| ObjectiveManganese is an essential trace element to maintain the function of human body.Long-termenvironmental exposure or occupational excessive exposure to manganese can causenormal cell function abnormal,oxidativestress,mitochondrial dysfunction,autophagy disorder,nerve function damage and neurodegenerative diseases.Autophagy is a highly conserved life process in eukaryotic cells,which can remove misfolded proteins,damaged organelles and maintain homeostasis of the internal environment in cells.Abnormal autophagy is closely related to neurodegeneration.mTOR and ULK1 are the key molecules in the autophagy signaling pathway.Phosphorylation of mTOR directly negatively regulates ULK1,and the decrease of phosphorylated ULK1 leads to the inhibition of autophagy.PP2 A is the most important serine/threonine protein phosphatase in eukaryotic cells that regulates various physiological processes such as autophagy,apoptosis,and cell cycle by its proteindephosphorylation.Catalytic subunit C methylation offers its synthase activity.The methylation modification of PP2 Ac is regulated by leucine carboxymethyl transferase 1(LCMT1)and protein methyl esterase(PPME-1),respectively.However,whether PP2 A and its methylation are involved in Mn-induced nerve cell injury,oxidative stress and abnormal autophagy,as well as the underlying mechanisms are still unclear.In this study,we constructed avitro model of manganese exposure to N2 a cells to study the effect of manganese exposure on autophagy of nerve cells,oxidative stress,and changes of PP2 Ac methylation.PPME inhibitor(ABL-127),antioxidant acetylcysteine(NAC)and LCMT1over-expression cell line were applied to explore the mechanismof PP2 Ac methylation in Mn-inhibited nerve cells autophagy.Methods1.Detection of autophagy induced by Mn in N2 a cells.N2a cells were exposed to different concentrations of Mn(0～2000 μmol/L)for 2 h,4 h,6 h,8 h,12 h,and 24 h.Lysosome probe was diluted proportionally,and the number and distribution of lysosomes in N2 a cells were observed and photographed by fluorescence microscope.The fluorescence intensity of lysosomal staining in N2 a cells was measured with an enzyme marker after treated with Mn for 6 h,12 h,24 h.and quantified the results to determine the time/concentration-effect.The cell viability of N2 a cells treated with Mn was detected by Alama Blue.The expressions of autophagy marker proteins(p62,LC3-Ⅱ/I),PP2Ac-related proteins(PP2Ac,demethylated PP2 Ac,methylated PP2Ac)and mTOR-related proteins(mTOR,pmTOR,ULK1,p ULK1)were detected by Western blot in N2 a cells.2.ABL-127 intervention alleviated Mn-inhibited autophagy in N2 a cellsThe cell activity was measured by Alama Blue after incubation with 0.5μM ABL-127 and Mn for 12 h.The expressions of autophagy marker proteins(p62,LC3-Ⅱ/I),PP2Ac-related proteins(PP2Ac,demethylated PP2 Ac,methylated PP2Ac)and mTOR-related proteins(mTOR,pmTOR,ULK1,p ULK1)were detected by Western blot in N2 a cells.3.The effect of LCMT1 overexpression on Mn-inhibited autophagy in N2 a cells.PCDNA3.0 and PCDNA3.0-LCMT1 mouse gene plasmids were synthesized fromthe Miaoling plasmid platform.DH5α competent cells were used to transformand amplify the plasmids.We transferred the plasmids into N2 a cells,harvested and identified the LCMT1-PCDNA3.0 N2 a cell line and PCDNA3.0 N2 a cell line.The cell viability was measured by Alama Blue after PCDNA-N2 a cells and LCMT1-N2 a cells were exposure to Mn for 12 h.The expressions of autophagy marker proteins(p62,LC3-Ⅱ/I),PP2Ac-related proteins(PP2Ac,demethylated PP2 Ac,methylated PP2Ac)and mTOR-related proteins(mTOR,pmTOR,ULK1,p ULK1)were detected by Western blot in PCDNA-N2 a cells and LCMT1-N2 a cells.4.Effect of antioxidant(NAC)on Mn-inhibited autophagy in N2 a cells.ROS assay kit was used to detect the intracellular ROS levels of individual Mn treated groups,ABL-127,NAC intervention groups in N2 a cells and PCDNA-N2 a cells and LCMT1-N2 a cells.The cells were pretreated with 1 mM NAC 2 h before exposure to Mn for 12 h,the cell viability was detected by Alama Blue,and the expressions of autophagy marker proteins(p62,LC3-Ⅱ/I)were detected by Western blot.Results1.Autophagy inhibition in N2 a cells exposure to Mn:(1)With the increase of Mn concentration,the morphology of N2 a cells shrinked and the number of protrusions decreased.The viability of N2 a cells showed a dose-dependent decreasing trend,compared with the control group,the difference was statistically significant(P<0.05).(2)Mn exposure led to lysosome production increase,with the increase of Mn exposure concentration and time,the numeber of lysosomes increased.Compared with the control group,the difference was statistically significant(P<0.05).(3)Different concentrations of chloroquine intervened N2 a cells for 2 h and 4 h,the most suitable treatment concentration was 50 μmol/L,and the most suitable intervention time was 2 h.