PHAGOSOME DYNAMICS IN THE RETINAL PIGMENT EPITHELIUM

长期就业：在未来30年，近800万人将 由于年龄相关性黄斑变性而导致严重的视力丧失 (AMD)除非我们在理解上取得重大进展， 疾病过程。目前，我们的研究重点是阐明机制 调节AMD的发作。视网膜的进行性变性 作为AMD特征的色素上皮（RPE）和布鲁赫膜可能 结果是由于脂褐素在RPE溶酶体中积累，就像组织 在溶酶体贮积病中，溶酶体充血后出现功能障碍。 溶酶体生物学的最新进展提出了脂褐素假说 对AMD来说尤其及时和有吸引力。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 必要的RPE功能，提出了一个双管齐下的方法来评估 组织病理学的老化黄斑和检查细胞功能，在人类 体外RPE。五个具体目标是：目标1和2 -检查黄斑 白色（60-80岁）的捐赠者，以确定（a）是否 黄斑变性风险较大的个体（即，白人与 蓝眼睛的人比棕色眼睛的人具有更高的脂褐素浓度 （B）相同年龄的眼睛的存活数量之间的关系 光感受器和RPE脂褐质的量;目的3和4 -确定 脂褐素是否抑制吞噬体消化或营养物质的关键事件 摄取（例如酸化、溶酶体融合、酸性水解酶的作用）; 目的5 -区分溶酶体扩增和累积效应 氧化损伤的影响。 方法：包括供体固定切片的显微镜检查 眼睛和视频显微镜与计算机辅助图像分析，以测量 脂褐素浓度。相同年龄但不同的人类RPE细胞 将从黄斑区和赤道区培养脂褐质内容物 供体眼睛的功能，以及与营养吸收和形成 并测定吞噬溶酶体的功能。猪RPE细胞饲养 不同数量的乳胶珠将被用作另一种模型， 扩大的溶酶体体积。内体的内吞作用和酸化将 测定为（125）I转铁蛋白和（59）Fe的摄取。的动力学 将检查吞噬溶酶体的形成，并观察脂褐质，年龄， 和视网膜位点上的这一点以及所有RPE功能将被分析。 重要性和健康意义：这些实验将系统地 探索脂褐素积累和RPE之间的可能关系 功能协调发展的如果我们发现更多的证据表明脂褐素的积累可能是 AMD的早期事件， （目前正在开发）可以提供一个额外的指数， AMD风险，一种多因素疾病。最后，这些研究将扩大 我们对吞噬体参与的RPE细胞基本功能的了解 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.