Effect And Mechanism Of Reelin Signal Transduction Pathway On The Abnormal Phosphorylation Of Tau Protein Induced By Aluminum In PC12 Cells

Aluminum（Aluminum,Al）is recognized as a strong neurotoxin,which can lead to a variety of neurodegenerative diseases（ND）such as Parkinson’s disease（PD）and Alzheimer’s disease（AD）.AD is a kind of dementia characterized by insidious onset and progressive progression,which is commonly seen in elderly people.In recent years,more and more evidences have shown that abnormal phosphorylation of tau protein was an important mechanism that constituted the characteristic pathological changes of AD,and abnormal phosphorylation of tau protein induced by aluminum was closely related to cognitive dysfunction in patients with AD.It was found that the levels of serum aluminum and abnormal tau protein phosphorylation were significantly increased in occupational workers with aluminum exposure,and the individual cognitive function was significantly decreased.Some experimental such as animals SD rats,zebrafish,rabbits,cats and others that exposed to Al could occur some changes in neural behavior,learning and memory dysfunction,cognitive decline and other neuropsychiatric symptoms similar to AD.In conclusion,Al is closely related to the abnormal phosphorylation of tau protein,but the exact mechanism has not been clarified.Even more remarkably,the protein Reelin,Apolipoprotein E receptor-2（Apo ER2）,Very low density lipoprotein receptor（VLDLR）and Disabled-1（Dab-1）protein were studied.The Reelin signal transduction pathway formed by phosphorylation of VLDLR and articular protein（Disabled-1）played an important role in regulating tau phosphorylation.A large number of studies have found that the decreased expression of Reelin protein significantly affects the abnormal phosphorylation of tau.And some classical experiments have also confirmed the presence of a large number of abnormal phosphorylation of tau in the brain of Reeler mice and Apo ER2/VLDLR knockout rats.In addition,in vivo and in vitro studies have found that aluminum can significantly reduce the expression of Reelin and induce abnormal expression of related components of Reelin signal transduction pathway.Taken together Reelin signal transduction pathway is highly likely to be an important mechanism of abnormal tau phosphorylation induced by aluminum,but few reports have been reported.In this study,rat adrenal pheochromocytoma-derived cells（PC12）were treated with Aluminum maltolate（Al（mal）₃）to prepare Aluminum neurotoxic cell model.To observe the effect of Al（mal）₃ on the phosphorylation of tau protein in PC12 cells.By adding Melatonin（MT）and Streptozotocin（STZ）,to observe the effect of changes in the expression of Reelin signal transduction pathway related molecules on the phosphorylation of tau protein.PartⅠStudy on the effects of exposure to aluminum maltolate on tau protein in PC12 cellsObjectiveTo investigate the effects of exposure to aluminum maltolate on the tau protein in PC12cells.MethodsRat adrenal pheochromocytoma（PC12）cells were treated with high-glucose DMEM medium with final concentrations of 0μmol/L,50μmol/L,100μmol/L,200μmol/L,and400μmol/L aluminum maltolate（Al（mal）₃）,respectively,for 12 h,24 h,and 48 h,the relative cell viability of PC12 was assessed by Cell Counting Kit-8（CCK-8）to determine the final dose and treated time of Al（mal）₃.PC12 cell viability was detected by CCK-8 assay.By using the method of Western blot（WB）,the protein expression levels of tau 5,P-tau231,P-tau262 and P-tau396 were determined.Results1.Determination of Al（mal）₃ dose and timeCompared with control,there were no significant changes in the cell viability of PC12cells among groups induced by different concentrations of Al（mal）₃ which exposure time was 12 h（P<0.05）;after 24 h and 48 h of Al（mal）₃ exposure,the cell viability of PC12 cells significantly decreased in a dose-dependent manner,and the cell viability of PC12 cells in200μmol/L Al（mal）₃ group and 400μmol/L Al（mal）₃ group significantly decreased（P<0.05）.Based on toxicological studies and related studies,cytotoxicity experiments are performed at concentrations of 70 to 80%for cell viability.The results of CCK-8 showed that the viability of PC12 cells exposed to 200μmol/L Al（mal）₃ for 24 h was（76.83±0.06）%,so the final determined dose was 200μmol/L Al（mal）₃,the duration of