Pathogenesis Of Post-stroke Depression Based On Transcriptome Sequencing

Post-stroke Depression(PSD)is one of the common complications after stroke,which seriously affects the functional prognosis of stroke patients and is associated with high mortality.Existing studies have shown that the pathogenesis of PSD is mainly related to neurotransmitters,neuroanatomical location,neurotrophic factors,inflammatory reaction,excitatory neurotoxicity mediated by glutamate,and psychosocial factors.Selective serotonin reuptake inhibitors(SSRIs)and selective serotonin norepinephrine reuptake inhibitors(SNRIs)are the main treatments,but these drugs have poor clinical efficacy and side effects.The main reason is that most of the existing studies on PSD are based on mental disorders,no matter in the basic research of the mechanism of occurrence or clinical transformation and drug treatment.PSD is significantly associated with stroke.In order to get out of the dilemma of the current treatment of post-stroke depression,it is necessary to reveal the specific pathogenesis of PSD and find new therapeutic targets.This can also provide new ideas for the prevention and treatment of stroke,which has important theoretical significance and clinical value.It is currently believed that the hippocampus is closely related to human emotion and cognition.The dorsal hippocampus is involved in the regulation of cognitive function,while the ventral hippocampus is closely related to the maintenance of emotion.Studies have found that the hippocampus contains a large number of steroid receptors.After stroke,inflammatory reaction will lead to the release of a large number of glucocorticoids,which will then act on the steroid receptors in the hippocampus,eventually leading to neurogenesis disorder and hippocampal degeneration,resulting in depression.In recent years,with the development and application of various high-throughput omics technologies,they have played an important role in revealing the mechanism of disease occurrence and development.Whole transcriptome sequencing is a deep sequencing technology for transcriptome,which has been widely used in differential gene expression level analysis,gene function analysis and novel gene discovering.At present,a few studies have used gen chip to explore the pathogenesis of post-stroke depression.These studies were conducted on blood samples from patients and lacked mouse brain tissue,particularly in areas associated with post-stroke deprssion.Therefore,in order to explore the differential genes in the hippocampal brain region between PSD and normal mice,we studied the hippocampal tissue of post-stroke depression mice and normal control mice by the whole transcriptome sequencing technology.Then the possible pathogenesis of the found differential genes was studied.On the basis of previous studies,we further used single cell sequencing technology to study the hippocampal tissues of post-stroke depression mice and chronic social defeat stress mice.We identify the differential genes of PSD and CSDS by informatics data analysis.It can provide a new target for the prevention and treatment of post-stroke depression.PartⅠ Differential genes analysis and related mechanism between post-stroke depression mice and normal miceObjective1.Differentially expressed genes in hippocampus of post-stroke depression mice and normal mice was identified by whole transcriptome sequencing.2.Observe the changes of expressions of related autophagy proteins FEZ1,SCOC,ULK1 and NBR1 after overexpression or interference of mi R-129-5p in hippocampal neurons and the effect of mi R-129-5p on hippocampal neurons.To investigate the relationship between mi R-129-5p and autophagy related proteins FEZ1/SCOC/ULK1/NBR1,and observe the behavioral changes of mice with post-stroke depression after overexpression or interference of mi R-129-5pMethods1.We injected ET-1(endothelin-1)into the medial prefrontal cortex of C57 mice using microsyringes.It caused a medial prefrontal cortex infarction.Magnetic resonance diffusion was used to detect infarct lesions in mice 48 hours after operation.One week after operation,behavioral tests were performed to detect whether the mice had depressed behaviors,including elevated cross test,open field test,tail suspension test,forced swimming.2.Differentially expressed genes(DEGs)in the hippocampus of PSD and normal mice were screened by whole transcriptome sequencing,and the Differentially expressed genes were verified by RT-q PCR and Western-blotting.3.Vitro cell experiments were performed to observe the effect of mi R-129-5p on neuronal autophagy:Lentivirus overexpression or interference of mi R-129-5p infected hippocampal neurons.m RFP-GFP-LC3 fusion protein was used to detect the effect of mi R-129-5p on neuronal autophagy.4.The dual luciferase reporter system vector was used to construct the reporter gene vectors of mi R-129-5p binding site and mutant site in the 3 ’untranslated region(3’UTR)of FEZ1,SCOC,ULK1 and NBR1 genes,respectively.This experiment was conducted to observe the regulatory relationship between mi R-129-5p and FEZ1,SCOC,ULK1 and NBR1.5.Lv-mir-129-5p-puro and no-load control lentivirus were injected into the hippocampus of PSD mice by microsyringes.Then,we performed open field test,elevated cross test and tail suspension test to detect the behavioral changes of experimental mice in each group.6.Hippocampal tissues were extracted from the Normal,PSD,PSD + control virus,and PSD + Overexpressing mi R-129-5P virus groups mice.The effect of mi R-129-5p on the expression of FEZ1,SCOC,ULK1 and NBR1 was observed by q PCR,Western blot and immunohistochemistry.Results1.A mouse model of poststroke depression was successfully establishedMagnetic resonance imaging showed high signal shadow in the m PFC area of mice in the post-stroke depression group,which proved that there was infarction in the m PFC area.2.Whole transcriptome sequencing was used to identify genes differentially in hippocampus of post-stroke depression mice and normal mice.In the PSD