Mechanisms of age-related RPE dysfunction and CNV

描述（由申请人提供）：年龄相关性黄斑变性（AMD）是老年人失明的主要原因。该疾病以视网膜色素上皮（RPE）功能障碍和囊肿形成为开始，并将进一步发展为亚RPE空间的慢性炎症、地理萎缩和脉络膜新生血管（CNV）。尽管近年来在遗传学研究和临床治疗方面取得了进展，但其病因尚不清楚，目前尚无有效的治疗干性AMD的方法。我们最近建立了一种AMD动物模型，使用核因子红细胞2相关因子2 （Nrf2）缺失的敲除小鼠。Nrf2-/-小鼠出现amd样视网膜病理，包括年龄依赖性RPE变性、自发性CNV和补体相关蛋白亚RPE沉积。在我们的前期研究中，我们进一步证明了Nrf2是RPE自噬和巨噬细胞活化的重要调节因子。我们发现nrf2缺陷的RPE细胞体内自噬液泡和脂褐素的积累增加，表明溶酶体的降解效率较低。在使用氯喹（一种溶酶体功能和RPE自噬抑制剂）的慢性毒性模型中，Nrf2-/-小鼠出现了更为严重的RPE病理。通过差异基因表达实验，我们发现Nramp1是Nrf2的下游基因，具有控制自噬体形成的新功能。在实验性CNV模型中，Nrf2-/-小鼠的病变大小和巨噬细胞浸润明显高于相同处理的野生型小鼠。与RPE共培养时，Nrf2-/-巨噬细胞表现出比野生型细胞更强的促炎反应。基于这些发现，我们假设Nrf2是针对年龄依赖性RPE功能障碍和随后的CNV的多种保护机制的重要调节因子。我们将以Nrf2-/-小鼠为模型，进一步研究RPE变性的机制，并探讨浸润性巨噬细胞介导的慢性炎症的作用。特异性Aim 1将确定Nrf2是否通过依赖于nramp1的机制调节RPE自噬。特异性目的2将确定抑制RPE自噬是否会加速Nrf2-/-小鼠amd相关病理的发展，并检查RPE自噬途径与外泌体蛋白输出之间的功能相互作用。特异性Aim 3将确定Nrf2是否调节实验性CNV中极化巨噬细胞的激活。这些研究结果将揭示AMD病理生物学的新机制，并进一步确定Nrf2作为干预的分子靶点。

DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is the leading cause of blindness in elderly people. The disease starts with dysfunction of the retinal pigment epithelium (RPE) and formation of drusen, and will further progress to chronic inflammation in the sub-RPE space, geographic atrophy and choroidal neovascularization (CNV). Despite the recent progresses in genetic research and clinical treatment of exudative AMD, its etiology is largely unclear and currently there is no effective cure for dry AMD. We have recently developed an animal model of AMD using knockout mice deficient of nuclear factor erythroid 2-related factor 2 (Nrf2). The Nrf2-/- mice developed AMD-like retinal pathology including age-dependent RPE degeneration, spontaneous CNV and sub-RPE deposit of complement-related proteins. In our preliminary studies, we further demonstrated that Nrf2 is an important regulator of RPE autophagy and macrophage activation. We found that Nrf2-deficient RPE cells had increased accumulation of autophagic vacuoles and lipofuscin in vivo, indicating less efficient degradation by lysosome. Substantially more severe RPE pathology was developed in Nrf2-/- mice in a chronic toxicity model using chloroquine, an inhibitor of lysosome function and RPE autophagy. With differential gene expression assay, we identified Nramp1 as a downstream gene of Nrf2 with novel functions of controlling autophagosome formation. In a model of experimental CNV, Nrf2-/- mice showed significantly increased lesion size and macrophage infiltration than the wild type mice received the same treatment. When co-cultured with RPE, Nrf2-/- macrophages showed more robust pro-inflammatory responses than the wild type cells. Based on these findings, we hypothesize that Nrf2 is an essential regulator of multiple protective mechanisms against age-dependent RPE dysfunction and subsequent CNV. Using Nrf2-/- mice as an established model, experiments will be performed to further examine the mechanisms of RPE degeneration and to explore the roles of chronic inflammation mediated by infiltrating macrophages. Specific Aim 1 will determine whether Nrf2 regulates RPE autophagy by Nramp1-dependent mechanisms. Specific Aim 2 will determine whether inhibiting RPE autophagy will accelerate the development of AMD-related pathology in Nrf2-/- mice, and examine the functional interactions between RPE autophagy pathway and exosomal protein export. Specific Aim 3 will determine whether Nrf2 regulates polarized macrophage activation in experimental CNV. Results from these studies will reveal new mechanisms of the pathobiology of AMD and further define Nrf2 as a molecular target for intervention.