Cone Opsin-Ligand Interactions and Photoreceptor Health

描述（由申请人提供）：本项目的主要目的是了解当类维生素A代谢出现问题时导致感光细胞变性的潜在事件，并开发防止视锥细胞变性的方法。在Leber先天性黑蒙2型（LCA 2）中，视色素（11-顺式视网膜）的天然发色团的产生受到抑制。在LCA 2的小鼠模型中，视锥细胞迅速死亡。对短波长敏感（SWS 1）的视锥细胞损害最严重。这种模式与LCA 2的发病机制大致相似。来自其他实验室的研究表明，基因治疗可以恢复视力，但在治疗前受到不可逆的视锥细胞损失的限制。早期给予小鼠模型外源性11-顺式视网膜可提高视锥细胞存活率，但该实验室最近的工作表明，当小鼠受到室内光照时，这是无效的。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方面很重要，（2）11-顺式视网膜不会成为治疗LCA 2的解决方案，因为正常的光照条件会抵消其益处。由于视锥视蛋白是组成性活性的，但与11-顺式视黄醇一起失活，因此该项目的假设是活性视锥视蛋白水平的增加导致LCA 2中的视锥变性。因此，当内源性11-顺式视网膜受到限制时，使视锥视蛋白失活的光不敏感小分子将对视锥细胞具有保护作用。初步数据表明，β紫罗酮，截断类似物的11-顺式视网膜，提高生存的中/长波长敏感（M/LWS）锥，但不SWS 1锥。与该假设一致，β紫罗酮是M/LWS视锥细胞视蛋白的反向激动剂，但是是SWS 1视锥细胞视蛋白的激动剂。该提案旨在提高LCA 2小鼠模型中所有视锥细胞类型的存活率，以便在发育后期重新引入缺失的基因仍然可以改善视力;鉴定可以使视锥细胞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;并确定这些化合物对细胞培养物和动物模型中视锥视蛋白的运输和翻译后修饰的影响。荧光显微镜、视网膜电图、质谱和生物化学方法将用于评估供试化合物对视锥细胞存活、功能和视蛋白性质的影响。视蛋白反向激动剂的使用在改善与受损的类维生素A加工相关的其他视觉问题（例如Stargardt's、色素性视网膜炎和老化）的光感受器存活方面可能具有更广泛的适用性。 公共卫生相关性：视锥细胞用于日常明亮的光线和颜色视觉，但眼睛中维生素A加工的缺陷会导致视锥细胞变性，这就是Leber先天性黑蒙2型的情况。该项目的目标是了解这种退化过程，并通过靶向锥视色素的蛋白质成分来开发保持锥细胞存活的治疗方法。这种方法可以有效减缓与其他视觉缺陷相关的视杆和/或视锥感光细胞死亡，所述其他视觉缺陷例如Stargardt病、色素性视网膜炎和由异常维生素A代谢引起的衰老。

DESCRIPTION (provided by applicant): 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 biochemical 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. PUBLIC HEALTH RELEVANCE: Cones are used for daily bright light and color vision, but defects in Vitamin A processing in the eye can lead to cone cell degeneration, which is what happens with Leber Congenital Amaurosis type 2. The goals of this project are to understand this degeneration process and to develop therapeutic approaches to keeping cones alive by targeting the protein components of cone visual pigments. This approach may be effective in slowing down rod and/or cone photoreceptor cell death associated with other visual defects such as Stargardt's disease, retinitis pigmentosa, and aging that arise from abnormal Vitamin A metabolism.