Study On The Involvements Of 14-3-3 Proteins In The Pathogenesis Of Parkinson's Disease

PART ONE Distribution of 14-3-3 protein isoforms in rat's substantia nigra, striatum corpora, and PC12 cellObjective To investigate the distribution profile of 14-3-3 isoforms in rat's substantia nigra, striatum corpora and PC12 cell, and to provide morphological bases for exploring potential involvements of 14-3-3 protein in the pathogenesis of Parkinson's disease. Methods Immunohistochemical/immunofluorescence and immunoblotting methods were utilized.Results Immunohistochemical stains showed that only the four 14-3-3 protein isotypesγ,ε,θandζare positively immunoreactive in rat's substantia nigra and striatum corpora, immunofluorescence stains showed all the six 14-3-3 isotypes are positively immunoreactive in PC12 cells. Under light microscope, the immunoreactive stain mainly localized in cytoplasm, some prominences also showed positive reactivity, there showed no evident positive stain in the nuclei. Western blot showed that the content orders of positive 14-3-3 isoforms in rat's substantia nigra and striatum corpora wereε>γ>ζ>θ, while in PC12 cells the order wasγ>ε>β>ζ>η>θ.Conclusion In rat's substantia nigra and striatum corpora, there existγ,ε,θandζ14-3-3 protein isotypes which might be involved in the pathogenesis of Parkinson's disease. The highest content of isotpyeεimplied it might play the central role among the four isotypes. There exist six 14-3-3 isoforms in PC12 cells, and the distribution of these isotypes in PC12 cells are similar as that in rat's substantia nigra and striatum corpora. This suggest that PC12 cell is suitable for being used as a cell model to investigate the effects of 14-3-3 protein.PART TWO Changes of 14-3-3 protein expression in SNpc in 6-OHDA induced rat's PD models and in PD cell models induced by MPP+Objective To observe the changes of 14-3-3 protein expression in substatia nigra in 6-OHDA induced PD rat's models and the changes of 14-3-3 protein in MPP+ induced PD cell model, and to explore the relationships between the changes of 14-3-3 protein expression and the death of dopaminergic cells.Methodsâ‘ PD rat's models were constructed by Stereotactic injection of 6-OHDA into unilateral substantia nigra, the success of models were tested by the rotational behavior induced by emetomorphine. RT-PCR and Western blot techniques were used to estimate the changes of 14-3-3 expression at the mRNA level and at the protein expression level respectively.â‘¡PD cell models were set up by MPP+ induced PC12 cells, the effects of MPP+ on the expression of 14-3-3 protein and on the viability of PC12 cells were determined by Western blot and MTT test respectively.Resultsâ‘ RT-PCR showed that there was no evident difference between control group and PD group in the expression of 14-3-3 mRNA in rat's substatia nigras of PD model induced by 6-OHDA. Western blot showed that the four positively immunoreactive 14-3-3 protein isoforms all decreased dramatically.â‘¡T he decrease of 14-3-3 protein expression was in time-dependent manner. 6 hours after treated with MPP+ the level of 14-3-3 proteins showed no evident decrease (P>0.05). The level of 14-3-3 protein reduced significantly 12 hours after treatment (P<0.05) and decreased extremely significantly after 48 hours (P<0.01). The changes of viability of PC12 cells showed the same tendency as the 14-3-3 proteins.Conclusion The levels of 14-3-3 protein expression were downregulated both in substantia nigra of PD rat model and in PC12 cells induced by MPP+, 14-3-3 protein expression and cell viability of PC12 cells showed the same decrease tendence. These results suggest that the downregulation of 14-3-3 proteins expression might involve in the mechanism of cell death in PC12 cells induced by MPP+.PART THREE The construction of pcDNA 3.1(+)-14-3-3 plasmid and the setup of cell line overexpressing 14-3-3 proteinsObjective To construct the plasmids containing human 14-3-3 DNA and to set up the cell line overexpressing 14-3-3 protein for next assays. Methods DNA recombination technique was utilized to construct pcDNA3.1(+)- 14-3-3 plasmids, then the plasmids constructed and the pEGFP plasmids were cotransfected into PC12 cells; immunofluorescence and immunoblotting methods were used to test the expression of 14-3-3 protein in PC12 cells.Results DAN sequencing showed that the base sequence was the same as the DNA consulted in Genebank (NM₀06761). After cotransfected with pcDNA3.1(+)-14-3-3 and pEGFP plasmids into PC12 cells, the cells expressing enhanced green fluorescence protein were very few at the first day; but at 2～6d, the cells expressing EGFP became more and more and the green fluorescence became stronger and stronger. Western blot showed that the level of 14-3-3 protein in plasmid transfection group increased evidently compared with the control group and the empty plasmids group.Conclusions The pcDNA3.1 (+)-14-3-3 and the cell line overexpressing 14-3-3 protein was constructed successfully.PART FOUR Overexpression of 14-3-3 protein alleviates the toxicity of MPP+ to PC12 cellObjective To investigate the protective effects of the 14-3-3 protein overexpression on the injury of PC12 cell induced by MPP+. Methods For expression in mammalial cells, pcDNA3.1 (+)-14-3-3 plasmid was constructed and transfected into PC12 cell with LipofectamineTM 2000. The overexpression of transfected 14-3-3 gene in PC12 cell was determined by immunofluorescence and Western blotting. The effects of 14-3-3 overexpressing on the cells viability, apoptotic ratio of PC12 cell treated with MPP+ were measured by MTT assay, flow cytometry analysis and DAPI stainning respectively.Results The expression of 14-3-3 protein in transfection group(1.19±0.06) increased evidently compared with control group (0.75±0.05). MTT assay and flow cytometry analysis shows that the overexpression of 14-3-3 protein increases the cell viability (transfection group:0.78±0.06,MPP+ group:0.54±0.07), and inhibits cell apoptosis (transfection group: (11.87±3.26)%,MPP+ group:(36.30±2.39)% ) of PC12 induced by MPP+. Conclusion The overexpression of 14-3-3 protein could protect PC12 cells against the toxicity induced by MPP+.PART FIVE Study on the mechanisms of 14-3-3 protein overexpression protecting PC12 cells against MPP+ toxicityObjective To explore the potential mechanisms of 14-3-3 protein overpression protecting PC12 cells against the MPP+ toxicity.Methods Cell line of overexpressing 14-3-3 protein was constructed by DNA recombination technique. PD cell model was set up by 0.1 mM MPP+ induced PC12 cells. Western blot and microplate reader were used to examine the expression levels of BAD and p-BAD proteins and the activities of SOD and GSH-Px respectively.Results Western blot showed that the level of BAD protein in PC12 cells was decreased evidently by MPP+. The overexpression of 14-3-3 protein increased the level of p-BAD protein significantly. In the meantime, the overexpression of 14-3-3 protein could enhance the activitied of SOD and GSH-Px.Conclusion 14-3-3 protein overexpression protected PC12 cells against MPP+ toxicity. These effects were carried out through increasing the p-BAD protein expression ,enhancing the activities of SOD and GSH-Px, and so inhibiting apoptosis.