Effects And Underlying Mechanisms Of ?-Synuclein On Iron Metabolism-Related Proteins In MES23.5 Cell And Astrocytes

Parkinson's disease(PD)is the second common neurodegenerative disease.In China,the prevalence of PD is about 1.7% in people over 65 years old.The main clinical manifestations of PD are rest tremor,bradykinesia,rigidity and postural instability.Many studies have shown that oxidative stress,mitochondrial dysfunction,inflammation,endoplasmic reticulum(ER)stress,and abnormal iron accumulation may cause damage to dopaminergic neurons,but the exact mechanism is still unclear.The main neuropathological hallmark of PD is the degeneration of dopaminergic neurons in the substantia nigra(SN)and the formation of lewy body(LB).As the most important neuropathological manifestation of PD,aggregated ?-synuclein constitutes the main component of LB,also a key factor in the pathogenesis of PD.ER stress is one of the core toxic mechanisms of ?-synuclein.In PD patients,aggregation of ?-synuclein is often accompanied by iron deposition.Previous studies in our laboratory had demonstrated that oxidative stress induced by iron promoted phosphorylation and abnormal aggregation of ?-synuclein in vitro,which could be partially blocked by antioxidants.Iron regulatory proteins(IRPs)can upregulate the expression of ?-synuclein by acting on iron response element(IRE)of its mRNA(it was supposed to be a functional IRE);Degradation of ?-synuclein was inhibited by iron-mediated blockage of the nuclear transcription factor 2(Nrf2)/heme oxygenase(HO-1)pathway.Cells overexpressing ?-synuclein were more susceptible to iron damage,whereas cells with ?-synuclein knockdown were resistant to iron induced oxidative stress.These evidences indicated a close relationship between intracellular iron deposition and?-synuclein.Alpha-synuclein can spread between neurons and other types of cells in a "prion-like" manner,contributing to its pathological propagation.Extracellular ?-synuclein can be taken by adjacent neurons,which disrupts mitochondrial membrane potential,induces apoptosis,also activates glial cells to release large amounts of pro-inflammatory factors and further damage neurons.There are several mechanisms of ?-synuclein transmission between cells:intracellular ?-synuclein can bind to the cell membrane to form vesicles for secretion outside the cell,and then can be degraded by proteases and lipases to release ?-synuclein into the extracellular matrix.Extracellular ?-synuclein can be transmitted in adjacent cells via endocytosis and receptor-mediated internalization.The spread of intercellular ?-synuclein can also establish tunnel nanotubes between cells.However,the effect of ?-synuclein transmitted between cells on cellular iron metabolism has not been reported.In this study,western blots were used to detect iron metabolism-related protein expression,includingdivalent metal transposter1(DMT1),ferroportin 1(FPN1),and iron regulatory protein 1/2(IRP1/2);real-time quantitative PCR was used to detect the expression of hepcidin mRNA in different concentrations of ?-synuclein-treated MES23.5 dopaminergic cells.MES23.5dopaminergic cells were pretreated by the endocytic inhibitor Dynasore,the autophagy-inducer Rapamycin,the ER-activator thapsigargin(TG)and the ER stress blocker Salubrinal to detect ER stress-related protein C/EBP homologous protein(CHOP),X-box binding protein 1s(XBP-1s),activation of transcription factor 4(ATF-4),phosphorylated eukaryotic translation initiation factor2?(p-eIF2?),and cAMP-response element binding protein(CREB)to elucidate the effects of extracellular ?-synuclein on iron metabolism-related proteins and to explore whether these effects were associated with ER stress.The results were shown as follows:1.The cell activity assay kit(Cell Counting Kit-8,CCK8)results showed that the cell viability remained unchanged when MES23.5 cells were treated for 24 h with 0-20 ?g/ml?-synuclein.1 ?g/ml or 5 ?g/ml ?-synuclein that had no effect on cell viability were chosen for the following experiments.2.MES23.5 cells were treated with 1 ?g/ml or 5 ?g/ml ?