Gut dysbiosis induces the development of depression-like behavior through abnormal synapse pruning in microglia-mediated by complement C3

Abstract

Background: Remodeling eubiosis of the gut microenvironment may contribute to preventing the occurrence and development of depression. Mounting experimental evidence has shown that complement C3 signaling is associated with the pathogenesis of depression, and disruption of the gut microbiota may be an underlying cause of complement system activation. However, the mechanism by which complement C3 participates in gut-brain crosstalk in the pathogenesis of depression remains unknown.

Results: In the present study, we found that chronic unpredictable mild stress (CUMS)-induced mice exhibited obvious depression-like behavior as well as cognitive impairment, which was associated with significant gut dysbiosis, especially enrichment of Proteobacteria and elevation of microbiota-derived lipopolysaccharides (LPS). In addition, peripheral and central complement C3 activation and central C3/CR3-mediated aberrant synaptic pruning in microglia have also been observed. Transplantation of gut microbiota from CUMS-induced depression model mice into specific pathogen-free and germ-free mice induced depression-like behavior and concomitant cognitive impairment in the recipient mice, accompanied by increased activation of the complement C3/CR3 pathway in the prefrontal cortex and abnormalities in microglia-mediated synaptic pruning. Conversely, antidepressants and fecal microbiota transplantation from antidepressant-treated donors improved depression-like behaviors and restored gut microbiome disturbances in depressed mice. Concurrently, inhibition of the complement C3/CR3 pathway, amelioration of abnormal microglia-mediated synaptic pruning, and increased expression of the synapsin and postsynaptic density protein 95 were observed. Collectively, our results revealed that gut dysbiosis induces the development of depression-like behaviors through abnormal synapse pruning in microglia-mediated by complement C3, and the inhibition of abnormal synaptic pruning is the key to targeting microbes to treat depression.

Conclusions: Our findings provide novel insights into the involvement of complement C3/CR3 signaling and aberrant synaptic pruning of chemotactic microglia in gut-brain crosstalk in the pathogenesis of depression. Video Abstract.

Keywords: Complement C3; Depression; Fecal microbiota transplantation; Gut microbiota; Microglia; Synaptic pruning.

Fig. 1

Study design for the whole experiment. SPF Specific pathogen-free, PBS Phosphate-buffered saline, XYS Xiaoyaosan, CUMS Chronic unpredicted mild stress, FLX Fluoxetine, ABX Antibiotics, FMT Fecal microbiota transplantation

Fig. 2

XYS, as potential antidepressant, alleviated CUMS-induced depression/anxiety -like behaviors, and cognitive impairment. A After 1 week of acclimation, mice were randomly divided into four groups as follows: Control, CUMS, CUMS + XYS, and CUMS + FLX. B Daily body weight changes throughout the entire duration of the study. C Sucrose preference test (SPT). D Open field test(OFT) (Total distance moved). E OFT (Frequency in center zone). F Tail suspension test (TST). G Elevated plus-maze (EPM). H Novel object recognition test (NORT). I Y-maze. J Movement trajectories of mice in OFT, EPM and NORT. Data represent the mean ± SEM (n = 10 per group). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus the Control group; ^#P < 0.05, ^##P < 0.01 versus the CUMS group

Fig. 3

XYS suppressed CUMS-induced colonic inflammatory response, improved the barrier function. A H&E-stained colon sections (× 200). B Transmission electron microscopy (× 10,000, 1um). C Histological scores of colons. The level of D IL-1β; E TNF-α; F IL-6; G IL-10 in the serum. Data represent the mean ± SEM (n = 6 per group). ^***P < 0.001 versus the Control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus the CUMS group

Fig. 4

Depression-like behaviors are accompanied by dysbiosis in gut homeostasis, and antidepressants restore gut homeostasis. A Alpha diversity shown by Shannon index. B PCoA. C Gut microbiota change at phylum level (barplot). D Gut microbiota change at phylum level (circos). E,F Cladogram (Gut microbiota change at the family level). The relative abundances of G: Bacteroides spp.; H Klebsiella spp.; I Lactobacillus. J Changes in gut microbial metabolites(PLS-DA). K Spearman’s correlation analysis of microbiome and metabolome. Data represent the mean ± SEM (n = 6 per group). ^**P < 0.01, ^***P < 0.001 versus the Control group; ^#P < 0.05, ^###P < 0.001 versus the CUMS group

Fig. 5

Complement C3/CR3 activation and complement-involved microglia-mediated aberrant synaptic pruning are important pathological manifestations of depression. A LPS in serum. B C3 in serum. C The protein expression level of C3 in PFC. D Immunofluorescence of microglia and CR3. E The expression of IBA-1 protein in PFC. F The expression of CR3 protein in PFC. G The protein expression level of SYN and PSD95 in PFC. Data represent the mean ± SEM (n = 6 per group for ELISA; n = 3 per group for IF and WB). ^**P < 0.01, ^***P < 0.001 versus the Control group; ^#P < 0.05, ^###P < 0.001 versus the CUMS group

