The Role Of TREM2 And A192T Mutant In The Pathogenesis Of Alzheimer's Disease

Objective:Alzheimer's disease(AD)is the most common type of dementia in the elderly people,and the abnormal aggregation of ?-amyloid peptide(A?)is considered to be related to AD.Triggering receptor expressed on myeloid cells 2(TREM2),which is mainly expressed in microglia,macrophages,and other myeloid cells,could participate in A? removal through enhancing the phagocytosis of microglia.Inflammatory response and the function of microglia are closely related to the pathogenesis of Alzheimer's disease(AD).Genome-wide association study(GWAS)found that some TREM2 mutations significantly increased the risk of AD.But the mechanism remains unclear.The aim of this study was to investigate the effect of TREM2 and its mutations on the pathogenesis of AD.Methods:(1)The changes of cytokines in the brain tissues of AD model mice(APP/PS1)and wild type mice(WT)at different ages were detected by meso scale discovery(MSD).(2)The expression positions of A? and TREM2 in brain tissues of mice were detected by polychromatic immunofluorescence labeling.(3)The expression levels of TREM2,A? and microglia marker(Iba-1)in the brain tissues of AD model mice(APP/PS1)and wild type mice(WT)at different ages were detected by Western blot.(4)The correlation analysis between the expression levels of A?,TREM2 and cytokinesin the brain tissues of APP/PS1 mice were performed via Spearman analysis method.(5)PCR and whole exome sequencing were used to detect the TREM2 gene mutations in the peripheral blood samples of AD patients in Han population.(6)Three TREM2 or its mutant plasmids were constructed on the background of pEGFP-N1 vector,and then transfected into HEK293 cells.(7)The intracellular metabolism and glycosylation of TREM2 expression were determined by Western blot and sandwish ELISA.(8)Cell viabilty was determined via CCK-8 kit.Results:(1)MSD results showed that in the same age,compared with WT mice,there was no significant difference in the expression level of cytokines(TNF-a,IL-1p,IL-6,IL-12,IL-4 and IL-10)in the brain tissues of AD model mice(APP/PS1).(2)Polychromatic immunofluorescence and Western blot results showed that in the same age,the expression levels of A?(A? plaques,A? oligomers and A?42)in brain tissue of AD model mice(APP/PS1)was significantly increased compared with that of WT mice(APP/PS1:43.52±8.8,WT:19.6 ± 2.55,P<0.05).In APP/PS1 mice,the expression level of A? increased with age;In APP/PS1 mice over 16 weeks old,the expression of TREM2 increased with age,and the expression of TREM2 increased significantly at 24 weeks old compared with 3 weeks old(APP/PS1:2.22 ± 0.71,WT:5.45:1±1.28,p<0.05);In APP/PS1 mice,compared with WT mice the expression level of microglia marker(Iba-1)was significantly increased at 16 weeks old(APP/PS1:0.0446 ± 0.0126,WT:0.0212 ± 0.0062,P<0.05).(3)In AD model mice(APP/PS1),there was significantly positive correlation between cytokines(TNF-a and IL-1?),A?(A?oligomers and A?42)and TREM2(TNF-a and IL-1? with A?,correlation coefficient r>0.5,p<0.05;TNF-a and IL-1? with TREM2,correlation coefficient r>0.5,p<0.05;TREM2 with A?,correlation coefficient r>0.5,p<0.01).(4)Through PCR and whole exome sequencing,the TREM2 mutation p.A192T was found from peripheral blood samples of AD patients in Han population.(5)Western blot results showed that the main bands of TREM2-FL-EGFP were located near 50 kDa,and the main bands of TREM2-CTF-EGFP were located near 35 kDa.The glycosylated bands of TREM2 were located near the range of 50-80 kDa.In TREM2-R47H group,compared with TREM2-WT group,the expression levels of TREM2 in cytoplasm and sTREM2 in supernatant were significantly increased(WT:621 ± 158.99,R47H:1178.78±102.05,p<0.05),and there was no significant change in glycosylation.In TREM2-A192T group,compared with TREM2-WT group,the expression levels of sTREM2 in the supernatant was significantly increased(WT:621 ± 158.99,A192T:832.11 ± 110.67,P<0.05),and the residual glycosylation bands increased significantly after treatment with glycosylase(WT:0.22 ± 0.27,A192T:0.81 ± 0.23,P<0.05).In TREM2-A192T group,cell viabilty increased significantly(WT:32.53 ± 2.03,A192T:65.6 ± 12.66,P<0.05).Conclusion:(1)TREM2 p.A192T mutant in Han population patients was found,and the expression plasmids were constructed.(2)In AD model mice(APP/PS1),TREM2 was positively correlated with cytokines(TNF-a and IL-1?)and pathologic A(3??which suggested that TREM2 might involve in AD through the process of inflammation.(3)In HEK293 cell,A192T mutation increased the expression level of sTREM2 and was related to the change of glycosylation modification of TREM2.A192T mutation improved the cell viability,suggesting that this mutant might not decrease the cell viabilty in AD pathogenesis.