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

项目摘要/摘要 该项目的目标是确定视网膜固有视觉周期的三种蛋白质，它们维持 日光条件下圆锥体的灵敏度。在上一次供资期间，私人投资委员会发现-- RGR视蛋白和视黄醇脱氢酶-10(RDH10)的表达赋予细胞摄取能力 从介质中上调全反式视黄醇(AtROL)并释放11-顺式视黄醇(11cROL)，仅在暴露于 可见光。同样的atROL到11cROL的转化活性已经被Müller单元观察到， 尽管之前没有报道过依赖于光。圆锥体，而不是杆状，拥有11cROL- 脱氢酶(11cRDH)活性，使它们能够利用Müler细胞释放的11cROL来 产生11个发色团，从而再生光漂白的锥体视蛋白。长期目标 本项目的主要目的是了解在 哺乳动物的日光暴露。鉴于视锥细胞对人类视觉的重要性，解决这个问题 我们的知识赤字肯定在NEI的任务范围内。具体目标1是确定 RGR视蛋白和Rdh10在视网膜固有视觉周期中的作用。这将通过协作方式实现 使用生化和生理学方法进行研究。目标1的中心假设是RGR Opsin和Rdh10组成了Müler细胞中11cROL生成器，并通过 这些蛋白质是由光驱动的。具体目标2是确定11cRDH允许锥体 从11cROL中再生视觉色素。这将使用cdna表达筛选来完成 短链脱氢酶/还原酶(SDR)家族的测试成员，在锥体中表达。 这项研究的预期结果是鉴定出三种共同构成 固有的视网膜视觉周期。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关于视锥视色素再生的知识 天亮了。此外，它们还将为进一步的基因和功能生化研究打开大门。 视网膜视觉周期，包括识别遗传性视网膜病变新易感基因的可能性 和黄斑病。人类RGR基因突变已被认为与视网膜炎有关 一小部分病例出现色素变性。虽然人们很早就意识到无脊椎动物，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出现过。这个 因此，Pi发现光驱动视锥视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.