Ferroxidases in RPE iron transport

RPE 铁转运中的铁氧化酶

• 批准号: 6931023
• 负责人: JOSHUA L DUNAIEF
• 金额: $ 31.7万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6931023
• 关键词: Adenoviridae; angiogenesis; biological models; confocal scanning microscopy; ferroxidase; gene induction /repression; gene targeting; genetically modified animals; immunoelectron microscopy; in situ hybridization; ion transport; iron; laboratory mouse; model design /development; protein localization; retina degeneration; retinal pigment epithelium; transfection /expression vector; vascular endothelial growth factors

DESCRIPTION (provided by applicant): Iron is necessary in the retina for oxidative phosphorylation, membrane biogenesis and many other metabolic processes, but can also produce oxidative stress if improperly regulated, leading to cell death. Iron plays a significant role in retinal degeneration, as follows: 1) Iron toxicity causes retinal degeneration following direct entry of iron into the eye carried by an intraocular foreign body 2) Iron has been implicated in the RCS rats' retinal degeneration, in which the mechanism of iron overload is indirect. 3) Human AMD retinas have more iron than age-matched controls, suggesting that iron overload may play a role in AMD pathogenesis. 4) Inherited defects in iron metabolism result in retinal degeneration in Friedreich's ataxia, pantothenate kinase associated neuropathy, and aceruloplasminemia. The focus of the current proposal is to understand the functions in ocular iron homeostasis of the ferroxidases ceruloplasmin (Cp) and its homologue hephaestin (Heph). In Aim 1, the retinal localization of Cp and Heph will be assessed. In Aim 2, an RPE-specific Heph knockout will be generated. This will determine whether RPE Heph expression is necessary for RPE iron homeostasis. It should also provide a mouse model of retinal degeneration that like our Cp-/-Heph-/- mice, has subretinal neovascularization, but unlike Cp-/-Heph-/- mice, has a normal lifespan. In addition it will provide reagents for generating RPE-specific knockouts for the eye research community. In Aim 3, the retinal degeneration in Cp-/-Heph-/- and Cp-/-RPE-specific Heph-/- mice will be characterized. Both oxidative stress and mechanisms of neovascularization will be studied. In Aim 4 an RPE in vitro system will be used to determine whether Cp and Heph mediate RPE iron import or export, and assess the polarity of transport. These studies are important because: 1) The mechanism of retinal degeneration in aceruloplasminemia is not known. 2) The retinal functions of Cp and Heph are not known. 3) Our preliminary studies show that Cp-/-Heph-/- mice will provide a model for several features of AMD, including subretinal neovascularization. 4) These mice will provide a model of neurodegeneration caused by age-dependent increases in iron levels, which recent reports suggest may contribute to the pathogenesis of Alzheimer's and Parkinson's diseases and AMD. 5) These mice will provide the opportunity to test iron chelators and antioxidants as potential treatments/preventions for those age-related diseases.

描述（由申请人提供）：铁是视网膜氧化磷酸化、膜生物发生和许多其他代谢过程所必需的，但如果调节不当，也会产生氧化应激，导致细胞死亡。铁在视网膜变性中起着重要作用，其原因如下：1)眼内异物携带铁直接进入眼内，铁中毒导致视网膜变性2)铁与RCS大鼠视网膜变性有关，其铁超载机制是间接的。3)人类黄斑变性视网膜的铁含量高于同龄对照组，提示铁超载可能在黄斑变性的发病机制中发挥作用。4)铁代谢的遗传性缺陷导致弗里德赖希共济失调视网膜变性、泛酸激酶相关神经病和乙酰纤溶酶血症。本研究的重点是了解氧化铁酶铜蓝蛋白（Cp）及其同源酶hephaestin （Heph）在眼铁稳态中的功能。在Aim 1中，将评估Cp和Heph的视网膜定位。在Aim 2中，将产生rpe特异性Heph敲除。这将决定RPE Heph表达对于RPE铁稳态是否必要。它也应该提供一种视网膜变性的小鼠模型，像我们的Cp-/- heph -/-小鼠一样，有视网膜下新生血管，但不像Cp-/- heph -/-小鼠，有正常的寿命。此外，它还将为眼科研究界提供生成rpe特异性敲除的试剂。在Aim 3中，将对Cp-/-Heph-/-和Cp-/- rpe特异性Heph-/-小鼠的视网膜变性进行表征。氧化应激和新血管形成的机制将被研究。在Aim 4中，RPE体外系统将用于确定Cp和Heph是否介导RPE铁的进口或出口，并评估运输的极性。这些研究是重要的，因为：1)急性纤溶酶血症视网膜变性的机制尚不清楚。2) Cp和Heph的视网膜功能尚不清楚。3)我们的初步研究表明，Cp-/- heph -/-小鼠将为AMD的几个特征提供模型，包括视网膜下新生血管。4)这些小鼠将提供由年龄依赖性铁水平升高引起的神经退行性变模型，最近的报道表明，铁水平升高可能有助于阿尔茨海默病、帕金森病和AMD的发病机制。这些小鼠将提供机会来测试铁螯合剂和抗氧化剂作为治疗/预防那些与年龄有关的疾病的潜在方法。