The Effect Of Mutated DJ-1 Protein To Cellular Mitochondrial Function And Expression Profiles Of Genes And The Correlation Analysis Of Genotype And Phenotype Of AREP

Partâ… The effect of mutated DJ-1 protein to cellular mitochondrial function and expression profiles of genesDJ-1 gene is one of the pathogenic genes that are responsible for autosomal recessive early-onset parkinsonism(AREP)and its effect to the pathogenesis of Parkinson's disease(PD)is still unknown.In our prophase works,a novel DJ-1 mutation(L10P)was found.To elucidate the effect of L10P mutated DJ-1 protein to the pathogenesis of PD,we investigated the function of DJ-1 on the following three aspects:1.To generate HEK293 monoclone cell lines which are stably expressing Flag-tagged wild-type and L10P mutated DJ-1 protein, G418(800μg/ml)was used when pCMV-Tag 2A-Flag-DJ-1,pCMV-Tag 2A-Flag-DJ-1-L10P were transfected.To confirm the HEK293 monoclone cell lines which can stably express Flag-tagged wild-type and L10P mutated DJ-1 protein,we identified the transfected plasmids were conformed in the gDNA,could be transcripted in the RNA level and expressed protein with the Flag tag.We got the HEK293 monoclone cell lines which were stably expressing empty vector,Flag-tagged wild-type and L10P mutated DJ-1 protein successfully.2.Using spectrophotometer,flow cytometry and electron microscope to investigate cell viability,reactive oxygen species(ROS), mitochondrial transmembrane potential,complexâ… activity and mitochondfial morphous of the HEK293 monoclone cell lines which are stably expressing wild-type and L10P mutated DJ-1 protein.Compared with the cell lines expressing empty vector,we found the ROS was increased,the cell viability,mitochondrial transmembrane potential, complexâ… activity were reduced in these cell expressing L10P mutated DJ-1 protein.We also found mitochondria in these cells were swelling and some mitochondria were vacuolar degeneration.These phenomena were more obvious when rotenone was used.But in the cell expressing wild-type DJ-1,ROS was lower,the cell viability,mitochondrial transmembrane potential,complexâ… activity were higher than other cell lines,especially under the induction of rotenone.It suggested that L10P mutated DJ-1 protein probably loss the ability of anti-oxidative stress and affect the normal function of mitochondria.3.To identify genes for which expressions are abnormally regulated by L10P mutated DJ-1 protein,DNA microarray analyses were carried out using HEK293 monoclone cell lines which are stably expressing empty vector,wild-type and L10P mutated DJ-1 protein.Compared with the cell lines expressing empty vector,we found expression levels of 14 and 28 genes in expressing wild-type DJ-1 protein cells and expression levels of 14 and 9 genes in expressing L10P mutated DJ-1 protein cells increased and decreased,respectively.Compared with the cell lines expressing wild-type DJ-1 protein,we found expression levels of 59 and 27 genes in expressing L10P mutated DJ-1 protein cells increased and decreased,respectively.These genes were classified genes related to signal transduction,cell adhesion,regulation of transcription,regulation of cell cycle,protein modification,apoptosis,oxidative stress and neurotoxicity.It suggested that L10P mutated DJ-1 protein probably affects the normal function of these pathways via changing these associated gene express level to participate the pathogenesis of PD. Partâ…¡The correlation analysis of genotype and phenotype of autosomal recessive early-onset parkinsonismThree loci,for autosomal recessive early-onset parkinsonism(AREP) have been mapped thus far(PARK2,PARK6 and PARK7)and all the three pathogenic genes have been cloned,that is parkin,PINK1 and DJ-1. To elucidate the correlation between genotype and phenotype of AREP, we investigated the following three aspects:1.After establishing the methods of real-time PCR to screen parkin gene rearrangement and DNA direct sequencing to detect the ATP13A2 gene mutation,we consummated the mutation analysis of parkin,PINK1, DJ-1 and ATP13A2 gene in 30 families with AREP.In our study, mutations in the parkin,PINK1,DJ-1 and ATP13A2 genes account for up to 46.7%,6.7%,3.3%of Chinese families with AREP,respectively.The digenic inheritance of PINK1 and DJ-1 genes accounts for up to 3.3%.2.We studied three families with the mutations of parkin,PINK1 and DJ-1 gene respectively,with a dopamine transporter ligand ¹¹C-CFT positron emission tomography(PET).A marked bilaterally and symmetrically decrement of ¹¹C-CFT uptake was found in all these patients,and putamen as well as caudate nucleus was affected.We also found asymptomatic parkin and PINK1 heteroygotes showed a mild but significant decreae in ¹¹C-CFT uptake,suggesting a sub-clinical disease process in parkin and PINK1-heterozygotes,but this phenomenon was not found in the DJ-1-heteroygotes. 3.Detailed analyse the clinic feature of the patients in the 30 families with AREP and then compare the clinic feature according to the genotype.No significant feature was found to differentiate the genotype.