Mechanisms for Light-driven Chromophore Synthesis by Müller Cells to Regenerate Cone Opsin and Maintain Cone Sensitivity

项目总结/摘要 这个项目的目标是确定三种内在视网膜视觉周期的蛋白质，这些蛋白质维持了视网膜的视觉周期。 视锥细胞在日光条件下的敏感性。在上一个资助期间，PI发现，共同- RGR视蛋白和视黄醇脱氢酶-10（Rdh 10）的表达赋予细胞摄取 从培养基中增加全反式视黄醇（atROL）并释放11-顺式视黄醇（11 cROL），仅在暴露于 可见光Müller细胞也观察到了相同的atROL至11 cROL转化活性， 尽管以前没有报道过光依赖性。视锥细胞，而不是视杆细胞，具有11 cROL- 脱氢酶（11 cRDH）活性，使他们能够利用Müller细胞释放的11 cROL， 制备11 cRAL发色团，从而再生光漂白的视锥蛋白。远景目标 这个项目的目的是了解过程中，使连续锥视觉， 哺乳动物的日光暴露。鉴于视锥细胞对人类视觉的重要性， 我们知识的不足肯定是NEI使命的一部分。具体目标1是确定 RGR视蛋白和Rdh 10在视网膜固有视觉周期中的作用这将通过一个合作 使用生物化学和生理学方法进行研究。目标1的中心假设是RGR 视蛋白和Rdh 10构成了Müller细胞中的11 cROL发生器， 这些蛋白质由光驱动。具体目标2是鉴定允许视锥细胞 从11 cROL再生视色素。这将使用cDNA表达筛选来完成， 测试在视锥细胞中表达的短链脱氢酶/还原酶（SDR）家族的成员。 这项研究的预期结果是鉴定出三种蛋白质，它们共同组成了 内在视网膜视觉周期这些结果相当于揭示了一种新的代谢途径， 视网膜。它们将极大地扩展我们对视锥细胞色素再生的认识， 日光此外，他们将打开大门，进一步遗传和功能生化研究，进入 视网膜视觉周期，包括识别遗传性视网膜病新易感位点的潜力 和黄斑病变人类RGR基因的突变已经与视网膜炎有关 在一小部分病例中有色素沉着。虽然长期以来一直认为无脊椎动物，如 昆虫利用光来再生它们的视觉色素，这在脊椎动物中从未被证明过。的 因此，PI发现光驱动视锥细胞色素的再生是一种新颖的， 重大发现。

Project Summary / Abstract The goal of this project is to identify three proteins of the intrinsic retinal visual cycle that maintain the sensitivity of cones under daylight conditions. During the previous funding period, the PI found that co- expression of RGR opsin and retinol dehydrogenase-10 (Rdh10) confers upon cells the capacity to take up all-trans-retinol (atROL) from the medium and release 11-cis-retinol (11cROL), only on exposure to visible light. The same atROL to 11cROL conversion activity has been observed by Müller cells, although not previously reported as light dependent. Cones, but not rods, possess an 11cROL- dehydrogenase (11cRDH) activity that allows them to utilize the 11cROL released by Müller cells to make 11cRAL chromophore and thereby regenerate photobleached cone opsins. The long-term goal of this project is to understand the processes that enable continuous cone vision during daylight exposure in mammals. Given the importance of cones to human vision, addressing this deficit in our knowledge is certainly within the NEI mission. Specific Aim 1 is to determine the roles of RGR opsin and Rdh10 in the intrinsic retinal visual cycle. This will be accomplished in a collaborative study using biochemical and physiological methodologies. The central hypothesis for Aim 1 is that RGR opsin and Rdh10 comprise the 11cROL-generator in Müller cells, and that formation of 11cROL by these proteins is driven by light. Specific Aim 2 is to identify the 11cRDH that allows cones to regenerate visual pigment from 11cROL. This will be accomplished using a cDNA expression-screen to test members of the short-chain dehydrogenase/reductase (SDR) family that are expressed in cones. The expected outcome of this study is the identification of three proteins that together comprise the intrinsic retinal visual cycle. These results amount to the uncovering of a new metabolic pathway in the retina. They will greatly extend our knowledge about the regeneration of cone visual pigment in daylight. Also, they will open the door to further genetic and functional biochemical studies into the retinal visual cycle, including the potential to identify new susceptibility loci for inherited retinopathies and maculopathies. Mutations in the human RGR gene have already been associated with retinitis pigmentosa in a small subset of cases. While it has been long appreciated that invertebrates, such as insects, use light to regenerate their visual pigments, this has never been shown for vertebrates. The PI's finding that light drives regeneration of cone visual pigment is therefore a novel and important discovery.