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由视杆或视锥感光细胞中的视蛋白视色素吸收光子。这导致11-顺式-视黄醇（11-cis-RAL）发色团异构化为全反式-视黄醇（all-trans-RAL），其衰变产生脱辅基视蛋白和游离的全反式-RAL。当脱辅基视蛋白与另一个11-顺式-RAL重组时，其恢复光敏感性。11-cis-RAL的合成是通过视网膜色素上皮细胞（RPE）中称为视觉周期的酶途径进行的。越来越多的证据表明，视锥细胞可能通过第二种被称为交替视觉周期的假设途径，获得M？ller细胞中视觉发色团的另一个来源。该途径被认为在日光条件下特异性地向视锥细胞提供视觉发色团。我们确定了交替视觉循环的关键异构酶（异构酶-2）为二氢神经酰胺去饱和酶-1（DES 1）。通过DES 1的视黄醇异构化速率比通过RPE视觉循环的Rpe 65催化的异构化速率快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，其中视网膜和RPE在物理上分离。本研究拟对DES 1的视-视色素加工活性进行表征，并研究其在交替视觉周期中的作用。我们最近做了重要的初步观察，异构酶-2活性的DES 1是有力的刺激可见光。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它们的视觉色素。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：DES 1蛋白内部维生素A的自由基阳离子吸收550 nm的光子。除了DES 1的深入生物化学表征外，我们还将研究培养细胞和活的转基因小鼠中视觉发色团的光刺激再生。

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.