Structural and functional integrity and microenvironment of RPE cells

描述(申请人提供)：视网膜色素上皮(RPE)是脊椎动物视网膜的一个多功能和不可或缺的组成部分。RPE细胞曲折的顶端和基底膜提供了较大的表面积，允许这些细胞与其局部微环境之间进行快速的物质交换。RPE被认为是老年性黄斑变性(AMD)的主要病变部位，AMD是一种跨越整个光感受器-RPE-脉络膜毛细血管复合体的疾病。RPE最初受损的根本原因尚不清楚。由于RPE细胞具有主动的转运作用，我们认为膜转运的减少足以对膜的特化、蛋白质转运和/或分泌造成许多不利影响。这些效应可以通过细胞间的直接相互作用和/或扩散传递给邻近的细胞。氯离子细胞内通道4(CLIC4)是一种顶端RPE蛋白，在其他类型的上皮细胞的囊泡吐出过程中起着重要作用。为了研究CLIC4的生理相关性，我们建立了两种新的啮齿动物模型，在这种模型中，CLIC4可以被选择性地从RPE细胞中原位抑制(即体内转染法和条件性基因敲除小鼠)。在这两个模型系统中，年轻人都表现出几个细胞自主和非细胞自主的特征，这些特征不仅相互映射，而且模仿AMD的特征。为了建立在这些发现的基础上，我们将对这些动物进行全面的特征描述，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CLIC4的囊泡运输功能失调是导致突变动物微绒毛畸形和视网膜脱离的原因(目标2)。最后，我们将检验CLIC4对于RPE细胞产生的分子的分泌是重要的这一假设(目标3)。分泌不平衡可能会导致邻近组织的萎缩。几种创新技术、细胞培养和最先进的动物模型将结合使用来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，最终有助于更好地诊断早期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.