Study On Early Molecular Changes Of Parkinson’s Disease Transgenic Mice Based On Quantitative Phosphorylation Proteomics

Parkinson’s disease(PD)is one of the common neurodegenerative diseases,and its incidence is second only to Alzheimer’s disease(Alzheimer’s disease,AD).With the increase of age,the incidence of PD increases year by year.Abundant studies have shown that PD is related to genetic factors,environmental factors,age factors and other factors.The main pathological changes of PD are the loss and death of dopaminergic neurons in the substantia nigra pars compacta(SNpc).Meanwhile,the formation of Lewy bodies,the activation of microglia,the release of inflammatory mediators and the abnormal aggregation of α-synuclein have been suggested to participate in aggravating the damage of neurons.Patients suffering from PD will typically experience a range of motor and non-motor symptoms during the course of their illness.The diagnosis of PD is currently dependent on the presence of motor deficits,including tremor,slowness of movement(bradykinesia),rigidity,and postural instability.Comparing the motor symptoms,non-motor feathers that include hyposmia,sleep disorders,depression and constipation appears earlier in PD.Thus,developing new methods in early diagnosis and treatment of PD is the major scientific questions in basic science and clinical applications.Recently,numerous non-motor symptoms-related measurements,such as medical imaging,blood test and cerebrospinal fluid test,played an important role in early diagnosis of PD.Nevertheless,the molecular mechanism underlying the pathophysiology of early PD remains poorly unknown.Historically,post-translational protein modifications(PTMs),especially phosphorylation,have been considered a pivotal factor in SNpc dopaminergic neuronal death during PD progression.Therefore,we applied quantitative phosphoproteomics in two biological replicates to analyze SNpc tissue protein samples from early-stage alpha-synuclein transgenic mice in this study.And this elucidation of dysregulated phosphoproteins has implications for understanding the pathophysiological mechanism of dopaminergic neuron degeneration and for developing novel therapeutics for early PD.Objective: A30 P and A53 T mutations in the gene encoding α-synuclein—a presynaptic protein—are the most frequently identified genetic causes of PD.Aberrant α-synuclein likely plays central roles in dopaminergic neuronal death and motor symptoms in PD.Previously published studies have suggested that the aberrantα-synuclein accumulations could directly induce the changes of proteins phosphorylation in the pathogenesis of PD.However,the molecular mechanism underlying the abnormal α-synuclein protein-induced loss of dopaminergic neurons remains poorly understood.This study investigated the protein phosphorylation profile in early-stage PD through phosphoproteomics analyses of tissue samples from the substantia nigra pars compacta(SNpc)of 6-month-old α-synuclein transgenic mice(A30P/A53 T double-mutant human α-synuclein;hm~2α-SYN-39 strain),in order to know the mechanism of mechanism of protein phosphorylation in early-stage PD,find diagnostic biomarkers and neuroprotective therapies for PD.Materials and Methods: Firstly,twenty transgenic mice(strain hm~2α-SYN-39)were randomly divided into two PD groups(PD1 and PD2,n = 10 mice for each group),and twenty wild-type mice divided into two control(CON1 and CON2,n = 10 mice for each group)groups.Rotarod test was employed to evaluate the motor function in all mice.Secondly,the tissue samples from midbrain SNpc of mice were isolated,and then the tissue proteins were extracted.The protein samples were subjected to high performance liquid chromatography fractionation,affinity enrichment,and LC–MS/MS.Thirdly,bioinformatics analysis of phosphoproteomics dataset was performed.Fourthly,the expression of p25 protein was detected by western blotting in primary cortical neurons,which was stimulated by MPP~+ at 0 h,0.5 h and 1h.Fifthly,the protein levels of p25 and tyrosine hydroxylase(TH)protein were quantified by immunoblotting in MPTP-treated mice.Results: Firstly,5351 phosphorylation sites in 2136 phosphoproteins were quantified in total.Of these,357 upregulated sites in 245 proteins and 50 downregulated sites in46 proteins were differentially phosphorylated between α-synuclein transgenic and wildtype mice.Secondly,bioinformatics analyses,including Gene Ontology(GO), Kyoto Encyclopedia of Genes and Genomes(KEGG)and motif analyses,were used to elucidate the molecular and cellular mechanisms underlying double-mutant humanα-synuclein overexpression of PD transgenic mice.Thirdly,scansite-based computational analysis and prediction of differentially quantitated phosphoproteins identified the neuronal protein cyclin-dependent kinase 5(Cdk5)as the most significantly enriched kinase.Fourthly,biochemical experiments suggested that the p25/Cdk5 pathway was activated in an MPP~+-induced cell culture model and MPTP-induced mouse model.Moreover,Cdk5 could directly phosphorylate the Ank2 protein at Ser1889 in vitro.Conclusion: Firstly,the global protein phosphorylation changes in midbrain SNpc tissue were observed in transgenic mice with early-stage PD.Bioinformatics analyses of dysregulated phosphoproteins identified the biological process and cellular signaling pathway involved in the pathophysiology of PD.Secondly,the aberrant p25/Cdk5 signaling pathway is likely involved in SNpc dopaminergic neuronal loss.