The Role Of DMT1 In The MPP~+ And 6-OHDA-induced Iron Accumulation In MES23.5 Cells

Parkinson's disease(PD) is the second most prevalent neurodegenerative disease just after Alzheimer's disease.There are about 1.72 billion people affected by PD in our country. The incidence is close to 1%in the people over 55.Along with the aging of our society,PD is becoming an overwhelming problem.The exact etiology and pathology of PD are still unclear.Since the exact etiology of the PD is unknown,all of the efferts used in the clinic today aims to alleviate the symptoms.It is of great important solving the problem of whate causes PD,in order to prevent and treat this diseae.Iron plays a crucial role in the PD pathogenesis.Excessive free iron,especially ferrous iron could generate hydroxyl radical(OH·) through Fenton reaction.The OH·could damage proteins,nuclear and mounts of unsaturated fatty acids,which finally lead to the cell death.There are mounting evidences suggesting the role of apoptosis in the PD pathosenesis.Aberrant accumulation of iron in the substantia nigra(SN) might be the vital reason of the neurodegeneration of the dopaminegic neurons.However,the reason for this selective accumulation in the SN is still unclear.The classical transferrin/tranferrin receptor (Tf/TfR) pathway was reported unchanged in PD.In the present study we investigated the role of DMT1,a newly discovered iron transporter,in the iron accumulation in the SN of PD.Western blots,real time PCR,flow cytometry,confocal laser scanning microscopy and other multiple methods were applied to investigate the role of DMT1 in the MPP⁺ and 6-OHDA-induced iron accumulation in MES23.5 cells.The results were as follows:1.An increased ferrous iron influx was oberserved in MES23.5 cells after MPP⁺(5μmol/L) treatment for 24h(P＜0.01),resulted in a reduction of the mitochondrial transmembrane potential(△Ψm),an increase in reactive oxide species(ROS) generation,the activativation of caspase-3 and DNA fragmentation.Iron chelator deferoxamine mesylate(DFO) could fully abolish this effect.2.An increased expression of the DMT1(-IRE) protein and mRNA were observed in the MPP⁺-treated MES23.5 cells,while there was no change in the DMT1(+IRE). 3.IRP2 mRNA level was observed decreased in these cells,while there was no change in IRP1 mRNA.4.An increased ferrous iron influx was oberserved in MES23.5 cells after 6-OHDA (10μmol/L) treatment for 24h(P＜0.01),resulted in a reduction of the△Ψm,an increase in ROS generation,the activativation of caspase-3 and DNA fragmentation.Iron chelator DFO could fully abolish this effect.5.An increased expression of the DMT1(+IRE) protein and mRNA were observed in the 6-OHDA-treated MES23.5 cells,while there was no change in the DMT1(-IRE).6.Both IRP1 and IRP2 mRNA and protein were observed increased in these cells.7.pSilencer1.0U6-IRP1-1,pSilencer1.0U6-IRP1-2,pSilencer1.0U6-IRP2-1 and pSilencer1.0U6-IRP2-2 siRNA expression vectors were successfully constructed,taking the interference consequences targeting the IRP1 and IRP2.RT-PCR showed that the interference efficiency of pSilencer1.0U6-IRP1-1 or pSilencer1.0U6-IRP2-1 were best for IRP1 or IRP2 separately,when applied with the mass/volume ratio of 1:5 of the plasmid/ liposome and for 48h.IRP1 protein levels were significantly suppressed about 71±6%,in MES23.5 cells with IRP1 RNA interfering.IRP2 protein levels were decreased by 65±5% in MES23.5 cells with IRP2 RNA interfering.8.DMT1(+IRE) protein and mRNA levels were both decreased in MES23.5 cells with IRP1 or IRP2 RNA interfering;IRP1 or IRP2 knockdown partially blocked the up-regulation of DMT1(+IRE) mRNA level in MES23.5 cells with 6-OHDA treatment.In the present study,we investigated the involvement of DMT1 that accounted for the ferrous iron accumulation in MPP⁺ and 6-OHDA-treated MES23.5 cells.In the treated cells, a significant influx of ferrous iron was observed.This resulted in a decreased△Ψm,an increase ROS generation,the activativation of caspase-3,untimately lead to the cell's apoptosis.All the changes could be prevented by iron chelator DFO.MPP⁺ could up-regulate the expression of DMT1(-IRE) in an IRE/IRP-independent manner.The upregulation of DMT1(+IRE) were IRE/IRP dependent in the 6-OHDA treated MES23.5 cells.Although the two neurotoxins regulated the expression of DMT1 in diffrent mechanisms,they both could induce the cell apoptosis by the upregulation of DMT1 and the uptake of Fe²⁺.