Study Of CRMP2 Protein Involved In The Effects Of Early Stress On Adult Stress Resilience And The Neuroplasticity Mechanisms

Part 1 Effect of maternal separation on behavior of adult male and female SD rats and its effect on stress responsiveness after CUMS stress Objective:Early-life social-environmental factors are important for the normal developmental process,different degrees of early-life stress experience can have different impacts on adult behaviors and stress responsiveness,and there may be differences between different genders.The aim of this study was to investigate the effects of different degrees of early maternal separation stress on the behavior of adult male and female rats;and the effects of different degrees of early separation stress on stress responsiveness in male and female rats after CUMS stress.Method:Maternal separation(MS)was executed during postnatal day(PND)4-PND11.The newborn rats were divided into three groups,no separation group(NS),maternal separation 15 min group(MS15)and maternal separation 360 min group(MS360).After weaning at PND21,the rats were housed by gender and group.Rats were weighed at PND56,and a part of the male and female rats were applied for behavioral tasks[MS15(M,n=8;F,n=8);MS360(M,n=8;F,n=8);NS(M,n=8;F,n=8)],including forced swimming test(FST),sucrose preference test(SPT)and open field test(OFT).Another part of the rats were given chronic unpredictable mild stress(CUMS)for 4consecutive weeks from PND56,and the groups included NSNC group(no MS and no CUMS),CUMS group,MS15+CUMS group,MS360+CUMS group,at PND84 body weight and behavioral tasks were measured,[NSNC(M,n=8;F,n=8);CUMS(M,n=8;F,n=8);MS15 + CUMS(M,n=10;F,n=10);MS360 + CUMS(M,n=10;F,n=10)].After the end of the tests,the results were statistically analyzed to compare the behavioral differences between male and female rats under different stress conditions.Results:(1)The comparison of body weight and behavioral performance at PND56At PND56 before CUMS,the body weight of rats in the three group were measured.Results of two-way ANOVA [sex and stress conditions(MS15,MS360,NS)]showed that the sex,but not the exposure of MS affected the body weight of the rats.Two-way ANOVA analysis of SPT performance showed that neither sex nor early-lifestress experience had effect on the sucrose preference index,there was no interaction effect between sex and stress conditions.The comparison of FST immobility time showed that sex had no effect on immobility time,while stress status had an effect on immobility time.The immobility time of MS360 group in male and female rats was significantly higher than that in MS15 group and NS group(P<0.01).There was no statistically significant difference between the NS group and the MS15 group.The comparison of OFT performance,main effect of sex and interactions were not significant(P>0.05),while the main effect of stress status was significant(P<0.05).Compared with the NS group and the MS15 group,the total moving distance,total speed,distance in center,and duration in center of the MS360 group were significantly decreased(P<0.05).There was no significant difference between MS15 group and NS group(P>0.05).(2)The comparison of body weight and behavioral performance after CUMS at PND84At PND 84 after CUMS,results of two-way ANOVA [sex and stress conditions(MS15+CUMS,MS360+CUMS,CUMS,NSNC)] showed that both sex and stress conditions had impact on the body weight(P<0.05)without an interaction between sex and stress conditions(P>0.05).Further analysis showed that male rats had higher body weight compared to female rats,MS360+CUMS group showed lower body weight when compared to NSNC group(P<0.05).The comparison of SPT and FST performance showed that there was neither significant effect of sex nor stress conditions on the performance,and no interaction effect existed.The OFT comparison showed that the interaction between sex and stress status was significant(P<0.05),and the main effects of stress status were significant(P<0.05)in rearing frequencies,total distance and velocity.Comparison of distance in the center and duration in the center,the main effect of stress status was significant(P<0.05),while main effect of sex and interaction were not significant(P>0.05).Further analysis showed that in male rats,MS360+CUMS group had the worst performance in OFT,followed by CUMS group,MS15+CUMS group and NSNC group had the best performance and there was no significant difference between them;in female rats,behavioral performance in CUMS group was the worst,followed by the MS36+CUMS group,MS15+CUMS group and NSNC group performed best and there was no significant difference between the two.Conclusion:The present study supports the notion that early life environment is important to the development of mental health,different degrees of MS experience export differentimpacts and permanently modify the behavior in adulthood.Prolonged MS experience make male rats more vulnerable to adult CUMS,while make female rats more resilient to CUMS than subjects without MS experience.Brief early life MS can increase the resilience to adult CUMS exposure.Part 2 Effects of maternal separation combined with adult CUMS stress on Akt/GSK3?/CRMP2 signaling pathway and cytoskeletal protein in hippocampus of male SD rats Objective:To investigate the effects of different degrees of maternal separation(MS)on the Akt/GSK3?