Cone opsins in photoreceptor degeneration

描述（由申请人提供）：类视黄醇异构酶，RPE65和卵磷脂-视黄醇酰基转移酶（LRAT）在视网膜色素上皮（RPE）中的11-顺式视网膜循环中很重要。这两种基因的突变都会导致Leber先天性黑朦（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，早期视锥细胞变性，随后是视杆细胞变性。Rpe65-/-和Lrat-/-两种缺乏11-顺式视网膜的模型概括了主要的病理特征。然而，小鼠模型和人类患者早期锥体变性的机制尚不清楚。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亡得快得多。同样，患者早期蓝锥细胞功能丧失的原因也不清楚。本应用程序的目的是使用Lrat-/-模型确定LCA快速锥体变性的机制。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。我们基于我们的初步数据提出了这一假设，该数据显示短波（SW）视蛋白比中/长波视蛋白更容易聚集/积累触发内质网（ER）应激。在目的1中，我们将通过基因删除S-视蛋白或M-视蛋白来解剖S和M视蛋白对Lrat-/-小鼠锥体变性的相对贡献。在目标2中，我们将确定为什么发色团剥夺导致s -视蛋白聚集而不是m -视蛋白聚集的结构基础。我们在短波视蛋白家族（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却没有。由于芳香残基通过相互作用指导蛋白质的自组装，在蛋白质的聚集中起着重要作用，我们假设s -视蛋白中富含苯丙氨酸的区域负责其在Lrat-/-锥体中的聚集。我们设计了两个子目标来检验这一假设。Sub-aim 2;通过基因靶向将S-opsin（70-125）中的富苯丙氨酸区域与M-opsin（86-141）的同源区域交换。亚目标2B，通过破坏富苯丙氨酸区域之间的相互作用，抑制S-opsin与N-（1,4 -二氢- 1,4 -二氧基-2-萘基）- l -色氨酸（NQTrp）的聚集。我们期望这些研究能够明确RPE65/LRAT-LCA中s -锥比m -锥退化更快的机制。

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.