ m.

Fig. 3 |. Transcriptome analyses revealed altered ribosome pathways in GFP–(GR)₁₀₀ mice.

a, Hierarchical clustering of differentially expressed genes (FDR < 0.01) in brains of 1.5-month-old mice expressing GFP or GFP–(GR)₁₀₀ (n = 4 per group). b, MA plots of up- and downregulated genes (FDR < 0.01) in brains of 1.5-month-old mice expressing GFP–(GR)₁₀₀ compared to GFP controls (n = 4 per group). c, Network diagram of the green module identified as the top module by WGCNA. The primary cluster of protein–protein interactions, highlighted in pink, consisted entirely of ribosomal proteins (n = 4 per group).

Fig. 4 |. Expression of GFP–(GR)₁₀₀ impaired canonical and non-canonical translation.

a,b, Triple-immunofluorescence staining for GFP–(GR)₁₀₀, eIF3η and either S6 or TIA-1 in HEK293T cells expressing cytoplasmically aggregated or diffuse GFP–(GR)₁₀₀ under basal conditions (n = 3 independent experiments). Scale bars, 5 μ m. c, Quantification of the percentage of GFP- or GFP–(GR)₁₀₀-expressing HEK293T cells containing TIA-1-positive stress granules 24 h and 48 h post-transfection (n = 3 independent experiments). d, Immunohistochemical analysis of TIA-1 in the cortex and hippocampus of six-month-old (G₄C₂)₂, (G₄C₂)₆₆ and (G₄C₂)₁₄₉ mice (n = 3 per group). Insets, higher magnification showing examples of cytoplasmic TIA-1 inclusions, which are indicated by the arrows. Scale bars, 20 μ m. e, Double-immunofluorescence staining for poly(GR) and TIA-1 in the cortex of 6-month-old (G₄C₂)₆₆ and (G₄C₂)₁₄₉ mice (n = 3 per group). Scale bars, 10 μ m. f, Double-immunofluorescence staining for GFP and puromycin in HEK293T cells expressing GFP, GFP–(GR)₁₀₀ or GFP–(GA)₁₀₀ to examine the production of newly translated proteins (n = 3 independent experiments). Scale bars, 5 μ m. g, Double-immunofluorescence staining for poly(GR) and puromycin in the cortex of 1.5-month-old mice expressing GFP–(GR)₁₀₀ (n = 3). Scale bars, 20 μ m. The arrows indicate poly(GR)-expressing cells. h, Immunoblot of the indicated proteins to examine the effect of GFP or GFP–(GR)₁₀₀ on (G₄C₂)₆₆ RAN translation of poly(GA) and poly(GP) in HEK293T cells co-expressing (G₄C₂)₆₆ and either GFP or GFP–(GR)₁₀₀ (n = 3 independent experiments). Cropped blots are shown in full in Supplementary Fig. 10a. Data are presented as mean ± s.e.m. In c, ****P < 0.0001; ***P = 0.0007; NS, P = 0.9710; two-way ANOVA; Tukey’s post hoc analysis.