The Role And The Mechanism Of SAM And DNMT3a In Regulating DNA Methylation Modification In Perioperative Neurocognitive Disorders

Background:Perioperative neurocognitive disorders(PND),characterized by cognitive impairment such as memory,attention,orientation,and even personality changes and decline in social behavior,is one of the most common and awkward complications of the central nervous system in elderly surgical patients.The incidence of PND in elderly patients over 65 years old is as high as 56%.As aging population expands in China,the number of surgery evens for the elderly is increasing.In several major types of surgeries,such as malignant tumors,orthopedics,the proportion of elderly patients over 60 years old accounts for 30.4%-65.9%of whole surgical cases,and the adverse events caused by PND are becoming more and more common.Unfortunately,there is no cure for PND yet.Deeply exploring the mechanism of postoperative neurocognitive impairment in elderly patients is the key to prevent and treat PND.Our previous studies showed that the accumulation of homocysteine(Hcy)in the hippocampus after surgery is a critical factor for PND,but the specific mechanism is still unclear.Hcy is located at the "cross road" of methionine cycle.The accumulation of Hcy could affect the metabolism of methyl donor S-adenosylmethionine(SAM)and methyltransferase inhibition S-adenosylhomocysteine(SAH)and the function of methylation modification.It needs to be explored how methionine cycle changes during perioperative period,and whether methylation is related to postoperative cognitive impairment,and its potential mechanism in PND.On the other hand,SAM,as a methyl donor,can participate in multiple methylation modification functions(DNA,RNA or histone),among which DNA methylation is an epigenetic mechanism catalyzed by DNA methyltransferases(DNMTs).Changes of the methylation modification in the promoter region could affect the expression of target genes.A number of studies have shown that DNA methylation plays an important role in aging,neurodegenerative diseases,learning and memory.However,few studies have systematically investigated the function of gene-wide DNA methylation in aged mice after surgery.It is still unclear how does the DNA methylation modification function change and what is the underlying mechanisms in brain after surgery.Therefore,we selected 18-month-old mice as the research objects and conduct experiments using behavioral,molecular biology,AAV,RRBS,RNA-Seq,electrophysiology,Etc.Clarify changes of methionine cycle and methylation function in PND;Describe changes of DNA methylation profile and transcription profile in PND using a combination of multi-omics methods;and preliminarily clarified the role and molecular mechanism of methyltransferases and methyl donors in the prevention and treatment of PND by regulating DNA methylation.Provide new strategies and new targets for the prevention and treatment of PND.Experiment 1:Changes of methionine cycle and methylation after surgery in hippocampus in aged miceObjective:To explore the temporal changes of methionine cycle and methylation level in hippocampal tissue after anesthesia and surgery in miceMethods:1.To establish PND model,18-month-old C57BL/6 male mice were subjected to laparotomy under 1.4%isoflurane anesthesia,Open field test,Barnes maze and fear conditioning test were used to assess the learning and memory abilities after surgery.2.Enzyme-linked immunosorbent assay(Elisa)was used to observe the changes of methionine cycle(Hcy,SAM and SAH)in the hippocampus at the time of 1,3 and 7 days after surgery.The SAM/SAH ratio was calculated to assess the methylation status.Results:1.Compared with control group,exposure to anesthesia and surgery impaired the learning ability,spatial memory and contextual fear memory in 18month-old mice(P<0.01),but did not impair the motor ability.2.Compared with control group,the content of SAM in hippocampus was significantly decreased at day 1 and day 3 after surgery,while the content of SAH was significantly increased at day 1 and day 3(P<0.001);meanwhile the ratio of SAM/SAH was decreasing at 1 day and 3days after surgery.Conclusion:These experiments clarified that exposure to anesthesia and surgery would disrupt methionine cycle metabolism in the hippocampus,which resulting in decreased methyl donor SAM and elevated methyltransferase inhibitor SAH.The decrease in the SAM/SAH ratio indicates a decrease in the overall methylation status in hippocampus.Experiment 2:Changes and mechanisms of DNA methylation modification in the hippocampus of PND miceObjective:To screen and verify the essential methyltransferases during perioperative period using the transcriptome,explore potential mechanisms of methyltransferases in PND.Methods:1.Full-length transcriptome analysis was used to detect the differentially expressed genes(DEGs)between control and surgery groups,the critical DNA methyltransferases were screened by KEGG and GO analysis.2.qRT-PCR and Western Blot were used to verify the critical