The Study Of Ubiquitin-proteasome Pathway Regulates Aluminum-induced Hyperphosphorylated Tau Protein In Vitro

Objective:1.To investigate whether the ubiquitin-proteasome pathway and GSK-3?,PP2A are involved in the regulation of abnormal phosphorylation of tau protein by aluminum trichloride.2.To study whether the ubiquitin-proteasome pathway regulates the abnormal phosphorylation of tau caused by aluminum trichloride in cells of different species.Methods:1.Mouse-derived N2a cell line and human-derived SH-SY5Y cell line were selected as experimental materials and treated with trichloride aluminum,with aluminum ion concentrations of 0.5,1,and 2 mmol/L as low-dose groups,the middle-dose group and high-dose group respectively and no treatment in the control group.In the logarithmic phase of cell growth,the cell morphology was observed under an optical microscope after 24 hours of exposure,cell viability was measured using cck-8,and cell samples were collected,and total protein was extracted.The expression of tau-5,pThr181,pThr231,pSer262,pSer396,Hsp70 and CHIP proteins in N2a and SH-SY5Y cells were detected by Western blotting,and the expression of ubiquitin?Ub?,GSK-3?and PP2A were detected by ELISA,respectively.2.N2a and SH-SY5Y cells were treated with proteasome inhibitor MG132?5?mol/L?for 6h,and exposed to trichloride aluminum,divided into control group,MG132?5?mol/L?group,1mmol/LAl³⁺group,MG132?5?mol/L?+1 mmol/LAl³⁺group.The expression of tau-5,pThr181,pThr231,pSer262,pSer396,Hsp70 and CHIP proteins in N2a and SH-SY5Y cells were detected by Western blotting.The expression of ubiquitin?Ub?in N2a and SH-SY5Y cells were detected by ELISA.Results:1.Observation under light microscope showed that with the increase of exposure dose,the both of cell numbers decreased gradually,the synapse retracted,and the cell bodies became round.Cell viability assay results showed that:?1?With the increase of exposure dose,the viability of N2a cells gradually decreased.The cell viability of the medium and high dose groups was significantly lower than that of the control group?P<0.05?.?2?With the increase of exposure dose,the viability of SH-SY5Y cells gradually decreased.The cell viability of the medium and high dose groups was significantly lower than that of the control group?P<0.05?.2.The results of total tau and phosphorylated tau after treatment with different concentrations of trichloride aluminum solution for 24h show that:?1?As the concentration of aluminum increases,the expression of tau-5?total tau??pSer262 and pSer396 protein was gradually increased,and the expression levels of various proteins in the medium and high dose groups were significantly higher than those in the control group?P<0.01?.The expression of pThr231 protein in the high dose group was significantly higher than that in the control group?P<0.01?;and the expression of pThr181 protein in N2a cells did not change significantly with the increase of the concentration of aluminum?P>0.05?.?2?With the increase of aluminum dosage,the expression levels of tau-5?total tau?,pThr181,pThr231,and pSer396 proteins in SH-SY5Y cells showed a tendency to increase,and the expression levels of each protein in the medium and high dose groups were significantly higher than the control?P<0.05?.The expression level of pThr262 protein in SH-SY5Y cells did not change significantly with the increase of the concentration of aluminum?P>0.05?.3.The results of GSK-3?and PP2A detection after treatment with different concentration of aluminum chloride solution for 24h showed that:?1?With the increase of aluminum concentration,the expression of GSK-3?protein in N2a cells did not change significantly?P>0.05?;the level of PP2A protein gradually decreased,and the PP2A expression level in the high-dose group was significantly lower than that in the control group?P<0.01?.?2?With the increase of aluminum concentration,the expression of GSK-3?protein in SH-SY5Y cells was gradually increased,and the expression level of GSK-3?in high-dose group was significantly higher than that in the control