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（sub-RPE）存款形成的细胞和分子途径尚不清楚，这是年龄相关性黄斑变性（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。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存款形成中起着关键作用。然而，PUFA的饮食摄入量和促进沉积物形成的信号通路之间的联系尚不清楚。因此，我们的目标是确定致病机制，脂质，包括多不饱和脂肪酸，调节存款形成。 过氧化物酶体增殖物激活受体（PPARs）是作为脂质传感器的核受体。在这三种亚型中，PPAR <$/d介导膳食脂肪酸（包括n-6 PUFA）对基因表达的调节作用，并刺激过氧化物酶体（参与脂肪酸氧化的细胞器）的增殖。PPAR转录活性与胆固醇和脂质流出、细胞外基质（ECM）合成改变和线粒体功能障碍相关分子的上调相关;这些分子家族也与存款形成相关。考虑到这一点，我们提出了一个必然的问题：饮食脂质激活RPE细胞中的PPAR/d信号通路是否刺激存款形成？ 在我们的初步研究中，我们发现RPE细胞培养物暴露于n-6 PUFA的天然和氧化衍生物导致与存款形成相关并调节其形成的分子表达的深刻细胞变化。这些变化包括（1）ECM分子胶原IV的合成和分泌增加;（2）胆固醇和脂质流出调节基因ABCA 1和CD 36的表达增加;（3）受损线粒体和活性氧的积累;（4）PPAR <$/d的激活;（5）调节ECM分子和脂质分泌的PPAR <$/d特异性靶基因的上调。 基于这些初步的数据，我们假设饮食中的n-6脂肪酸通过激活PPARm/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。沉积物的积累需要通过RPE的脂质和胆固醇分泌以及通过RPE的ECM分子的失调合成，导致脂质的捕获。我们进一步提出，额外的“压力源”对过氧化物酶体增殖物激活受体/d途径（即，老化、氧化剂）导致这些过程的上调，并进一步损害由线粒体功能障碍介导的RPE细胞功能。为了验证这一假设，我们将使用人RPE细胞和小鼠沉积物模型的细胞培养试验组合来研究PPARs在存款形成中的作用。我们还将研究在存款形成的小鼠模型中，降低PPAR/d的活性是否可以减缓AMD的进展。 公共卫生相关性：视网膜相关性黄斑变性（AMD）影响30%的65岁以上的个体，并且是西方世界视力丧失的主要原因。在这三种临床亚型中，早期的“干燥”形式的疾病影响超过85%的患者群体。发生这种疾病的危险因素包括高龄、饮食中摄入脂质和氧化损伤。这项资助的目的是研究导致早期“干性”AMD表型病理学的饮食脂质所使用的信号传导途径，特别是（1）富含脂质和蛋白质的细胞外沉积物的产生和积累，以及（2）脂质代谢处理细胞器的功能变化。我们还建议调查其他AMD危险因素，年龄和氧化损伤对疾病发展的调节作用。目前，对于早期“干性”AMD没有可用的治疗选择。了解存款形成的机制和识别信号通路的关键疾病的开始和进展，将开辟新的途径，治疗策略，在早期'干' AMD。

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.