IL-6 Enhances the Activation of PI3K-AKT/mTOR-GSK-3β by Upregulating GRPR in Hippocampal Neurons of Autistic Mice

Abstract

Autism spectrum disorder (ASD) is a neurological disorder associated with brain inflammation. The underlying mechanisms could be attributed to the activation of PI3K signaling in the inflamed brain of ASD. Multiple studies highlight the role of GRPR in regulating ASD like abnormal behavior and enhancing the PI3K signaling. However, the molecular mechanism by which GRPR regulates PI3K signaling in neurons of individuals with ASD is still unclear. In this study, we utilized a maternal immune activation model to investigate the effects of GRPR on PI3K signaling in the inflamed brain of ASD mice. We used HT22 cells with and without GRPR to examine the impact of GRP-GRPR on the PI3K-AKT pathway with IL-6 treatment. We analyzed a dataset of hippocampus samples from ASD mice to identify hub genes. Our results demonstrated increased expression of IL-6, GRPR, and PI3K-AKT signaling in the hippocampus of ASD mice. Additionally, we observed increased GRPR expression and PI3K-AKT/mTOR activation in HT22 cells after IL-6 treatment, but decreased expression in HT22 cells with GRPR knockdown. NetworkAnalyst identified GSK-3β as the most crucial gene in the PI3K-AKT/mTOR pathway in the hippocampus of ASD. Furthermore, we found that IL-6 upregulated the expression of GSK-3β in HT22 cells by upregulating GRP-GRPR. Our findings suggest that IL-6 can enhance the activation of PI3K-AKT/mTOR-GSK-3β in hippocampal neurons of ASD mice by upregulating GRPR.

Keywords: Autism; GRPR; Hippocampus; IL-6; PI3K.

Fig. 1

GRPR is increased in DG of the hippocampus of MIA offspring. (A) In the three-chamber social test, social interaction of Poly-ic group showed significantly less than control group (n = 8, *** p < 0.001). (B) In the open field experiment, the time of mice in the Poly-ic group staying in the central area was reduced compared with that in the control group (n = 8, ** p < 0.01). (C) The number of buried beads in the Poly-ic group was higher than that in the control group (n = 8, ** p < 0.01). (D) In the cross elevated test, the mice in the Poly-ic group spent significantly less time in the open arm area than those in the control group, while the time spent in the closed arm area increased (n = 8, ** p < 0.01). Representative images of GRPR in DG (E) and CA1 (F) of the hippocampus and amygdala (G). Scale bar, 50 μm. (H) Count the number of GRPR + neurons in each group of three independent visual fields (100µm 2) (Data are mean ± SEM, n = 6, * p < 0.05)

Fig. 2

The increase of GRPR is related to the activation of PI3K-AKT pathway in ASD mice. Immunostaining shows the expression of GRPR and p-AKT in the control group and the Poly-ic group mice were co-localized (green represents GRPR, red represents p-AKT, blue represents nucleus) in DG (A) and CA1 (B) of the hippocampus of ASD mice. Scale bar = 100 μm. (C) Representative images showed the expressions of GRPR, PIK3R3, AKT, p-AKT, mTOR in hippocampus of control and ASD mice, and (D) quantified by ImageJ. Data are mean ± SEM ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001)

Fig. 3

IL-6 increases GRPR and activates PI3K-AKT/mTOR pathway in vitro. (A) Immunohistochemistry shows that there is a difference in the expression of IL-6 in the coronal section of the brain between the control group mice and the Poly-ic group mice (the scale is 1 mm). (B) The average unit area optical density of IL-6 in the brain of mice in Poly-ic group was significantly higher than that in the control group ( n = 3). (C) Compared with the control group, the expression of IL-6 in the brain tissue of mice in Poly-ic group increased significantly ( n = 3, ** p < 0.01). After stimulated with IL-6 at a concentration of 60 ng/ml for 24 h, GRPR positive neurons of HT22 cells (D) and primary neurons (E) were increased compared with cells without IL-6 treatment (scale 20 μm). (F) Representative images showed the expressions of GRPR, PIK3R3, AKT, p-AKT, mTOR in HT22 cells after treated with IL-6 at indicated concentrations and (G) quantified by ImageJ (* p < 0.05, ** p < 0.01)

Fig. 4

Reducing GRPR in HT22 cells can inhibit PI3K-AKT/mTOR pathway activation. (A) Constructing the GRPR Knockdown (KD) cells strain. (B) Representative images showed blots of different proteins (PIK3R3, AKT, p-AKT, mTOR) in GRPR WT and GRPR KD cells after IL-6 treatment in different concentrations (0ng/ml, 20ng/ml, 40ng/ml) for 24 h. (C–E) Relative expression of PIK3R3, p-AKT and mTOR in GRPR WT and GRPR KD cells after IL-6 stimulation in different concentrations (0ng/ml, 20ng/ml, 40ng/ml) for 24 h. Data are mean ± SEM ( n = 3, * p < 0.05, ** p < 0.01)

Fig. 5

mRNA expression of GRPR was increased and related to PI3K-AKT/mTOR signaling pathway activation in the hippocampus of ASD mice. Expression of GRPR in the dorsal hippocampus (A), GRPR in ventral hippocampus (B) and anterior cingulate cortex (C) of samples from GSE178403. ** p < 0.01. (D) The enrichment network of DEGs identified using network-based analysis, the colors of nodes are positively correlated with the fold change of DEGs in the signaling pathway. (E) ORA-generated heatmaps of core enrichment genes in the PI3K-AKT and mTOR signaling pathway upregulated in ASD mice from the Poly-ic group of orsal hippocampus samples from GSE178403

Fig. 6

Gsk3b was a hub gene in the PI3K-AKT/mTOR signaling pathway activated by IL-6-STAT3-GRPR. Zero-order interaction network of genes involved in neuron projection (A) and dendrite (B) identified by network-based analysis. (C) The gene expression correlation between GRPR and Gsk3b. Pearson correlation coefficients (r) and p values were calculated and shown. (D) qPCR for Gsk3b in the entire hippocampus of MIA male offspring ( n = 5). (E) qPCR for Gsk3b in the HT22 and HT22-GRPR KD with or without IL-6 treatment ( n = 5). (F) After treated with or without Stattic (STAT3 inhibitor, 10 µM, 4 h), HT22 cells were stimulated by IL-6 (40 ng/ml, 24 h) to detect the mRNA expression of GRP and GRPR. (G) After treated with or without Stattic (STAT3 inhibitor, 10 µM, 4 h), HT22 cells were stimulated by recombinant mouse GRP (100 nM, 24 h) to detect the mRNA expression of Gsk3b. (H) Schematic diagram of IL-6 promoting GRP-GRPR and Gsk3b through STAT3. (I) mRNA expression of GRPR and Gsk3b in HT22-WT and HT22-GRPR KO cells after GRP (100 nM, 24 h) treatment. Representative data from three independent experiments were shown (* p < 0.05, ** p < 0.01, *** p < 0.01)