Fig. 6

Transplantation of dysregulated gut microbiota directly induces development of depression-like behaviors. A After 1 week of acclimation, mice were randomly divided into four groups as follows: Control, Control + FMT(CUMS), CUMS and CUMS + FMT(XYS). B Body weight (SPF mice). C SPT (SPF mice). D OFT (Total distance moved) (SPF mice). E TST (SPF mice). F EPM (SPF mice). G Y-maze (SPF mice). H Movement trajectories of mice in OFT, EPM (SPF mice). I Study design for the GF mice. J OFT (GF mice). K TST (GF mice). L NORT (GF mice). M EPM (GF mice). N Movement trajectories of mice in OFT, EPM (GF mice). Data represent the mean ± SEM (n = 10 per group for SPF mice; n = 6 per group for GF mice). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus the Control group; ^#P < 0.05, ^##P < 0.01 versus the CUMS group

Fig. 7

CUMS-FMT contributed to colonic inflammatory response, intestinal barrier damage; XYS-FMT suppressed CUMS-induced colonic inflammatory response, improved the barrier function. A HE (SPF mice). B Transmission electron microscopy (SPF mice). C IL-1β (SPF mice). D IL-6 (SPF mice). E TNF-α (SPF mice). F IL-10 (SPF mice). G HE (GF mice). H Transmission electron microscopy (GF mice). I IL-1β (GF mice). J IL-6 (GF mice). K TNF-α (GF mice). L IL-10 (GF mice). Data represent the mean ± SEM (n = 6 per group). ^**P < 0.01, ^***P < 0.001 versus the Control group; ^#P < 0.05, ^##P < 0.01 versus the CUMS group

Fig. 8

CUMS-FMT mediated disturbance of the gut microbiota in mice; XYS-FMT regulated the composition of gut microbiota. A Alpha diversity shown by Shannon index. B PCoA (Control versus Control + FMT(CUMS)). C PCoA (CUMS versus CUMS + FMT(XYS)). D Gut microbiota change at phylum level (barplot). E Gut microbiota change at phylum level (circos). F,G Cladogram (Control versus CUMS)). H,I (Control versus Control + FMT(CUMS)). J,K Cladogram (CUMS versus CUMS + FMT(XYS)). L Gut microbiota change at phylum and genus level of GF after FMT. Data represent the mean ± SEM (n = 6 per group). ^**P < 0.01, ^***P < 0.001 versus the Control group; ^#P < 0.05, ^###P < 0.001 versus the CUMS group

Fig. 9

Dysregulated gut microbiota induced complement C3 activation and microglia-mediated aberrant synaptic pruning. A LPS in serum (SPF mice). B C3 in serum (SPF mice). C LPS in serum (GF mice). D C3 in serum (GF mice). E WB. F Immunofluorescence of microglia and CR3. G The expression of C3 protein (SPF mice). F The expression of C3 protein (GF mice). I The expression of IBA-1 protein (SPF mice). J The expression of CR3 protein (SPF mice). K The expression of IBA-1 protein (GF mice). L The expression of CR3 protein (GF mice). M The protein expression level of SYN (SPF mice). N The protein expression level of SYN (GF mice). O The protein expression level of PSD95 (SPF mice). P The protein expression level of PSD95 (GF mice). Data represent the mean ± SEM (n = 6 per group for ELISA; n = 3 per group for IF and WB). ^**P < 0.01, ^***P < 0.001 versus the Control group; ^#P < 0.05, ^###P < 0.001 versus the CUMS group

Fig. 10

Inhibition of abnormal synaptic pruning is the key to targeting microbes to treat depression. Gut microbiota contributes to regulating the gut-brain axis and maintaining health, while its alteration (increase of Proteobacteria and decreased of microbial diversity) due to CUMS is related to depression and its adverse consequences on cognition. XYS supplementation is thought to decrease Proteobacteria and regulated the composition and metabolism of gut microbiota (1), thereby, contribute to gut barrier function and immune homeostasis (2); this attenuates the translocation of components of Gram-negative bacteria (3), which decreases the peripheral inflammatory tone and inhibits activation of C3/CR3 (4) and microglia-mediated abnormal synaptic pruning in the CNS (5). Therefore, the supplement of XYS beneficially impacts on depression, via restoration of gut microbiota and its regulatory role in the gut-brain axis, suggesting that inhibition of abnormal synaptic pruning is the key to targeting microbes to treat depression