Sigma-1 receptor counteracts non-cell-autonomous poly-PR-induced astrocytic oxidative stress in C9orf72 ALS

Abstract

C9orf72-associated amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are characterized by the accumulation of toxic dipeptide repeat proteins (DPRs) generated from G₄C₂ hexanucleotide repeat expansions. Among these, the arginine-rich poly-PR (proline-arginine) species is the most neurotoxic, eliciting glial activation and neuroinflammation via non-cell-autonomous mechanisms. Although growing evidence implicates glial cells, particularly astrocytes, in disease progression, the molecular pathways linking neuron-derived poly-PR to astrocyte-mediated oxidative stress remain poorly understood. We demonstrate that exogenous poly-PR induces robust NOX4 expression and hydrogen peroxide (H₂O₂) production in astrocytes through activation of the IKK/IκB/NF-κB p65 signaling pathway. Mechanistically, poly-PR promotes nuclear translocation of p65 and enhances its binding to the NOX4 promoter, thereby amplifying astrocytic oxidative stress. Overexpression of the Sigma-1 receptor (Sigma-1R), an endoplasmic reticulum-resident chaperone, significantly attenuates poly-PR-induced NOX4 transcription and reactive oxygen species (ROS) production by interacting with p65 and blocking its nuclear translocation, independently of upstream p65 phosphorylation. Notably, clemastine, a clinically approved Sigma-1R agonist, suppresses astrocytic NOX4 expression by disrupting p65 binding to the NOX4 promoter. In a mouse model of C9orf72 ALS, Sigma-1R deficiency exacerbates poly-PR-induced neurodegeneration, astrogliosis, and NOX4 upregulation, whereas Sigma-1R sufficiency confers neuroprotection and anti-inflammatory effects. This study identifies Sigma-1R as a critical modulator of non-cell-autonomous poly-PR toxicity and establishes its activation as a potent suppressor of astrocyte-derived oxidative stress. Our findings uncover a previously unrecognized glial mechanism driving C9orf72 ALS pathogenesis and support Sigma-1R activation, via clemastine, as a promising therapeutic strategy to mitigate neuroinflammation and disease progression.

Keywords: Astrocyte; C9orf72 ALS; NOX4; Non-cell-autonomous poly-PR; Sigma-1R.

Graphical abstract

Fig. 1

Exogenous, but not overexpressed, poly-PR induces NOX4 expression and increases H₂O₂ production in astrocytes. (A) RT-qPCR analysis revealed no significant difference in NOX4 mRNA expression between astrocytes expressing EGFP and those expressing EGFP-PR₄₂ at 24 h. (B) Western blot analysis showed no significant change in NOX4 protein levels in astrocytes overexpressing either EGFP or EGFP-PR₄₂. (C) Quantification of NOX4 protein levels from three independent experiments performed on biologically independent replicates, all yielding similar results. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t-test. (D) RT-qPCR analysis demonstrated a time-dependent increase in NOX4 mRNA levels in astrocytes following exogenous poly-PR₂₀ peptide treatment. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001). N = 3 biologically independent experiments with consistent results. (E) Western blot analysis revealed increased NOX4 protein expression in astrocytes treated with exogenous poly-PR₂₀ peptides. (F) Quantification of NOX4 protein levels from three biologically independent experiments, yielding similar results. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗p < 0.05, ∗∗p < 0.01). (G) Schematic diagram illustrating the experimental design for H₂O₂ detection following poly-PR₂₀ treatment. (H, I) Extracellular (H) and intracellular (I) H₂O₂ levels were measured using a luminescence-based H₂O₂ assay (see Methods). Astrocytes were treated with varying concentrations of poly-PR₂₀ for 24 or 48 h prior to measurement. Data are presented as mean ± SEM and analyzed using two-way ANOVA followed by Tukey's multiple comparisons test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). N = 3 biologically independent experiments with consistent results. (J) Schematic representation showing that exogenous poly-PR, secreted from poly-PR-expressing motor neurons or astrocytes, can be internalized by naïve astrocytes, leading to NOX4 upregulation and increased H₂O₂ production.