exposure was 24 h.2.Effects of Al（mal）₃ alone on the expression of total tau protein and the protein of each phosphorylation site in PC12 cellsCompared with the control group,the expression of tau5 protein in PC12 cells in Al（mal）₃ group increased（P<0.05）;Compared with the control group,the expression of P-tau396 protein in PC12 cells in Al（mal）₃ group was significantly increased（P<0.05）;Compared with the control group,the expression of P-tau262 in PC12 cells in Al（mal）₃group was significantly increased（P<0.05）;Compared with the control group,the expression of P-tau231 protein in PC12 cells in Al（mal）₃ group was significantly increased（P<0.05）.ConclusionAl（mal）₃ can cause overexpression of tau5 protein and tau protein excessive phosphorylation at Ser396,Thr231 and Thr262 sites in PC12 cells.PartⅡStudy on the effects of exposure to aluminum maltolate single-handled or in combination with the different interventions on the Reelin signal transduction pathway in PC12 cellsObjectiveTo investigate the effects of exposure to aluminum maltolate single-handled or in combination with the different interventions on the Reelin signal transduction pathway in PC12 cells.MethodsPC12 cells and treatment were same with those of partΙ.PC12 cells were divided into the following 7 groups:0μmol/L Al（mal）₃ group（control group）,200μmol/L Al（mal）₃group,100μmol/L Streptozotocin（STZ）group,50μmol/L Melatonin（MT）group,Al（mal）₃+STZ group,Al（mal）₃+MT group,0.5%Ethanol group（solvent control group）.MT was the agonist of Reelin and anhydrous ethanol was the solvent of MT.STZ was the antagonist.In the combined exposure group,PC12 cells were pretreated with specific intervention agent for 2h,and then Al（mal）₃ was added until the final concentration was 200μmol/L.After 24 h of culture,the shape and amounts of PC12 cells was observed by inverted microscope.PC12cell viability was detected by CCK-8 assay.Using the measures of Quantitative real-time PCR（q RT-PCR）,the m RNA expression levels of Reelin,VLDLR,Apo ER2,Dab-1 and GSK3βwere determined.Using the method of ELISA,the protein expression levels of Reelin,Dab-1 and GSK3βwere determined.The protein expression levels of VLDLR and Apo ER2 were detected by WB.Results1.Effect of Al（mal）₃ alone or combined with intervention agent on PC12 cell activity after 24 h exposureCompared with the control group,the cell viability of PC12 in 100μmol/L STZ group was not significantly changed（P<0.05）.Compared with control group,the cell viability of PC12 in 50μmol/L MT group was not significantly changed（P<0.05）.Compared with control group,the cell viability of PC12 cells in 0.5%Ethanol group was not significantly changed（P<0.05）.Compared with Al（mal）₃ group,there was significantly decreased in cell viability of PC12 in Al（mal）₃+STZ group（P<0.05）.The cell viability of PC12 in Al（mal）₃+MT group was significantly increased（P<0.05）.2.The effects of exposure to Al（mal）₃ on the Reelin signal transduction pathway in PC12 cellsCompared with control,treatment with Al（mal）₃ significantly downregulated the protein and m RNA expression of Reelin,Apo ER2,VLDLR and Dab-1 in PC12 cells（P<0.05）.After exposed to Al（mal）₃,the protein expression of GSK3βin PC12 cells was notably upregulated（P<0.05）.3.The effects of exposure to Al（mal）₃ in combination with the intervention on the Reelin signal transduction pathway in PC12 cellsCompared with control,the protein and m RNA expressions of Reelin,Apo ER2,VLDLR and Dab-1 were downregulated in PC12 cells treated with STZ（P<0.05）.The protein and m RNA exoressions of GSK3βwere notably upregulated（P<0.05）.Compared with Al（mal）₃,the protein and m RNA expressions of Reelin and VLDLR were notably downregulated in PC12 cells treated with Al（mal）₃+STZ（P<0.05）.Compared with Al（mal）₃,the protein and m RNA expressions of GSK3βwas notably increased in PC12 cells treated with Al（mal）₃+STZ（P<0.05）.Compared with control,the protein and m RNA expressions of Reelin,Apo ER2,VLDLR and Dab-1 were notably in PC12 cells treated with MT（P<0.05）.The protein exoression of GSK3βwas obviously downregulated in PC12 cells treated with MT（P<0.05）.Compared with Al（mal）₃,the protein and m RNA expressions of Reelin,Apo ER2,VLDLR and Dab-1 were obviously upregulated in PC12 cells treated with Al（mal）₃+MT（P<0.05）.Compared with Al（mal）₃,the protein and m RNA expressions of GSK3βwere obviously downregulated