Structural and functional integrity and microenvironment of RPE cells

描述（由申请人提供）：视网膜色素上皮（RPE）是脊椎动物视网膜的多功能和不可缺少的组成部分。RPE细胞的卷曲的顶端和基底质膜提供了大的表面积，允许这些细胞与其局部微环境之间的快速物质交换。RPE被认为是年龄相关性黄斑变性（AMD）的主要病变部位，AMD是一种具有跨越整个光感受器-RPE-脉络膜毛细血管复合体的病理的疾病。RPE最初损坏的根本原因仍不清楚。由于RPE细胞具有积极的运输作用，我们认为膜运输的下降足以对膜特化、蛋白质运输和/或分泌造成许多不利影响。这些效应可以通过直接的细胞-细胞相互作用和/或扩散传递到相邻细胞。氯化物胞内通道4（CLIC 4），一种顶端RPE蛋白，在其他上皮细胞类型的囊泡胞吐中具有重要作用。为了研究CLIC 4的生理相关性，我们开发了两种新的啮齿动物模型，其中CLIC 4可以从RPE细胞原位选择性抑制（即，体内转染和条件性敲除小鼠）。在这两个模型系统中，年轻人表现出几种细胞自主和非细胞自主的特征，不仅相互反映，而且模仿AMD的标志。基于这些发现，我们将对这些动物进行全面表征，以更好地模拟AMD的疾病进展（目标1）。此外，我们将直接测试我们的模型，即CLIC 4的失调的囊泡运输功能是导致突变动物中微绒毛畸形和视网膜脱离的原因（目的2）。最后，我们将检验CLIC 4对于RPE细胞产生的分子的分泌是重要的假设（目的3）。不平衡的分泌可导致邻近组织萎缩。几种创新技术，细胞培养和最先进的动物模型将结合使用，以解决这些相互关联的问题。这些研究将丰富我们对RPE的基本认识，并最终导致更好地诊断早期AMD和合理设计治疗方案。

DESCRIPTION (provided by applicant): The retinal pigment epithelium (RPE) is a multifunctional and indispensable component of the vertebrate retina. The convoluted apical and basal plasma membranes of RPE cells provide a large surface area that allows rapid material exchange between these cells and their local microenvironment. The RPE is considered to be the primary lesion site of age-related macular degeneration (AMD), a disease that has pathology spanning the entire photoreceptor-RPE-choriocapillaris complex. The root cause of the initial damage to the RPE remains unclear. Because the RPE cells have an active transport role, we propose that a decline in membrane trafficking is sufficient to cause numerous adverse effects on membrane specialization, protein trafficking, and/or secretion. These effects could be conveyed to neighboring cells through direct cell-cell interaction and/or diffusion. Chloride intracellular channel 4 (CLIC4), an apical RPE protein, has an important role in vesicular exocytosis in other epithelial cell types. To study the physiological relevance of CLIC4, we have developed two novel rodent models in which CLIC4 can be selectively suppressed from RPE cells in situ (i.e., in vivo transfection, and conditional knockout mice). In both of these model systems, young adults manifest several cell autonomous and non-cell autonomous features that not only mirror each other, but also mimic the hallmarks of AMD. To build upon these findings, we will conduct a comprehensive characterization of these animals to better model the disease progression of AMD (Aim 1). Furthermore, we will directly test our model that the dysregulated vesicular trafficking function of CLIC4 is what causes the microvillar dysmorphogenesis and retinal detachment in the mutant animals (Aim 2). Finally, we will test the hypothesis that CLIC4 is important for the secretion of molecules produced by RPE cells (Aim 3). Imbalanced secretion may lead to atrophy in the adjacent tissues. Several innovative techniques, cell cultures, and state-of-the-art animal models will be used in combination to address these inter-related questions. These studies will enrich our fundamental understanding of the RPE and ultimately lead to better diagnosis of early AMD and rational design of treatments.