| Background:Major depression disorder(MDD)is a major mental disorder,which is caused by gene-environment interactions.The global incidence of MDDis about 3.1%,and the prevalence of MDD is about 6.87% in China,which has about 90 million patients.Based on the in-depth studies of the pathogenesis of depression,we found that depression is not only a kind of neuropsychiatric disease related to neurotransmitter disorder,but also a complex comorbidity system with multiple pathogenic factors.And,the effects of gut microbiota dysbiosis on complex diseases has become an international focus in recent years.Associated studies focusing on dysbiosis of gut microbiota in the pathophysiologic mechanisms of psychiatry,nerve development,nerve regeneration and neurodegenerative diseases are in the ascendant.Gut microbiota palys an importance role in maintaining the balance of organism.Many studies have confirmed that gut microbiota can infulence the brain function through the microbiota-gut-brain axis.Our previous studies have found that the patients with MDD showed changes in composition of gut microbiota.After transplanting the fecal samples from MDD individuals on germ-free(GF)mice,the gut microbiome remodeling mice exhibit significant depressive-like behaviors.These results demonstrated that dysbiosis of the gut microbiome may have a causal role in the development of depression and provided a new clue for the study of depression.Howerver,the underlying mechanisms mediating gut microbiota effects on brain function and behavior are complex and still remain obscure.Post-translational modifications(PTMs),such as phosphorylation,are crucial regulators of protein functions and signaling and can be used as important instruments to understand the molecular pathways and signaling networks in pathological processes.Phosphorylation is the process of transferring the phosphate group of ATP to the residues of serine,threonine or tyrosine by protein kinases.Phosphorylation,as a kind of molecular switch,can dynamically regulate enzyme activity and protein-protein interactions,and can form a series of cascade reactions of protein phosphorylation to quickly transmit signals.The phosphoproteomic profiling of postmortem dorsolateral prefrontal cortex(DLPFC)tissue from MDD patients was performed previously and identified a total of 90 significant phosphoproteins,and these significant phosphoproteins were mainly involved in synaptic structure and functional remodeling.Therefore,we propose that dysbiosis of the gut microbiome may have a causal role in the development of depression via modulating protein phosphorylation.Objective:To explore whether intestinal microbial disorders induce the occurrence and development of depression through the change of protein phosphorylation modification level,and then further analyze the differential phosphorylation protein to find the key pathogenic pathway and potential intervention targetMethods:1.To construct the mouse gut microbiome remodeling mice model,and the behavioral tests were conducted two weeks after transplanting the fecal samples,followed by samples collection.2.we used global phosphoproteomic analysis of hippocampus tissue by the tandem-mass-tag(TMT)labeling combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS)to identify dysregulated protein phosphorylation-dependent signaling and other biological processes disrupted in GF mice model(GF vs.SPF;n = 8)and gut microbiome remodeling mice model(the ‘depression microbiota’ vs.the ‘healthy microbiota’ recipient mice;n = 6).The fold-change ratio and p-value were calculated for each phosphorylation site.Cutoff values of fold-change above 1.5 and below 0.67 with p-value < 0.05 were considered to be up-regulated and down-regulated,respectively.3.We performed Motif analysis for the significant phosphopeptides,and predicted the upstream kinases.Moreover,we performed gene ontology(GO)annotations,functional pathway and network analysis and protein-protein interactions for the significant phosphoproteins.4.Western Blotting was used to further verify the key phosphoproteins(CaMKII-α,HSPA8,PKC and FAK).5.Integrated analysis of the present phosphoproteomic results,our previous metabolomic data in gut microbiome remodeling mice,the phosphoproteomic profiling of stressed rats and MDD postmortem brains were performed to further identify and characterize the metabolic mechanisms of gut microbial dysbiosis caused depressionResults:1.GF mice exhibited significantly decreased immobility time in the forced swimming test(FST)and increased center distance proportion in the open field test(OFT)relative to SPF mice,indicating reduced depression-like and anxiety-like behaviors,respectively.In contrast,as compared to the ’healthy control’ recipient mice,the ’depression microbiota’ recipient mice displayed significant depression-like and anxiety-like behaviors as evidenced by an increased immobility time in the FST and a decreased center distance proportion in the OFT,respectively.2.As a result,327 phosphosites in 237 proteins were identified as significant in GF vs.SPF comparation,and 478 phosphosites in 334 proteins were identified as significant in ’depression microbiota’ vs.’healthy control’ recipient mice comparation.3.In the GF vs.SPF comparation,four Ser phosphorylated consensus motifs(i.e.,Rxxs,sP,sxE and sxD)were identified.The Rxxs was most conserved sequence that can be targeted by CaMKII,PKA and PKC.In addition,three