Impact of Lipofuscin in Retinal Pigment Epithelial Cells

描述(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色素的细胞。事实上，已经证明RPE脂褐素的一个主要成分，荧光团A2E，可以介导蓝光损伤，并可以在细胞膜上发挥类似洗涤剂的活性。为了追踪导致蓝光照射的A2E培养的RPE死亡的事件，正在研究单线态氧的参与和A2E活性光氧化产物的形成。人们正在探索将DNA作为这些活性中间体的靶子，并正在测试维生素E等抗氧化剂防止蓝光对携带A2E的RPE造成损害的能力。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bc l-2的调节，现在的工作是确定caspase-9是caspase的起始者。在导致细胞死亡程序激活的上游信号方面，人们正在研究与DNA损伤相关的P53激活，以及应激蛋白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。 A2E在细胞的溶酶体隔间积聚，即使在没有光照的情况下，当积聚到一定水平时也会损害RPE细胞。这种影响正在通过洗涤剂样的溶酶体膜破坏来研究。决定这一行为的A2E的结构特征也将被检查。在A2E生物合成的研究中，人们正在探索促进或损害A2E形成的条件(强光、抗氧化剂处理、NADPH耗竭)。A2E是由前体A2-PE在RPE细胞的溶酶体隔室内通过酶介导的机制产生的这一前提也正在研究中。为了重新探讨与人类RPE脂褐素的组成相关的问题，正在鉴定A2E的其他光异构体，并正在探索A2E以外的全视网膜凝聚产物的存在。 这项工作的长期目标是开发治疗方法，以减少A2E的形成，破坏RPE细胞内形成的分子，或对抗其积累的有害影响。

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.