Mechanisms of age-related RPE dysfunction and CNV

描述(申请人提供)：老年性黄斑变性(AMD)是导致老年人失明的主要原因。该病始于视网膜色素上皮(RPE)功能障碍和玻璃体形成，并将进一步发展为RPE下间隙的慢性炎症、地理性萎缩和脉络膜新生血管(CNV)。尽管渗出型AMD的遗传学研究和临床治疗最近取得了进展，但其病因很大程度上仍不清楚，目前还没有有效的治疗干性AMD的方法。我们最近利用缺乏核因子红系相关因子2(NRF2)的基因敲除小鼠建立了AMD的动物模型。Nrf2-/-小鼠出现类似AMD的视网膜病变，包括年龄依赖性RPE变性、自发性CNV和补体相关蛋白在RPE下沉积。在我们的初步研究中，我们进一步证明了Nrf2是RPE自噬和巨噬细胞激活的重要调节因子。我们发现，Nrf2缺陷的RPE细胞体内自噬空泡和脂褐素的积累增加，表明溶酶体的降解效率较低。在使用溶酶体功能和RPE自噬抑制剂氯喹的慢性毒性模型中，在Nrf2-/-小鼠中出现了更严重的RPE病理。通过基因差异表达分析，我们确定Nramp1是Nrf2的下游基因，具有控制自噬小体形成的新功能。在实验性CNV模型中，与接受相同治疗的野生型小鼠相比，Nrf2-/-小鼠表现出明显的病变大小和巨噬细胞浸润。当与RPE共培养时，NRF2-/-巨噬细胞表现出比野生型细胞更强烈的促炎反应。基于这些发现，我们假设Nrf2是多种保护机制的重要调节者，以对抗年龄依赖性RPE功能障碍和随后的CNV。以Nrf2-/-小鼠作为已建立的模型，将进行实验以进一步研究RPE退变的机制，并探索由巨噬细胞渗入介导的慢性炎症的作用。具体目标1将确定Nrf2是否通过Nramp1依赖的机制调节RPE自噬。特定目的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，并研究RPE自噬途径与胞外蛋白输出之间的功能相互作用。具体目标3将确定Nrf2是否调节实验性CNV中极化的巨噬细胞激活。这些研究的结果将揭示AMD病理生物学的新机制，并进一步将Nrf2确定为干预的分子靶点。

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.