The Mechanism Of CHIP Regulated On The Hyperphosphorylated Tau Induced By AlCl₃ In N2a

Objective:1.To investigate the regulation of different expression of CHIP on abnormal phosphor-ylation sites of tau protein in N2a cells induced by aluminum.2.To study the specific roles of different domains of CHIP in the regulation of abnormal phosphorylated tau protein in N2a cells induced by aluminum.Methods:1.Mice neuroblastoma cells?N2a?were selected as experimental materials.The loga-rithmic growth phase cells were infected with 1 mmol/L AlCl₃ and transfected with CHIP plasmid.The experiment was divided into two parts:one was to observe the effect of CHIP overexpression on tau protein.The experimental groups were as follows:normal control group,1 mmol/L AlCl₃ group,empty plasmid group,CHIP plasmid transfection group,CHIP+1 mmol/L AlCl₃ group.To observe the effect of CHIP interference on tau protein:normal control group,1 mmol/L AlCl₃ group,CHIPshRNA negative control group,CHIPshRNA group,CHIPshRNA+1 mmol/L AlCl₃ group;2.Choose the same cell line as above and carry out 1mmol/LAlCl₃ poisoning and plas-mid transfection in different domains of CHIP.The experiment is divided into two parts:one is to observe the role of CHIP??U-box?in regulating abnormal phosphorylation of tau pro-tein induced by aluminum in CHIP.The experimental groups are:normal control group,1mmol/L AlCl₃ group,empty plasmid group,CHIP??U-box?plasmid transfection group,CHIP??U-box?+1mmol/L AlCl₃ group;To observe the role of CHIP??TPR?in the regula-tion of abnormal phosphorylation of tau protein induced by aluminium in CHIP.The exper-imental groups were normal control group,1mmol/L AlCl₃ group,empty plasmid group,CHIP??TPR?plasmid transfection group and CHIP??TPR?+1mmol/L AlCl₃ group.After the treatment,the cells were collected and the total protein samples were extracted.The pro-tein expression levels of tau-5,pThr181,pThr231,pSer262,pSer396,CHIP,Hsp70 and Ub were detected by Western blot.The interaction between CHIP and tau-5,pThr181,pThr231,pSer262,pSer 396,Hsp70 and Ub was detected by immunoprecipitation.Result:1.Overexpression of CHIP in N2a cells1.1 Immunoblotting results showed that the expression levels of CHIP,Ub,pThr231,pSer262,pSer 396 protein were significantly higher than those of control group?P<0.05?;the expression levels of CHIP and Ub protein were significantly higher than those of control group?P<0.05?;the expression levels of pThr231,pSer262,pSer 396 protein were signifi-cantly lower than those of control group?P<0.05?.Factorial analysis showed that CHIP over-expression in N2a cells interacted with AlCl₃ exposure in the expression changes of CHIP,Ub and pThr231,pSer262 and pSer 396 proteins.1.2 Immunoprecipitation results showed that Hsp70,Ub and tau protein pThr231,pSer262,pSer 396 phosphorylation sites were detected by WB,indicating the interaction between CHIP and Hsp70,Ub,tau protein and tau protein at each phosphorylation site.2.CHIP interference in N2a cells2.1 Immunoblotting results showed that the expression levels of CHIP,Ub,pThr231,pSer262 and pSer 396 were significantly higher than those of the control group?P<0.05?;the expression levels of CHIP and Ub were significantly lower?P<0.05?,and the expressions of pThr231,pSer262 and pSer 396 were significantly higher?P<0.05?.Factorial analysis showed that CHIP interference and AlCl₃ exposure in N2a cells interacted with the expres-sion of CHIP,Ub and pThr231,pSer262 and pSer 396 proteins.3.CHIP??U-box?domain plasmid transfection in N2a cells3.1 Immunoblotting results showed that the expressions of CHIP,Ub,pThr231,pSer262 and pSer 396 protein were significantly higher than those of the control group?P<0.05?;the expression levels of pThr231,pSer262 and pSer 396 protein were significantly higher in CHIP??U-box?domain transfection?P<0.05?.3.2 Immunoprecipitation results showed that no interaction between Hsp70,Ub and tau protein pThr231,pSer262,pSer 396 phosphorylation sites and CHIP was detected by WB in CHIP??U-box?transfection group and CHIP??U-box?+1 mmol/L AlCl₃ group.Myc-CHIP was detected in all immunoprecipitation complexes.4.Transfection of CHIP?TPR?domain plasmid in N2a cells4.1 Immunoblotting results showed that compared with the control group,the expres-sion levels of CHIP,Ub,pThr231,pSer262,pSer 396 were significantly increased in the1mmol/L AlCl₃ group?P<0.05?;the expression levels of pThr231,pSer262 and pSer 396were significantly increased in the CHIP??TPR?domain transfection group?P<0.05?.4.2 Immunoprecipitation results showed that Hsp70 was detected by WB and interac-tion between Ub and CHIP was detected by anti-Myc immunoprecipitation complex.Myc-CHIP was detected in CHIP??TPR?plasmid transfection group,CHIP??TPR?+1mmol/L AlCl₃ group,and no interaction between pThr231,pSer262,pSer 396 and CHIP was detected in CHIP??TPR?plasmid transfection group,CHIP??TPR?+1 mmol/L AlCl₃ group.Conclusion:1.CHIP participates in the regulation of abnormal phosphorylation of tau protein in N2a cells induced by aluminum,and focuses on the regulation of pThr231,pSer262 and pSer396 sites of tau protein.2.Different domains of CHIP play important roles in the regulation of tau protein ab-normal phosphorylation in N2a cells induced by aluminum,and both of them are indispen-sable.