【作者】 宋宁；

【导师】 谢俊霞；

【作者基本信息】 青岛大学 ， 生理学， 2008， 博士

【摘要】 帕金森病(Parkinson’s disease,PD)是一种多发于中老年的中枢神经系统退行性疾病。PD的神经病理学基础是黑质(substantia nigra,SN)致密带多巴胺(dopamine,DA)能神经元的变性脱失以及由此而导致的纹状体DA含量降低。迄今为止,PD的病因学仍未完全明了,基因变异(如PARK1,2,5,6,8等)和环境因素(如杀虫剂、毒品、金属离子等)都被认为参与了PD的发病。越来越多的证据表明铁在PD的发病中是一个关键因素,研究表明在残存的DA能神经元内铁含量明显高于对照组。目前,PD中铁选择性沉积的原因仍不清楚。本实验室的前期工作证实,二价金属离子转运体(divalent metal transporter 1,DMT1)的表达异常,可能参与了PD中SN区铁的聚积。脑细胞铁平衡取决于细胞铁转入和转出两个环节,而DMT1仅是转入蛋白,如果铁转出蛋白的功能正常,DMT1的表达异常将无法导致细胞内的铁聚积。因此本研究我们拟探讨铁转出蛋白ferroportin1(FP1)和hephaestin(HP)在PD SN异常铁聚积中的作用。FP1是一种具有十个跨膜区的铁转出蛋白。HP是与铜蓝蛋白结构和功能类似的一种亚铁氧化酶,只有一个跨膜区域,必须与PP1协同作用使铁转出细胞。目前FP1和HP在脑细胞中的功能和调节仍知之甚少。我们的在体实验证实在6—羟基多巴胺(6-hydroxydopamine,6-OHDA)制备的PD模型大鼠SN区,FP1和HP的表达下降可能参与了SN的铁聚积。本研究选用体外培养的腹侧中脑(ventralmesencephalic,VM)神经元和MES23.5细胞系,应用免疫荧光、分子生物学、激光共聚焦扫描技术、流式细胞仪技术等多项研究方法来探讨了FP1和HP在6-OHDA导致的细胞铁聚积中的作用;并进一步探讨了其表达调控的深入机制。结果表明:1.体外培养的VM神经元和MES23.5细胞上均有FP1和HP的共表达。2.10μmol/L 6-OHDA处理24 h后,体外培养的VM神经元和MES23.5细胞铁的转出功能明显降低,FP1和HP蛋白和mRNA表达水平均有明显降低。3.100μg/ml或1 mg/ml枸橼酸铁胺(ferric ammonium citrate,FAC)处理24 h后,体外培养的VM神经元和MES23.5细胞FP1mRNA表达水平明显增加,且1 mg/ml处理组比100μg/ml处理组增加得更明显,HP mRNA表达水平没有变化。4.10μmol/L 6-OHDA处理24 h后,体外培养的VM神经元和MES23.5细胞中铁调节蛋白(iron regulatory protein,IRP)1和IRP2蛋白和mRNA的表达水平都有明显上调。5.成功构建了pSilencer-IRP1和pSilencer-IRP2 siRNA表达载体。特异性的沉默IRP1或IRP2的表达后,IRP1蛋白水平下降了71±6%;IRP2蛋白水平下降了65±5%。6.干涉IRP1或IRP2后,FP1蛋白和mRNA表达水平与空载体对照组相比都明显上调。10μmol/L 6-OHDA处理24 h后,FP1mRNA的表达水平仍有降低,但降低的幅度明显减轻;HP mRNA的表达下降被完全逆转,其表达出现了上调。7.成功构建了pSilencer-FP1和pSilencer-HP siRNA表达载体。特异性的沉默FP1或HP的表达后,FP1蛋白水平下降了57±3%;HP蛋白水平下降了61±5%。8.FP1干涉后,100μmol/L铁孵育4 h后细胞内铁含量明显增加,细胞内增加的铁上调了L型铁蛋白mRNA水平,进一步增加了细胞内高铁导致的乳酸脱氢酶(lactate dehydrogenase,LDH)释放和活性氧物质(reactive oxide species,ROS)的生成,降低了线粒体跨膜电位(mitochondrial transmembrane potential,△ψm)。HP干涉后,除了使△ψm降低得更明显外,对细胞内的铁水平、L型铁蛋白的表达、LDH的释放和ROS的生成都没有影响。铁离子螯合剂去铁胺1mmol/l预孵育3 h可以完全阻断细胞内高铁引起的LDH的释放、ROS的生成和△ψm的下降。9.成功构建了pIRES-FP1和pIRES-HP-FP1表达载体。pIRES-FP1转染的MES23.5细胞中,FP1蛋白水平是空载体对照组的3.38±0.06倍;pIRES-HP-FP1转染MES23.5细胞中,FP1蛋白水平是空载体转染组的2.86倍±0.04,HP蛋白水平是空载体转染组的3.21±0.06倍。10.高表达FP1和HP的MES23.5细胞铁转出功能都明显增强。11.pcDNA3.1-HP-Flagtag转染的MES23.5细胞中,HP蛋白水平是空载体对照组的3.81±0.07倍。高表达HP的MES23.5细胞铁转出功能也明显增强。12.高表达FP1和/或HP能够转出更多的铁,在铁负载状态下能够降低细胞内的铁水平,阻断了高铁引起的L型铁蛋白mRNA水平的上调和FP1的表达上调,从而阻断或减轻了细胞内高铁导致的LDH释放和ROS的生成,在一定程度上恢复△ψm。上述结果表明,铁转出蛋白FP1和HP在体外培养的VM神经元和MES23.5细胞中部有表达;FP1和HP的表达降低参与了6-OHDA导致的铁聚积;6-OHDA诱导的FP1和HP的表达降低不是细胞内高铁的结果;细胞内IRPs可能参与了6-OHDA对FP1和HP的表达调节;FP1的表达下降会增加细胞内的铁水平,从而加重细胞内高铁引起的氧化应激损伤,表现为LDH释放和ROS生成的增加、△ψm的下降;高表达FP1和/或HP能够转出更多的铁,减少LDH的释放和ROS的生成,在一定程度上恢复△ψm,因此能保护细胞免受高铁引起的氧化应激的损伤。本研究为FP1和HP参与脑铁代谢提供最直接的证据,为阐明PD SN内铁选择性聚积的原因提供部分的实验基础,并为铁特异性地损伤多巴胺能神经元提供更加详实和有力的理论依据,并为药物干预提供可能的作用靶点。更多还原

【Abstract】 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.更多还原