-synuclein for 24 h.DMT1 protein levels were up-regulated(32.26%)in 5 ?g/ml ?-synuclein-treated group,although there was an upward trend in 1 ?g/ml ?-synuclein-treated group compared with the control group(P<0.01);FPN1 protein levels were down-regulated(25.14% and 26.75%),respectively,in 1?g/ml and 5 ?g/ml ?-synuclein-treated group compared with the control group(P<0.01).The results indicated that ?-synuclein can up-regulate DMT1 protein expression and down-regulate FPN1 protein expression.3.MES23.5 cells were treated with 1 ?g/ml or 5 ?g/ml ?-synuclein for 24 h.Hepcidin mRNA levels were up-regulated(3.05 folds)in 5 ?g/ml ?-synuclein-treated group,although there was an upward trend in 1 ?g/ml ?-synuclein-treated group compared with the control group(P<0.01).The results indicated that ?-synuclein can up-regulate hepcidin mRNA expression.4.MES23.5 cells were treated with 1 ?g/ml or 5 ?g/ml ?-synuclein for 24 h.IRP1 protein levels were up-regulated(25.47%)in 5 ?g/ml ?-synuclein-treated group,although there was an upward trend in 1 ?g/ml ?-synuclein-treated group compared with the control group(P<0.01).IRP2 protein expression were up-regulated(37.94% and 60.27%),respectively,in1 ?g/ml and 5 ?g/ml ?-synuclein-treated group compared with the control group(P<0.01).The results indicated that ?-synuclein can up-regulate the expression of IRP1 and IRP2 proteins.5.The results of CCK8 showed that the cell viability was significantly decreased whenMES23.5 cells were treated for 24 h with 50 ?M Dynasore,however,the cell viability was unchanged when MES23.5 cells were treated with 10 ?M Dynasore.MES23.5 cells were pretreated with Dynasore(10 ?M)for 30 min and then treated with ?-synuclein(5 ?g/ml)for24 h.Compared with the ?-synuclein-treated group,the levels of hepcidin mRNA,IRP1 protein,IRP2 protein and DMT1 protein were significantly down-regulated,and FPN1 protein levels were significantly up-regulated in the ?-synuclein/Dynasore group(P<0.01).Compared with the control group,hepcidin mRNA levels were unchanged;IRP1 protein levels were significantly down-regulated;IRP2 protein levels were unchanged;DMT1protein levels were significantly down-regulated,suggesting that Dynasore can completely block ?-synuclein-induced expression of hepcidin mRNA,IRP1 protein,IRP2 protein and DMT1 protein.Compared with the control group,FPN1 protein leves were down-regulated in ?-synuclein/Dynasore group,suggesting that Dynasore could not completely block?-synuclein-induced FPN1 down-regulation.The levels of hepcidin mRNA and iron-metabolism-related proteins were unchanged with Dynasore treatment.6.The results of CCK8 showed that the cell viability remained unchanged when MES23.5cells were treated with 100 nM rapamycin for 24 h.MES23.5 cells were pretreated with rapamycin(100 nM)for 30 min and then treated with ?-synuclein(5 ?g/ml)for 24 h.Hepcidin mRNA levels were up-regulated with ?-synuclein treatment.Compared with?-synuclein group,the hepcidin mRNA expression levels further were up-regulated,the protein levels of IRP1 and DMT1 were down-regulated significantly,FPN1 protein levels were up-regulated in rapamycin/?-synuclein-treated group.Compared with the control group,the mRNA levels of hepcidin were significantly up-regulated,IRP2 protein levels were unchanged,IRP1 protein and DMT1 protein levels were significantly down-regulated FPN1 protein levels were significantly up-regulated in ?-synuclein/rapamycin-treated group(P<0.01).7.MES23.5 cells were treated with 1 ?g/ml or 5 ?g/ml ?-synuclein for 24 h.ER stress-related proteins,CHOP,ATF-4 and XBP-1s levels were up-regulated in the 1 ?g/ml?-synuclein group(41.47%,48.97% and 61.51%),CHOP,ATF-4 and XBP-1s levels were up-regulated in the 5 ?g/ml treatment group(39.66%,1.54 folds,58.31% and 20.30%),respectively,compared with control group(P<0.01).The above results indicated that extracellular ?