The Involvement Of FP1 And HP In 6-hydroxydopamine Induced Iron Accumulation In The Ventral Mesencephalic Neurons And MES23.5 Cells

Parkinson's disease(PD) is an adult-onset progressive neurodegenerative disorder. Neuropathological hallmarks of the disease include the degeneration and loss of dopaminergic neurons in the substantia nigra(SN) and the subsequent dopamine(DA) depletion in the striatum.Although the causes of PD are not known,both genetic mutations(such as PARK 1,2,5,6,8 gene) and environmental factors(pesticide,toxin, metal) are considered to be involved.Amounting evidence demonstrate that iron plays a key role in the pathogenesis of PD.In PD patients,individual dopaminergic neuron showed increased iron levels compared to the control.To date,it is still unknown what causes this selective iron accumulation in the SN of PD.Our previous studies demonstrated that divalent metal transporter 1(DMT1) up-regulation was involved in the nigral iron accumulation in PD animal models.For maintenance of a balanced iron homeostasis in brain cells,the cellular iron influx,as well as iron efflux must be tightly regulated.Since DMT1 is an iron importer,in the present study,we set out to investigate the roles of iron exporters-ferroportinl(FP1) and hephaestin(HP) in the iron accumulation of PD.FP1 was a multiple transmembrane domain protein identified in several cell types involved in the export of cellular iron.HP,a multicopper ferroxidase,is a membrane bound ceruloplasmin homologue.In the presence of iron transporter FP1 and a ferroxidase HP,the newly released ferrous iron could be oxidized to its ferric form,which then binds to transferrin.The roles of FP1 and HP in cellular iron homeostasis in the CNS are still unknown.Our in vivo experiments suggested that decreased FP1 and HP might account for the cellular iron accumulation in the SN of 6-hydroxydopamine(6-OHDA) lesioned rats.Using immunofluorescence,molecular biology,laser confocal scanning microscopy,flow cytometry and other methods,in the present study we investigate the role of FP1 and HP in 6-OHDA induced iron accumulation in primary cultured ventral mesencephalic(VM) neurons and MES23.5 cells.The mechanisms underlying the regulation of FP1 and HP by 6-OHDA were investigated.The results were as follows:1.Both FP1 and HP were expressed on primary cultured VM neurons and MES23.5 cells.2.Iron efflux was decreased with 10μmol/L 6-OHDA treatment for 24 h in primary VM neurons or MES23.5 cells.FP1 and HP were down-regulated on both protein and mRNA levels in these cells.3.FP1 mRNA level was up-regulated with 100μg/ml or 1 mg/ml ferric ammonium citrate(FAC) treatment for 24 h in primary VM neurons and MES23.5 cells.There was a more dramatic up-regulation in 1 mg/ml group compared to 100μg/ml group.HP mRNA level showed no response to 100μg/ml or 1 mg/ml FAC treatment.4.Iron regulatory protein(IRP) 1 and IRP2 were up-regulated on both protein and mRNA levels with 10μmol/L 6-OHDA treatment for 24 h in primary VM neurons and MES23.5 cells.5.pSilencer-IRP1 and pSilencer-IRP2 siRNA expression vectors were successfully constructed.IRP1 protein levels were significantly suppressed about 71±6%,in MES23.5 cells with IRP1 RNA interfering.IRP2 protein levels were significantly suppressed about 65±5%in MES23.5 cells with IRP2 RNA interfering.6.FP1 protein and mRNA levels were both up-regulated in MES23.5 cells with IRP1 or IRP2 RNA interfering.IRP1 or IRP2 knockdown partially blocked the down-regulation of FP1 mRNA level and reversed the down-regulation of HP mRNA level in MES23.5 cells with 6-OHDA treatment.7.pSilencer-FP1 and pSilencer-HP siRNA expression vectors were successfully constructed.FP1 mRNA and protein levels were significantly suppressed about 57±3% in MES23.5 cells with FP1 RNA interfering.HP protein level was significantly suppressed about 61±5%in MES23.5 cells with HP RNA interfering.8.Cellular iron levels were further increased with FP1 knockdown in MES23.5 cells with 100μmol/L iron incubation for 4 h.The increased intracellular iron further induced up-regulation of ferritin L mRNA level,lactate dehydrogenase(LDH) leakage, reactive oxide species(ROS) generation,and reduction of mitochondrial ransmembrane potential(△Ψm).HP knockdown further reduced△Ψm and had no effects on the intracellular iron level,as well as ferritin L mRNA level,LDH leakage and ROS generation.Pretreatment of deferoxamine mesylate for 3 h could fully abolish ron induced LDH leakage,ROS generation and△Ψm reduction.9.pIRES-FP1 and pIRES-HP-FP1 expression vectors were successfully constructed. FP1 mRNA and protein levels were significantly increased 4.72±0.772 and 3.38±0.06 folds,respectively,in MES23.5 cells with pIRES-FP1 transfection.In cells with pIRES-HP-FP1 transfection,FP1 mRNA and protein levels were significantly increased 8.42±1.591 and 2.86±0.04 folds,respectively;HP mRNA and protein level was significantly increased 5.49±0.211 and 3.21±0.06 folds,respectively.10.Iron efflux was enhanced in MES23.5 cells with over-expression of FP1 and HP.11.HP mRNA and protein levels were significantly increased to 5.62±0.121 and 3.81±0.07 fold,respectively,in MES23.5 cells with pcDNA3.1-HP transfection.Iron efflux was also enhanced in MES23.5 cells with over-expression of HP.12.High expression of FP1 and/or HP could lower cellular iron level and thus block the up-regulation of ferritin L and FP1 mRNA level under iron overloaded conditions. ROS generation was then partially suppressed and△Ψm was partially restored,due to more iron efflux out of the cells.The above results suggest that FP1 and HP are co-localized on primary VM neurons and MES23.5 cells;down-regulations of FP1 and HP are involved in 6-OHDA induced iron accumulation in these cells.Down-regulations of FP1 and HP are not the secondary event due to increased intracellular iron level.IRPs are involved in 6-OHDA induced down-regulations of FP1 and HE FP1 knockdown results in cellular iron accumulation,thus aggravates iron induced oxidative stress indicated by LDH leakage, ROS generation and△Ψm reduction.Over-expression of FP1 and/or HP can transport more iron outsides,thus protect cells from iron induced oxidative stress indicated by suppression of LDH leakage and ROS generation,as well as△Ψm restoration.Our findings provide direct evidence that both FP1 and HP are responsible for the iron efflux process in brain cells and elucidate their involvement and underlying mechanisms in PD iron accumulation.We aim to put powerful evidence to iron induced dopaminergic neurons injury in PD and further show some light on the development of potential clinical approaches.