PHAGOSOME DYNAMICS IN THE RETINAL PIGMENT EPITHELIUM

远景目标：未来30年，将有近800万人 由于年龄相关性黄斑变性而经历严重的视力丧失 (AMD)，除非我们对 疾病过程。目前，我们的研究主要集中在阐明机制上 调节AMD的发病。视网膜进行性退行性变 色素上皮(RPE)和Bruch膜是AMD的特征，可能 由于脂褐素在RPE溶酶体中积聚，就像组织一样 在溶酶体储存疾病中，溶酶体充血后会出现功能障碍。 溶酶体生物学的最新进展使脂褐素假说成为可能 对于AMD来说，尤为及时和诱人。我们相信 了解导致视力丧失的早期细胞事件将极大地 提高减缓或最终预防疾病的可能性。 具体目的：检验积累的脂褐素破坏 基本RPE函数，提出了一种双管齐下的方法来评估 人老年性黄斑的组织病理学及细胞功能检测 体外培养的RPE。五个具体目标是：目标1和目标2-检查黄斑 对白人(60-80岁)的捐赠者进行调查，以确定(A) 黄斑变性风险较高的个体(即，患有 蓝色眼睛)比棕色眼睛有更高的脂褐素浓度 同龄眼球与(B)存活人数的关系 光感受器和RPE脂褐素的量.AIMS 3和4.测定 脂褐素是否抑制吞噬小体消化或营养中的关键事件 摄取(例如酸化、溶酶体融合、酸性水解酶的作用)； 目标5-区分溶酶体扩张和累积的影响 氧化损伤的影响。 方法：包括对供体的固定切片进行显微镜检查。 计算机辅助图像分析的眼睛和视频显微镜测量 脂褐素浓度。年龄相同但不同的人RPE细胞 将从黄斑和赤道区培养脂褐素含量 以及与营养摄取和形成相关的功能 并对吞噬小体的功能进行检测。饲喂猪视网膜色素上皮细胞 不同数量的乳胶珠将被用作另一种型号的巨大 溶酶体体积扩大。内吞体的内吞作用和酸化将 测定为摄取~(125)I转铁蛋白和~(59)Fe。反应的动力学 将检查吞噬酶小体的形成，以及脂褐素、年龄、 并对视网膜上的这个部位和所有的RPE功能进行分析。 意义和与健康相关：这些实验将系统地 探讨脂褐素蓄积与RPE的可能关系 功能。如果我们发现进一步的证据表明脂褐素的堆积可能 AMD的早期事件及其非侵入性测量方法 (目前正在开发中)可以为评估提供额外的指标 AMD的风险，这是一个多因素的情况。最后，这些研究将延伸到 我们对吞噬小体所涉及的RPE细胞基本功能的了解 正在处理。

LONG-RANGE OBJECTIVES: In the next 3 decades, nearly 8 million people will experience serious visual loss due to age-related macular degeneration (AMD) unless major advancements are made in our understanding of the disease process. Currently our research focuses on elucidating mechanisms modulating the onset of AMD. The progressive degeneration of the Retinal Pigment Epithelium (RPE) and Bruch's membrane, which characterizes AMD, may result from lipofuscin accumulation in RPE lysosomes, just as tissue dysfunction follows lysosomal engorgement in lysosomal storage diseases. Recent advances in the biology of lysosomes makes the lipofuscin hypothesis for AMD particularly timely and attractive. It is our belief that understanding the early cellular events leading to vision loss will greatly improve the possibility of slowing, or ultimately preventing the disease. SPECIFIC AIMS: To test the hypothesis that accumulated lipofuscin disrupts essential RPE functions, a two-pronged approach is proposed to evaluate the histopathology of the aging macula and to examine cell functions in human RPE in vitro. The five Specific Aims are: Aims 1 and 2 - Examine the macula of donors who are white (aged 60-80 years) to determine (a) whether individuals at greater risk for macular degeneration (i.e., whites with blue eyes) have greater lipofuscin concentrations than those with brown eyes of the same age and (b) the relationship between number of surviving photoreceptors and amount of RPE lipofuscin; Aims 3 and 4 - Determine whether lipofuscin inhibits key events in phagosome digestion or nutrient uptake (e.g. acidification, lysosome fusion, action of acid hydrolases); Aim 5 - Distinguish effects due to lysosomal expansion and to cumulative effects of oxidative damage. METHODS:These include microscopic examination of fixed sections of donor eyes and videomicroscopy with computer-assisted image analysis to measure lipofuscin concentration. Human RPE cells of the same age but varying lipofuscin content will be cultured from the macular and equatorial regions of donor eyes, and functions related to nutrient uptake and the formation and function of the phagolysosome will be assayed. Porcine RPE cells fed varying amounts of latex beads will be used as another model of vastly expanded lysosomal volume. Endocytosis and acidification of endosomes will be assayed as uptake of (125)I transferrin and (59)Fe. The kinetics of phagolysosome formation will be examined, and effects of lipofuscin, age, and retinal site on this and all RPE functions will be analyzed. SIGNIFICANCE AND HEALTH-RELATEDNESS: These experiments will systematically explore possible relationships between lipofuscin accumulation and RPE functions. If we find further evidence that lipofuscin accumulation may be an early event in AMD then noninvasive methods for its measurement (currently in development) could provide an additional index in assessing risk for AMD, a multifactorial condition. Finally these studies will extend our knowledge of essential RPE cell functions involved in phagosome processing.