Functional Characterization of RGR-opsin in Retinal Muller Cells

 描述(申请人提供)：光感受器中的视紫红质或视锥视色素捕获光子时，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)，从而激活色素。不久后，ALL-TRAL分裂，留下对光不敏感的凋亡素。为了再生视素色素，释放的全反式必须转化回11-顺式，然后与凋亡素重组。视觉生色团的合成发生在视网膜的色素上皮(RPE)和Müler细胞，两者都毗邻光感受器。在RPE和Müller细胞中，介导这种转化的生化途径是不同的。RPE细胞以缓慢的速度产生11-顺式缩醛，被认为在昏暗的光线下为视杆和视锥提供发色团。Müller细胞主要产生发色团前体11-顺式视黄醇(11-cis-rol)，该物质被视锥而不是视杆所利用，以合成11-顺式视黄醇。在明亮的 轻的米勒细胞翻转类维甲酸的速度比RPE细胞高得多。RGR-视蛋白是一种存在于视网膜色素上皮细胞和Müler细胞中的非视觉视蛋白。RGR基因敲除突变的小鼠表现出视觉色素再生缓慢，并在RPE和视网膜中积累视黄酸酯。视黄酸酯是视黄醇(维生素A)的脂溶储存形式。初步研究表明，RGR-opsin对RPE中视黄酸酯的光依赖动员起作用。RGR-opsin在Müler细胞中的功能目前尚不清楚，这是目前研究的主题。这个项目测试了一个广泛的假设，即RGR-opsin以一种光依赖的方式调节RPE和Müler细胞之间视觉维甲酸的流动。例如，通过刺激RPE中视黄酸酯的水解，RGR-opsin耗尽RPE细胞中异构酶的底物(RPE65)，同时为Müller细胞中的异构酶提供全反式ROL底物(DES1)。这项研究将在表达选定的视黄醇加工蛋白的培养哺乳动物细胞和转基因小鼠的眼睛组织上进行。实验方法包括维甲酸的液相色谱分析，动物组织和培养细胞的单光子和双光子共聚焦显微镜，以及活体小鼠眼球和视网膜外植体的视网膜电图术。

 DESCRIPTION (provided by applicant): Capture of a photon by a rhodopsin or cone-opsin pigment in a photoreceptor isomerizes the 11-cis-retinaldehyde (11-cis-RAL) chromophore to all-trans-retinaldehyde (all-trans-RAL), which activates the pigment. Shortly afterwards, the all-trans-RAL dissociates, leaving behind light-insensitive apo-opsin. To regenerate the opsin pigment, the released all-trans-RAL must be converted back to 11-cis-RAL, which then recombines with apo-opsin. Synthesis of visual chromophore takes place in the retinal pigment epithelium (RPE) and Müller cells of the retina, both adjacent to photoreceptors. The biochemical pathways that mediate this conversion are different in RPE and Müller cells. RPE cells produce 11-cis-RAL at a slow rate, and are thought to provide chromophore for both rods and cones under dim light. Müller cells mainly produce the chromophore- precursor, 11-cis-retinol (11-cis-ROL), which is utilized by cones, but not rods, to synthesize 11-cis-RAL. In bright light, Müller cells turn-over retinoids at a much higher rate than do RPE cells. RGR-opsin is a non-visual opsin located in both RPE and Müller cells. Mice with a knock-out mutation in the rgr gene exhibit slow regeneration of visual pigments and accumulate retinyl esters in the RPE and retina. Retinyl esters are lipid-soluble storage forms of retinol (vitamin A). Preliminary studies suggest that RGR-opsin effects light-dependent mobilization of retinyl esters in the RPE. Nothing is known about the function of RGR-opsin in Müller cells, which is the subject of the current study. This project tests the broad hypothesis that RGR-opsin regulates the flow of visual retinoids between RPE and Müller cells in a light-dependent fashion. For example, by stimulating hydrolysis of retinyl esters in the RPE, RGR-opsin depletes substrate for the isomerase in RPE cells (Rpe65) while providing all-trans-ROL substrate for the isomerase in Müller cells (DES1). This study will be performed on cultured mammalian cells expressing selected retinoid-processing proteins, and ocular tissues from genetically modified mice. Experimental methods include liquid chromatographic analysis of retinoids, single- and two-photon confocal microscopy of animal tissues and cultured cells, and ex vivo electroretinography of live mouse eyecups and retina explants.