Long-term Administration With Fluoxetine Induces Changes In Microtubule Plasticity Mediated By CRMP2

1、Objective:Depression is a neuropsychiatric disease with a high risk of relapse.We intended to study the changes in microtubule scaffold plasticity induced by long-term antidepressant administration to explore the possible mechanism of depression relapse.2、Methods:PC12 cells were cultured and divided into normal control group（NC）and fluoxetine(Flu,10^-6mol/L)group.CCK8 was used to analyze the changes of cell activity in 3 days.The fluorescence expression of CRMP2 and Tubulin between the two groups on the 1st,2nd and 3rd day was detected by immunofluorescence technique.RT-PCR and Western Blotting were used to detect the m RNA expression and protein content of CRMP2 and Tubulin on the 1day,2day and 3day.The interaction between those two proteins was detected by immunoprecipitation（Co-IP）technique to verify the phenomenon of fluorescence co-localization.Finally,CRMP2inhibitor（SB216763,SB,10μM）and agonist（Wortmannin,WT,5μM）were treated for3 days.The changes of CRMP2 and Tubulin fluorescence expression,m RNA and protein content were analyzed by the above experimental methods.3、Results:1.In NC group,the cell activity showed a downward trend after continuous culture for 3 days.There was significant difference in cell activity between the 2 day（93.5±3.5%）and the 3 day（71.4±4.4%）（P<0.000）.2.The extension of protuberance was obvious in Flu treatment group and NC group on the 1day of continuous treatment,but the neurite junction was significantly inhibited in Flu group on the 3day of treatment.On the 1 and 2 day of SB treatment,the processes of the cells extended slowly and the intercellular connections were sparse,but the cells formed network-like connections on the 3day.However,during the continuous treatment of WT for 3 days,the cell processes grew vigorously and the cell junctions were tightly and orderly.3.Compared with NC group,the m RNA expression of CRMP2 was significantly increased on the 1 day（1.31±0.08,P=0.030）and the 2 day（1.60±0.07,P=0.001）in Flu group,but decreased on the 3 day（0.46±0.04,P=0.001）.The Tubulin of Flu group increased on the 1 day（1.35±0.05,P=0.017）and the 2 day（1.63±0.05,P=0.001）,and also decreased on the 3 day（0.64±0.07,P=0.026）.4.Compared with NC group,the CRMP2 of Flu group increased significantly on the 1 day（0.84±0.26,P=0.004）,but there was no significant difference between the2 day（0.67±0.23,P=0.266）and NC group,and the 3 day（0.23±0.11,P=0.026）protein content was lower.The expression of Tubulin protein was the same as that of CRMP2 protein on the 1day（1.34±0.23,P=0.041）,the 2day（0.73±0.29,P=0.123）,and the 3day（0.21±0.09,P=0.015）.5.The fluorescence results showed that there was fluorescence co-localization of Tubulin and CRMP2.Immunoprecipitation further confirmed the interaction between CRMP2 and Tubulin proteins.4、Conclusion:1.Long-term treatment with fluoxetine can cause plastic changes of scaffold microtubule structure.It effectively improves the plastic change in the early stage of use,but causes the damage of stent microtubules in the later stage.2.There is protein-protein interaction between CRMP2 and Tubulin,and CRMP2 mediates the changes of microtubule plasticity in cell scaffolds induced by fluoxetine.