Study Of CRMP2 DNA Methylation Involved In The Effects Of Depressive-like Behavior And The Neuroplasticity Mechanisms

Part 1 DNA methylation at the CRMP2 promoter region between the hippocampus and prefrontal cortex in a CUMS depression modelObjective:Current evidence supports the idea that CRMP2 may contribute to the etiology of depression.However,the regulatory mechanisms underlying the role of CRMP2 remain unclear.DNA methylation alteration is generally acknowledged to be involved in the development of depression.The aim of this study was to explore the relationship between the expression and DNA methylation of CRMP2 in the hippocampus and prefrontal cortex of a rat depression model.Method:The SD rats were randomly divided into two groups:the control group,the CUMS group.CUMS was used to establish a rat depression model,body weight and behavioral tests(sucrose preference test,open field test and forced swimming test)were used to evaluate the effects of stress.Real-time PCR and Western blotting were used to test CRMP2 mRNA and protein expression,respectively,in the hippocampus and prefrontal cortex of rats.DNA methylation levels of the CRMP2 promoter were analyzed by bisulfite sequencing PCR(BSP).Results:(1)Body weight and behavioral testsBefore the CUMS procedures,the body weight of the rats in each group did not have a significant difference,and there was also no significant difference in sucrose preference rate,immobility time in forced swimming test and total distance traveled,rearing frequency,velocity in open field test.After 4 weeks of CUMS,the CUMS group showed a lower body weight(P<0.05)and sucrose preference rate(P<0.05),lower total distance traveled(P<0.05),rearing frequency(P<0.05),velocity(P<0.05)in open field test;and increased immobility time(P<0.05)in forced swimming test compared with the control group.(2)CRMP2 mRNA and protein expression levelChronic stress significantly decreased the CRMP2 mRNA expression level in both the hippocampus(P<0.05)and prefrontal cortex(P<0.05)in the CUMS group compared with the control group.Compared with the control group,the CRMP2 protein expression level was significantly downregulated in the hippocampus(P<0.05)and prefrontal cortex(P<0.05)of the CUMS group.(3)DNA methylation level in CRMP2 promoter regionThe DNA methylation level at the CRMP2 promoter region in the hippocampus of the CUMS group increased compared with that in the control group(P<0.05).However,no differences in CRMP2 promoter DNA methylation in the prefrontal cortex were observed(P>0.05).Conclusion:(1)These results provide evidence for regional differences in the regulation of DNA methylation in the CRMP2 promoter between the hippocampus and prefrontal cortex during stress;(2)Changes in DNA methylation of the CRMP2 promoter in the hippocampus provide novel insight to aid in the understanding of the etiology and mechanisms underlying depression.Part 2 Effect of CUMS and fluoxetine treatment on DNA methylation of the CRMP2 promoter region and cytoskeletal protein in hippocampus of ratsObjective:DNA methylation is a process catalyzed by DNA methyltransferase enzymes(DNMTs).Chronic stress appears to alter DNA methylation and DNMTs in the emotion related brain regions.DNA methylation in the CRMP2 promoter is a potential regulatory mechanism for stress induced depressive-like behaviors.CRMP2 is known to regulate the microtubule dynamics.The aim of this study was to explore the effect of CUMS and fluoxetine treatment on the expression of CRMP2 and cytoskeletal protein in hippocampus of rats,to elucidate the possible mechanism of CRMP2 DNA methylation in depression and antidepressant treatment.Method:The SD rats were randomly divided into three groups:the control group,the CUMS group and the fluoxetine group.CUMS was used to establish a rat depression model.At the end of the CUMS procedure,normal saline was administered daily to the CUMS group rats for 4 weeks by intraperitoneal injection.Fluoxetine was diluted in normal saline and intraperitoneally administered to the fluoxetine group rats.Body weight and behavioral tests(sucrose preference test,open field test and forced swimming test)were used to evaluate the effects of CUMS and fluoxetine treatment.Immunohistochemistry(IHC)technique was used to detect the level of CRMP2 in rat hippocampus.DNA methylation levels of the CRMP2 promoter were analyzed by bisulfite sequencing PCR(BSP).Western blotting was used to test CRMP2 and pCRMP2 proteins expression and the expression of Try-tubulin and Acet-tubulin proteins in the hippocampus of rats.Co-immunoprecipitation(Co-IP)was used to detect the interaction between CRMP2 and α-tubulin in rat hippocampus.Results:(1)Body weight and behavioral testsBefore the CUMS procedure,no significant difference was observed among the three groups,but the CUMS group and the fluoxetine group were significantly different from the control group following 4 weeks of CUMS.The CUMS group and the fluoxetine group rats showed a lower index of body weight(P<0.05)and sucrose preference(P<0.05),decreased distance traveled(P<0.05),rearing frequency(P<0.05)and velocity(P<0.05)in the open field test and an increased immobility time in the forced swimming test(P<0.05).Compared with the CUMS group,fluoxetine treatment significantly increased the body weight(P<0.05),sucrose preference(P<0.05),and the distance traveled(P<0.05),rearing frequency(P<0.05)and velocity(P<0.05)in the open field test and decreased immobility time in the forced swimming test(P<0.05).