Soman induces endoplasmic reticulum stress and apoptosis of cerebral organoids via the GRP78-ATF6-CHOP signaling pathway

Abstract

Soman is an organophosphorus compound that induces neurotoxicity. In addition to its direct toxic effects resulting from acetylcholine accumulation, neurotoxicity may also be exacerbated by inducing endoplasmic reticulum (ER) stress. In light of the current scarcity of appropriate in vitro assessment models, in the present study, we used cerebral organoids derived from human pluripotent stem cells, a new tool for investigating the mechanisms of neurotoxicity, to investigate soman-induced ER stress. The results demonstrated that soman significantly suppressed acetylcholinesterase activity and activated the GRP78-ATF6-CHOP (i.e. glucose-regulated protein 78-activating transcription factor 6-C/EBP homologous protein) ER stress cascade, driving apoptosis in cerebral organoids. Pharmacological inhibition of ER stress by pre-treating cerebral organoids with the ER stress inhibitor 4-phenylbutyric acid prior to soman exposure attenuated apoptotic signaling and downregulated GRP78, ATF6 and CHOP expression. Parallel in vivo validation utilized a rat model with subcutaneous soman exposure, focusing on hippocampal and striatal ER stress markers. Consistent with the in vitro findings, soman-exposed rats exhibited marked ER stress activation in brain regions critical for neurotoxicity. This study establishes ER stress as a key contributor to soman-induced neurotoxicity and highlights cerebral organoids as a physiologically relevant model for organophosphorus compound research. We propose ER stress modulation as a potential therapeutic strategy to mitigate neurotoxic outcomes.

Keywords: ATF6; ER stress; cerebral organoids; organophosphates; soman.

Fig. 1

Characterization of iPSCs and cerebral organoids. (A) Showing the process of generating cerebral organoids from iPSCs. Scale bar = 200 μm. (B) Characterization of iPSCs by immunofluorescence staining of pluripotent stem cell markers SOX2 and OCT4. Scale bar = 200 μm. (C–G) Characterization of iPSCs‐derived cerebral organoids. The mature organoids showed the differentiated cells and organization structures. (C) GALC positive oligodendrocyte and GFAP positive astrocyte. (D) TBR1 positive immature neurons and MAP2 positive mature neurons. (E) β‐III tubulin positive neurons and PAX6 positive neural epithelial progenitor cells. (F) FOXG1 positive marked forebrain. (G) Reelin positive neurons indicated the surficial cortex. CTIP and SATB2 displayed early born (CTIP2) and late born (SATB2) neurons. n = 3. Scale bar = 200 μm.

Fig. 2

Effect of soman on AChE activity and cell apoptosis in cerebral organoids. (A) Schematic diagram of soman exposure. (B) Effects of soman on AChE activity in cerebral organoids. (C) TEM revealed cell apoptosis after soman exposure. Scale bar = 1 μm. (D) TUNEL staining shows that apoptosis cells (red) increased after soman exposure compared to the control group. Scale bar = 200 μm. (E) Quantitative analysis of TUNEL staining. Data are represented as the mean ± SEM and n = 3 for each group. Statistical analysis utilized unpaired t‐test. **P < 0.01 vs. control group.

Fig. 3

Soman activates ER stress in cerebral organoids. (A, B) Immunofluorescence for GRP78 (green) and CHOP (red) in cerebral organoids after soman exposure for 24 h. Scale bar = 200 μm. (C–E) Changes in mRNA expression of ER stress related genes GRP78, ATF6 and CHOP induced by soman in cerebral organoids. (F–J) Images of western blots for GRP78, ATF6 and CHOP. (G–K) Quantitative analysis of data from western blots. Data are represented as the mean ± SEM, n = 3 for each group. Statistical analysis utilized one‐way ANOVA followed by the Tukey's test. *P < 0.05, **P < 0.01 vs. control group.

Fig. 4

4‐PBA protected from cell apoptosis in cerebral organoids by inactivating ER stress. (A) TUNEL stain for soman exposure and 4‐PBA intervention before soman exposure. Scale bar = 200 μm. (B) Quantitative analysis of TUNEL staining. (C–E) Images of western blots for GRP78, ATF6 and CHOP. (F–H) Quantitative analysis of data from western blots. Data are represented as the mean ± SEM, n = 3 for each group. Statistical analysis utilized one‐way ANOVA followed by the Tukey's test. *P < 0.05, **P < 0.01 vs. control group, ^# P < 0.05, ^## P < 0.01 vs. soman group.

Fig. 5

ER stress induced by soman in rats' brains. Changes of ER stress related mRNAs levels in hippocampus (A) and striatum (B) after soman exposure. Data are represented as the mean ± SEM and n = 6 for each group. Statistical analysis utilized one‐way ANOVA followed by the Tukey's test. *P < 0.05, **P < 0.01 vs. control group. Changes of CHOP and GRP78 protein in hippocampus (C) and striatum (E) of rats followed by soman exposure. The densitometric analysis of CHOP and GRP78 in hippocampus (D) and striatum (F). Data are represented as the mean ± SEM and n = 4 for each group. Statistical analysis utilized unpaired t‐test. *P < 0.05, **P < 0.01 vs. control group.

Fig. 6

Schematic model for ATF6/GRP78/CHOP pathway in ER stress. ATF6‐GRP78 were bound on ER. The polymers split upon soman activation, and ATF6 was carried into the Golgi apparatus where it was cleaved. The ATF6 fragment entered the nucleus, where it increased CHOP gene expression and ultimately caused cell death.