Impact of Lipofuscin in Retinal Pigment Epithelial Cells

描述（由申请人提供）：在一些视网膜疾病（包括黄斑变性的萎缩形式）中，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，已经表明，RPE脂褐质的主要成分，荧光团A2 E，可以介导蓝光损伤，并可以在细胞膜上发挥去污剂样活性。为了追踪导致蓝光照射的A2 E负载培养的RPE死亡的事件，正在研究单线态氧的参与和A2 E的反应性光氧化产物的形成。DNA作为这些活性中间体的目标正在被探索，并且抗氧化剂如维生素E保护A2 E负载的RPE免受蓝光损伤的能力正在被测试。已经表明细胞死亡程序涉及半胱天冬酶蛋白酶的活化和线粒体蛋白Bcl-2的调节，现在的工作是针对鉴定半胱天冬酶-9作为起始半胱天冬酶。在导致细胞死亡程序激活的上游信号传导方面，正在研究与DNA损伤相关的p53激活，以及应激激酶途径的组分。与线粒体相关的促凋亡蛋白的作用也正在研究中。 A2 E在细胞的溶酶体区室中积累，即使在没有照明的情况下，当积累到一定水平时也会损害RPE细胞。这种作用正在研究的洗涤剂样破坏的溶酶体膜。决定这种行为的A2 E的结构特征也将被检查。在A2 E生物合成的研究中，正在探索A2 E的形成可能加速或受损的条件（强光、抗氧化剂处理、NADPH耗竭）。A2 E是通过RPE细胞的溶酶体隔室内的酶介导的机制从前体A2-PE产生的前提也正在被检查。为了重新审视与人类RPE脂褐素的组成相关的问题，正在鉴定A2 E的其他光异构体，并且正在探索除A2 E之外的全反式视网膜缩合产物的存在。 这项工作的长期目标是开发减少A2 E形成的疗法，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.