(2)Immunohistochemistry(IHC)The results of IHC showed that compared with control group,CRMP2 expression in CUMS group was downregulated in CA1 region(P<0.05),and fluoxetine treatment significantly upregulated the CRMP2 expression in CA1 region(P<0.05).Compared with control group and fluoxetine group,CRMP2 expression in CUMS group was decreased in DG region(P<0.05).(3)BSPThe DNA methylation level at the CRMP2 promoter region in the hippocampus of the CUMS group increased compared with that in the control group,and fluoxetine treatment significantly decreased the DNA methylation level at the CRMP2 promoter region(P<0.05).(4)Western blotCompared with control group,the CRMP2 protein expression was significantly downregulated in the hippocampus of CUMS group,and CUMS significantly increased the expression of pCRMP2 protein.Fluoxetine treatment significantly upregulated the protein expression of CRMP2 in rats from the fluoxetine group,and fluoxetine treatment significantly decreased the expression of pCRMP2 protein(CRMP2 P<0.05;pCRMP2 P<0.05).The DNMT1 and DNMT3a protein levels in the CUMS group was significantly increased compared with that in the control group and the fluoxetine group(DNMT1 P<0.05;DNMT3a P<0.05).CUMS significantly reduced the expression of Tyr-tubulin protein,and increased the expression of Acet-tubulin protein,and treatment with fluoxetine reversed these changes(Try-tubulin P<0.05;Acet-tubulin P<0.05).(5)Co-immunoprecipitation(Co-IP)The results of Co-IP showed that there was an interaction between CRMP2 andα-tubulin in rat hippocampus.Compared with the control group and the fluoxetine group,the interaction between CRMP2 and α-tubulin was significantly decreased in the hippocampus of CUMS group(P<0.05).Conclusion:(1)DNMT1 and DNMT3 regulate the DNA methylation level of CRMP2 gene,and the DNA methylation of CRMP2 gene is associated with the pathogenesis of depression;(2)CUMS can significantly decrease the interaction between CRMP2 andα-tubulin,and derease the microtubule dynamics.The results suggested that the interaction between CRMP2 and α-tubulin may play an important role in the pathogenesis of depression.Part 3 Effect of DNA methylation in hippocampus on depressive-like behavior and cytoskeletal protein in ratsObjective:Dysfunctional activity and expression of the different DNMT isoforms may contribute to depression development.The changes of DNMTs expression may provide a possible mechanism for the changes of DNA methylation of depression candidate genes.CRMP2 mediates the development of depression by regulating microtubule dynamics.The aim of this study was to explore the effect of DNMTs inhibitor 5-aza on depressive-like behavior and cytoskeletal protein of rats.Method:The SD rats were randomly divided into two groups:the CUMS+Nacl group and the CUMS+5-aza group.CUMS was used to establish a rat depression model.After CUMS,normal saline was administered to the hippocampus CA1 of CUMS+Nacl group rats by microinjection.5-aza was diluted in normal saline and administered to the hippocampus CA1 of CUMS+5-aza group rats by microinjection.Behavioral tests(sucrose preference test,open field test and forced swimming test)were used to evaluate the effects of drugs.Global DNA methylation was quantified using 5-mC ELISA.Western blotting was used to test CRMP2 and pCRMP2 proteins expression and the expression of Try-tubulin and Acet-tubulin proteins in the hippocampus of rats.Co-immunoprecipitation(Co-IP)was used to detect the interaction between CRMP2 and α-tubulin in rat hippocampus.Results:(1)behavioral testsCompared with the CUMS+Nacl group,the CUMS+5-aza group exhibited significantly increased the distance traveled(P<0.05),rearing frequency(P<0.05)and velocity(P<0.05)in the open field test and decreased immobility time in the forced swimming test(P<0.05).(2)ELISACompared with the CUMS+Nacl group,the CUMS+5-aza group showed a reduction of 5-mC level in hippocampus CA1 of rats(P<0.05).(3)Western blotCompared with the CUMS+Nacl group,5-aza significantly upregulated the protein expression of CRMP2 and decreased the expression of pCRMP2 protein in hippocampus CA1 of rats(CRMP2 P<0.05;pCRMP2 P<0.05).Compared with the CUMS+Nacl group,5-aza significantly increased the expression of Tyr-tubulin protein,and decreased the expression of Acet-tubulin protein in hippocampus CA1 of rats(Try-tubulin P<0.05;Acet-tubulin P<0.05).(4)Co-immunoprecipitation(Co-IP)The results of Co-IP showed that there was an interaction between CRMP2 andα-tubulin in rat hippocampus CA1.Compared with the the CUMS+Nacl group,the interaction between CRMP2 and α-tubulin was significantly increased in the hippocampus CA1 of CUMS+5-aza group(P<0.05).Conclusion:Hypomethylation by injection of 5-aza into hippocampus CA1 can induce antidepressant-like effects,and increase the expression of CRMP2 and microtubule dynamics.The results suggested that DNA methylation may be involved in regulating the microtubule dynamic-associated protein expression,and mediate the depressive-like behavior.