/CRMP2 signaling pathway and cytoskeletal protein in hippocampus of adult male SD rats;and the effects of different degree of MS combined with CUMS stress on the Akt/GSK3?/CRMP2 signaling pathway and cytoskeletal protein in hippocampus of adult male SD rats after CUMS.Method:Co-immunoprecipitation(Co-IP)was used to detect the interaction between CRMP2 and tubulin as well as actin in hippocampus.Immunofluorescence(IF)was used to detect the co-expression of CRMP2 and tubulin as well as actin in hippocampus.q-RT-PCR was used to detect the effects of different MS stress status(NS,MS15,MS360)on the expression of Akt/GSK3?/CRMP2 signaling pathway and cytoskeletal protein m RNA in hippocampus,and the application of WB was to detect the effects of different MS stress status on Akt/GSK3?/CRMP2 signaling pathway protein levels and phosphorylation levels as well as the expression of Tyr-tubulin and Acet-tubulin proteins in rat hippocampus.Rats were sacrificed after adult CUMS stress.Immunohistochemistry(IHC)technique and q-RT-PCR and WB techniques were used to detect the effects of different degrees of MS stress combined with CUMS stress(NSNC,CUMS,MS15+CUMS,MS360+CUMS)on the expression of Akt/GSK3?/CRMP2 signaling pathway and cytoskeletal protein in rat hippocampus.Results:(1)Effects of different degrees of MS status on Akt/GSK3?/CRMP2 signaling pathway and cytoskeletal protein in hippocampusThe results of Co-IP showed that there was an interaction between CRMP2 and tubulin in rat hippocampus,but no interaction with actin was detected.IF showed in CA1,CA3 and DG of hippocampus,CRMP2 was co-localized with tubulin and actin.Before adult CUMS stress,MS360 and MS15 did not alter the m RNA expression levels of Akt,CRMP2,tubulin and actin.The m RNA expression level of GSK3?in MS15 and MS360 group was significantly decreased compared to NS group(P<0.05).MS360 significantly reduced the expression level of P-Akt,but had no effect on the level of Akt;MS15 and MS360 decreased the expression level of GSK3 ?,while only MS360 reduced the level of P-GSK3?.MS360 significantly increased the level of P-CRMP2,but had no significant effect on total CRMP2 protein levels,and had no significant effect on actin levels.MS360 significantly reduced the expression level of Tyr-tubulin,and increased the expression level of Acet-tubulin,while MS15 had no effect on the expression;MS360 and MS15 did not alter the expression levels of tubulin.(2)Effects of different degrees of MS status combined with CUMS on Akt/GSK3?/CRMP2 signaling pathway and cytoskeletal protein in hippocampusThe results of IHC showed that compared with NSNC group and MS15+CUMS group,CRMP2 expression in CUMS group and MS360+CUMS group was down-regulated in CA1,CA3 and DG region,the difference was statistically significant.In addition,comparing the expression levels of P-CRMP2,when compared to the NSNC group and the MS15+CUMS group,the P-CRMP2 levels in the CUMS group and the MS360+CUMS group was up-regulated in the CA1,CA3,and DG region,and the difference was significant(P<0.05).Comparing the results of q-RT-PCR,there was no significant difference in m RNA levels of Akt,tubulin and actin in the four groups(P>0.05).The comparison of m RNA expression of GSK3? showed that compared to the NSNC group,the GSK3 ? levels in the CUMS group and the MS360+CUMS group decreased significantly,while the MS15+CUMS group and the NSNC group had no significant difference.The comparison of CRMP2 m RNA levels showed that compared with the NSNC group,the CUMS group and the MS360+CUMS group had lower CRMP2 m RNA levels than the MS15+CUMS group;there was no significant difference between the MS15+CUMS group and the NSNC group.Comparing the WB results,CUMS significantly reduced the protein expression levels of P-Akt,but had no significant effect on the overall expression of Akt;MS360+CUMS also decreased the expression of P-Akt,while MS15+CUMS had no influence.Both CUMS and MS360+CUMS significantly reduced the levels of P-GSK3?,while MS15+CUMS had no significant effect.Although the difference did not reach the statistical standard,the GSK3?levels of CUMS group and MS360+CUMS group decreased when compared to NSNC and MS15+CUMS group.The CUMS and MS360+CUMS groups significantly increased P-CRMP2 levels;the CRMP2 levels in the CUMS and MS360+CUMSgroups decreased when compared to the NSNC and MS15+CUMS groups(P<0.05).There was no significant difference in actin expression between the four groups,and no significant difference was found in tubulin levels.The comparison of Tyr-tubulin showed that compared with NSNC and MS15+CUMS,the Tyr-tubulin levels of CUMS group and MS360+CUMS group decreased,while the levels of Acet-tubulin protein levels in the two groups increased,the difference reached statistical standard(P<0.05).There was no significant difference between MS15+CUMS and NSNC group(P>0.05).Conclusion:There is an interaction between rat hippocampal CRMP2 and tubulin.Early long-term MS can significantly alter the expression of phosphorylated protein in hippocampal Akt/GSK3?/CRMP2 signaling pathway,increase the expression level of P-CRMP2,and decrease the expression of microtubule dynamic protein,while short-term MS has no significant effect.Early long-term MS combined with CUMS stress and the simple CUMS stress can significantly reduce the expression of CRMP2,affect the expression of phosphorylated protein in hippocampal Akt/GSK3?