group,21 mi RNAs were significantly up-regulated and 32 mi RNAs were significantly down-regulated.Among them,we find the mi R-129-5p gen which we’re interested in.The four genes around the mi R-129-5p gene are FEZ1,NBR1,ULK1 and SCOC whichi are closely related to autophagy in the nervous system.q PCR and Western blot results showed that the expression of mi R-129-5p was significantly decreased,while the expression of FEZ1,NBR1,ULK1 and SCOC were significantly increased in the hippocampus of PSD mice.3.Observe the effect of mi R-129-5p on neuronsMi R-129-5p can minimize autophagy in neuronal cells.4.Mi R-129-5p regulated autophagy related proteins FEZ1,SCOC,ULK1 and NBR1 in vitroWe used q PCR,Western blot and immunofluorescence to detect that the overexpression of mi R-129-5p caused a decrease in the expressions of FEZ1,SCOC,ULK1 and NBR1,while interference with mi R-129-5p caused a significant increase in the expressions of FEZ1,SCOC,ULK1 and NBR1.Dual luciferase reporter system test showed that FEZ1,SCOC,ULK1 and NBR1 were the target genes of mi R-129-5p.Immunoprecipitation assay showed that SCOC,ULK1 and NBR1 proteins could directly bind to FEZ1 protein.5.Effect of mi R-129-5p on the behavior of post-stroke depression miceThere was no significant difference between mi R-129-5p +PSD mice and no-load control virus +PSD mice in the behavioral detection results of elevated cross test,open field test and tail suspension test,but the indicators showed a significant upward trend.6.Observe the regulatory relationship between mi R-129-5p and the expression of FEZ1,SCOC,ULK1 and NBR1 in mouse hippocampusThe expression levels of FEZ1,SCOC,ULK1 and NBR1 in hippocampus were significantly decreased after mi R-129-5p overexpression in mice by q PCR,Western blot and immunohistochemistry.Conclusion1.A mouse model of post-stroke depression was successfully established,and the differential expression of micro RNAs and m RNAs was revealed in the PSD model group.A total of 21 micro RNAs were significantly up-regulated and 32 micro RNAs were significantly down-regulated.Mi R-129-5p and autophagy-related genes FEZ1,NBR1,ULK1 and SCOC can be used as targets for further study.2.We found that SCOC/ULK1/NBR1 protein can directly bind to FEZ1 to form protein complex,and all four FEZ1/SCOC/ULK1/NBR1 proteins are the target genes of mi R-129-5p.Overexpression of mi R-129-5p can effectively restore the behavioral of post-stroke depressed mice and reduce the expression of FEZ1/SCOC/ULK1/NBR1 proteins.These findings may provide new ideas for the research and treatment of PSD.PartⅡ Preliminary study on transcriptome differential genes between post-stroke depressed mice and chronic social defeat stress miceObjectiveIn order to find differentially expressed cells and their related genes,single cell sequencing technology was used to analyze the hippocampus of post-stroke depression mice and chronic social defeat stress mice.Methods1.Establish a mouse model of post-stroke depression: The method was the same as before.2.Establish a chronic social defeat stress model.CD1 male mice were used to fight with C57 mice for 5 minutes every day for 10 consecutive days.After the fight,C57 mice were subjected to social interaction experiment to screen out the model mice of social defeat stress.3.Single cell sequencing was performed on the hippocampus of post-stroke depression group and social defeat stress mice.Then,PCA and t-SNE were performed for dimensionality reduction,and cell clusters were defined.KEGG and GO enrichment analysis were performed for the specfic genesResults1.Social interaction: CSDS mice had significantly lower interaction times than controls(p<0.01);Elevated cross test,open field test and tail suspension test were used to detect CSDS mice and PSD mice,and the results showed that both CSDS and PSD mice had depression-like behaviors.2.Thirty cell clusters were obtained according to individual cell expression profiles,Detection of known cell type markers led us to identify 6 major cell types.The DEGs of these6 types of brain cells were compared between PSD and CSDS,and the DEGs were analyzed by GO and KEGG enrichment analysis.The GO analysis showed that: The DEGs were mainly involved in ATP metabolism,potassium and calcium ion transport,dendritic development,learning and memory,response to stress in multicellular organisms,dopamine receptor signaling pathway,adenylate cyclase-dopamine receptor signaling pathway,regulation of ion transmembrane transport,glutamate receptor signaling pathway and other biological processes.KEGG analysis showed that DEGs were mainly involved in cytokine receptor interaction,endocytosis,nicotine addiction,TGFβsignaling pathway,neurotrophin signaling pathway,long-term potentiation,oxidative phosphorylation,melanin production,circadian rhythm,ligand-receptor interaction in neural tissues and other biological processes.3.Among the six brain cell types,inhibitory neurons contained the most DEGs.We found the specific gens are Drd1(dopamine receptor D1)and Drd2(dopamine receptor D2),which were highly expressed in CSDS and low expressed in PSD.4.The inhibitory neurons was subdivided into 15 subclusters(C0-C14),and then each subcluster of PSD and MDD was compared to find the DEGs.The expression of Drd1 in the C8 subgroup was significantly different.GO analysis showed that Drd1 was involved in synaptic vesicle transport and endocytosis,and KEGG analysis showed that Drd1 was involved in calcium signaling pathway.ConclusionSingle cell sequencing results showed that Drd1 was significantly down-regulated in inhibitory neurons of PSD and up-regulated in inhibitory neurons of CSDS.Drd1 was involved in synaptic vesicle transport,endocytosis and calcium signaling pathways.It is speculated that the differences may be related to the better effect of antidepressants in patients with major depressive disorder than in patients with post-stroke depression.It also provides us with new ideas and directions for further research on the mechanism of post-stroke depression and drug treatment.