Roflupram Promotes Alpha-synuclein Clearance In Experimental Models Of Parkinson’s Disease

Objective:The abnormal aggregation of α-synuclein(α-syn)is the key factor leading to the death of dopaminergic neurons in the substantia nigra.Current studies suggest that promoting the clearance of α-syn reduces neuronal damage in the treatment of Parkinson’s disease(PD).Our previous studies have confirmed that roflupram(ROF)reduces the loss of dopaminergic neurons in the substantia nigra and alleviates the motor function in MPTP-treated mice.However,whether ROF promotes the clearance of α-syn against neuronal damage is still unclear.Therefore,we aimed to explore whether ROF promotes the clearance of α-syn and its underlying mechanism in the rotenone(ROT)-treated experimental models of PD.Methods:(1)To investigate the protective effect of ROF in SH-SY5Y cells induced by ROT:The rate of cell viability,apoptosis and toxicity were measured by MTT,flow cytometry and lactate dehydrogenase(LDH)detection kit,respectively.The mitochondrial morphology and function were determined by Mito-tracker,etramethylrhodamine,Ethyl Ester,Perchlorate(TMRE),ATP detection kit and Western blot.The contents of 3-Nitrotyrosine(3-NT)and malondialdehyde(MDA)were detected respectively by the ELISA kit of 3-NT and MDA assay kit to measure the changes of oxidative stress.(2)To explore the mechanism of ROF on promoting α-syn clearance:The lysosomal fluorescence intensity was measured by LYT dye and the levels of various proteins(lysosomal relative protein,SIRT1,and α-syn)were detected by Western blot.The ratio of NAD+/NADH was measured by the kit of NAD+/NADH.Moreover,we explored whether SIRT1 inhibitor(EX527)and lysosomal inhibitors(pepstatin A and chloroquine)could block the protective effect of ROF.(3)To verify the protective effect and mechanism of ROF in C57BL/6J male mice treated with ROT:Pole,rotarod and balance beam test were used to evaluate motor function.The level of superoxide dismutase(SOD)activity was detected by the kit of SOD.The levels of different proteins in substantia nigra of ROT-treated mice were determined by Western blot.Results:(1)Silencing PDE4B decreases cell death rate in SH-SY5Y cells exposed to ROT.ROF(10 μM)enhanced cell viability and decreased the level of cytotoxicity and the expression of cleaved caspase-3 in SH-SY5Y cells induced by ROT(2 μM).ROF increased mitochondrial area,which ameliorated the damage of mitochondrial morphology.ROF promoted the protein expression of cytochrome c oxidase Ⅳ(COX Ⅳ)and mitochondrial transcription factor A(TFAM)and increased the levels of mitochondrial membrane potential(MMP)and ATP.ROF decreased the mitochondrial production of reactive oxygen species(ROS)and the levels of 3-NT and MDA.ROF increased the expression level of thioredoxin reductasel(TrxR1)in SH-S Y5 Y cells induced by ROT.(2)ROF decreased the protein level of α-syn and increased the level of lysosomal-associated membrane protein 1(LAMP1)and mature cathepsin D(mCTSD).ROF significantly increased the ratio of NAD+/NADH and the expression level of SIRT1.However,SIRT1 specific inhibitor(EX527)blocked the protective effect of ROF in SH-SY5Y cells,which reduced the rate of cell viability and increased the level of cytotoxicity.EX527 also decreased the levels of MMP,ATP and TrxR1.In addition,EX527 significantly decreased the expression level of LAMP 1 and mCTSD.EX527 increased the protein level of α-syn,while had no effect on the mRNA expression.Pepstatin A and chloroquine significantly decreased cell viability and reduced the number of lysosomes.Moreover,pepstatin A increased the protein expression of α-syn.(3)In ROT-treated mice,ROF significantly improved motor function.ROF reversed the reduction of tyrosine hydroxylase(TH),TFAM,COX Ⅳ and TrxR1 expression and SOD activity.ROF promoted the expression of SIRT1,LAMP1 and mCTSD.In addition,ROF decreased the protein level of α-syn.Conclusion:In the experimental PD models induced by ROT,ROF improved cell viability and reversed the damage of mitochondrial dysfunction and oxidative stress in SH-SY5Y cells.In addition,ROF attenuated motor deficits in ROT-treated mice.These protective effects of ROF were exerted via activating NAD+/SIRT1 pathway to enhance lysosomal function and promote the clearance of α-syn.