An Experimental Study On Glyceraldehyde-3-phosphate Dehydrogenase Overexpression And Aggregation In The Pathogenesis Of Parkinson's Disease

PART 1 The Effect of Rotenone on Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) Expression, Subcellular Localization andAggregationSection 1 The Effect of Rotenone on GAPDH Expression, Subcellular Localizationand Enzyme ActivityObjective To investigate the effect of rotenone on GAPDH expression, subcellularlocalization and enzyme activity.Methods Nerve growth factor (NGF) differentiated PC12 cells were treated withrotenone for different periods.The cell apoptotic rates were measured using flow cytometryby FITC-Annexin V/PI staining; Real time-PCR and western blot were used to detect theexpression of GAPDH mRNA and protein level; Subcellular fractionation and western blotwas used to study the subcellular location of GAPDH in cells; GAPDH glycolysis activitywas measured by spectrophotometry.Results Flow cytometry analysis indicated that rotenone induced PC12 cell apoptosis in atime-dependent manner.Real-time PCR and western blot showed that GAPDH mRNA andprotein level were both up-regulated in a time-dependent manner; while spectrophotometrydemonstrated that GAPDH glycolysis activity were decreased after rotenone exposure.Subcellular fractionation and western blot data showed that GAPDH protein was increasedin the nuclear and mitochondria fractions.Conclusions Rotenone might induce GAPDH overexpress, suppress GAPDH glycolyticactivity and induce GAPDH enrich in the nuclear and mitochondria fractions, which mayplay an important role in rotenone induced dopaminergic neuron death. Section 2 Rotenone induce GAPDH aggregation and its possible mechanismObjective To explore the possible mechanism involved in rotenone induced GAPDHaggregation in dopaminergic neurons.Methods Nerve growth factor induced PC12 cells were treated with rotenone.LaserScanning Confocal Microscope (LSCM) was used to observe the GAPDH distribution andcomformational change after rotenone treatment.Non-reduced SDS-PAGE Was used todetect the disulfide-bonded GAPDH in cells.The Triton X-100 soluble and insolubleGAPDH were separated and detected by western blot analysis.Results GAPDH distributed uniformly in the cytosol and seldom GAPDH were locatedin the nuclear in control cells.After rotenone treatment, GAPDH expression was muchhigher, some GAPDH positive aggregates were observed in the cytosol.Non-reducedSDS-PAGE also indicated that rotenone induced a time-dependent disulfide-bondedGAPDH overexpression.After rotenone exposing, both Triton X-100 soluble and insolubleGAPDH were increased.Inclusion Rotenone can induce GAPDH aggregation.The possible mechanism may dueto the increase of disulfide-bonded GAPDH and Triton X-100 insoluble GAPDH.This maybe an important mechanism involved in rotenone induced cell apoptosis and inclusionbodies formation.PART 2 The effect of targeting inhibition of GAPDH expression byRNA interference on rotenone induced cell deathObjective To investigate the effect of GAPDH siRNA on rotenone induced cell apoptosis.Methods The small interfering RNA (siRNA) specific targeted to GAPDH gene was constructed by chemical synthesis.SiRNA was transfected into nerve growth factordifferentiated PC12 cells using lipofectamine2000.Transfection efficiency was detectedusing FAM-labeled siRNA.Interfere efficiency was identified by real-time PCR andwestern blot analysis.Laser scanning confocal microscope was used to study the locationand aggregation of GAPDH.The mitochondria membrane potential changes of transfectedor unvalidated negative siRNA transfected cells after rotenone exposure were detected byrhodamine 123 staining using flow cytometry.Results Real-time PCR and western blot analysis showed that GAPDH siRNA canefficiently suppress GAPDH expression.LSCM found that GAPDH became aggregated inthe cytosol and nuclear translocation in rotenone treated PC12 cells; while GAPDH siRNAtreated cells has lesser GAPDH aggregation and nuclear translocation.As detected by flowcytometry, down-regulation of GAPDH significantly decreased rotenone inducedmitochondrial membrane potential collapse, compared with unvalidated negative control.Conclusions Down-regulation of GAPDH suppresses rotenone induced GAPDHaggregation and nuclear translocation, decreases rotenone induced mitochondrialmembrane potential collapse and cells apoptosis, which may play a role in rotenoneinduced dopaminergic neuron death.PART 3 GAPDH Expression and Activity in Rotenone-induced PD RatsSection 1 Rotenone-induced PD models: a comparison between subcutaneousinjection and stereotactic injectionObjective To explore and compare the rotenone-induced PD models: subcutaneousinjected models and stereotactic injected models.Method Rotenone was injected subcutaneously at the back or stereotactically at the VTA and SN of the rat brain.The behaviors features of animals were investigated using bar test,grid test and apomorphine-induced rotations.HE pathology, the immunoreactivity oftyrosine hydroxylase,α-synuclein, and ubiquitin