Gut microbiota from sigma-1 receptor knockout mice induces depression-like behaviors and modulates the cAMP/CREB/BDNF signaling pathway

Abstract

Introduction: Depression is a common mental disorder that affects approximately 350 million people worldwide. Much remains unknown about the molecular mechanisms underlying this complex disorder. Sigma-1 receptor (Sig-1R) is expressed at high levels in the central nervous system. Increasing evidence has demonstrated a close association between the Sig-1R and depression. Recently, research has suggested that the gut microbiota may play a crucial role in the development of depression.

Methods: Male Sig-1R knockout (Sig-1R KO) and wild-type (WT) mice were used for this study. All transgenic mice were of a pure C57BL/6J background. Mice received a daily gavage of vancomycin (100 mg/kg), neomycin sulfate (200 mg/kg), metronidazole (200 mg/kg), and ampicillin (200 mg/kg) for one week to deplete gut microbiota. Fecal microbiota transplantation (FMT) was conducted to assess the effects of gut microbiota. Depression-like behaviors was evaluated by tail suspension test (TST), forced swimming test (FST) and sucrose preference test (SPT). Gut microbiota was analyzed by 16s rRNA and hippocampal transcriptome changes were assessed by RNA-seq.

Results: We found that Sig-1R knockout induced depression-like behaviors in mice, including a significant reduction in immobility time and an increase in latency to immobility in the FST and TST, which was reversed upon clearance of gut microbiota with antibiotic treatment. Sig-1R knockout significantly altered the composition of the gut microbiota. At the genus level, the abundance of Alistipes, Alloprevotella, and Lleibacterium decreased significantly. Gut microbiota dysfunction and depression-like phenotypes in Sig-1R knockout mice could be reproduced through FMT experiments. Additionally, hippocampal RNA sequencing identified multiple KEGG pathways that are associated with depression. We also discovered that the cAMP/CREB/BDNF signaling pathway is inhibited in the Sig-1R KO group along with lower expression of neurotrophic factors including CTNF, TGF-α and NGF. Fecal bacteria transplantation from Sig-1R KO mice also inhibited cAMP/CREB/BDNF signaling pathway.

Discussion: In our study, we found that the gut-brain axis may be a potential mechanism through which Sig-1R regulates depression-like behaviors. Our study provides new insights into the mechanisms by which Sig-1R regulates depression and further supports the concept of the gut-brain axis.

Keywords: BDNF; FMT; SIGMAR1; antibiotic; depression-like behaviors; gut microbiota.

Figure 1

Antibiotic treatment reverses Sig-1R knockout-induced depression-like behaviors. (A) The ABX treatment flowchart by Figdraw. (B) Latency (s) to immobility in the TST [For ABX treatment, F(1, 13) = 11.50, p = 0.0048, two-way ANOVA]. (C) Immobility time (s) in the TST [For ABX treatment, F(1, 13) = 6.068, p = 0.0285, two-way ANOVA]. (D) Latency (Sec) to immobility in the FST [For ABX treatment, F(1, 13) = 5.950, p = 0.0298, two-way ANOVA]. (E) Immobility time (s) in the FST [For ABX treatment, F(1, 13) = 13.37, p = 0.0029, two-way ANOVA]. (F) The sucrose preference index (SPI) in the SPT [For ABX treatment, F(1, 10) = 37.09, p = 0.0001, two-way ANOVA]. ^**p < 0.01. ^*p < 0.05. N.S.: not significant. WT, n = 8; KO, n = 8; WT + ABX, n = 6; and KO + ABX, n = 6.