Ser and one Thr phosphorylated consensus sequences(i.e.,sP,Rxxs,sxxE and tP)were detected in the ’depression microbiota’ vs.’healthy control’ recipient mice comparation.Proline located in the +1 position of Ser sites was the most conserved residues,and this consensus sequences can be targeted by GSK-3,ERK1/2 and CDK5.4.Gut microbiota dysbiosis may influence brain function through glutamatergic neurotransmitter disturbances,Moreover,CAMKII-CREB signaling pathway in neurons,in response to these disturbances,was the primary common perturbed cellular process between the GF vs.SPF comparation and the ’depression microbiota’ vs.’healthy control’ recipient mice comparation.Western blotting results showed that in the hippocampus of ’depression microbiota’ vs.’healthy control’ model,there was no difference in the expression of CaMKII-α,hspa8,PKC and FAK protein.5.According to the data of phosphorylated protein and metabolite in the depression model of "humanized" mice,brain tissue of depression animal model and brain tissue of autopsy patients with depression,the results showed that intestinal microbial disorders may induce related behavior phenotypes through related pathways mediated by lipid metabolism and amino acid metabolism of the host;depression may be related to axon guidance Dysfunction..Conclusions:1.The level of protein phosphorylation in hippocampus of mice with intestinal microbiological disorder changed significantly,and the intestinal microbiological disorder may be induced by the change of protein phosphorylation to induce depression.2.There is glutamatergic neurotransmitter dysfunction in depression like behavior mice induced by intestinal microbiological disorder.Camkii-creb signaling pathway is the most significant common pathway.3.For the first time,it was found that the disorder of intestinal flora would cause the disorder of spliceosome function and ncbp1,a key protein,might be a new specific pathogenic target for the disorder of intestinal microorganisms to affect the central function and behavior.4.Integrating the data of phosphorylated protein and metabolite in the depression model of "humanized" mice,brain tissue of depression animal model and the data of phosphorylated modified proteomics in the autopsy brain tissue of depression patients,it was found that the disorder of lipid metabolism and amino acid metabolism and the function of nerve axon guidance may be the key way to mediate the depression induced by intestinal microorganisms.5.The research provides a new perspective for exploring the pathological mechanism of depression,and provides a possible theoretical basis and potential new target for exploring the treatment of depression based on intestinal microbial intervention.Background: With the establishment of the Human Microbiome Project(HMP),more and more researches have been carried out to get further understanding of how the microbiome impacts human health and disease.The research on intestinal gut microorganisms has become the focus of life science research all over the world,which is of great significance to human health and diseases.Our previous studies have proved that gut microbial dysbiosis can cause depression-like behaviors.Reduced OB volumes have been associated with olfactory dysfunction in patients with major depressive and post-traumatic stress disorders.Moreover,a previous study using GF mice found that microbiota can modulate the physiology of the olfactory epithelium,resulting in a thinner cilia layer and the reduced expression of genes related to the olfactory transduction pathway.Thus,we hypothesized that commensal microbiota dysbiosis may influence the biochemistry of the OB and affect olfaction through host-microbe interactions.Emerging evidence has indicated that microbes can influence the host’s olfactory signaling systems by producing metabolites that function as odorants.To further explore the molecular mechanisms of OB dysfunction that may be influenced by the presence of gut microbiota,we conducted this study.Objective: 1.To determine whether the absence of gut microbiota can affect the olfactory function in mice.2.To explore the molecular underpinnings of OB dysfunction that may be influenced by the presence of commensal microbiota.Methods: We used a buried food pellet test(BFPT)to investigate and compare olfactory functions in adult,male,germ-free(GF)and specific-pathogen-free(SPF)mice,then examined and compared the metabolomic profiles for olfactory bulbs(OBs)isolated from GF and SPF mice to uncover the mechanisms associated with olfactory dysfunction.Results: We found that the absence of commensal microbiota was able to influence olfactory function and the metabolic signatures of OBs,with 38 metabolites presenting significant differences between the two groups.These metabolites were primarily associated with disturbances in glycolysis,the tricarboxylic acid(TCA)cycle,amino acid metabolism,and purine catabolism.Finally,the commensal microbiota regulation of metabolic networks during olfactory dysfunction was identified,based on an integrated analysis of metabolite,protein,and m RNA levels.Conclusions: This study demonstrated that the absence of commensal microbiota may impair olfactory function and disrupt metabolic networks.These findings provide a new entry-point for understanding olfactory-associated disorders and their potential underlying mechanisms. |
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