The Role of Muller Cells in Visual Pigment Regeneration

描述（由申请人提供）：光感知的第一个事件是视蛋白视觉色素在杆状或锥状感光细胞中吸收光子。这导致11-顺式视黄醛（11-顺式视黄醛）发色团异构化为全反式视黄醛（all-trans-RAL），后者衰变生成载视蛋白和游离的全反式视黄醛。当载脂蛋白视蛋白与另一个11-顺式ral重组时，其光敏性得以恢复。11-顺式ral的合成是通过视网膜色素上皮细胞（RPE）中称为视觉循环的酶途径进行的。越来越多的证据表明，视锥细胞可能通过另一种被称为交替视觉周期的假设途径，获得了另一种视觉发色团的来源。这条通路被认为在日光条件下为视锥细胞提供视觉发色团。我们确定了交替视觉循环（异构酶-2）的关键异构酶是二氢神经酰胺去饱和酶-1 （DES1）。在RPE视觉循环中，DES1催化的视黄醇异构化速率比Rpe65催化的视黄醇异构化速率快300倍。在临床上，完全位于视网膜内的交替视觉周期可以保护光感受器免受视网膜脱离后的快速变性，视网膜和RPE在物理上分离。本研究旨在描述DES1的视觉-类维甲酸加工活性，并研究其在交替视觉周期中的作用。我们最近进行了重要的初步观察，DES1的异构酶-2活性在可见光下受到强烈刺激。这一结果表明，在日光条件下，脊椎动物（包括人类）在其视网膜中捕获光能以再生视觉发色团。一段时间以来，人们已经知道昆虫和其他无脊椎动物利用光能再生视觉色素。光有助于动物发色团再生的观察是前所未有的。此外，这一过程似乎是通过一种新的生化机制发生的：DES1蛋白内的维生素a自由基阳离子吸收550 nm的光子。除了对DES1进行深入的生化表征外，我们还将在培养细胞和转基因活小鼠中研究光刺激下视觉发色团的再生。

DESCRIPTION (provided by applicant): The first event in light perception is absorption of a photon by an opsin visual pigment in a rod or cone photoreceptor cell. This causes isomerization of the 11-cis-retinaldehyde (11-cis-RAL) chromophore to all-trans-retinaldehyde (all-trans-RAL), which decays to yield apo-opsin and free all-trans-RAL. Light sensitivity is regained by apo-opsin when it recombines with another 11-cis-RAL. Synthesis of 11-cis-RAL is carried out by an enzyme pathway called the Visual Cycle in cells of the retinal pigment epithelium (RPE). Accumulating evidence suggests that cones may have access to another source of visual chromophore in M�ller cells through a second hypothesized pathway called the Alternate Visual Cycle. This pathway is thought to provide visual chromophore specifically to cones under daylight conditions. We identified the critical isomerase of the Alternate Visual Cycle (isomerase-2) as dihydroceramide desaturase-1 (DES1). The rate of retinol isomerization by DES1 is 300-fold faster than the rate of isomerization catalyzed by Rpe65 of the RPE Visual Cycle. Clinically, the Alternate Visual Cycle, which is located entirely within the retina, may protect photoreceptors from rapid degeneration following retinal detachment, where the retina and RPE become physically separated. This proposal is to characterize the visual-retinoid processing activity of DES1, and to study its role in the Alternate Visual cycle. We recently made the important preliminary observation that the isomerase-2 activity of DES1 is potently stimulated by visible light. This result suggests that under daylight conditions, vertebrates (including humans) capture light energy in their retinas to regenerate visual chromophore. It has been known for some time that insects and other invertebrates regenerate their visual pigments using light energy. The observation that light contributes to chromophore regeneration in animals is unprecedented. Further, this process appears to take place through a novel biochemical mechanism: absorption of a 550-nm photon by a radical-cation of vitamin A inside the DES1 protein. Besides in-depth biochemical characterization of DES1, we will study light-stimulated regeneration of visual chromophore in cultured cells and in live, genetically modified mice.