Figure 2

Sig-1R KO gut microbiota induced depression-like behaviors. (A) The FMT treatment flowchart. By Figdraw. (B) Latency (s) to immobility in the TST. F(2, 18) = 6.048, p = 0.0098. (C) Immobility time (s) in the TST. F(2, 18) = 14.20, p = 0.0002. (D) Latency (s) to immobility in the FST. F(2, 15) = 2.884, p = 0.0871. (E) Immobility time (s) in the FST. F(2, 15) = 13.60, p = 0.0004. (F) The sucrose preference index (SPI) in the SPT. F(2, 15) = 0.6173, p = 0.5523. ^**p < 0.05. N.S.: not significant. WT-FWT, n = 7; WT-FKO, n = 7; and KO-FWT, n = 7. (G) The correlation between the gut microbiota and depressive-like behaviors in recipient mice was analyzed using Spearman’s correlation analysis. The red color represents a positive correlation; the blue color represents a negative correlation. Asterisk-marked columns indicated significant correlation. ^*0.01 < p ≤ 0.05, ^**0.001 < p ≤ 0.01, and ^***p ≤ 0.001.

Figure 3

Sig-1R knockout induced dysbiosis of the gut microbiota. (A) ACE index and (B) Simpson index of the gut microbiota. (C) PCoA and (D) hierarchical clustering of 16S rRNA gene sequences. (E) The LEfSe analysis of the gut microbiota from the phylum level down to the genus level. (F) Composition of the gut microbiota at the genus level. (G–I) The relative abundance of Alistipes, Alloprevotella, and Lleibacterium. ^**p<0.01. WT, n = 6; KO, n = 6. (J) The correlation between the gut microbiota and depressive-like behaviors in WT and KO mice was analyzed using Spearman’s correlation analysis. The red color represents a positive correlation; the blue color represents a negative correlation. Asterisk-marked columns indicated significant correlation. ^*0.01 < p ≤ 0.05, ^**0.001 < p ≤ 0.01, and ^***p ≤ 0.001.

Figure 4

FMT alters the gut microbiota in recipient mice. (A) The gut microbiota PCoA analysis of four groups of mice. (B) ACE index and (C) Simpson index of the gut microbiota. (D) PCoA and (E) hierarchical clustering of 16S rRNA gene sequences. (F) The LEfSe analysis of the gut microbiota from the phylum level down to the genus level. (G) Composition of the gut microbiota at the genus level. (H–J) The relative abundance of Alistipes, Alloprevotella, and Lleibacterium. ^**p < 0.01. WT-FWT, n = 6; WT-FKO, n = 6.

Figure 5

cAMP/CREB/BDNF signaling mediated depression-like behaviors in Sig-1R knockout mice. (A) PCA of the RNA-seq data. (B) Volcano plots of the DEGs from RNA-seq, with 241 upregulated and 204 downregulated. Red color intensity signifies upregulation of gene expression, and green signifies downregulation. (C) Mapping of the RNA-seq data to the KEGG pathway (KEGG). The vertical axis represents the pathway names, while the horizontal axis represents the enrichment factor. The size of the point indicates the number of DEGs in the pathway, and the color of the point corresponds to a different Q-value range. (D) PPI network analysis. The darker color indicates a higher core degree. (E) cAMP levels in WT and KO group. (F) Western blotting and statistical analysis of cAMP/CREB/BDNF signaling pathway in WT and KO group. (G) cAMP levels in FMT recipient mice. (H) Western blotting and statistical analysis of cAMP/CREB/BDNF signaling pathway in FMT recipient mice. (I,J) RT-qPCR for the expression of neurotrophic factors CTNF, TGF-β, and NGF [CTNF F(3, 12) = 9.073, p = 0.0021; TGF-β F(2, 15) = 2.413, p = 0.1174. F(2, 15) = 2.395, p = 0.1192. One-way ANOVA]. ^**p < 0.05. N.S.: not significant.

Figure 6

Gut microbiota from Sigma-1 receptor knockout mice induces depression-like behaviors and modulates the cAMP/CREB/BDNF signaling pathway. Sig-1R knockout induced increased depression-like behaviors in mice with decreased neurotrophic factors, which may be mediated by gut microbiota. The microbiota from Sig-1R knockout mice was sufficient to cause a depression-like phenotype and a reduction in neurotrophic factors. Gut microbiota may regulate the expressions of neurotrophic factors through the cAMP/CREB/BDNF signaling pathway. By Figdraw.