Ferroxidases in RPE Iron Transport

描述（由申请人提供）：铁是视网膜氧化磷酸化、膜生物发生和视黄醇异构化所必需的，但如果调节不当，也会产生氧化应激，导致细胞死亡。这可能导致视网膜疾病如下：1)铁中毒引起快速视网膜变性，由眼内异物携带铁直接进入眼睛。2)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。3)铁氧化酶铜蓝蛋白（Cp）的遗传性缺陷导致视网膜铁积累和早发性黄斑变性。4) Cp及其同系物hephaestin （Heph）突变的小鼠具有年龄依赖性视网膜铁过载和变性，其特征与AMD相似，包括视网膜下新生血管。后两点表明Cp和Heph对视网膜健康很重要。来自其他器官的证据表明，Cp或Heph可以与质膜铁转运蛋白铁转运蛋白（Fpn）合作，将铁从细胞中输出。本研究的目的是增加对Cp、Heph和Fpn在视网膜铁稳态中的作用以及分泌激素hepcidin （Hepc）对它们的调节的理解。Hepc在视网膜（以及肝脏）中产生，并触发Fpn的内化和降解。Hepc可能作为视网膜细胞感知铁过量（如光感受器）的信息来降解Fpn并限制铁从RPE和Muller细胞转移。我们现有的Cp/Heph双突变体和Hepc-/-小鼠表明，这三种蛋白质对视网膜铁稳态和健康至关重要，但提供的信息很少关于蛋白质在视网膜内的具体功能。条件小鼠敲除技术（lox/cre）提供了确定这些蛋白在特定视网膜细胞类型中的功能以及细胞间铁转运如何执行和调节的机会。在Aim1中，Heph的光感受器特异性功能，一个可能的“铁释放阀”，以防止PR铁过载，将在Cp-/-背景下使用Heph条件敲除。在Aim 2中，将使用RPE和光受体特异性条件敲除小鼠来研究Fpn的铁转运功能。在Aim 3中，将在敲除小鼠和条件敲除小鼠中研究Hepc的视网膜功能。这些研究的重要意义在于：1)它们将提供关于Heph， Fpn和Hepc的细胞类型特异性功能以及控制视网膜铁稳态的细胞间铁转运途径的新信息。2)条件敲除小鼠可能为AMD的几个特征提供模型，包括视网膜下新生血管，同时避免了我们现有的Cp/Heph双突变小鼠的寿命限制的脑铁过载。公共卫生相关性：关于视网膜铁转运机制的拟议工作与视网膜健康相关，因为铁超载与年龄相关性黄斑变性（AMD）和其他视网膜疾病有关。根据我们之前的工作，由铁转运蛋白突变引起的小鼠模型预计具有AMD的特征，包括视网膜下新生血管、光受体和RPE死亡、脂褐素积累和补体级联激活。这些模型将为理解视网膜铁调节机制和测试视网膜疾病的潜在治疗方法提供一个平台。

DESCRIPTION (provided by applicant): Iron is necessary in the retina for oxidative phosphorylation, membrane biogenesis and retinol isomerization, but can also produce oxidative stress if improperly regulated, leading to cell death. This can contribute to retinal disease as follows: 1) Iron toxicity causes rapid retinal degeneration following direct entry of iron into the eye carried by an intraocular foreign body. 2) Human AMD retinas have more iron than age-matched controls, suggesting that iron overload may play a role in AMD pathogenesis. 3) Inherited defects in the ferroxidase ceruloplasmin (Cp) result in retinal iron accumulation and early onset macular degeneration. 4) Mice with mutation in Cp and its homolog hephaestin (Heph) have an age-dependent retinal iron overload and degeneration with a number of features similar to AMD, including subretinal neovascularization. The latter two points indicate that Cp and Heph are important for retinal health. Evidence from other organs suggests that Cp or Heph can cooperate with the plasma membrane iron transporter ferroportin (Fpn) to export iron from cells. The goal of this proposal is to increase understanding of the roles of Cp, Heph and Fpn in retinal iron homeostasis and their regulation by the secreted hormone hepcidin (Hepc). Hepc is produced in the retina (as well as the liver) and triggers internalization and degradation of Fpn. Hepc may serve as a message from retinal cells sensing iron excess (such as photoreceptors) to degrade Fpn and limit iron transfer from RPE and Muller cells. Our existing Cp/Heph double mutant and Hepc-/- mice indicate that these three proteins are critical for retinal iron homeostasis and health, but provide little information about the specific functions of the proteins within the retina. Conditional mouse knockout technology (lox/cre) affords the opportunity to determine how these proteins function within specific retinal cell types and how intercellular iron transfer is executed and regulated. In Aim1, the photoreceptor-specific functions of Heph, a possible "iron release valve" to prevent PR iron overload will be investigated using a Heph conditional knockout on a Cp-/- background. In Aim 2, the iron transport function of Fpn will be investigated using RPE and photoreceptor-specific conditional knockout mice. In Aim 3, the retinal function of Hepc will be investigated in knockout and conditional knockout mice. These studies are important because: 1) They will provide new information about the cell-type specific functions of Heph, Fpn and Hepc and the routes of intercellular iron transfer that control retinal iron homeostasis. 2) The conditional knockout mice are likely to provide models for several features of AMD, including subretinal neovascularization while avoiding the lifespan-limiting brain iron overload in our existing Cp/Heph double mutant mice. PUBLIC HEALTH RELEVANCE: The proposed work on the mechanisms of retinal iron transport is relevant to retinal health since iron overload has been implicated in age-related macular degeneration (AMD) and other retinal diseases. The mouse models resulting from mutation of iron transporters are expected, based on our previous work, to have features of AMD, including subretinal neovascularization, photoreceptor and RPE death, lipofuscin accumulation, and activation of the complement cascade. These models will provide a platform for understanding the mechanisms of retinal iron regulation and testing potential therapeutics for retinal disease.