The Effect Of P25/Cdk5-related Signaling Pathway In Tau Protein Hyperphosphorylation In SH-SY5Y Cells Induced By Aluminum

Objectives: To study the effects of aluminum on the level of tau phosphorylation in SH-SY5Ycells，and the role of p25/Cdk5-related signal pathways in the process of tau phosphorylationinduced by aluminum.Methods:1. To establish the Al-induced neuronal damage cell model in cultured SH-SY5Y cells. Theexperiments was divided into four groups: blank control group, 200μM of AlCl₃group, 400μMof AlCl₃group, 800μM of AlCl₃group. After 48 hours exposure, the CCK-8 kit detection wasused to determine the cell viability; the content of the total tau protein , Tau [pS396], Tau[pT231], Tau[pT181] were detected by ELISA; the concentration of intracellular Ca2+wasobserved by flow cytometry after Flu-3AM labeled the target cells. Western-blot was used todetect the p25/p35 protein levels and ELISA to detect the content of calpain and Cdk5;2. After using CDK5-specific inhibitor: Roscovitine to antagonist Cdk5,the content of celltotal tau protein, Tau [pS396], Tau [pT231], Tau[pT181] were detected by ELISA in order toanalysis their effects to tau phosphorylation.Results:1. CCK8 detection shows: with the concentration of aluminum chloride increased, theSH-SY5Y cell viability decreased gradually. The cell viability of 400μM of AlCl₃and 800μM OfAlCl₃groups were significantly lower than the blank control group （P<0.05）. Under the lightmicroscope observation, with the role of the aluminum chloride concentration increased, theSH-SY5Y cell damage also gradually increased.2. The total tau protein and phosphorylation of tau protein results shows: the contents of thetotal tau protein of all groups have no difference. Compare with the control group, thephosphorylation level of tau protein at Ser396,Thr231 and Thr181 sites of the 200μM, 400μMand 800μM of AlCl₃groups was significantly higher （P<0.05）.3. p25/Cdk5 signaling pathway results displays: the concentration of intracellular calciumwas significantly increased followed by the aluminum chloride increased （P<0.05）; the contents of Cdk5 and calpain were significantly increased （P<0.05）. Using Western-blot to analysis theexpression of related protein, the results showed that p25/p35 protein expression levels of800μM ofAlCl₃group was significantly higher than the control group （P <0.05）.4. There was neither toxic effects nor morphological damage on SH-SY5Y when theRoscovitine concentrations was 10μM. After Roscovitine antagonistic Cdk5, the phosphorylationlevel of tau protein at ser396,Thr231 and Thr181 sites of SH-SY5Y exposure by aluminumchloride was significantly lower than 400μM of AlCl₃group （P <0.05）.Conclusions:1. Aluminum chloride can reduce the SH-SY5Y cell viability and it can cause toxicity toSH-SY5Y cells.2. Aluminum chloride enables to cause excessive phosphorylation of the SH-SY5Y cells tauprotein at Ser396, Thr231 and Thr181 sites.3. The mechanism of the excessive phosphorylation of tau protein in SH-SY5Y caused byaluminum may be relevant with the p25/Cdk5 related signaling pathways: After aluminum goesinto the cells, the intracellular calcium concentration increases, so as the calpain, which canupgrade the expression of p25 protein, then actives Cdk5, leading to the tau proteinhyperphosphorylation.