Cone Opsin-Ligand Interactions and Photoreceptor Health

项目摘要/摘要 这个项目的广泛目标是了解导致光感受器的潜在事件 当类维甲酸代谢出现问题时出现退化，并开发防止视锥细胞的方法 不会退化。Leber先天性肥大症2型(LCA2)，一代本地人 视觉色素(11顺式视网膜)的生色团受到抑制。在LCA2小鼠模型中，锥体细胞死亡 迅速地。短波敏锥(SWS1)的损伤最为严重。这种模式大体上 与LCA2的发病机制相似。来自其他实验室的研究已经证明了这种基因 治疗可以恢复视力，但似乎受到治疗前不可逆转的视锥细胞丢失的限制。早些时候 给予外源性11-顺式视网膜-小鼠模型可改善视锥细胞存活率，但 该实验室最近的研究表明，当小鼠受到 房间里有灯光。这些结果表明：(1)视锥细胞视蛋白/11-顺式视网膜相互作用在 预防视锥细胞死亡，和(2)11顺式视网膜不是治疗LCA2的解决方案，因为正常 较轻的条件会抵消其好处。因为视锥细胞视蛋白在结构上是活跃的，但随着 11顺式视网膜，这个项目的假设是活性视锥视蛋白水平的增加导致 LCA2的锥体退行性变。因此，使锥体视蛋白失活的光不敏感小分子 当内源性11顺式视网膜受限时，保护锥体细胞。初步数据表明，贝塔 紫罗兰酮，一种截断的11顺式视网膜类似物，提高了中/长波长敏感症的存活率 (M/LWS)圆锥体，而非SWS1圆锥体。与假设一致，β紫罗兰酮是一种反向激动剂。 对M/LWS锥体视蛋白有拮抗作用，但对SWS1视锥视蛋白有激动剂作用。这项建议旨在改善 LCA2小鼠模型中所有锥体类型的存活，使缺失基因稍后重新引入 正在开发中的仍然可以改善视力；识别可以使视锥视蛋白失活的新化合物； 确保它们不会严重阻碍野生型小鼠的视力；并确定它们的影响 关于细胞培养中锥体视蛋白的运输和翻译后修饰的化合物 动物模型。荧光显微镜、视网膜电描记术、质谱学和生化 将使用各种方法来评估测试化合物对锥体细胞存活、功能和视蛋白的影响 属性。使用视蛋白反向激动剂可能在改善光感受器方面具有更广泛的适用性 与视黄醇处理受损相关的其他视觉问题的存活率，如Stargardt‘s， 视网膜色素变性和衰老。

Project Summary / Abstract The broad objective of this project is to understand the underlying events that lead to photoreceptor degeneration when problems with retinoid metabolism arise and to develop methods to prevent cones from degenerating. In Leber Congenital Amaurosis type 2 (LCA2), generation of the native chromophore of visual pigments (11-cis retinal) is inhibited. In mouse models for LCA2, cone cells die rapidly. Damage is most severe with short wavelength sensitive (SWS1) cones. This pattern roughly parallels the pathogenesis of LCA2. Studies from other laboratories have demonstrated that gene therapy can restore vision but appears limited by irreversible cone loss prior to the treatment. Early administration of an exogenous source of 11-cis retinal to mouse models improved cone survival, but recent work from this laboratory demonstrated that this was ineffective when mice were subjected to room light. These results suggest that (1) cone opsin/11-cis retinal interactions are important in preventing cone death, and (2) 11-cis retinal will not be the solution to treating LCA2 because normal light conditions negates its benefits. Because cone opsins are constitutively active but deactived with 11-cis retinal, the hypothesis for this project is that the increased levels of active cone opsins lead to cone degeneration in LCA2. Thus, light-insensitive small molecules that deactivate cone opsins will be protective to cone cells when endogenous 11-cis retinal is limited. Preliminary data indicate that beta ionone, a truncated analog of 11-cis retinal, improved survival of middle/long wavelength-sensitive (M/LWS) cones but not SWS1 cones. Consistent with the hypothesis, beta ionone is an inverse agonist to M/LWS cone opsins but an agonist to SWS1 cone opsins. This proposal aims to improve the survival of all cone types in mouse models for LCA2 such that reintroduction of the missing gene later in development can still improve vision; identify new compounds that can deactivate cone opsins; ensure that they will not severely impede vision in wild-type mice; and determine the impact of these compounds on trafficking of and post-translational modifications to cone opsins in cell culture and animal models. Fluorescence microscopy, electroretinography, mass spectrometry, and biochemicial methods will be used to assess the effects of test compounds on cone cell survival, function, and opsin properties. The use of opsin inverse agonists may have broader applicability in improving photoreceptor survival for other visual problems associated with compromised retinoid processing such as Stargardt's, retinitis pigmentosa, and aging.