Dysregulation of PPARα in RPE degeneration

项目摘要/摘要 尽管抗血管内皮生长因子疗法对与年龄相关的湿型患者显示出令人印象深刻的好处 黄斑变性(AMD)，目前还没有有效的治疗干性AMD，这是一个主要的临床需求没有得到满足。视网膜 色素上皮(RPE)和视网膜功能障碍及变性是干性视网膜病变的主要病理特征 AMD。线粒体功能缺陷和脂质代谢紊乱被认为是RPE的关键 干性AMD这些病理过程中的致病作用。然而，细胞外信号调节失调的分子机制 AMD患者的RPE中的脂代谢难以捉摸。过氧化物酶体增殖物激活受体α(PPARα)是一种 转录因子。它调节脂质代谢，因此，pparα激动剂被用于临床治疗 血脂异常。尽管我们最近的研究表明PPARα在视网膜中具有保护作用，但这种联系 PPARα与AMD的发病机制尚不清楚。我们的初步研究表明，PPARα 干性AMD和两种动物模型中干性AMD患者视网膜和RPE中的水平均下调 部分AMD表型。此外，在视网膜色素上皮细胞中激活或表达PPARα部分地保护了 视网膜和RPE对抗氧化应激诱导的RPE和视网膜损伤。我们已经证明了PPARα 仅基因敲除(KO)就可导致与年龄相关的ERG下降，视网膜变性，RPE细胞形态异常， RPE细胞体积增大，RPE屏障受损，小胶质细胞/巨噬细胞对RPE的黏附增加。 PPARαKO还可诱导视网膜色素上皮和视网膜色素上皮细胞膜脂积聚。因此，我们假设 PPARα是视网膜色素上皮中脂肪酸氧化和脂质平衡的主要调节因子，对 维持RPE和视网膜的正常结构和功能。在这个项目中，我们将使用新生成的 RPE特异性PPARα条件性KO(PPARα-CKO)小鼠和表达PPARα的转基因(PPARα-TG)小鼠 在RPE中为拟议的研究。我们将分析RPE屏障功能、RPE细胞形态和 细胞大小、ERG、视网膜和光感受器细胞层厚度、视网膜下炎症和脂质堆积 在正常饮食和高脂高胆固醇饮食条件下，α-Cko小鼠视网膜色素上皮和Bruch‘s膜 饮食，以揭示是否仅在视网膜色素上皮细胞中进行PPARα消融会导致视网膜和视网膜色素上皮病变，这将是 HFC饮食加速和加剧了这种情况。我们还将确定PPARα消融是否会减少粮农组织和 增加RPE中的糖酵解。将对PPARα-CKO RPE进行蛋白质组学分析以鉴定酶 和脂结合蛋白在PPARα-Cko小鼠视网膜色素上皮中的表达水平发生了变化。此外，我们将调查是否 PPARα-TG小鼠表现出减轻，而PPARα-CKO小鼠表现出更严重的视网膜色素上皮和视网膜损伤。 氧化应激。我们还将探索PPARα激动剂非诺贝特对RPE和RPE的治疗潜力。 具有某些AMD表型的两种遗传小鼠模型的视网膜功能障碍和变性。建议数 研究将确定RPE中脂代谢的新调节机制，并有可能导致 治疗干性老年性黄斑变性的口服降脂药物的再利用。

PROJECT SUMMARY/ABSTRACT Although anti-VEGF therapies have shown impressive benefits for patients with wet form age-related macular degeneration (AMD), there is no effective treatment for dry AMD, a major unmet clinical need. Retinal pigment epithelium (RPE) and retina dysfunction and degeneration are the major pathological features in dry AMD. Deficient mitochondrial function and disturbed lipid metabolism in the RPE are believed to play key pathogenic roles in these pathologies of dry AMD. However, the molecular mechanism for the dysregulation of lipid metabolism in the RPE with AMD is elusive. Peroxisome Proliferator-Activated Receptor α (PPARα) is a transcription factor. It regulates lipid metabolism, and thus, PPARα agonists are used clinically to treat dyslipidemia. Although our recent study showed that PPARα has a protective role in the retina, the association of PPARα with the pathogenesis of AMD remains unknown. Our preliminary studies demonstrated that PPARα levels are down-regulated in the retina and RPE of human donors with dry AMD and in two animal models with partial AMD phenotypes. Furthermore, activation or expression of PPARα in the RPE partially protected the retina and RPE against oxidative stress-induced RPE and retina damage. We have demonstrated that PPARα knockout (KO) alone resulted in age-related ERG decline, retinal degeneration, abnormal RPE cell morphology, enlarged RPE cell size, impaired RPE barrier, and increased microglia/macrophage adherence to the RPE. PPARα KO also induced lipid accumulation in the RPE and Bruch’s membrane. Thus, we hypothesize that PPARα is a major regulator of fatty acid oxidation (FAO) and lipid homeostasis in the RPE, and essential for maintaining normal structure and function of the RPE and retina. In this project, we will use our newly generated RPE-specific PPARα conditional KO (PPARα-CKO) mice and transgenic (PPARα-Tg) mice expressing PPARα in the RPE for the proposed studies. We will analyze changes in RPE barrier function, RPE cell morphology and cell size, ERG, retinal and photoreceptor cell layer thicknesses, subretinal inflammation, and lipid accumulation in the RPE and Bruch’s membrane of PPARα-CKO mice under a regular diet or high-fat, cholesterol-rich (HFC) diet, to reveal if PPARα ablation in the RPE alone will induce retina and RPE pathologies, which will be accelerated and exacerbated by the HFC diet. We will also determine if PPARα ablation will decrease FAO and increase glycolysis in the RPE. Proteomic analysis of PPARα-CKO RPE will be performed to identify enzymes and lipid-binding proteins with changed levels in the RPE of PPARα-CKO mice. Further, we will investigate if PPARα-Tg mice will show alleviated, while PPARα-CKO mice will show more severe, RPE and retinal injury by oxidative stress. We will also explore the therapeutic potential of PPARα agonist fenofibrate against RPE and retinal dysfunction and degeneration in two genetic mouse models with some AMD phenotypes. The proposed studies will identify a new regulation mechanism for lipid metabolism in the RPE and has the potential to lead to the repurposing of an oral lipid-lowering drug for the treatment of dry AMD.