Dyrk1a Regulates LAG3 DNA Methylation In The Pathogenesis Of Parkinson’s Disease

Objective: Based on the patient’s clinical data,the influence of peripheral blood cell immune disorder and serum LAG3 on Parkinson’s disease is discussed.This study also investigates the effects of Dyrk1 a on phosphorylation of α-synuclein,as well as its regulation of LAG3 protein in the pathological process of PD at the animal and cellular level.And to explore the mechanism of Dyrk1 a regulating LAG3 protein.Methods: 1)We collected the clinical data of 47 PD patients and 36 physical examination controls,used a blood analyzer to count the cells,calculated the ratio of peripheral blood cells of each experimental subject.The Elisa method was used to detect LAG3 in the serum(s LAG3).And the relationship between variables and PD was determined through single-factor and multi-factor analysis.The receiver operating characteristic curve(ROC)was used to evaluate the diagnostic value of the index.2)45 C57 mice were intraperitoneally injected with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)at30mg/kg/d for 6 days.Simultaneously,the 15 mice were intraperitoneally injected with sterile saline at the same volume for 6 days.Then we performed the behaviour test.Subsequently,we divided the remaining 39 mice into 3 groups: mice intraperitoneally injected with Harmine,the Dyrk1 a inhibitor,at 30mg/kg/d for 5 days,mice intraperitoneally injected with Harmine at 15mg/kg/d for 5 days,mice intraperitoneally injected with equal volumes of solvent without Harmine for 5 days.The normal controls were intraperitoneally injected with equal volumes of solvent without Harmine for 5 days at the same time.After obtaining the midbrain of all the mouse,we used immunohistochemistry(IHC)and Western Blot(WB)methods to detect tyrosine hydroxylase(TH),Dyrk1 a,Ser129 phosphorylated α-synuclein(α-syn)and LAG3.We also used reverse transcription real-time fluorescent quantitative PCR(RT-q PCR)to detect the mRNA of LAG3,and used Bisulfite Sequencing PCR(BSP)to detect the methylation level of the LAG3 promoter region.3)MN9D cells were transfected with plasmids to establish a cell model of Dyrk1 a overexpression and silence.Dyrk1 a,Ser129phosphorylated α-syn and LAG3 were detected by immunocytochemistry(ICC)and WB methods.RT-q PCR was used to detect the mRNA of LAG3.We also use BSP to detect the methylation level of LAG3 promoter region.Results: 1)Our study found that platelet-basophil ratio(PBR),lymphocyte-basophil ratio(LBR),neutrophil-basophil ratio(NBR),and s LAG3 were increased in PD patients(P<0.05),while monocyte-neutrophil ratio(MNR)was decreased in PD patients(P<0.05).However,all these variables were not correlated with Hoehn-Yahr stage,duration and onset age of PD(P>0.05).And after multivariate analysis,it was found that only s LAG3 was associated with PD(P<0.05).The best cut-off for distinguishing PD and control in s LAG3 is 9.43ng/ml,and the sensitivity and specificity were 51.1% and 97.2%.2)We found that the phosphorylatedα-syn and the LAG3 protein decreased and the TH and methylation level of the LAG3 promoter increased in PD model mice treated with Dyrk1 a inhibitor compared with PD models(P<0.05).The score of suspension test also increased in PD model treated with Harmine(P < 0.05).The methylation level of LAG3 promoter is negatively correlated with mRNA of LAG3(P<0.05).3)The level of Dyrk1 a increased in the overexpression group,while the level of Dyrk1 a decreased in the 1279 site silence group.Compared with control group,the level of phosphorylated α-syn,LAG3 protein and mRNA levels increased and LAG3 promoter methylation level decreased in the overexpression group(P<0.05),while the level of phosphorylated a-syn,LAG3 protein and mRNA dropped and LAG3 promoter methylation level increased in 1279 site silence group(P <0.05).The methylation level of LAG3 gene is negatively correlated with its mRNA(P < 0.05).Conclusion: 1)This study found that PBR,LBR,NBR,and s LAG3 increased in the peripheral blood of PD patients,while MNR decreased in the peripheral blood of PD patients.s LAG3 is an independent risk factor for PD and has certain diagnostic value.2)Using the Dyrk1 a inhibitor Harmine for the treatment of MPTP-induced mouse model of PD led to that the phosphorylation level of α-syn decreased,the methylation level of LAG3 gene increased,the mRNA and protein level of LAG3 decreased,the content of TH increased,the DA neurons were protected and the behavioral performance of mice were improved.3)By establishing Dyrk1 a overexpression and silence in vitro cell models,it was found that Dyrk1 a phosphorylates α-syn and regulated the level of LAG3 mRNA and protein by affecting the methylation level of the promoter region of LAG3 gene.