Lipid Activated Nuclear Receptors in Age-Related Macular Degeneration

描述(申请人提供)：视网膜下色素上皮(SubRPE)沉积形成的细胞和分子途径尚不清楚，这是老年性黄斑变性(AMD)早期干性形式的标志，也是老年人视力丧失的主要原因。流行病学研究发现，年龄、n-6多不饱和脂肪酸(PUFA)饮食摄入量和氧化剂损伤是AMD的危险因素。此外，包括我们自己在内的几个实验室的研究表明，调节脂肪和胆固醇的加工和分泌的途径在沉积物的形成中发挥着关键作用。然而，多不饱和脂肪酸的饮食摄入量与促进沉积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多不饱和脂肪酸在内的脂类调节沉积物形成的致病机制。过氧化物酶体增殖物激活受体(PPAR)是作为脂质感受器的核受体。在这三种异构体中，PPAR？/d介导了包括n-6多不饱和脂肪酸在内的膳食脂肪酸对基因表达的调节作用，并刺激了参与脂肪酸氧化的细胞器--过氧化物体的增殖。PPAR转录活性与与胆固醇和脂质外流相关的分子上调、细胞外基质(ECM)合成改变和线粒体功能障碍有关；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：RPE细胞中PPAR？/d信号通路的饮食脂质激活是否刺激沉积形成？在我们的初步研究中，我们发现，RPE细胞培养物暴露于n-6多不饱和脂肪酸的天然和氧化衍生物时，细胞内与沉积形成相关的分子的表达和调节发生了深刻的变化。这些变化包括：(1)ECM分子IV型胶原合成和分泌增加；(2)胆固醇和脂质外流调节基因ABCA1和CD36表达增加；(3)受损线粒体和活性氧积累；(4)PPAR？/d激活；(5)调节ECM分子和脂质分泌的PPAR？/d特异性靶基因上调。基于这一初步数据，我们假设，饮食中的n-6脂肪酸通过激活PPAR？/d和促进过氧化物体的增殖来刺激沉积物中发现的分子的产生。沉积物的积累需要RPE分泌脂类和胆固醇，RPE失调合成ECM分子，导致脂类被捕获。我们进一步提出，PPAR？/d途径上的额外‘应激源’(即，AGE、氧化剂)导致这些过程的上调，并通过线粒体功能障碍进一步损害RPE细胞的功能。为了验证这一假设，我们将使用人类RPE细胞的细胞培养分析和小鼠沉积模型相结合的方法来研究PPAR在沉积形成中的作用。我们还将在沉积形成的小鼠模型中研究PPAR？/d活性降低是否可以减缓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.