-synuclein can induce ER stress in cells.8.MES23.5 cells were treated with 0.1 ?M and 1 ?M TG for 24 h.Compared with the control group,CHOP protein levels were up-regulated(37.36% and 71.15%),XBP-1s protein levels were up-regulated(96.02% and 1.55 folds),respectively,p-eIF2? protein levels were up-regulated(34.75%)in 1 ?M TG group(P<0.01),which suggested TG can cause intracellular ER stress.Compared with the control group,the hepcidin mRNA levels were up-regulated(3.28 folds and 3.7 folds),IRP1 protein levels were up-regulated(3 foldsand 5.4 folds)in the 0.1 ?M and 1 ?M TG-treated-groups,the IRP2 protein was up-regulated(46.23%)in the 0.1 ?M group,DMT1 protein levels were up-regulated(53.59% and 68.17%)and FPN1 protein levels down-egulated(20.41% and 52.26%)in the 0.1 ?M and 1 ?M TG-treated-groups(P<0.01).The above results indicated that ER stress was involved in the regulation of iron metabolism-related proteins.9.The results of CCK8 showed that the cell viability was unchanged when MES23.5 cells were treated for 24 h with Salubrinal(10 ?M).MES23.5 cells were pretreated with Salubrinal(10 ?M)for 30 min and then treated ?-synuclein(5 ?g/ml)for 24 h.Compared with ?-synuclein group,CHOP protein levels were significantly down-regulated,hepcidin mRNA,IRP1 protein and IRP2 protein levels were significantly down-regulated in the?-synuclein/Salubrinal-treated group.Compared with the control group,CHOP protein,IRP1 protein and IRP2 protein levels were unchanged,the hepcidin mRNA levels were significantly up-regulated in the ?-synuclein/Salubrinal group.These results indicated that Salubrinal can block ER stress,as well as the up-regulation of IRP1 and IRP2 caused by?-synuclein.However,the up-regulation of hepcidin mRNA levels could not be fully blocked.These results suggested ER stress was involved in the regulation of hepcidin and IRPs induced by ?-synuclein.10.MES23.5 cells were treated with 1 ?g/ml or 5 ?g/ml ?-synuclein for 24 h,p-CREB protein levels were up-regulated(98.18%)in 5 ?g/ml ?-synuclein-treated group,although there was an upward trend in 1 ?g/ml ?-synuclein-treated group compared with the control group(P<0.05).MES23.5 cells were pretreated with Salubrinal(10 ?M)for 30 min and then treated ?-synuclein(5 ?g/ml)for 24 h.p-CREB protein levels were significantly down-regulated,compared with the ?-synuclein-treated group.The above results indicated that CREB phosphorylation might be partially involved in the ER stress-mediated IRPs regulation induced by ?-synuclein.11.Astrocyte cells were treated with 1 ?g/ml or 5 ?g/ml ?-synuclein for 24 h.Hepcidin mRNA,IRP1,IRP2,DMT1,FPN1 protein levels were unchanged in 1 ?g/ml and 5 ?g/ml?-synuclein-treated group compared with the control group.These results indicated that extracellular ?-synuclein could up-regulate the expression of DMT1 and down-regulate the expression of FPN1 in MES23.5 cells.Hepcidin mRNA levels,as well as IRP1 and IRP2 protein levels were also up-regulated,suggesting that extracellular ?-synuclein might regulate DMT1 and FPN1 protein expression by hepcidin and IRPs.Dynasore blocked a-synuclein-induced hepcidin and IRPs up-regulation.The autophagy inducer rapamycin can block the up-regulation of IRPs induced by ?-synuclein,however,hepcidin mRNA levels were further increased,suggesting that autophagy might affect the levels of hepcidin mRNA.These results suggested that extracellular a-synuclein can enter the cell causing the above changes.ER stress-related proteins CHOP,ATF4,XBP-1s and p-eIF2? proteins were up-regulated in ?-synuclein-treated MES23.5 cells,and the results were consistent with the effects of ER stress inducer TG.The ER stress blocker Salubrinal also blocks the up-regulation of hepcidin and IRPs caused by ?-synuclein.Phosphorylation of CREB could be partially blocked by Salubrinal,suggesting that it might be involved in the regulation of IRPs by ER stress.According to the results of the blocking experiments,the above results indicate that extracellular ?-synuclein may enter the cell,induce ER stress and up-regulate hepcidin and IRPs,partially mediated by CREB phosphorylation.This study explored the relationship between iron metabolism-related proteins and extracellular ?-synuclein in MES23.5 cells,providing new experimental evidence for further elucidation of the mechanism of iron metabolism and ?-synuclein interactions in the pathogenesis of PD.