group?P<0.01?.However,the level of PP2A protein gradually increased.The expression of PP2A in the lower,middle and high dose groups was significantly lower than that in the control group?P<0.05?,and the PP2A expression level in the high dose group was significantly lower than that in the low dose group?P<0.01?.4.After treatment with different concentration of aluminum chloride solution for24h,the detection results of key factors of ubiquitin-proteasome pathway showed that:?1?With the increase of aluminum concentration,the expression of Ub,CHIP and Hsp70 protein in N2a cells gradually Increased.The expression of CHIP protein was significantly higher in the medium and high dose groups than in the control group?P<0.01?.The levels of CHIP protein in the medium and high dose groups were significantly higher than those in the low dose group?P<0.05?;the Ub and Hsp70proteins in the medium and high dose groups The expression level was significantly higher than that of the control group?P<0.05,P<0.01?.?2?With the increase of aluminum concentration,the expression of Ub,CHIP and Hsp70 protein in SH-SY5Y cells increased gradually.The expression levels of each protein in the medium and high dose groups were significantly higher than those in the control group?P<0.05?;the expression levels of CHIP and Hsp70 protein in the high dose group were higher than those in the low dose group?P<0.05?;the Ub protein expression levels were significantly higher in the high dose group Higher than low dose group?P<0.01?.5.Proteasome inhibitor MG132 was used as treating cells in logarithmic growth phase for 6h,and then treated with 1mmol/LAl³⁺for 24h.Western blot results showed that:?1?In N2a cells,the expression of tau-5,pThr231,pSer262 and pSer396 protein in N2a cells treated with proteasome inhibitor MG132 was significantly higher than that in the control group?P<0.05?,but the pThr181 protein was not significantly different from that in the control group?P>0.05?;Hsp70 protein expression level was significantly higher than the control group?P<0.01?,Ub,CHIP protein expression levels compared with the control group did not change significantly?P>0.05?.The expression levels of tau-5,pSer262 and pSer396 in MG132?5?mol/L?+1mmol/LAl³⁺group were significantly higher than those in 1mmol/L Al³⁺group?P<0.05?,while the expression levels of pThr181 and pThr231 were similar to 1mmol/L.There was no significant difference in the Al³⁺group?P>0.05?;the Ub,CHIP and Hsp70 protein expression levels were significantly higher than those in the 1 mmol/L Al³⁺group?P<0.05?.?2?In SH-SY5Y cells,the expression levels of pThr181,pThr231,and pSer396proteins in SH-SY5Y cells after treatment with proteasome inhibitor MG132 were significantly higher than those in the control group?P<0.05?,but the expression levels of tau-5 and pSer262 protein were not changed compared with the control group?P>0.05?;CHIP protein expression was significantly higher than the control group?P<0.01?,Ub,Hsp70 protein expression was significantly higher than the control group?P<0.05?.The expression levels of tau-5,pThr231 and pSer396 in MG132?5?mol/L?+1 mmol/LAl³⁺group were significantly higher than those in 1mmol/L Al³⁺group?P<0.05?,while the expression levels of pThr181 and pSer262protein were changed with 1 mmol/L.There was no significant difference in the Al³⁺group?P>0.05?.The Ub,CHIP and Hsp70 protein expression levels were significantly higher than those in the 1 mmol/L Al³⁺group?P<0.05?.Conclusion:1.GSK-3?and PP2A participate in the synthesis of tau protein abnormal phosphorylation induced by aluminum trichloride.PP2A was predominant in N2a cells,while GSK-3?and PP2A in SH-SY5Y cells were involved in the synthesis of tau hyperphosphorylation induced by aluminum trichloride.2.The ubiquitin-proteasome pathway is involved in the degradation of tau phosphorylation induced by aluminum trichloride,but it is site-specific in cells of different species origin.In N2a cells,UPP mainly regulates Ser262 and Ser396phosphorylation sites,while in SH-SY5Y cells,UPP mainly regulates Thr231 and Ser396 phosphorylation sites.