Study On The Mechanism Of PP2Ac Methylation Regulation In Manganese Induced N2A Cell Injury

ObjectiveMethionine,as an essential amino acid in human body,can promote the growth and protection of nervous system.lt not only has antioxidant effect,but also as a methyl donor.Methionine can affect DNA and protein methylation modification.lt has been suggested that manganese staining not only causes tau protein hyperphosphorylation in nerve cells but also promotes apoptosis.Tau protein phosphorylation homeostasis is regulated by protein krinases and protein phosphatases,many of which have been shown to be involved in manganese induced neurotoxicity.PP2A and its methylation modification in the protein phosphatase family have important significance in cell signal transduction and phosphorylation,thus affecting cell proliferation,differentiation and apoptosis,and are closely related to neurodegenerative diseases.However,whether PP2A and its met:hylation regulation are involved in mangane-induced neurotoxicity and its potential regulatory mechanism remains unclear.In this study,the protective effect of methionine on the regulation of PP2Ac methylation on Mn induced nerve cell injury and its regulatory mechanism were studied at the molecular level.MethodsNeuroblastoma cells(N2a)were divided into three groups and treated as follows:?In the methionine-free medium,N2a cells was treated with mang-anese alone for 24h and to establish tau protein hyperphosphorylation and apoptosis model;?N2a cells were treated with manganese and methionine for 24 h.?N2a nerve cells were incubated with manganese and PPME1 specific inhibitor ABL-127 for 24h.After the treatment,the morphological changes of cells were observed by inverted microscope.The activity of cells was detected by the method of blade azure.Cell apoptosis was detected by apoptosis kit.The expression of phosphorylated at different sites of tau protein in cells was analyzed by Western Blot;the methylation and demylation of PP2Ac proteins,the phosphorylation of PP2Ac proteins and the expression of LCMT1,PPME1 proteins were detected by Western B.lot.ROS and GSSG in cells were detected by reactive oxygen assay kit,GSH and GSSG assay kit.The contents of SAM and SAH in cells and the changes of SAM/SAH ratio were determined by HPLC.Results1.In the absence of methionine,manganese exposure alone can induce the damage of neuroblastoma cells:?Which is manifested as dose-dependent reduction of nerve cell activity and induction of apoptosis.?With the increase of manganese concentration,tau protein phosphorylation at sites Thsl99,Ser396,Ths 202 and Ser 404 increased in N2a cells,showing a dose effect relati-onship.Compared with the group without manganese,the difference was statistically significant(p<0.05).At the same time,manganese exposure can also induce the increase of PP2Ac demethylation,PPME1 and PP2Ac phosp-horylatedprotein expression in cells,while the decrease of PP2Ac methylation and LCMT1 protein expression.?Mn induced N2a cell injury is related to oxidative stress:with the increase of manganese concentration,fluorescence brightness and intracellular ROS content are increased,while reduced glu-tathione GSSG,and presents a dose effect relationship.The difference is statistically significant compared with the group without manganese(p<0.05).?With the increase of manganese concentration,the intracellular SAM content and SAM/SAH ratio decreased,and SAH increased.2.The effect and mechanism of methionine supplementation on manganese induced N2a nerve cell injury:?In the test concentration range(0?20mg/L),methionine has no toxic effect on cells,and cell activity increases with the increase of concentration,with 10mg/L promoting cell activity as the most obvious.Compared with the methionine deficient group,methionine supp-lementation protected the apoptosis of N2a cells induced by manganese.?At the same concentration of manganese exposure,methionine supplementation can reduce the expression of tau phosphorylated protein at sites Thsl99,Ser396,Ths202 and Ser404.Meanwhile,the decreased PP2Ac demethylation,PPME1 and PP2Ac phosphorylated protein expressions.while the PP2Ac methylation and LCMT1 protein expressions were significantly increased,with statistically significant differences(p<0.05).?Supplementation of methionine can reduce the fluorescence brightness and intracellular ROS content,while increase the content of intracellular antioxidant GSSG content.Compared with the meth-ionine deficiency group,the difference is statistically significant(p<0.05).?Methionine on methionine metabolism cycle impact results show:no manganese exposure,methionine supplement can increase the content of SAM in cells and SAM/SAH ratio,and reduce the SAH content;At 1000 ?mol/L manganese exposure,meth ionine reversed the manganese-induced decrease in SAM content and SAM/SAH values.Compared with the methionine deficiency group,the difference was statistically significant(p<0.05).3.The effect and mechanism of ABL-127 on tau protein hyper phosp-horylation and apoptosis in manganese induced nerve cells:?In the range of test concentration,ABL-127 treatment alone has no toxic effect on N2a cells,and with the increase of concentration,the cell activity increases,with 0.5nmol/L as the highest cell activity,and the difference is statistically significant(p<0.05).?The 0.5nmol/L ABL-127 concentration was used to interfere with the experiment of manganese staining in cells,and the results showed that ABL-127 supplementation could restrain the cell injury and apoptosis.?In the same concentration of manganese,Thr199,Ser396,Thr202,Ser 404 sites of tau protein phosphorylated protein expression decreased.The demethylation of PP2Ac,PPME1 and PP2Ac phosphorylated protein were decreased,while the methylation of PP2Ac,LCMT1 protein expression were increased,compared with the ABL-127 deficient group,the differences is statistically significant(p<0.05).?After the same concentration of manganese treatment,the addition of ABL-127 can reduce the cell fluorescence intensity and ROS content,showing a dose-effect relationship;At the same time,it can increase the content of GSSG in cells.Compared with the ABL-127 deficient group,the differences were statistically significant(p<0.05).Conclusions1.This study confirmed that methionine can reduce the nerve cells activity damage,tau protein hyperphosphorylation and cell apoptosis by manganese inducting.2.Methionine may regulate PP2Ac methylation through methionine cycle and antioxidant,and reduce abnormal phosphorylation of tau protein,oxidative stress and cell apoptosis by Mn causing.3.PPME1 inhibitor ABL-127 can directly inhibit PP2Ac demylation,increase the activity of PP2A,and thereby reducing Mn induced abnormal phosphorylation of tan protein,oxidative stress and cell apoptosis.of manganese induced nerve cells N2a.