Lipid Activated Nuclear Receptors in Age-Related Macular Degeneration

描述（由申请人提供）：视网膜下色素上皮（亚rpe）沉积形成的细胞和分子途径是年龄相关性黄斑变性（AMD）早期“干”形式的标志，也是老年人视力丧失的主要原因，目前尚不清楚。流行病学研究已经确定年龄、n-6多不饱和脂肪酸（PUFA）饮食摄入和氧化损伤是AMD的风险因素。此外，包括我们自己在内的几个实验室的研究表明，调节脂质和胆固醇加工和分泌的途径在沉积物形成中起着关键作用。然而，膳食摄入PUFAs与促进沉积物形成的信号通路之间的联系尚不清楚。因此，我们的目标是确定包括PUFAs在内的脂质调节沉积形成的致病机制。过氧化物酶体增殖激活受体（PPARs）是一种核受体，具有脂质传感器的作用。在这三种亚型中，PPAR¿/d介导膳食脂肪酸（包括n-6 PUFAs）对基因表达的调节作用，并刺激参与脂肪酸氧化的过氧化物酶体（细胞器）的增殖。PPAR转录活性与胆固醇和脂质外排相关分子的上调、细胞外基质（ECM）合成改变和线粒体功能障碍相关；这些分子家族也与沉积物的形成有关。考虑到这一点，我们提出了一个必然的问题：膳食脂质激活RPE细胞中的PPAR¿/d信号通路是否刺激沉积形成？在我们的初步研究中，我们发现RPE细胞培养物暴露于天然和氧化的n-6 PUFAs衍生物中，导致与沉积物形成相关和调节沉积物形成的分子表达发生深刻的细胞变化。变化包括：(1)ECM分子胶原IV的合成和分泌增加；(2)胆固醇和脂质外排调节基因ABCA1和CD36表达增加；(3)受损线粒体和活性氧的积累；(4) PPAR¿/d的活化；(5)调控ECM分子和脂质分泌的PPAR¿/d特异性靶基因上调。基于这些初步数据，我们假设饮食中的n-6脂肪酸通过激活PPAR¿/d和增加过氧化物酶体的增殖来刺激沉积物中分子的产生。沉积物的积累需要RPE分泌脂质和胆固醇，RPE合成ECM分子的失调导致脂质被捕获。我们进一步提出PPAR¿/d通路上的额外“压力源”（即年龄、氧化剂）导致这些过程的上调，并进一步损害RPE细胞功能，由线粒体功能障碍介导。为了验证这一假设，我们将结合人类RPE细胞和小鼠沉积模型进行细胞培养试验，以研究ppar在沉积形成中的作用。我们还将研究PPAR¿/d活性的降低是否可以减缓沉积形成小鼠模型中AMD的进展。

DESCRIPTION (provided by applicant): The cellular and molecular pathways of sub-retinal pigment epithelial (sub-RPE) deposit formation, the hallmark of the early 'dry' form of age-related macular degeneration (AMD), and the leading cause of vision loss in the elderly, are not known. Epidemiology studies have identified age, n-6 polyunsaturated fatty acid (PUFA) dietary intake and oxidant injury as risks for AMD. Further, studies from several laboratories, including our own, suggest that pathways regulating lipid and cholesterol processing and secretion play a pivotal role in deposit formation. However, the link between dietary intake of PUFAs and signaling pathways that promote production of deposits formation is not known. Our goal therefore is to identify the pathogenic mechanisms by which lipids, including PUFAs, regulate deposit formation. Peroxisome proliferator activating receptors (PPARs) are nuclear receptors that act as lipid sensors. Of the three isoforms, PPAR¿/d mediates the regulatory effects of dietary fatty acids, including n-6 PUFAs, on gene expression and stimulates the proliferation of peroxisomes, organelles involved in fatty acid oxidation. PPAR transcriptional activity is coupled with upregulation of molecules associated with cholesterol and lipid efflux, altered extracellular matrix (ECM) synthesis, and mitochondrial dysfunction; these families of molecules are also associated with deposit formation. With this in mind, we asked a corollary question: does dietary lipid activation of the PPAR¿/d signaling pathway in RPE cells stimulate deposit formation? In our preliminary studies, we found that exposure of RPE cell cultures to native and oxidized derivatives of n-6 PUFAs resulted in profound cellular changes in the expression of molecules associated with and regulating deposit formation. The changes included (1) increased synthesis and secretion of ECM molecule collagen IV; (2) increased expression of cholesterol and lipid efflux regulatory genes ABCA1 and CD36; (3) accumulation of damaged mitochondria and reactive oxygen species; (4) activation of PPAR ¿/d; and (5) upregulation of PPAR¿/d specific target genes regulating ECM molecules, and lipid secretion. Based on this preliminary data, we hypothesize that dietary n-6 fatty acids stimulate production of molecules found in deposits through activation of PPAR¿/d and increased proliferation of peroxisomes. Accumulation of deposits requires lipid and cholesterol secretion by the RPE and dysregulated synthesis of ECM molecules by the RPE, leads to trapping of lipids. We further propose that additional 'stressors' on the PPAR¿/d pathway (i.e., age, oxidants) lead to upregulation of these processes and further compromise RPE cell function, mediated by mitochondrial dysfunction. To test this hypothesis we will use a combination of cell culture assays with human RPE cells and mouse models of deposits to investigate the role of PPARs in deposit formation. We will also investigate if decreasing activity of PPAR¿/d can slow the progression of AMD in murine models of deposit formation. PUBLIC HEALTH RELEVANCE: Age-related macular degeneration (AMD) afects 30% of individuals over the age of 65 years and is the leading cause of vision loss in the Western World. Of the three clinical sub-types, the early 'dry' form of the disease effects over 85% of the patient population. Risk factors for developing the disease include advanced age, dietary intake of lipids and oxidant injury. The objective of this grant is to investigate signaling pathways used by dietary lipids that cause phenotypic pathology of early 'dry' AMD, specifically (1) production and accumulation of lipid and protein rich-extracellular deposits and (2) functional changes in lipid metabolic processing cellular organelles. We also propose to investigate the modulating effect of additional AMD risk factors, age and oxidant injury, on disease development. Currently there are no therapeutic options available for early 'dry' AMD. Understanding the mechanisms underlying deposit formation and identifying signaling pathways key to disease initiation and progression will open up new avenues for therapeutic strategies in early 'dry' AMD.