in the Al（mal）₃+MT group（P<0.05）.Compared with control,the protein and m RNA expressions of Reelin,Apo ER2,VLDLR and Dab-1 were no obvious changes in PC12 cells treated with Ethanol（P>0.05）.ConclusionAl（mal）₃ can cause a significant decrease in the expression of Reelin,Apo ER2,VLDLR and Dab-1 in PC12 cells,meanwhile the protein expression of GSK3βwas increased.When adding STZ,the expression of Reelin,VLDLR and Dab-1 were obviously decreased in PC12cells.Conversely,when adding MT,the expression of Reelin,Apo ER2,VLDLR and Dab-1were obviously increased in PC12 cells.As the signal initiator of Reelin signal transduction pathway,the expression changes of Reelin can also cause the changes of the expression of related receptors in Reelin signal pathway.PartⅢStudy on the mechanism of Reelin signaling pathway on abnormal phosphorylation of tau protein induced by aluminum in PC12 cellsObjectiveTo explore the effect and mechanism of Reelin signal transduction pathway on the abnormal hyperphosphorylation of tau protein induced by aluminum in PC12 cells.MethodsPC12 cells and treatment were same with those of partΙ.The m RNA expression of tau was determined by q RT-PCR.The protein expression levels of tau 5,P-tau231,P-tau262 and P-tau396 were detected by WB.Results1.The effects of exposure to Al（mal）₃ on the tau m RNA in PC12 cellsCompared with control group,the expression of tau m RNA of PC12 cells in Al（mal）₃group was significantly increased（P<0.05）.Compared with Al（mal）₃ group,the expression of tau m RNA of PC12 cells in Al（mal）₃+STZ group had no significant changes（P>0.05）;the expression of tau m RNA of PC12 cells in Al（mal）₃+MT group significantly decreased（P<0.05）.2.The effects of exposure to Al（mal）₃ in combination with the intervention on the tau protein in PC12 cellsCompared with control group,the protein of expression of tau5,P-tau396,P-tau262and P-tau231 was increased in Al（mal）₃ group（P<0.05）.Compared with control group,the m RNA of expression of tau5 was increased in Al（mal）₃ group（P<0.05）.There were no significant changes in the expression of tau5 protein in MT group（P>0.05）.Compared with Al（mal）₃ group,the expression of tau5,P-tau396,P-tau262 and P-tau231 had no significant changes in 0.5%Ethanol group（P>0.05）.Compared with control,the protein expressions of tau5,P-tau396,P-tau262 and P-tau231 were notably upregulated in PC12 cells treated with STZ（P<0.05）.Compared with Al（mal）₃ group,the protein expression of tau5,P-tau262 and P-tau231was significantly upregulated in PC12 cells treated with Al（mal）₃+STZ（P<0.05）.Compared with Al（mal）₃group,the expression of P-tau396 protein was increased in Al（mal）₃+STZ,but there were no statistically significant differences（P>0.05）.Compared with control,the protein expressions of tau5,P-tau396,P-tau262 and P-tau231 were notably downregulated in PC12 cells treated with MT（P<0.05）.Compared with Al（mal）₃ group,the protein expression of tau5,P-tau396,P-tau262 and P-tau231was notably upregulated in PC12 cells treated with Al（mal）₃+MT（P<0.05）.ConclusionThe abnormal expression of Reelin signal transduction pathway may be the important mechanism of the excessive phosphorylation of tau protein in PC12 cells caused by aluminum,which is as follows:Aluminum causes a decrease in Reelin expression,which in turn causes a decrease expression in VLDLR and（or）Apo ER2 and Dab-1.As follows,the expression of GSK3βwas increased,leading to tau protein phosphorylation.Conclusions:1.Aluminum can significantly increase the expression of tau5,P-tau396,P-tau262 and P-tau231 in PC12 cells.2.Aluminum can significantly decrease the m RNA and protein expressions of Reelin,VLDLR,Apo ER2 and Dab-1,and increase the protein expression of GSK3βin PC12 cells.3.Abnormal expression of Reelin signal transduction pathway played an important role in hyperphosphorylation of tau protein which was induced by aluminum.When STZ down-regulated the expression of Reelin,the expressions of Apo ER2,VLDLR and Dab-1 were decreased,and the expression of GSK3βwas increased,leading to a significant increase in the abnormal phosphorylation level of tau protein.When MT up-regulated the expression of Reelin,the expressions of Apo ER2,VLDLR and Dab-1 were significantly increased,the expression of GSK3βwas decreased,and the expression level of abnormal phosphorylation of tau protein was significantly decreased.