Roles Of PKC? In The Phosphorylation And Mitochondrial Translocation Of P66shc In 6-OHDA Induced PD Rats And PC12 Cells

Objective:To investigate the modulation of protein kinase C?(PKC?)on phosphorylation and mitochondrial translocation of adaptor protein p66 shc in 6-OHDA induced Parkinson's disease(PD)in vivo and in vitro models,and to observe the effect of lipoic acid(LA)on this pathway.Methods: PD rat model was established as follws: 40 male SD rats were randomly divided into Sham group(inject 4 ?L of 0.02% ascorbic acid solution in the right striatum)and PD group(inject 20 ?g 6-OHDA solution in the right striatum which soluted in 4 ?L 0.02% ascorbic acid solution).The behavioral tests were determined by Apomorphine(APO)-induced rotation.The tyrosine hydroxylase protein was detected by immunohistochemistry to quantify the numbers of dopaminenergic neuron.The protein levels of PKC?,Pin1,p66 shc and phospho-p66 shc in substantia nigra and the protein level of PKC? and p66 shc in mitochondria was detected by Western Blotting.The co-localization of PKC? and p66 shc in mitochondria were detected by confocal microscope.The cellular model was established by exposure of 6-OHDA to PC12 cells.The experiments were divided into four groups,the Control group without treatment with 6-OHDA,the 6-OHDA group treated with 75 ?M 6-OHDA,the Rottlerin group treated with 75 ?M 6-OHDA and 0.1 ?M rottlerin(PKC? inhibitor),and the LA group treated with 75 ?M 6-OHDA and 0.1 ?M LA.Cell viability was tested by the assay of CCK-8.The protein levels of PKC?,Pin1,p66 shc and phospho-p66 shc in PC12 cells and the protein level of PKC? and p66 shc in mitochondria of PC12 cells were detected by Western Blotting.The co-localization of mitochondrial PKC? and p66 shc was detected by confocal microscopy.The level of reactive oxygen species(ROS)were detected by the ROS assay kit.Mitochondial membrane potential was detected by the mitochondria membrane potential assay kit.The apoptosis of PC12 cells was determined by flow cytometry.Results:The results obtained from animal invesgation showed that the behavior was significantly different from Sham group(P<0.05);The immunhistochemistry stain of Tyrosine hydroxylase(TH)showed significant loss of TH positive neurons in Substantia nigra pars compacta(SNpc)in PD group compared to Sham group,suggesting the decreased of dopaminergic neurons.Western Blotting showed that the protein levels of PKC?,Pin1 and phospho-p66 shc protein in the right substantia nigra of PD group was significantly higher than Sham group(P < 0.05),and the protein levels of mitochondrial PKC? and p66 shc of SNpc in PD group were higher than Sham group(P <0.05).Confocal micoroscope showed the PKC? and p66 shc are colocalized with mitochondria.The results obtained from cultured PC12 cells showed that the cell viability of PC12 cells were significantly dcreased at the concentrations of 75 ?M 6-OHDA and above for 24 h of incubation with a manner of dose dependence.Compared to Control group,the survival rate was significantly declined in 6-OHDA group as,Meanwhile,the declined survival rate in 6-OHDA group can be prevented by Pre-treatment of PKC? inhibitor Rottlerin and LA(P <0.05);The protein levels of PKC?,Pin1 and phospho-p66 shc were significantly increased in 6-OHDA group compared to Control.Meanwhile,pre-treatment of Rottlerin or LA can significantly attenuated the upregulation of those protein above in 6-OHDA group by(P <0.05);The protein levels of mitochondrial PKC? and p66 shc of 6-OHDA group were significant higher than Control,while the Rottlerin and LA can downregulate the protein levels of mitochondrial PKC? and p66 shc compared to 6-OHDA group(P <0.05);The mitochondrial translocation of PKC? and p66 shc occurred in 6-OHDA group detected by confocal microscope,while Rottlerin and LA treatments can block this translocation.significantly increased ROS and apoptosis rate were detected in 6-OHDA group d,while decreased mitochondrial membrane potential was observed in the cells treated by 6-OHDA(P <0.05).Interestingly,theses above changes can be significantly inhibited by Rottlerin or LA treatments.Conclusion: p66 shc was phosphated and translocated to mitochondria together with PKC? in the SNpc of PD rat models and PC12 cells induced by 6-OHDA,which company with oxidative stress injury.Rottlerin and LA could attenuated the toxicity of 6-OHDA.