Fig. 2

NF-κB p65 regulates NOX4 transcription via promoter binding and facilitates poly-PR₂₀-induced NOX4 expression. (A–E) RT-qPCR analysis of NOX4 mRNA levels in astrocytes following overexpression of the transcription factors SOX2 (A), FOS (B), OCT1 (C), SP3 (D), and NF-κB p65 (E). Only p65 significantly enhanced NOX4 mRNA expression. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t-test (∗∗∗p < 0.001). N = 3 biologically independent experiments with consistent results. (F) Schematic diagram illustrating the experimental design for assessing NOX4 mRNA expression, protein levels, and luciferase reporter activity following poly-PR₂₀ treatment and p65 overexpression. (G–I) Co-treatment with poly-PR₂₀ and p65 overexpression further increased NOX4 mRNA expression (G) and protein levels (H, I) compared to poly-PR₂₀ treatment alone. Data are presented as mean ± SEM and analyzed using two-way ANOVA followed by Tukey's multiple comparisons test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). N = 3 biologically independent experiments with consistent results. (J) Schematic representation of predicted NF-κB p65-responsive elements within the NOX4 promoter and design of luciferase reporter constructs (NOX4/FL-pGL3: −710 to +1; NOX4/PI-pGL3: −210 to +1). (K) Luciferase reporter assay showing that p65 enhances transcriptional activity through the NOX4 promoter. Cells were co-transfected with HA-p65 and the indicated NOX4 reporter constructs. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗∗∗p < 0.0001). N = 3 biologically independent experiments with consistent results.

Fig. 3

Exogenous poly-PR₂₀ activates p65 phosphorylation and nuclear translocation in astrocytes via the IKK/IκB pathway. (A) Western blot analysis of phosphorylated p65 (p-p65) and total p65 in astrocytes treated with poly-PR₂₀. p65 phosphorylation was notably increased at 3 h post-treatment. (B) Quantification of the p-p65/p65 ratio from three biologically independent experiments, all yielding consistent results. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗p < 0.05). (C) Immunofluorescence staining showing nuclear translocation of p65 in astrocytes treated with FITC-poly-PR₂₀ for 3 h. (D) Confocal microscopy with Z-stack and line-scan analysis confirmed p65 nuclear localization. Red: p65; Green: FITC-poly-PR₂₀; Blue: DAPI. (E) Semi-quantitative analysis of nuclear versus cytosolic p65 using ImageJ (version 1.53t) from confocal images. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t-test (∗∗∗p < 0.001). Total number of cells analyzed: control group, n = 24; FITC-poly-PR₂₀ group, n = 24. N = 3 biologically independent experiments with consistent results. (F) Western blot analysis showing activation of IKKα/β phosphorylation following poly-PR₂₀ treatment. (G, H) Quantification of phosphorylated IKKα/β relative to total IKKα (G) and IKKβ (H) revealed a significant increase at 30 min post-treatment. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). N = 3 biologically independent experiments with consistent results. (I) Western blot analysis showing increased phosphorylation of IκBα in astrocytes following poly-PR₂₀ treatment. (J) Quantification of the p-IκBα/IκBα ratio showed a significant increase at 2 h. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). N = 3 biologically independent experiments with consistent results.

