Study On GAPDH Gene5’UTR Mutation And Over-expression In Parkinson’s Disease

Part1The study of Parkinson’s disease single nucleotide polymorphisms Section Ⅰ The correlation analysis of GAPDH gene single nucleotide polymorphismsObjectives:To explore the risk and correlationship between GAPDH gene and PD by a case-control study of PD.Methods:Genomic DNA was extracted from peripheral blood in265cases of idiopathic PD patients and269cases of normal people (sex and age-matched) by Peripheral Blood DNA Extraction kit. SNaPshot method was used to detect the gene sequence.Results:Risk and correlation was confirmed between the5’untranslated region of rs1136666polymorphism of GAPDH and PD pathogenesis. G nucleotide played a protect role in male case but not in female case in case-control study. And this SNP was statistically different in allele frequency study in the early-onset group of case-control study, but not in genotype. In the late-onset group, this SNP was statistically different both in allele frequencies and genotype.Conclusion:GAPDH polymorphism was retated to PD. Male and aging are risk factors for PD. Section Ⅱ The correlation analysis of NCAPD2, GAPDH family, PKP2P1, PPM1H, CD9, ETV6single nucleotide polymorphisms and PDObjectives:To explore the risk and correlationship between these genes and PD by a case-control study of PD.Methods:Genomic DNA was extracted from peripheral blood in265cases of idiopathic PD patients and269cases of normal people (sex and age-matched) by Peripheral Blood DNA Extraction kit. SNaPshot method was used to detect the gene sequence..Results:Polymorphism of rs7311174, rs2072374in NCAPD2gene which is upstream gene GAPDH was statistically correlative with PD, while the rs740850polymorphism in the PD patients was not statistically different from controls. rs2029721, rs4806173, rs12984928polymorphisms of GAPDH family were not statistically different in the PD case-control study. Subject who own rs10492243polymorphism have a high risk with PD in this study. rs2008134, rs342169, rs740839, rs732868were not statistically different in this case-control study.Conclusion:NCAPD2gene was strong associated with PD. Three polymorphisms of GAPDH family were not associated with PD in this study. Part2The study of GAPDH gene mutation with Parkinson’s disease Section I Intracellular oxidation level was increased in SH-SY5Y cells after rotenone interventionObjective:Oxidation change was observed after rotenone intervention in SH-SY5Y cells.Method:The cells were grouped according to the rotenone concentration gradient after SH-SY5Y cells culture. Appropriate intervention concentration was confirmed by MTT measure. Morphological change of SH-SY5Y cells were observed by light microscope after different rotenone concentration intervention. Intracellular oxidation level was measured by flow cytometry and DCFH-DA dye. Intracellular SOD activity level and lipid peroxidation level was separately detected by SOD Assay kit and TBARS Assay kit.Results:The optimal concentration of rotenone intervention was500nM and there is a concentration-dependent affection confirmed by MTT detection. Cell death was increase and the change of cell morphology was more obvious with rotenone concentration increase. Cells morphological changes occurred in200nM rotenone and half cell death in2uM rotenone. Intracellular ROS and lipid peroxidation were increase after500nM rotenone, but SOD activity level decreased.Conclusion:Rotenone intervention cells can elevate levels of intracellular oxidation and lipid peroxidation but reduce SOD level. Section II The effect of GAPDH gene5’untranslated region mutation on cell functionObjectives:Construct rs1136666mutation plasmid to explore the relationship between GAPDH gene mutation and PD.Methods:Clone rs1136666fragment and construct it into plasmid. Rotenone or/and plasimd was used to deal with SH-SY5Y cells. Seek appropriate intervention concentration of plasmid in SH-SY5Y cells. By real-time quantitative PCR, GAPDH mRNA expression was observed in each group. Intracellular SOD activity level and lipid peroxidation level was separately detected by SOD Assay kit and TBARS Assay kit. Western blot was used to observe GAPDH expression and apoptosis in each group. Apoptosis and oxidative stress levels were detected in each group by flow cytometry.Results:Real-time quantitative PCR showed the mRNA expression of GAPDH was reduced by transfected with GAPDH gene5’untranslated region mutation plasmid. A stronger apoptosis and increased levels of ROS was observed after GAPDH mutant plasmid transfection conbined rotenone treatment by flow cytometry. SOD level decreased and lipid peroxide level increased after transfection of GAPDH gene5’ untranslated region mutation plasmid transfection. Western blot shows GAPDH gene5’ untranslated region mutation plasmid transfection could cause apoptotic proteins expression increased, and anti-apoptotic proteins expression decreased, and increased GAPDH expression. The GAPDH mutations combined with rotenone could enhance apoptosis. Conclusion:GAPDH gene5’untranslated region mutation can cause abnormal GAPDH mRNA expression, protein folding disorders, abnormal GAPDH aggregation, eventually causing cell death. GAPDH gene5’untranslated region mutation conbined with rotenone can cause higher level of ROS and more severe apoptotic. Section Ⅲ The infection of GAPDH over-expression in SH-SY5Y cellsObjectives:GAPDH over-expression plasmid was constructed to explore the relationship between GAPDH gene over-expression and PD. Seek the cooperative affection of the GAPDH gene over-expression and GAPDH mutations in PD.Methods:SH-SY5Y cells were cultured and GAPDH over-expression plasmid was constructed. Rotenone or/and over-expression plasmid was transfected to SH-SY5Y cells according to groups. Seek appropriate intervention concentration of plasmid in SH-SY5Y cells. Intracellular SOD activity level and lipid peroxidation level was separately detected by SOD Assay kit and TBARS Assay kit. Western blot was used to observe cells GAPDH expression and apoptosis in each group. Neuronal cell apoptosis and oxidative stress levels were detected in each group by flow cytometry.Results:ROS levels and cell apoptosis was increased in GAPDH over-expression vector cells. SOD level decreased and lipid peroxide level increased after transfection of GAPDH over-expression mutation plasmid. When mutation and over-expression of GAPDH cooperate, this effect is enhanced. Western blot showed increased expression in GAPDH over-expression plasmid cells. Native PAGE showed GAPDH mutimers increased all in the groups of rot, GAPDH gene5’untranslated region mutation transfection and GAPDH over-expression mutation transfection.Conclusion:GAPDH over-expression could increase levels of intracellular oxidation and promote apoptosis. While over-expression and mutation co-exist, intracellular oxidation and apoptosis was stronger. Both GAPDH gene5’untranslated region mutation and GAPDH over-expression mutation can increase GAPDH mutimers.