CDK5 Inhibitory Peptide Prevents Loss Of Dopaminergic Neurons And Alleviates Behavioral Changes In Parkinson's Disease Model

BackgroundParkinson's disease(PD)is one of the most affected neurodegenerative diseases in the world.Deregulation of cyelin-dependent kinase 5(CDK5)is believed to play an important role in neurodegenerative diseases including PD.p25 is a cleavage peptide of p35,a physiologic activator of CDK5.p25 combines to CDK5 and leads to the hyperactivity of CDK5,which in turn hyperphosphorylates downstream substrates and leaded to neuroinflammation and apoptosis of neurons.Previously,we have demonstrated that adeno-associated virus serotype-9(AAV9)mediated CDK5 inhibitory peptide(CIP)inhibits the activity of CDK5/p25 complex and alleviates pathologic and behavioral changes in Alzheimer's disease mouse model.In this study,we evaluated whether AAV9-CIP protected dopaminergic neurons in 1-methyl-4-phe-nyl-1,2,3,6-tetrahydropyridine-probenecid(MPTP/p)induced PD mouse model.Purpose1.Establishment of virus vector AAV-GFP-CIP and control vector AAV-GFP?2.To observe the expression level,scope and duration of AAV9 virus vector in vivo.3.To observe whether CIP can alleviate the loss of dopaminergic neurons induced by MPTP/p in chronic PD mouse model and the possible protective mechanism.4.Behavior test was used to analyze whether CIP could improve the behavior index of chronic PD model.Method1.The corresponding virus vector and virus packaging were constructed by plasmid.2.Chronic PD model was constructed with MPTP propanecid regimen.3.The intracerebroventricular injection was performed with stereotactic locator,and the characteristics of intracranial expression after virus injection were observed.4.After grouping experiment,the content of dopaminergic neurons and the changes of protein expression in midbrain were analyzed by immunohistochemistry and Western blot.5.Behavioral examination and analysis of behavioral changes in each group of mice.6.Statistical analysis:all the data of this study were carried out by SPSS20.0 and GraphPad Prism 6.05 statistical analysis software.The comparison between the two groups was carried out by independent sample t-test analysis,and the test level was 0.05(P<0.05).6.Statistical analysis7.All date were analysied by Statistical software SPSS 20.0 analysisand GraphPad Prism 6.05.Comparison between two groups was conducted by Student't test.All the experimental data were expressed by the mean ? standard deviation(X ± SEM).P<0.05 was considered significant.Results1.We have successfully constructed the corresponding virus plasmid vector in the previous experiment.After the cell transfection experiment,we observed the expression of fluorescent labeled protein and Western blot analysis,and confirmed that the GFP-CIP and GFP products were obviously expressed.2.The tyrosine Hydroxylase tyrosine Hydroxylase in the model group was lower than that in the control group after the establishment of chronic PD model,indicating that the model was successful.2.The pathological section of the mesencephalon and the detection of tyrosine Hydroxylase in the model group were lower than those in the control group.3.Immunohistochemistry showed that the virus was widely expressed in the bilateral hippocampus,cortex and midbrain,and could still be detected after the injection on October.4.The results of grouping experiment showed that intraventricular injection of AAV-GFP-CIP one week before administration of MPTP/p could effectively alleviate the loss of dopaminergic neurons in the substantia nigra of the model rats,while the phosphorylated MEF2D in the control group and the treatment group was lower than that in the model group and the negative control group.The results suggest that CIP may play an important role by preventing neuroprotective factor MEF2D from being phosphorylated by CDK5/p25.5.Behavioral examination indicated that the changes of motor function and anxiety behavior in the treatment group were alleviated compared with the model group and the negative control group,suggesting that CIP could alleviate the behavioral changes of the chronic PD model rats.Conclusions1.AAV9 virus can be widely expressed in hippocampal,cortical and midbrain neurons after intracerebroventricular injection,and its expression in vivo can still detect the target protein after October,which is safe,effective,persistent and specific.2.The sustained and effective expression of CIP in dopaminergic neurons before MPTP/p was established could alleviate the CDK5/p25 hyperactivation and the loss of dopaminergic neurons caused by MPTP/p,as well as the anxiety behavior of chronic PD models.