DNA methyltransferases.3.AAV were used to overexpression or knockdown the expression of DNMT3a in dCA1,the effects on global DNA methylation level and cognitive function were observed by immunofluorescence,ELISA,Barnes maze and Fear conditioning test.Results:1.Compared with the control group,2645 DEGs were detected in the hippocampus after surgery.GO analysis suggested that total 12 methylation modification functions were changed after surgery.2.A total of 19 DEGs related to DNA methylation were extracted,and qRT-PCR and Western Blot showed that DNMT3a was the most significantly reduced DNA methyltransferase after surgery(p<0.01).The global DNA methylation level(5-mC)was also decreased after surgery.3.Knockdown of the DNMT3a expression in dCA1 could reduce the global DNA methylation level and impair learning and memory functions in aged mice.Conversely,overexpression of DNMT3a in dCA1 could recovery the cognitive impairment and decrease the global DNA methylation level after surgery.Conclusion:The decreased expression of DNMT3a in hippocampus could affect the global DNA methylation level after surgery,and DNMT3a is a critical DNA methyltransferase that causes postoperative cognitive impairment in aged mice.Experiment 3:DNMT3a alters Genome-Wide DNA methylation and transcriptome profile in PNDObjective:To explore the changes of DNA methylation profile and transcription profile after anesthesia and surgery and clarify the potential mechanism of DNMT3 a in PND.Methods:1.RRBS was performed in the hippocampus tissue of the contralateral cerebral hemisphere in the mice of experiment.Changes of DNA methylation profile was investigated,the differentially methylated regions(DMRs)were analyzed by bioinformatic analysis.2.The data of RNA-Seq in the mice of Experiment 2 were coanalyzed to clarity the differentially expressed genes and differentially methylated genes.Meanwhile the biological function changes was analysised by GO and qRTPCR was used to validate the expression of these genes.3.qRT-PCR was used to identify the expression changes triggered by AAV-Dnmt3a overexpression,the results with the RRBS test were co-analyzed to explore the potential mechanism of DNMT3a overexpression in PND.Results:1.The PND resulted in 7316 differentially methylated regions(DMRs)in aged mice,including 3563 hypermethylated regions and 3751 hypomethylated regions,compare to control group.Hypomethylated DMRs were enriched in promoter,exons,3’-UTR and 5’-UTR regions.2.By combined analysis,we found that a total of 323 genes were changed both in methylation modification and expression,among which hypomethylated&upregulated genes were related to endoplasmic reticulum stress and cell apoptosis,while hypermethylated and downregulated genes were related to synaptic plasticity and axonogenesis.3.DNMT3a overexpression could reduce the expression of apoptosis genes(Lrgl,Cebpb,Bok,Agt,Crlf1),and reverse the neuronal apoptosis after surgery.Conclusion:Anesthesia and surgery decreased the expression of DNMT3a and disrupted the homeostasis of DNA methylation modification in hippocampus.Abnormal DNA methylation modification activates the apoptosis and endoplasmic reticulum stress function and inhibits neuronal synaptic plasticity functions.DNMT3a overexpression prevents apoptosis and plays a protective role in PND.Experiment 4:The role and mechanism of SAM in the prevention and treatment of PNDObjective:To explore the potential protective effect and mechanism of SAM in PND.Methods:1.Different doses of SAM(25mg/kg,50mg/kg and 100mg/kg)were injected intraperitoneally before PND model,and the role of SAM in PND and the dose-effect relationship were assessed by open field test,Barnes maze and fear conditioning test.2.qRT-PCR and Western Blot was used to detect the mRNA level and protein expression of glutamatergic synapse NR2B.TBS was used to detect the methylation modification in gene body of Grin2b gene in the control group,surgery group and surgery+SAM group.Patch clamp technique was used to observe the changes of postsynaptic long-term potentiation(LTP)in three groups.3.Lentivirus and inhibitor of NR2B were used to further investigate the mechanism of SAM in PND.Results:1.100 mg/kg administration of SAM(i.p.)before surgery prevented the occurrence of PND(P<0.05).2.Compared with surgery group,supplementation of SAM significantly increased the mRNA level and protein expression of NR2B receptor,and SAM restored the disorder of methylation modification in the gene body region of Grin2b gene(Chr6:136171276-136173094)which were destroyed by anesthesia and surgery.SAM could reverse the decreasing of fEPSP Slope after surgery(p<0.01).3.Intervention of NR2B overexpression lentivirus reversed the cognitive impairment after surgery,while the protective effect of SAM was attenuated after the use of the NR2B receptor antagonist ifenprodil.Conclusion:SAM plays a protective role in PND by regulating the methylation modification function of Grin2b gene and increasing the expression of NR2B after surgery in the hippocampus.