Impact of Lipofuscin in Retinal Pigment Epithelial Cells

描述（由申请人提供）：在一些视网膜疾病中，包括萎缩性黄斑变性，视网膜色素上皮（RPE）细胞的死亡是导致视力损害的早期和关键事件。实验室的工作旨在确定处于危险中的RPE细胞是否是那些积累了临界水平的脂褐素的细胞。事实上，已经证明RPE脂褐素的主要成分，荧光团A2E，可以介导蓝光损伤，并可以对细胞膜施加类似洗涤剂的活性。为了追踪导致蓝光照射下A2E负载培养RPE死亡的事件，正在研究单线态氧的参与和A2E反应性光氧化产物的形成。人们正在探索DNA作为这些反应中间体的目标，而抗氧化剂（如维生素E）保护含有a2e的RPE免受蓝光损害的能力也在测试中。已经证明细胞死亡程序涉及caspase蛋白酶的激活和线粒体蛋白Bcl-2的调节，现在的工作是确定caspase-9作为启动caspase。就导致细胞死亡程序激活的上游信号而言，正在研究与DNA损伤相关的p53激活，以及应激激酶途径的组成部分。与线粒体相关的促凋亡蛋白的作用也在研究中。

DESCRIPTION (provided by applicant): In some retinal disorders including atrophic forms of macular degeneration, the death of retinal pigment epithelial (RPE) cells is an early and crucial event leading to visual impairment. Work in the laboratory is aimed at determining whether the RPE cells at risk are those that have accumulated critical levels of lipofuscin pigment. Indeed, it has been shown that a major constituent of RPE lipofuscin, the fluorophore A2E, can mediate blue light damage and can exert a detergent-like activity on cell membranes. To track the events that lead to the death of blue light-irradiated A2E-laden cultured RPE, the involvement of singlet oxygen and the formation of reactive photoxidative products of A2E are being investigated. DNA is being explored as a target of these reactive intermediates, and the ability of antioxidants such as vitamin E to protect against blue light damage to A2E-laden RPE is being tested. Having shown that the cell death program involves activation of caspase proteinases and modulation by the mitochondria protein Bcl-2, work is now directed towards identifying caspase-9 as the initiator caspase. In terms of the upstream signaling that leads to activation of the cell death program, activation of p53 in association with DNA damage is being investigated, as are components of the stress kinase pathway. The role of pro-apoptotic proteins, which associate with mitochondria, are also being studied. A2E accumulates in the lysosomal compartment of the cell and even in the absence of illumination, can damage RPE cells when amassed to certain levels. This effect is being investigated in terms of a detergent-like disruption of the lysosomal membrane. Structural features of A2E that determine this behavior are also to be examined. In studies of A2E biosynthesis, conditions (bright light, antioxidant-treatment, NADPH depletion) under which the formation of A2E may be accelerated or impaired are being probed. The premise that A2E is generated from the precursor A2-PE by enzyme-mediated mechanisms within the lysosomal compartment of the RPE cell is also being examined. To revisit questions related to the composition of human RPE lipofuscin, additional photoisomers of A2E are being identified and the presence of all-trans-retinal condensation products other than A2E is being explored. The long-term goals of this work are to develop therapies that would reduce the formation of A2E, destroy the formed molecule within the RPE cell, or counter the deleterious effects of its accumulation.