The Role of Muller Cells in Visual Pigment Regeneration

描述(申请人提供)：光感知的第一个事件是视蛋白视觉色素在视杆或视锥感光细胞中吸收光子。这导致11-顺式视黄醛(11-顺式)发色团异构化为全反式视黄醛(全反式视黄醛)，从而衰退产生凋亡素和游离全反式视黄醛。当凋亡素与另一个11顺时体重组时，它又恢复了对光的敏感性。在视网膜色素上皮(RPE)细胞中，11-顺式-醛的合成是通过一种称为视觉周期的酶途径来实现的。越来越多的证据表明，视锥细胞可能通过被称为交替视觉周期的第二个假设途径接触到M�ler细胞中视觉生色团的另一个来源。这一途径被认为是在白天条件下为锥体提供专门的视觉生色团。我们确定交替视觉周期的关键异构酶(异构酶-2)为二氢神经酰胺脱饱和酶-1(DES1)。DES1催化的视黄醇异构化速度是RPE视觉循环中RPE65催化的异构化速度的300倍。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快速退化的影响，在视网膜脱离后，视网膜和RPE在物理上是分开的。本研究拟对DES1的视维A酸类加工活动进行表征，并研究其在交替视觉周期中的作用。我们最近做了一个重要的初步观察，即DES1的异构酶-2活性被可见光有效地刺激。这一结果表明，在白天的条件下，脊椎动物(包括人类)在视网膜中捕捉光能，以再生视觉生色团。昆虫和其他无脊椎动物利用光能再生它们的视觉色素已经有一段时间了。光有助于动物发色团再生的观察是史无前例的。此外，这一过程似乎是通过一种新的生化机制发生的：DES1蛋白中维生素A的自由基阳离子吸收550 nm的光子。除了深入研究DES1的生化特性外，我们还将在培养细胞和活的转基因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.