in the brains were observed byimmunohistochemistry.Results Behavior investigation indicated that both rotenone subcutaneous injection andstereotactic injection can induce Parkinsonism features.Immunohistochemistry studysuggested that the tyrosine hydroxylase immunoreactivity were decreased in subtantia nigra.Acidophily inclusion bodies andα-synuclein-, ubiquitin-positive inclusions were found inrotenone-induced PD models.Organ toxicity was observed in subcutaneously injected PDmodels but not in stereotactically injected PD models.Conclusion Both rotenone subcutaneous injection and stereotactic injection are effectiveto induced PD models.Section 2 GAPDH expression and activity in rotenone-induced PD modelsObjective To observe the expression, distribution and enzyme activity of GAPDH inrotenone-induced PD rats.Methods Rotenone was injected subcutaneously at the back or stereotactically at theVTA and SN of the rat brain to induce PD models.Grid test, bar test andapomorphine-induced rotations were used to detect the behaviour changes inrotenone-induced PD models.Immunohistochemistry study was used to investigate theexpression and distribution of GAPDH in rotenone-induced PD models.GAPDH glycolyticactivity in substatia nigra, striatum and other brain areas were studied byspectrophotometry.Results In rotenone-induced PD models, GAPDH was up-regulated in the substatia nigraespecially in rotenone stereotactically injected PD models.Some neurons existed GAPDHnuclear translocation and aggregation.The GAPDH glycolytic activity was not only significantly decreased in the substatia nigra but also the peripheral organs such as the liverand kidney in subcutaneously rotenone administered rats.While there was only GAPDHactivity decrease in substatia nigra and striatum but not in peripheral organs in rotenonestereotacitcally injected models.Inclusions GAPDH protein is up-regulated and aggregated in the brain of rotenonetreated rats, but the glycolytic activity of GAPDH is decreased.PART 4 Effect of Deprenyl on Rotenone-Induced PD Models and ItsPossible MechanismSection 1 The protective effect of deprenyl on rotenone treated PC12 cells and thepossible mechanismObjective To investigate the protective effect of deprenyl on rotenone induced cell deathand its possible mechanism.Methods NGF differentiated PC12 cells were treated with deprenyl at differentconcentrations (1nM-1mM) instantly after rotenone treatment.Cell viability was measuredby MTT.The expression of GAPDH mRNA and protein were detected using real time-PCRand western blot.GAPDH glycolysis activity was measured by spectrophotometry.LaserScanning Confocal Microscope (LSCM) and subcellular fractionation and western blot wasused to study the subcellular location of GAPDH in deprenyi treated and untreated cells.The cell apoptosis were detected using flow cytometry.Results Cell viability declined in a concentration-dependent manner after rotenoneexposure, while 10nM-250μM deprenyl can partly inhibited rotenone induced cell death.The GAPDH mRNA and protein level were both significantlY decreased after deprenyltreatment compared with rotenone treated groups.Spectrophotometry analysis demonstrated that GAPDH glycolysis activity was increased after deprenyl exposure.Subcellular fractionation and western blot further indicated that deprenyl can reducerotenone induced GAPDH nuclear and mitochondria translocation.Flow cytometry alsoshowed that deprenyl exposure can reduce rotenone induced mitochondrial membranepotential collapse and cells apoptosis.Conclusions Deprenyl can reduce rotenone induced GAPDH overexpression, nuclear andmitochondria translocation, increase GAPDH glycolytic activity and protect cells fromapoptosis.Section 2 The effect of deprenyl on rotenone-induced PD ratsObjective To investigate the effect of deprenyl on rotenone-induced PD rats and itspossible mechanism.Methods Rotenone was injected subcutaneously at the back or stereotactically at theVTA and SN of the rat brain to induce PD models.Deprenyl were injected intraperitoneallyafter rotenone exposure.Grid test, bar test and apomorphine-induced rotations were used todetect the behaviour changes in animals.Immunohistochemistry study was used to studythe GAPDH expression and distribution in each group.GAPDH glycolytic activity insubstatia nigra, striatum and other brain areas were studied by spectrophotometry.Results In rotenone-induced PD models, GAPDH was up-regulated in the substatia nigraespecially in rotenone stereotactically injected PD models.Deprenyl can reduce rotenoneinduced GAPDH overexpression in rat brains.The GAPDH glycolytic activity wasdecreased in the substatia nigra, striatum and the peripheral organs in subcutaneouslyrotenone administered rats, while there was only GAPDH activity decreases in substatianigra and striatum but not in peripheral organs in rotenone stereotacitcally injected models.Inclusions Deprenyl can relieve GAPDH overexpression and the decrease of GAPDH glycolytic activity induced by rotenone exposure, and have protective effect onrotenone-induced PD rats.