IMPACT OF LIPOFUSCIN IN RETINAL PIGMENT EPITHELIAL CELLS

描述（来自申请人摘要的逐字描述）：衰老的一个显著特征 和一些遗传性视网膜变性是积累 视网膜色素上皮内脂褐素的自身荧光颗粒 细胞有间接证据表明年龄相关的 黄斑变性（AMD）和RPE脂褐素;然而， RPE细胞上的脂褐质积累尚未被直接测试。由于一个 在RPE脂褐质的成分中，荧光团A2 E表现出结构 和可能对细胞有害的光动力学特性， 假设这种荧光团在RPE细胞中的积累起着重要作用， 在AMD发病机制中的作用。这项工作的目的是了解 A2 E在RPE细胞中形成的机制，并确定 它在RPE细胞上的积累。高效液相色谱 HPLC分析，从人眼分离的RPE细胞中A2 E的量 将与捐献者的年龄、种族和性别相关。A2 E眼睛 也将被量化。为了建立一个细胞培养模型， 含A2 E的细胞，将呈现缺乏内源性A2 E的培养的RPE 在培养基中加入合成的A2 E。随后， 将表征细胞的A2 E，也将表征细胞内的A2 E。 A2 E的区室化。为了检验假设，A2 E，当存在于 临界浓度，可能对RPE细胞产生不利影响， 细胞内A2 E的作用，以（a）表现出洗涤剂样活性，（B）损伤细胞 作为光敏剂和（c）破坏溶酶体功能， 评估。还将确定A2 E介导的光毒性是否 涉及反应性氧化剂种类的产生和细胞凋亡形式的 RPE细胞死亡。黑色素对A2 E介导的细胞凋亡的保护作用 还将测试光毒性。最后，在与 A2 E的生物起源，我们将获得中间体形成的证据 化合物（A2-PE）在A2 E生物合成过程中。我们还将讨论 A2-PE/A2 E是否形成在感光体外节膜中， 与RPE细胞的溶酶体区室相反。的长期目标 这些研究是为了确定加速A2 E形成的条件， 目的：研究A2 E在AMD发病中的作用。如果能 证明了RPE细胞合成A2 E的积累在 就RPE细胞功能而言，治疗的目的可能是防止其 形成或破坏RPE细胞内形成的分子。

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.