Retinal Iron Homeostasis in Health and Disease

描述(申请人提供)：这个项目的主要目标是检验视网膜是遗传性血色病(HHC)中铁超载和铁诱导氧化损伤的目标组织的假设，遗传性血色病是最常见的遗传性疾病，其特征是铁在全身器官中积聚。HFE[组织相容性白细胞抗原I类蛋白参与铁稳态(FE)稳态]是一种编码细胞表面蛋白的基因，参与铁稳态的调节，它的突变导致了大约85%的这种疾病(经典的HHC)。其余病例是由另外四个基因(血凝素、海普西丁、转铁蛋白受体2和铁蛋白)突变引起的，这些基因也编码铁调节蛋白。血凝素(HJV)的突变导致幼年型HHC，在幼年型HHC中，器官中铁的过度积聚发生在比经典HHC更年轻的年龄。令人惊讶的是，关于视网膜是否在这种疾病中受到影响，人们知之甚少。我们首次发现所有五个与HHC相关的基因都在视网膜中表达：HFE仅在RPE中表达；Hjv在RPE和神经视网膜中表达；HJV在RPE、Muller细胞和感光细胞中表达；转铁蛋白受体2在整个视网膜中表达。更重要的是，我们有证据证明经典HHC小鼠模型(HFE-/-小鼠)的视网膜形态随年龄变化。此外，我们还发现与野生型RPE细胞相比，HFE-/-RPE细胞呈现出高增殖的表型。这些发现是新颖的，指出了铁状态和细胞增殖之间的有趣联系。我们的研究还表明，由于巨细胞病毒(CMV)和单纯疱疹病毒-1(HSV1)的感染，HFE、Hjv和Hepsidin在RPE和视网膜中的表达明显改变。这些有趣而重要的发现构成了拟议研究的基础。目的1是验证HHC与RPE和视网膜中铁超载有关的假说，并对其功能后果进行检验。这将使用两个不同的小鼠模型进行测试，一个代表经典的HHC(HFE-/-小鼠)，另一个代表幼年HHC(Hjv-/-小鼠)。目的2是验证这样一种假设，即HFe和HJV基因缺失的后果远远超出了铁积累和相关的氧化损伤。在这一特定目标下的研究将包括检测导致HFe-/-RPE细胞过度增殖表型、Hjv-/-RPE细胞上皮向间充质转化的分子事件以及Hjv-/-RPE细胞BMP信号的中断及其在Hjv-/-RPE细胞中的后果。目的3验证CMV和HSV1感染RPE和视网膜通过调节铁调节基因的表达来破坏铁稳态的假说。这将通过体外视网膜细胞原代培养和体内野生型和HHC小鼠模型进行研究。这些研究将对健康和疾病中维持视网膜铁稳态的分子机制以及高加索人最常见的遗传病HHC中视网膜的参与提供重要的见解。 公共卫生相关性：遗传性血色沉着症(HHC)是最常见的遗传性疾病，其特征是铁在各种系统器官中过度积聚，但视网膜中的铁状态在这种疾病中是否受到影响尚不清楚。然而，众所周知，视网膜中过多的铁积累会导致氧化损伤和视网膜退化。本项目中提出的研究将使用HHC转基因小鼠模型来研究HHC相关的视网膜铁状态、结构和形态的变化及其功能后果，并评估在感染巨细胞病毒和单纯疱疹病毒-1过程中各种铁调节蛋白在RPE和视网膜中的参与。

DESCRIPTION (provided by applicant): The primary goal of this project is to test the hypothesis that retina is a target tissue for iron overload and iron-induced oxidative damage in hereditary hemochromatosis (HHC), the most prevalent genetic disease characterized by iron accumulation in systemic organs. Mutations in HFE [Histocompatability leukocyte antigen class I-like protein involved in iron (FE) homeostasis], a gene coding for a cell surface protein involved in the regulation of iron homeostasis, are responsible for ~85% cases of this disease (classical HHC). The remaining cases arise from mutations in four other genes (hemojuvelin, hepcidin, transferrin receptor 2, and ferroportin), also coding for iron-regulatory proteins. Mutations in hemojuvelin (HJV) cause juvenile HHC in which excessive iron accumulation occurs in organs at a much younger age than in classical HHC. Surprisingly, little is known on whether retina is affected in this disease. We have shown for the first time that all five HHC-associated genes are expressed in the retina: Hfe exclusively in RPE; Hjv in RPE and neural retina; hepcidin and ferroportin in RPE, Muller cells, and photoreceptor cells; and transferrin receptor 2 throughout the retina. More importantly, we have evidence for age-dependent morphological changes in the retina in a mouse model of classical HHC (Hfe-/- mouse). In addition, we found that Hfe-/- RPE cells exhibit a hyperproliferative phenotype compared to wild type RPE cells. These findings are novel and point to an interesting link between iron status and cell proliferation. Our studies also show that the expression of Hfe, Hjv, and hepcidin is markedly altered in RPE and in retina as a result of infection with cytomegalovirus (CMV) and herpes simplex virus-1 (HSV1). These interesting and important findings form the basis for the proposed studies. Aim 1 is to test the hypothesis that HHC is associated with iron overload in RPE and retina with functional consequences. This will be tested using two different mouse models, one representing the classical HHC (Hfe-/- mouse) and the other representing the juvenile HHC (Hjv-/- mouse). Aim 2 is to test the hypothesis that deletion of HFE and HJV has consequences that go far beyond iron accumulation and associated oxidative damage. Studies under this specific aim will include examination of the molecular events responsible for the hyperproliferative phenotype of Hfe-/- RPE cells as well as for the epithelial-to-mesenchymal transition in Hjv-/- RPE cells and for the disruption of BMP signaling and its consequences in Hjv-/- retina. Aim 3 is to test the hypothesis that infection of RPE and retina with CMV and HSV1 disrupts iron homeostasis by modulating the expression of iron-regulatory genes. This will be investigated using primary cultures of retinal cells in vitro and with wild type and HHC mouse models in vivo. These studies will provide important insight into the molecular mechanisms involved in the maintenance of iron homeostasis in the retina in health and disease and on the involvement of the retina in HHC, the most prevalent genetic disease in Caucasians. PUBLIC HEALTH RELEVANCE: Hereditary hemochromatosis (HHC) is the most prevalent genetic disease characterized by excessive iron accumulation in various systemic organs, but whether the iron status in the retina is affected in this disease is not known. It is known however, that excessive iron accumulation in the retina causes oxidative damage and retinal degeneration. Studies proposed in this project will investigate the HHC-associated changes in the iron status, structure, and morphology of the retina and the functional consequences using transgenic mouse models of HHC, and assess the involvement of various iron-regulatory proteins in RPE and the retina during infection with cytomegalovirus and herpes simplex virus-1.