/CRMP2 signaling pathway,increase the expression level of P-CRMP2,and decrease the microtubule dynamics,while short-term MS combined with CUMS did not affect its expression.It suggests that the mechanism which long-term MS promotes stress vulnerability and short-term MS promotes stress resilience may be related to the regulation of hippocampal Akt/GSK3 ? /CRMP2 signaling pathway and cytoskeletal protein.Part 3 The effect of CRMP2 and P-CRMP2 levels alternation on behavioral performance and cytoskeletal proteins in SD rats Objective:To investigate the effect of hippocampus stereo injection of PI3 K inhibitor LY294002 on the phosphorylation levels of Akt/GSK3?/CRMP2 pathway protein and microtubule dynamics related protein,and the effect of CRMP2 phosphorylation levels alternation on rat behavioral performance.To investigate the effects of hippocampus stereo injection of LV-CRMP2-RNAi interfering virus on the CRMP2 expression and microtubule dynamics-related proteins expression levels in rat hippocampus,and the effects of CRMP2 expression alternation on behavioral performance in rats.Method:Male SD rats aged 6-8 weeks were divided into four groups,LY294002 group,solvent DMSO group,si CRMP2 group and si CON group.The LY294002 group and the DMSO group were firstly placed stereotactic tube on both sides of the hippocampal CA1 area(-3.5mm,±2.3mm,-2.6mm),after rats were rested for 2 weeks,LY294002(5m M,dissolved in DMSO)2ul per side was bilateral injected,2ul DMSO per side in the DMSO group.Behavioral tests were performed 30 min after injection,including SPT,FST and OFT.After the tests,rats were sacrificed.The m RNA expression levels of Akt,GSK3?,CRMP2,actin and tubulin were detected by q-RT-PCR.The Akt/GSK3?/CRMP2 pathway protein expression and phosphorylated protein levels,as well as expression of microtubule dynamics related proteins Tyr-tubulin and Acet-tubulin were detected by WB.In si CRMP2 group,LV-CRMP2-RNAi virus was injected into the hippocampal CA1 region(-3.5mm,±2.3mm,-2.6mm),and the si CON group was injected with negative control virus.After 2 weeks,behavioral tests were performed,including SPT,FST and OFT.The expression of GFP protein in hippocampal CA1 region was detected by IF.The m RNA expression level of CRMP2 was detected by q-RT-PCR.The protein expression levels of CRMP2,Tyr-tubulin and Acet-tubulin were detected by WB.Results:(1)Effects of stereo injection of LY294002 in hippocampus on behavioral performance and Akt/GSK3?/CRMP2 pathway protein in ratsThe injection of LY294002 in hippocampus significantly reduced the distance in center and duration in center in OFT,the difference was statistically significant(P<0.05).Although the difference of rearing frequencies,total distance and velocity did not reach the statistical standard,the LY294002 group still had a significant downward trend.The FST showed that immobility time of the LY294002 group increased,but the difference did not reach the statistical significance.SPT results showed no significant difference between the two groups(P>0.05).The results of q-RT-PCR showed that the m RNA expression levels of Akt,GSK3 ?,tubulin and actin in hippocampus of LY294002 group were not significantly different from those in DMSO group,while the m RNA expression level of CRMP2 was significantly lower than that in DMSO group(P<0.05).WB showed that LY294002 injection resulted in a decrease in the levels of P-Akt and P-GSK3 ?,but had no effect on the overall levels of Akt and GSK3 ?protein.LY294002 decreased the level of CRMP2,increased the expression level ofP-CRMP2,and decreased the level of Tyr-tubulin,and made the level of Acet-tubulin increased,suggesting that microscopic dynamics were impaired.(2)Effects of the interference of CRMP2 expression in hippocampus on behavioral performance in rats and the related mechanismsTwo weeks after hippocampus injection of CRMP2 interference lentivirus,FST showed that the immobility time of si CRMP2 group was significantly higher than that of si CON group(P<0.05).There was no statistical difference between the two groups in the sucrose preference index.The OFT results showed that compared with the si CON group,the rearing frequencies(P<0.01),total distance(P<0.05),and velocity(P<0.05)of the si CRMP2 group were significantly decreased.IF showed significant GFP fluorescence in the CA1 region,suggesting that the injection site is correct.The m RNA level of CRMP2 in hippocampus of si CRMP2 group was significantly decreased(P<0.05).The WB also showed that the expression of CRMP2 protein in si CRMP2 group was decreased,and the expression of Tyr-tubulin was also decreased,while the expression level of Acet-tubulin was increased,which was significantly different from that of si CON group(p<0.05).Conclusion:Alternation of phosphorylation levels of the Akt/GSK3?/CRMP2 pathway protein in rat hippocampus and elevation of P-CRMP2 levels may result in changes in microtubule dynamic-associated protein expression levels and impaired behavioral behavior in rats.Interfering with the expression of CRMP2 in rat hippocampus can also lead to changes in the expression of microtubule dynamic related proteins,and cause depression-like behavior in rats.