(4)Mn exposure causes the expression of phosphorylation mTOR,p62,LC3-Ⅱ/I increase,the expression of p ULK1 decreased,compared with control group,P<0.05,the difference was statistically significant.(5)Compared with the Mn alone group,the expression levels of autophagy marker protein p62 and LC3-Ⅱ/I increased in chloroquine intervention group,P<0.05,the difference was statistically significant.(6)Mn exposure decreased the expression of PP2 Ac methylated protein and increased the expression of demethylated PP2 Ac in N2 a cells.Compared with the control group,P<0.05,the difference was statistically significant.2.ABL-127 intervention alleviated Mn-inhibited in N2 a cells:(1)ABL-127 can protect Mn-induced N2 a cell damage,compared with the Mn alone group,the cell morphology was better at high concentrations,and the number of normal cells or protrusions reduced less;Except for the 0 μmol/L Mn group,the cell survival rate of the ABL intervention group increased,P<0.05,the difference was statistically significant.(2)Compared with the Mn alone group,the expression of phosphorylation mTOR,p62,LC3-Ⅱ/I decreased and the expression of p ULK1 increased in the ABL-127 intervention group,P<0.05,the difference was statistically significant;at the same time,the total PP2 Ac protein expression remained unchanged,demethylated PP2 Ac expression decreased,PP2 Ac methylated protein expression increased,P<0.05,the difference was statistically significant.3.Effects of LCMT1 overexpression on Mn-induced N2 a cell injury and autophagy:(1)Compared with the PCDNA-N2 a group,the LCMT1 gene expression level increased in LCMT1-N2 a group,LCMT1,methylated PP2 Ac protein expression level increased,and demethylated PP2 Ac protein expression level decreased in LCMT1-N2 a group,P<0.05,the difference was statistically significant.(2)Compared with the PCDNA-N2 a group,the cell morphology was better at high concentrations,and the number of normal cells or protrusions reduced more slowly;Except for the 0 μmol/L Mn group,the cell survival rate of the LCMT1-N2 a group increased,P<0.05,the difference was statistically significant,the above were consistent with the trend of ABL-127 intervention results.(3)Compared with the PCDNA-N2 a group,the expression ofphosphorylation mTOR,p62,LC3-Ⅱ/I decreased and the expression of p ULK1 increased in the LCMT1-N2 a group,P<0.05,the difference was statistically significant;at the same time,the total PP2 Ac protein expression remained unchanged,demethylated PP2 Ac protein expression decreased,methylated PP2 Ac protein expression increased,P<0.05,the difference was statistically significant,the above were consistent with the trend of ABL-127 intervention results.4.Oxidative stress participation in PP2 Ac methylation to regulate Mn-inhibited cell autophagy:(1)N2a cells were treated with NAC at different concentrations for 2 h.When NAC concentration was 2,4,8 and 10 mmol/L,N2 a cell activity was increased,P<0.05,the difference was statistically significant,and 1 mM NAC was the most suitable concentrationin N2 a cells.(2)Compared with the Mn alone group,except for the 0 μmol/L Mn group,the cell survival rate of the NAC intervention group increased,P<0.05,the difference was statistically significant.(3)The ROS level of N2 a cells increased after exposure to Mn for 12 h,and there was a dose-response relationship between the concentration of Mn and the ROS level in N2 a cells(P<0.05),the difference was statistically significant.Compared with the Mn alone group,except for the 0μmol/L Mn group,the intracellular ROS leveldecreasedin ABL-127 intervention group,P<0.05,the difference was statistically significant;Compared with the Mn alone group,except for the 0 μmol/L Mn group,the intracellular ROS level decreasedin NAC intervention group,P<0.05,the difference was statistically significant,Compared with the PCDNA-N2 a group,the intracellular ROS level decreasedin LCMT1-N2 a group,P<0.05,the difference was statistically significant.(4)Compared with the Mn alone group,the expressions of p62,LC3-Ⅱ/I decreased in NAC intervention group,P<0.05,the difference was statistically significant.Conclusions1.Manganese exposure inhibits N2 a cell autophagy,which is related to the activation of mTOR/ULK1 signaling pathway.Manganese decreased PP2 Ac methylation and increased demethylation.2.Inhibition of PP2 Ac demethylation by ABL-127 and upregulation of PP2 Ac methylation by LCMT1 overexpression can alleviate the inhibition of Mn-induced autophagy in N2 a cells.3.Inhibition of N2 a autophagy by PP2 Ac demethylation modification is related to the increase in ROS induced by manganese.ABL-127,NAC intervention,and LCMT1 overexpression all can reduce the cell damage and oxidative stress caused by Mn.The antioxidant NAC can alleviate the autophagy of N2 a cells inhibited by manganese. |
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