Research Of Cell Cycle Abnormol And Autophagy Of Dopaminergic Neurons Induecd By 6-OHDA

Aim: To study the role of cell cycle aberrant, autophagy in the death of dopaminergic neurons induced by 6-OHDA, prove the protective effect of Olomoucine, a cyclin dependent kinase inhibitor, on dopaminergic neurons and exploit the relative mechanisms.Methods: In this study, we established two PD models in vivo and in vitro. Rats model were established by stereotaxic administration of 6-OHDA into unilateral medial forebrain bundle(MFB). For Olomoucine intervention, 200 n mol Olomoucine dissolved in 100% dimethyl sulfoxide (DMSO) was injected into left lateral ventricle. Primary mesencephalic cell culture were prepared from ventral mesencephalic tissue of rat embryos. Cells were pretreated with Olomoucine for half an hour, followed by the treatment with 6-OHDA. Indirect immunofluorescence was used to detect the expression of Cyclin D1,Cyclin B1 ,PCNA, Cathepsin L and LC3; Total RNA was extracted from the left substantia nigra of rats'brain and Real Time PCR was used to detect the mRNA levels of Cyclin D1,Cyclin B1 ,PCNA, Cathepsin L and LC3; Apomorphine was used to induce the rotation of PD rats; Stereoinvestigator was used to count the number of TH positive cells.Results: Cyclin D, Cyclin B, PCNA and LC3 were aberrant expressed and the nuclear translocation of Cathepsin L happened in a part of doparminergic neurons. Olomoucine, one of the cyclin dependent kinase (CDK) inhibitors, which prohibits cells to enter S phase can improve the ethology of PD rat and protect dopaminergic neurons from death. Compared to the rats which did not use Olomoucine, data shows there were more TH positive neurons and the expression of LC3 was up-regulated during the early stage of lesion. More important, we found a intriguing result that nuclear translocation of Cathepsin L was delayed by Olomoucine treatment.Conclusion: These findings lead us to believe that cell cycle reentry may contribute to the death of dopaminergic neurons induced by 6-OHDA via nuclear translocation of Cathepsin L. In addition, the death of dopaminergic cells is autophagic related. However, the expression of LC3 during the early time indicates that autophagy may be a protection rather than a lethal factor.