Effects Of Chronic Stress In Adolescence On BDNF Methylation And Depression-like Behavior In The Prefrontal Cortex And Hippocampus Of Mice

1.BackgroundMajor depressive disorder(MDD)is a common mental illness,and adolescent depression is characterized by high recurrence rate and poor functional prognosis.Therefore,it is of great theoretical and practical significance to further explore the pathogenesis and therapeutic targets of adolescent depression.Early life stress(Stress)causes epigenetic changes,which is one of the main pathogenesis of depression.Both animal experiments and clinical studies have found that the occurrence of depression is closely related to specific DNA methylation.Brain-derived neurotrophic factor(BDNF)is a growth factor existing in the central nervous system.BDNF plays a key role in the development and function maintenance of the nervous system,and its dysfunction is involved in the pathogenesis of depression.In humans and rodents,BDNF promoter Ⅳ significantly promotes BDNF activity-dependent transcription,some studies suggest that changes in the methylation level of BDNF promoter Ⅳ are closely related to the occurrence of depression.DNA methyltransferase(DNMTs)works with DNA demethylation-related enzymes to maintain normal methylation levels.DNMTs is closely related to methylation of BDNF promoter Ⅳ and expression of BDNF.5-Aza-2’-deoxycytidine(5-AzaD)is a DNMT inhibitor that directly inhibits DNA methylation and induces rapid and sustained antidepressant action in a variety of depression-like animal models.Hypermethylation of the Ⅳ region of the BDNF promoter can affect the transcription process of BDNF and thus affect the expression of BDNF.It is not clear whether adolescent stress leads to short-term and long-term changes in the methylation of BDNF promoter Ⅳ,and what role does this change play in the pathogenesis of adolescent depression.In this study,chronic unpredictable mild stress(CUMS)was adopted to create an animal model,to investigate whether chronic stress at a specific age affects the behavior of mice and the expression of BDNF promoter Ⅳ,DNMTs,DNA demethylationrelated enzymes,and BDNF in the prefrontal cortex(PFC)and hippocampus(HIP)and the neural development of mice,and whether the above changes can be modulated after the treatment of 5-AzaD,to clarify the role of methylation of BDNF promoter Ⅳ in the occurrence of adolescent depressive disorders.2.Objectives2.1.1 To investigate the short-term and long-term effects of CUMS on depression-like behavior,methylation of BDNF promoter Ⅳ,DNMTs and demethylation related enzymes and BDNF expression in the PFC and HIP and hippocampal neurogenesis of adolescent mice.2.1.2 To discuss the effects of 5-AzaD on depression-like behavior,methylation of BDNF promoter Ⅳ,DNMTs and demethylation-related enzymes,BDNF expression,and neurodevelopment in the PFC and HIP of adulthood mice undergoing adolescent CUMS,in order to clarify the role of BDNF promoter Ⅳ methylation in depression.3.Materials and Methods3.1 Short-term and long-term effects of CUMS on behavior,BDNF promoter Ⅳmethylation in the PFC and HIP,and hippocampal neurogenesis in adolescent mice.3.1.1 Experimental animals and groupsSixty male C57 mice(21 days old)were divided into four groups:adolescent control group(AdoC),adolescent CUMS group(AdoS),adult control group(AduC)and adult CUMS group(AduS).After 7 days of adaptive feeding,mice in the control group received no stimulation,while mice in the stress group received 21 days of chronic stress.After modeling,the mice in the adolescent groups were subjected to behavioral tests and were sacrificed on the day after the tests.The adult mice were subjected to behavioral testsat 70 days,and the mice were sacrificed the day after the behavioral test.3.1.2 Experimental methods(1)Three weeks of CUMS.(2)After modeling,behavioral tests were conducted successively to evaluate depression-like behavior and spatial learning and memory ability of adolescent and adult mice.(3)DNA methylation analysis was used to detect the methylation level of BDNF promoter Ⅳ in the PFC and HIP of adolescent and adult mice.(4)Quantitative Real-time PCR(qPCR)was used to detect the expression of DNMTs and demethylationrelated enzymes in the PFC and HIP of adolescent and adult mice.(5)The expression of BDNF in the PFC and HIP of adolescent and adult mice were detected by Western blotting(WB).(6)Hippocampal neurogenesis was detected by immunofluorescence in adolescent and adult mice.3.2 Effects of adolescent CUMS and 5-AzaD on behavior,BDNF promoter Ⅳmethylation and neural development in the PFC and HIP of adult mice.3.2.1 Experimental animals and groupsFifty-four male C57 mice(21 days old)were divided into three groups:Control group(C),CUMS group(S)and CUMS+5-AzaD group(S+A).After one week of adaptation,mice in the Control group were not given CUMS,mice in the CUMS group were treated with CUMS for 21 days,and mice in the CUMS+5-AzaD group were given CUMS for 21 days and 5-AzaD treatment.3.2.2 Experimental methods(1)CUMS molding was carried out with the same method as in 3.1.2.(2)After modeling,behavioral testing and analysis were carried out in the same way as in 3.1.2.(3)DNA methylation analysis was used to detect the methylation level of BDNF promoter Ⅳ in the PFC and HIP of adult mice.(4)qPCR was used to detect the expression of DNMTs and demethylation-related enzymes in the PFC and HIP of adult mice.(5)WB was used to detect the BDNF expression in the PFC and HIP of adult mice.(6)Immunofluorescence and Golgi staining were used to detect neural development in the PFC and HIP of adult mice.4.Results4.1 Short-term and long-term effects of CUMS on behavior,BDNF promoter Ⅳmethylation in the PFC and HIP and hippocampal neurogenesis of adolescent mice.CUMS induced depression-like behavior and impairment of spatial learning and memory ability,increased expression of BDNF promoter Ⅳ methylation and methyltransferase,decreased expression of demethylation-related enzymes and BDNF in the PFC and HIP,and hippocampal neurogenesis disorders of adolescent and adult mice.4.2 Effects of adolescent CUMS and 5-AzaD on behavior,BDNF promoter Ⅳmethylation and neural development in the PFC and HIP of adult mice.5-AzaD improved depression-like behavior and spatial learning and memory impairment,reduced hypermethylation levels of BDNF promoter Ⅳ and elevated methyltransferase levels,and improved BDNF expression and neurodevelopmental disorders in the PFC and HIP of adult mice induced by adolescent CUMS.5.Conclusions5.1 CUMS induced depression-like behavior and the changes of BDNF promoter Ⅳmethylation,DNMTs and demethylation-related enzymes,BDNF expression in the PFC and HIP,and hippocampal neurogenesis in adolescent mice,and these changes persisted into adulthood.5.2 5-AzaD reduces hypermethylation of BDNF promoter Ⅳ in the PFC and HIP by modulating DNMTs levels,and improves depression-like behavior,BDNF expression,and neurodevelopment in adult mice undergoing adolescent CUMS.