Cone Opsin-Ligand Interactions and Photoreceptor Health

描述(申请人提供)：这个项目的主要目标是了解当视黄醇代谢出现问题时导致感光细胞退化的潜在事件，并开发防止视锥细胞退化的方法。在Leber先天性黑素血症2型(LCA2)中，视觉色素(11顺式视网膜)天然发色团的产生受到抑制。在LCA2的小鼠模型中，锥体细胞迅速死亡。短波敏锥(SWS1)的损伤最为严重。这种模式与LCA2的发病机制大致相似。其他实验室的研究表明，基因疗法可以恢复视力，但似乎受到治疗前不可逆转的视锥细胞丢失的限制。早期给予外源性11-顺式视网膜对小鼠的模型可以提高视锥细胞的存活率，但该实验室最近的工作表明，当小鼠受到室内光线的影响时，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防止视锥细胞死亡中起重要作用，(2)11-顺式视网膜不是治疗LCA2的解决方案，因为正常的光线条件会抵消其益处。由于视锥视蛋白在结构上是活跃的，但在11顺式视网膜中是失活的，因此该项目的假设是，活性视锥视蛋白水平的增加会导致LCA2的视锥退化。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起保护作用。初步数据表明，截短的11顺式视网膜类似物β紫罗兰酮可以提高中/长波长敏感(M/LWS)视锥细胞的存活率，但不能改善SWS1视锥细胞的存活率。与假设一致的是，β紫罗兰酮是M/LWS视锥蛋白的反向激动剂，而对SWS1视锥视蛋白是激动剂。这项建议旨在提高LCA2小鼠模型中所有视锥类型的存活率，以便在发育后期重新引入缺失的基因仍然可以改善视力；确定能够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；并确定这些化合物对细胞培养和动物模型中视锥视蛋白的运输和翻译后修饰的影响。将使用荧光显微镜、视网膜电描记术、质谱仪和生化方法来评估受试化合物对视锥细胞存活、功能和视蛋白特性的影响。视蛋白反向激动剂的使用可能在改善与视黄醇加工相关的其他视觉问题，如Stargardt‘s、视网膜色素变性和衰老的光感受器存活方面具有更广泛的适用性。 与公共健康相关：视锥细胞用于日常强光和颜色视觉，但眼睛中维生素A处理的缺陷会导致视锥细胞退化，这就是Leber先天性肥厚症2型患者所发生的情况。该项目的目标是了解这种退化过程，并开发通过靶向视锥视色素的蛋白质成分来保持视锥细胞存活的治疗方法。这种方法可能有效地减缓与其他视觉缺陷相关的视杆和/或视锥感光细胞死亡，如Stargardt病、视网膜色素变性和维生素A代谢异常引起的衰老。

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.