Fig. 4

Sigma-1R attenuates astrocytic NOX4 expression and H₂O₂ production. (A) Western blot analysis of NOX4 protein levels in astrocytes treated with exogenous poly-PR₂₀ following overexpression of Sigma-1R-GFP or GFP control. (B) Quantification of NOX4 protein levels revealed a significant reduction in NOX4 expression in Sigma-1R-overexpressing astrocytes compared to GFP controls under exogenous poly-PR₂₀ peptide treatment. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001). N = 3 biologically independent experiments with consistent results. (C) RT-qPCR analysis demonstrated decreased NOX4 mRNA expression in Sigma-1R-expressing astrocytes compared to GFP controls. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). N = 3 biologically independent experiments with consistent results. (D) Schematic diagram outlining the experimental design for assessing H₂O₂ production in astrocytes treated with exogenous poly-PR₂₀ and overexpressing Sigma-1R. (E, F) Intracellular (E) and extracellular (F) H₂O₂ levels were quantified using a luminescence-based H₂O₂ detection assay. Astrocytes overexpressing GFP or Sigma-1R-GFP were treated with poly-PR₂₀ for 24 or 48 h prior to measurement. Data are presented as mean ± SEM and analyzed using two-way ANOVA followed by Tukey's multiple comparisons test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). N = 3 biologically independent experiments with consistent results. (G) Schematic summary illustrating that Sigma-1R overexpression suppresses poly-PR-induced NOX4 expression and H₂O₂ production in astrocytes.

Fig. 5

Clemastine, a clinically used Sigma-1R agonist, reduces NOX4 expression and H₂O₂ production in astrocytes. (A) Immunoprecipitation analysis of BiP-Sigma-1R interaction following clemastine treatment. Sigma-1R agonist activity leads to dissociation from BiP, indicative of functional activation. (B) Quantification of BiP associated with Sigma-1R showed a significant reduction at 2.5 μM clemastine. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗p < 0.05, ∗∗∗∗p < 0.0001). N = 3 biologically independent experiments with consistent results. (C) CCK-8 assay assessing astrocyte viability following clemastine treatment. (D) Western blot analysis of NOX4 protein levels in astrocytes treated with exogenous poly-PR₂₀, with or without 2.5 μM clemastine. (E) Quantification of NOX4 protein levels revealed a significant reduction in clemastine-treated astrocytes under poly-PR₂₀ exposure. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). N = 3 biologically independent experiments with consistent results. (F) RT-qPCR analysis showed decreased NOX4 mRNA expression in clemastine-treated astrocytes under poly-PR₂₀ conditions. Data are presented as mean ± SEM and analyzed using one-way ANOVA followed by Tukey's multiple comparisons test (∗∗p < 0.01, ∗∗∗∗p < 0.0001). N = 3 biologically independent experiments with consistent results. (G) Schematic diagram illustrating the experimental timeline for H₂O₂ detection following clemastine and poly-PR₂₀ treatment. (H, I) Intracellular (H) and extracellular (I) H₂O₂ levels were measured using a luminescence-based detection assay. Astrocytes were pretreated with clemastine, followed by poly-PR₂₀ for 24 or 48 h. Data are presented as mean ± SEM and analyzed using two-way ANOVA followed by Tukey's multiple comparisons test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001). N = 3 biologically independent experiments with consistent results.

Fig. 6

Sigma-1R interacts with p65 and reduces its binding to the NOX4 promoter in astrocytes under exogenous poly-PR₂₀ peptide treatment. (A) Western blot analysis of phosphorylated p65 (p-p65) levels in astrocytes overexpressing Sigma-1R-GFP or GFP control, followed by exogenous poly-PR₂₀ peptide treatment. Astrocytes were transfected for 24 h and treated with exogenous poly-PR₂₀ peptide for 3 h. (B) Quantification of the p-p65/p65 protein ratio from three biologically independent experiments, each yielding consistent results. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t-test. (C) Schematic diagram illustrating the experimental timeline for the luciferase reporter assay following co-transfection of GFP or Sigma-1R-GFP with HA-p65, followed by exogenous poly-PR₂₀ peptide treatment. (D) Overexpression of Sigma-1R-GFP reduced p65-mediated activation of the NOX4 promoter. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t-test (∗p < 0.05). N = 3 biologically independent experiments with consistent results. (E) Schematic diagram illustrating the experimental timeline for the luciferase reporter assay after clemastine and poly-PR₂₀ treatment. (F) Clemastine treatment also reduced p65-driven NOX4 promoter activation. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t-test (∗p < 0.05). N = 3 biologically independent experiments with consistent results. (G) Immunoprecipitation analysis of p65 and Sigma-1R interaction in astrocytes treated with poly-PR₂₀ confirmed a physical interaction between Sigma-1R and p65. (H) Immunofluorescence analysis demonstrated that Sigma-1R overexpression in astrocytes attenuated p65 nuclear translocation in response to exogenous poly-PR peptide treatment. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t-test (∗∗∗∗p < 0.0001). Total number of cells analyzed: EGFP + poly-PR₂₀ group, n = 18; Sigma-1R-EGFP + poly-PR₂₀ group, n = 13. N = 3 biologically independent experiments with consistent results.

