The Role Of α-synuclein In Iron Metabolism Of BV2 Microglia Cell And Its Molecular Mechanism

Parkinson’s disease(PD)is a neurodegenerative disease of the central nervous system,which is characterized by bradykinesia,rigidity,resttremor and postural instability.The main pathologic features of PD are the damage of dopaminergic(DA)neurons in substantia nigra pars compactor(SNpc),and the presence of the acid inclusion body(lewy body,LBs)which α-synuclein(α-Syn)is the main component.Although the genetic,environmental and oxidative stress play a role,the exact pathogenesis of PD has not yet fully understood.In the pathogenesis of PD,iron plays a key role.Excessive deposition of iron may be involved in the PD DA neurons loss.Studies have shown that iron import protein divalent metal-ion transporter-1(DMT1)is highly expressed in the substantia nigra(SN)of PD patients and PD animal models,suggesting that abnormal expression of DMT1 may lead to iron accumulation in the region,eventually leading to neuron death.Ferroportin-1(FPN1)is currently known as the only iron export protein,FPN1 disregulation can cause cellular iron accumulation.Iron regulatory proteins(IRPs)are the most important factors in the regulation of post-transcriptional regulation of iron metabolism-related proteins and can bind to iron response element(IRE)of DMT1 and FPN1 m RNAs,thus regulating their expressions.Hypoxia-inducible factors(HIFs)are important factors of iron homeostasis.In addition to hypoxia,many other stimuli can activate HIFs,which combine with hypoxic response elements(HRE)of iron metabolism-related proteins to form transcriptional complexes that regulate their expressions.α-Syn is a 140-amino acid residue of the cytosolic neuronal protein,mainly synthesized in the synaptic terminal.It is reported that the main function of α-Syn is related to synaptic vesicle transport and synaptic vesicle regulation.α-syn as the main component of LBs,assembled in the SN to form insoluble aggregates and cause neurotoxicity.Neuropathological studies have found that abnormal aggregation of α-syn in SN is often accompanied by iron deposition.Recently,it has been found that α-syn is an iron reductase and can reduce Fe3+ to Fe2+.Therefore α-syn as an iron metabolism-related protein may participate in nigral iron accumulation in PD.However,the specific molecular mechanisms of α-syn regulating iron metabolism are still not very clear.Neurons and microglia have iron deposits in SN of PD.Microglia can engulf α-syn,then whether the engulfed α-syn could affect iron metabolism in microglia? In this study,using BV2 microglia cells treated with exogenous α-syn,we observed the iron reductase activity of both monomeric α-synuclein(M-α-syn)and oligomeric α-synuclein(O-α-syn)and investigated the effect of α-syn on iron metabolism of microglia.The results are as follows:1.BV2 microglia cell were treated with 0.5μmol/L,1μmol/L,2μmol/L,3μmol/L,4μmol /L,5μmol/L,6μmol/L,7μmol/L,8μmol/L M-α-syn for 24 h.The survival rate of the cells did not change in BV2 microglia cells with less than 5μmol/L,compared with the control group(P> 0.05,n = 6),Therefore,1μmol/L,3μmol/L,5μmol/L were selected as the concentration of the subsequent experiments.2.BV2 microglia cells were treated with 0.5μmol/L,1μmol/L,2μmol/L,3μmol/L,4μmol/L,5μmol/L,6μmol/L,7μmol/L 8μmol/L O-α-syn for 24 h.The survival rate of the cells did not change in BV2 microglia cells with less than 5μmol/L,compared with the control group(P>0.05,n=6),Therefore,1μmol/L,3μmol/L,5μmol/L were selected as the concentration of the subsequent experiments.3.The expressions of DMT1 were significantly up-regulated in BV2 microglia cells treated with 1μmol/L,3μmol/L,5μmol/L M-α-syn for 24 h compared with the control group(P<0.001,n=6).The expressions of FPN1 did not change compared with the control group in BV2 microglia cells(P>0.05,n=6).4.The expressions of IRP1 did not change in BV2 microglia cells treated with1μmol/L,3μmol/L and 5μmol/L M-α-syn for 24 h,compared with the control group(P>0.05,n=6).The expressions of HIF-2α did not change compared with the control group in BV2 microglia cells(P>0.05,n=6).5.The expressions of DMT1 was significantly up-regulated in BV2 microglia cells treated with 1μmol/L,3μmol/L,5μmol/L O-α-syn for 24 h compared with the control group(P<0.001,n=6).The expressions of FPN1 was significantly increased in 3μmol/L group compared with the control group in BV2 microglia cells(P <0.001,n=6).6.The expressions of IRP1 did not change in BV2 microglia cells treated with1μmol/L,3μmol/L and 5μmol/L O-α-syn for 24 h,compared with the control group(P>0.05,n=6).The expressions of HIF-2α did not change compared with the control group in BV2 microglia cells(P>0.05,n=6).7.Iron uptake was significantly increased in BV2 microglia cells treated with 1μmol/L M-α-syn for 24 h compared with the control group(P<0.01,n=6).Iron uptake was significantly increased in BV2 cells treated with 1μmol/L O-α-syn for 24 h compared with the control group(P<0.01,n=6).8.Iron release did not change in BV2 microglia cells treated with 1μmol/L M-α-syn for 24 h,compared with the control group(P>0.05,n=6).Iron release increased in BV2 microglia cells treated with 1μmol/L O-α-syn for 24 h,compared with the control group(P<0.01,n=6).9.BV2 microglia cell were treated with 100 μmol/L M-α-syn for 4h,the ferrireductase activity of M-α-syn increased compared with the control group(P<0.001,n=6).BV2 microglia cell were treated with 100μmol/L O-α-syn for 4h,the ferrireductase activity of O-α-syn significantly increased compared with the control group(P<0.001,n=6).The ferrireductase activity of the group of O-α-syn was higher than the group of M-α-syn(P<0.05,n=6).These results indicate that both M-α-syn and O-α-syn had iron reductase activity,but the activity of O-α-syn was stronger than M-α-syn.α-syn was involved in the iron metabolism in BV2 microglia cells.M-α-syn and O-α-syn increased DMT1 expressions and iron import.O-α-syn increased FPN1 expressions and iron export.The expressions of DMT1 and FPN1 in BV2 microglia were not related to the regulation of IRP1 and HIF-2α,but were related to the iron reductase activity of α-syn.This study provides a strong experimental evidence of α-syn participating in iron metabolism in microglia.