Detection The Role Of PINK1 Mutation And Oxidative Stress, Mitochondrial Dysfunction In The Pathogenesis Of Parkinson Disease

Parkinson's disease (PD) is a common neurodegenerative disorder which the cause of disease is still unknown, clinical and molecular genetic studies have led to the identification of different monogenic forms of PD. Several genes have been reported in the pathogenesis of familial PD. At least three loci for autosomal recessive early-onset parkinsonism (AREP) have been mapped so far (PARK2, PARK6 and PARK7) and all the three pathogenic genes have been cloned, those are parkin, PINK1 and DJ-1.PINK1 is the second most frequent AREP related causative gene which was cloned in 2004. It contains a 34-amino acid mitochondrial targeting motif and a highly conserved protein kinase domain, encode a mitochondrial protein which has kinase activity. The function of PINK1 is still unknown. Here we established stably expression of wild-type and R492X mutation PINK1 protein SH-SY5Y cell line, to detected the role of wild-type and R492X mutation, oxidative stress and mitochondrial dysfunction in the pathogenesis of PD with or without N- methyl -4-phenylpyridine (MPP⁺) induced cell injure. The research focus on the following issue:1. Using Lipofectamine transfection technique to establish stably expression of wild-type and R492X mutation PINK1 protein SH-SY5Y cell line; using DNA, RNA amplification and western blot method to verifying the expression of the gene respectively2. With or without MPP⁺ induced cell injure , using MTT method to detect the cells viability; flow cytometry analysis system to study cell apoptosis; hochest 33258 fluorescein stain to observe the morphological changes of cellular nucleus;3. Extracting mitochondrion and cytoplasm protein of the cultured cells , using Western blot methods to study the contents of cytochrome C in cytoplasm and mitochondrion respectively, immunofluorescence satining to observe the cytochrome C release under laser con-focu microscope;4. Using flow cytometry analysis system to detect the cellular reactive oxidative species (ROS), fluorospectrophotometer to detect mitochondrial membrane potential (△ψm ) and cellular Ca²⁺ concentration;5. 2,6-dichloroindophenol (DCIP) as the terminal electron acceptor using fluorospectrophotometer to detect the mitochondrial ComplexI activity;6. To observe the morphological changes in mitochondrial using electronic microscope.The study shows that, we successful to screening out stably transfected PINK1-pcDNA3.1- myc-his(-)B eucaryou vector SH-SY5Y cell line, improved the stably expression of wild-type and R492X mutation PINK1 through DNA, RNA amplification and western blot, indicated the successful construction of the stably expression of wild-type and R492X mutation PINK1 SH-SY5Y cell line. Under the basic condition, over expression of R492X mutation PINK1 cause increased in cytochrome C release and cell apoptosis rate, increased cellular Ca²⁺ concentration , deteriorated the mitochondrial membrane potential (p<0.05 ); under MPP⁺ induced cell injure, over expression of R492X mutation PINK1 cause decreased in cell viability, augmented in mitochondrial cytochrome C release and cell apoptosis rate, increased in cellular ROS production and Ca²⁺ concentration, deteriorated in mitochondrial membrane potential and Complex I activity, compare with the control group(p<0.05), severe mitochondrial swelling, diminishing or even absence of mitochondrial cristae;We firstly established stably expression of the wild-type and R492X mutation PINK1 protein SH-SY5Y cell line, laid the foundation for the further research; R492X mutation PINK1 protein cause increased in cytochrome C release and cell apoptosis , increased cellular Ca²⁺ concentration , deteriorated the mitochondrial membrane potential; under MPP⁺ induced cell injure, over expression of R492X mutation PINK1 cause seriously oxidative stress and mitochondrial dysfunction, decreased in cell viability, augmented in mitochondrial cytochrome C release and cell apoptosis rate, increased in cellular ROS production and Ca²⁺ concentration, deteriorated in mitochondrial membrane potential and Complex I activity; wild type PINK1 protein protected SH-SY5Y cell from mitochondrial cytochrome C release and decreased Complex I activity, it will be more sensitive to the environmental stress when PINK1 gene mutation, We further proved the co-effects of environmental and causative gene mutation in the pathogenesis of PD.