Fig. 7

SigmaR1^−/− mice exhibit exacerbated neurodegeneration and reduced survival compared to SigmaR1^+/+ mice. (A) Schematic diagram illustrating the experimental design for generating the poly-PR-induced C9orf72 ALS mouse model. (B) Genotyping (top) and Western blot analysis (bottom) confirming SigmaR1^+/+ and SigmaR1^−/− backgrounds. (C) Kaplan-Meier survival analysis showing that EGFP-Poly-PR₄₂ expression significantly reduced survival in SigmaR1^−/− mice (n = 10) compared to SigmaR1^+/+ mice (n = 10). (D, F) Representative images of whole-body (D) and brain size (F) in EGFP and EGFP-Poly-PR₄₂ mice. EGFP-Poly-PR₄₂-expressing mice exhibited visibly smaller body and brain sizes in both genotypes. (E) Quantification of body weight in SigmaR1^+/+ mice (EGFP: n = 11; EGFP-PR₄₂: n = 12) and SigmaR1^−/− mice (EGFP: n = 4; EGFP-PR₄₂: n = 8). (G) Quantification of brain length in EGFP or EGFP-Poly-PR₄₂-injected SigmaR1^+/+ mice (EGFP: n = 8; EGFP-PR₄₂: n = 5) and SigmaR1^−/− mice (EGFP: n = 4; EGFP-PR₄₂: n = 4). Data are presented as mean ± SEM and analyzed using two-way ANOVA followed by Šídák's multiple comparisons test (∗∗∗∗p < 0.0001).

Fig. 8

Neuronal poly-PR expression induces greater astrocyte activation and NOX4 upregulation in SigmaR1^−/− mice compared to SigmaR1^+/+ mice. (A) Immunofluorescence staining showing increased astrocyte activation, as indicated by GFAP-positive cells, in SigmaR1^−/− mice compared to SigmaR1^+/+ mice following EGFP-PR₄₂ expression. All confocal images were acquired under identical settings, and uniform brightness/contrast adjustments were applied. (B) Quantification of GFAP-positive astrocytes in EGFP-PR₄₂-injected SigmaR1^−/− mice (n = 4 per group) revealed a significant increase compared to SigmaR1^+/+ mice. Cell numbers were quantified using the Cell Detection function in QuPath software. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t-test (∗∗p < 0.01). (C) Immunofluorescence analysis showing intensity of NOX4 with GFAP-positive astrocytes. The number of colocalized signals was significantly higher in SigmaR1^−/− mice compared to SigmaR1^+/+ mice. (D) Quantification of NOX4 intensity in EGFP-PR₄₂-injected SigmaR1^−/− mice (n = 4 per group) confirmed a significant increase relative to SigmaR1^+/+ controls. NOX4 intensities were obtained using the Cell Detection function in QuPath software. Data are presented as mean ± SEM and analyzed using an unpaired two-tailed t-test (∗p < 0.05).

Fig. 9

Proposed model of Sigma-1R-mediated protection against exogenous poly-PR-induced oxidative stress in astrocytes. Neuron-derived poly-PR is released and internalized by astrocytes, where it activates the IKK/IκB/NF-κB pathway, driving p65 nuclear translocation, NOX4 transcription, and H₂O₂ production. Sigma-1R overexpression interacts with p65, limiting its nuclear translocation and binding to the NOX4 promoter, thereby suppressing astrocytic oxidative stress and neuroinflammation. This model underscores the non-cell-autonomous neuron-to-astrocyte mechanism contributing to C9orf72 ALS pathogenesis.