Cone opsins in photoreceptor degeneration

描述（由申请人提供）：类维生素A异构酶、RPE65和卵磷脂-视黄醇酰基转移酶（LRAT）对于视网膜色素上皮（RPE）中11-顺式-视黄醛的再循环很重要。任何一个基因的突变都会导致莱伯先天性黑蒙（LCA），这是一种遗传性视网膜退行性疾病，其特征是儿童时期视力严重丧失以及视锥细胞早期退化，随后是视杆细胞退化。主要病理特征由两种模型概括：Rpe65-/- 和 Lrat-/-，其中缺乏 11-cis-retinal。然而，小鼠模型和人类患者早期视锥细胞变性的机制尚不清楚。具体来说，尚不清楚为什么小鼠模型中的腹侧和中央视锥细胞比背侧视锥细胞死亡得更快。同样，目前还不清楚为什么患者早期蓝视锥细胞功能丧失。本应用的目的是使用 Lrat-/- 模型定义 LCA 中视锥细胞快速退化的机制。我们的中心假设是视锥细胞视蛋白决定 LCA 中视锥细胞光感受器变性的速率。我们根据初步数据制定了这一假设，该数据显示短波长（SW）视蛋白比中/​​长波长视蛋白更容易聚集/积累，从而触发内质网（ER）应激。在目标 1 中，我们将通过基因删除 S-视蛋白或 M-视蛋白来剖析 S 和 M 视蛋白对 Lrat-/- 小鼠视锥变性的相对贡献。在目标 2 中，我们将确定生色团剥夺导致 S-视蛋白而不是 M-视蛋白聚集的结构基础。我们在短波长视蛋白家族（SW1）中发现了一个富含苯丙氨酸的区域，但在中/长波长视蛋白家族中不存在。由于芳香族残基通过 ¿-¿ 相互作用指导自组装，在蛋白质聚集中发挥重要作用，因此我们假设 S-视蛋白中富含苯丙氨酸的区域负责其在 Lrat-/- 视锥细胞中的聚集。我们设计了两个子目标来检验这个假设。子目标2A；通过基因打靶将 S-视蛋白 (70-125) 中富含苯丙氨酸的区域与 M-视蛋白 (86-141) 的同源区域交换。子目标 2B，通过破坏富含苯丙氨酸的区域之间的 ¿-¿ 相互作用，抑制 S-视蛋白与 N-(1, 4-二氢-1, 4-二氧代-2-萘基)-L-色氨酸 (NQTrp) 的聚集。我们期望这些研究将明确为什么 RPE65/LRAT-LCA 中 S 锥体比 M 锥体退化更快的机制。 公共健康相关性：这些研究将确定 RPE65/LRAT-LCA 中 S 锥体比 M 锥体退化更快的机制。所提出的视锥细胞视蛋白聚集导致视锥细胞快速变性的机制可用于设计治疗 LCA 的新型治疗剂。例如，NQTrp 有潜力通过阻断视锥蛋白聚集来开发一类新型 LCA 治疗药物。

DESCRIPTION (provided by applicant): Retinoid isomerase, RPE65, and Lecithin-retinol acyltransferase (LRAT) are important in recycling 11-cis- retinal in retinal pigment epithelium (RPE). Mutations in either gene lead to Leber congenital amaurosis (LCA), an inherited retinal degenerative disease characterized by severe loss of vision in childhood and early degeneration of cones followed by rods. The main pathologic features are recapitulated by two models, Rpe65-/- and Lrat-/- in which 11-cis-retinal is lacking. However, the mechanisms responsible for early cone degeneration in both mouse models and human patients are not well understood. Specifically, it is unclear why ventral and central cones in mouse models die much more rapidly than dorsal cones. Similarly, it is unclear why blue cone function is lost early in patients. The objective of this application is to define the mechanism responsible for the rapid cone degeneration in LCA using the Lrat-/- model. Our central hypothesis is that cone opsin determines the rate of cone photoreceptor degeneration in LCA. We formulated this hypothesis based on our preliminary data showing the short-wavelength (SW) opsins are prone to aggregation/accumulation triggering endoplasmic reticulum (ER) stress than the medium/long-wavelength opsin. In Aim 1, we will dissect the relative contributions of S and M opsin to cone degeneration in Lrat-/- mice by genetically deleting S-opsin or M-opsin. In Aim 2, we will determine the structural basis on why chromophore deprivation causes S-opsin but not M-opsin aggregation. We identified a phenylalanine-rich region in the short-wavelength opsin family (SW1) but absent from the medium/long-wavelength opsin family. Since aromatic residues play a significant role in the aggregation of proteins by directing self-assembly via ¿-¿ interactions, we hypothesize that the phenylalanine-rich region in the S-opsin is responsible for its aggregation in Lrat-/- cones. We designed two sub-aims to test this hypothesis. Sub-aim 2A; swap the phenylalanine-rich region in the S-opsin (70-125) with the homologous region of M-opsin (86-141) by gene targeting. Sub-aim 2B, inhibit S-opsin aggregation with N- (1, 4-dihydro-1, 4-dioxo-2-naphthalenyl)-L-tryptophan (NQTrp) by disrupting the ¿-¿ interactions between the phenylalanine-rich region. We expect these studies will define the mechanism on why S-cones degenerate faster than M-cones in RPE65/LRAT-LCA. PUBLIC HEALTH RELEVANCE: These studies will define the mechanism on why S-cones degenerate faster than M-cones in RPE65/LRAT- LCA. The proposed mechanism that cone opsin aggregation is responsible for rapid cone degeneration can be exploited in designing novel therapeutic agents in the treatment of LCA. For example, NQTrp has the potential to serve as a lead for developing a new class of therapeutic drugs for LCA by blocking cone opsin aggregation.