IMPACT OF LIPOFUSCIN IN RETINAL PIGMENT EPITHELIAL CELLS

描述(逐字摘自申请者的摘要)：老龄化的一个显著特征 一些遗传性视网膜退行性变是积累的 视网膜色素上皮(RPE)中脂褐素的自发荧光颗粒 细胞。存在间接证据表明与年龄相关的 黄斑变性(AMD)和RPE脂褐素；然而， 脂褐素在RPE细胞上的蓄积尚未得到直接测试。从一开始 在RPE脂褐素的成分中，荧光团A2E显示出结构 以及可能对细胞有害的光动力学特性，它是 假设这种荧光团在RPE细胞中的积累起到了 在AMD发病机制中的作用。这项工作的目的是了解 A2E在RPE细胞中形成的机制以及确定 它在RPE细胞上的积聚。通过高效液相色谱 高效液相色谱法测定人眼RPE细胞中A2E的含量 将与捐赠者的年龄、种族和性别相关。眼睛里的A2E 来自AMD捐赠者的数据也将被量化。为了开发一种细胞培养模型 含A2E的细胞，缺乏内源性A2E的培养的RPE 在培养基中加入合成的A2E。随之而来的是，内化 A2E将由细胞和细胞内的A2E来表征 A2E的区隔。以检验假设，当A2E存在于 临界浓度，会对RPE细胞产生不利影响，倾向于 细胞内A2E的作用：(A)表现出类似洗涤剂的活性，(B)损伤细胞 作为光敏剂和(C)扰乱溶酶体功能， 已评估。还将确定A2E介导的光毒性 涉及活性氧化剂物种的产生和一种凋亡形式的 RPE细胞死亡。黑色素对A2E介导的损伤的保护作用 还将进行光毒性测试。最后，在与 A2E的生物发生，我们将获得中间体形成的证据 A2E生物合成过程中的化合物(A2-PE)。我们还将解决以下问题 光感受器外节膜上是否形成A2-PE/A2E 与RPE细胞的溶酶体室相对。的长期目标是 这些研究旨在确定加速体内A2E形成的条件。 并评价A2E在AMD发病机制中的作用。如果可以的话 证明RPE细胞积聚合成的A2E在 在RPE细胞功能方面，治疗的目的可能是预防其 在RPE细胞内形成或破坏形成的分子。

DESCRIPTION (Verbatim from applicant's abstract): A prominent feature of aging and of some inherited retinal degenerations is the accumulation of autofluorescent granules of lipofuscin in retinal pigmented epithelial (RPE) cells. Circumstantial evidence exists for an association between age-related macular degeneration (AMD) and RPE lipofuscin; however, the impact of lipofuscin accumulation on the RPE cell has not been directly tested. Since one of the constituents of RPE lipofuscin, the fluorophore A2E, exhibits structural and photodynamic properties that could be detrimental to cells, it is hypothesized that the accumulation of this fluorophore in RPE cells plays a role in the pathogenesis of AMD. The aim of this work is to understand the mechanisms by which A2E forms in the RPE cell and to determine the impact of its accumulation on the RPE cell. By high performance liquid chromatography (HPLC) analysis, the quantities of A2E in RPE cells isolated from human eyes will be correlated with the age, race and gender of the donors. A2E in eyes from AMD donors will also be quantified. To develop a cell culture model of A2E-containing cells, cultured RPE lacking endogenous A2E will be presented with synthetic A2E in the culture media. Subsequently, the internalization of A2E by the cells will be characterized, as will the intracellular compartmentalization of A2E. To test the hypothesis that A2E, when present at critical concentrations, can have adverse effects on RPE cells, the propensity of intracellular A2E to (a) exhibit detergent-like activity, (b) damage cells as a photosensitizing agent and (c) disrupt lysosomal function, will be evaluated. It will also be determined whether A2E-mediated phototoxicity involves the generation of reactive oxidant species and an apoptotic form of RPE cell death. The ability of melanin pigment to protect against A2E-mediated phototoxicity will also be tested. Finally, in studies related to the biogenesis of A2E, we will obtain evidence for the formation of an intermediate compound (A2-PE) during A2E biosynthesis. We will also address the issue of whether A2-PE/A2E is formed in the photoreceptor outer segment membrane as opposed to the lysosomal compartment of the RPE cell. The long-term goal of these studies is to define conditions that accelerate the formation of A2E in vivo and to evaluate the role of A2E in the causation of AMD. If it can be demonstrated that the amassing of synthetic A2E by RPE cells is significant in terms of RPE cell function, treatments could be aimed at preventing its formation or destroying the formed molecule within the RPE cells.