The Protein Modification By Reactive Oxidation Product Of Dopamine In The Pathogenesis Of Parkinson's Disease

Objective: To explore the formation of the reactive oxidation product of dopamine(ROPD),the abnormal oxidation metabolite of the neurotransmitter dopamine(DA),and the role of protein modification by ROPD in the pathogenesis of Parkinson's disease(PD).Methods: The formation of ROPD in the in vitro system was performed by analysis of the nucleophilic trapping product in the MS/MS database acquired from the DA,tyrosinase and trapping agent(eg.GSH,NAC and Cys)supplemented PBS or brain tissue homogenate incubation.Then,the proteolysis method was established and optimized to covert the ROPD-protein addcuts to ROPD-amino acid residue adducts.The protein internal standard were prepared and used to optimize the quantitative method for determination of ROPD-protein addcuts.The rats were injected with 6-OHDA or ROT to develop PD models and the PD models were validated by the TH expression determined using Western blot and immunofluorescence in combined with rotarod tests.The ROPD formed in the brain of PD model rats were characterized by UPLC-Q/Orbitrap MS after protein digestion.The results were verified by pure ROPD incubated with synthetic peptides.The striatum and substantia nigra samples of PD model rats were collected at a series of time points after 6-OHDA or ROT injection to investigate the time course of ROPD-protein adducts formation.The pure ROPD was infused into left substantia nigra pars compacta of rats to investigate the effect of covalent modification of protein by ROPD on dopaminergic neurons.Results: In vitro system,a total of 11 trapping products derived from the ROPD was found,such as 4-S-NAC-5,6-di OH-indole,7-S-GSH-animochrom,2-S-Cys-DA,4-S-NAC-5,6-di OH-indole,7-S-GSH-animochrom,2-S-Cys-DA.The results showed that dopamine o-quinone,5,6-indolequinone and aminochrome were produced by DA oxidation in vitro.The protein digestion protocol was established and optimized by using the tool compound genipin.BAS-NEM was used as a protein internal standard can effectively improve the accuracy of quantitative method.DOPAL-Cys,7-S-Cys-aminochrome and7-S-GSH-aminochrome were detected in PD model rat brain homogenates.Five adductsincluding DOPAL-Lys,DOPAL-Lys(Cyclizing),DOPAL-Gln,DOPAL-His and7-N-Lys-Aminochrome,were found in protease hydrolyzed brain samples of PD model rats,indicating the DOPAL and AC can formed by DA oxidation in PD model rats.The results show that DOPAL or AC can covalently bind with amino acid side chains containing primary amines of proteins through Schiff base reaction or nucleophilic addition.The reactivitiy as well as the mode of binding of DOPAL toward amino acid residues was verified by using the mimic peptide segment containing thiol or primary amine.Importantly,protein modification by DOPAL and AC in the substantia nigra and striatum of PD model rats was observed within about 10 min,and reached to peak at 6 h after injection of the modeling agent 6-OHDA or ROT.The content of the protein adduts at 6 h increased about 3-40 folds compared to that at 0 h(control).Research data indicate that the protein modification by ROPD was occurred much earlier than the large-scale loss of dopaminergic neurons(at 12 or 24 h),which may be a very early and a common event in the pathogenesis of PD triggered by various etiology.Direct injection of DOPAL into left substantia nigra pars compacta resulted in DOPAL-amino acid residue adducts formation and the significant loss of dopaminergic neurons in a dose-dependent manner,indicating that DOPAL covalent binding with protein causes degeneration of dopaminergic neurons.Conclusion: During the pathogenesis of PD,DOPAL and AC,abnormal oxidation products of DA,covalently modifies proteins in the substantia nigra and striatum with primary amine group of amino acid side chains via Schiff base reaction or nucleophilic addition.This early event may play an important role in the pathogenesis of PD,which provide rationales for the pathogenesis of PD and the potential drug target study.