Cone opsins in photoreceptor degeneration

描述（由申请人提供）：类维生素A异构酶、RPE 65和卵磷脂-视黄醇酰基转移酶（LRAT）在视网膜色素上皮（RPE）中回收11-顺式-视黄醛中是重要的。任何一个基因的突变都会导致Leber先天性黑蒙（LCA），这是一种遗传性视网膜变性疾病，其特征是儿童期视力严重丧失和视锥细胞早期变性，随后是视杆细胞。主要的病理学特征由两种模型Rpe 65-/-和Lrat-/-概括，其中缺乏11-顺式-视网膜。然而，在小鼠模型和人类患者中负责早期视锥细胞变性的机制还没有很好地理解。具体来说，目前还不清楚为什么小鼠模型中的腹侧和中央锥体比背侧锥体死得更快。同样，目前还不清楚为什么蓝锥功能在患者中早期丧失。本申请的目的是使用Lrat-/-模型定义LCA中快速视锥细胞变性的机制。我们的中心假设是视锥蛋白决定了LCA中视锥光感受器退化的速率。我们根据初步数据提出了这一假设，这些数据表明短波长（SW）视蛋白比中/长波长视蛋白更容易聚集/积聚，从而引发内质网（ER）应激。在目的1中，我们将通过遗传缺失S-视蛋白或M-视蛋白来剖析S和M视蛋白对Lrat-/-小鼠中视锥细胞变性的相对贡献。在目标2中，我们将确定为什么发色团剥夺导致S-视蛋白而不是M-视蛋白聚集的结构基础。我们确定了一个富含苯丙氨酸的区域在短波长视蛋白家族（SW 1），但不存在于中/长波长视蛋白家族。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起着重要作用，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-/-视锥中的聚集。我们设计了两个子目标来验证这一假设。子目标2A：通过基因靶向将S-视蛋白中富含苯丙氨酸的区域（70-125）与M-视蛋白的同源区域（86-141）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，用N-（1，4-二氢-1，4-二氧代-2-萘基）-L-色氨酸（NQTrp）抑制S-视蛋白聚集。我们期望这些研究将确定RPE 65/LRAT-LCA中S-锥比M-锥退化更快的机制。 公共卫生相关性：这些研究将定义RPE 65/LRAT-LCA中S-视锥细胞退化速度快于M-视锥细胞的机制。提出的锥视蛋白聚集导致快速锥变性的机制可以用于设计治疗LCA的新型治疗剂。例如，NQTrp有可能通过阻断视锥细胞视蛋白聚集来作为开发一类新的LCA治疗药物的先导。

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.