Functional Analysis of Novel Genes in Eye Development and Vision

眼睛发育和视力新基因的功能分析

基本信息

批准号：
10019996
负责人：
graeme j wistow
金额：
$ 112.92万
依托单位：
NATIONAL EYE INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10019996
关键词：
Age related macular degeneration Animal Model Apical Bioinformatics Blindness Bruch&apos s basal membrane structure Calcium Cell Culture Techniques Cell model Cells Centrosome Cholesterol Cilia Complex Defect Deposition Drusen Eye Eye Development Functional disorder Gene Expression Profiling Genes Genomics Human Hydroxyapatites Knockout Mice Labyrinth Lens Fiber Light Lipids Microglia Modeling Phenotype Photoreceptors Proteins RPE65 protein Retina Retinal Ganglion Cells Retinal Photoreceptors Serum Small Interfering RNA Structure of retinal pigment epithelium Therapeutic Tissues Transgenic Mice Up-Regulation Usher Syndrome Vision Visual Zinc age related base calcification capillary bed ciliopathy deprivation functional genomics gene product human model insight knock-down lens mineralization mouse model novel premature response retbindin therapeutic evaluation tool zinc-binding protein

项目摘要

Age-related macular degeneration (AMD) is a major cause of vision loss. We have developed a cell-culture model for serum-deprivation AMD using RPE-derived cells. In AMD, changes at Bruch's membrane and in the capillary bed are likely to restrict access of serum components to the RPE. We have shown that serum deprivation of RPE cells leads to a marked upregulation of cholesterol synthesis and transport and the accumulation of cholesterol in the RPE. This is strongly reminiscent of the accumulation of cholesterol RPE that we and others have seen in human AMD. Furthermore, serum-deprivation leads to depletion of intracellular zinc, while many zinc-binding proteins are induced. Analysis of gene expression in the cell model has led to the discovery of unexpected genes that are implicated in the hydroxyapatite mineralization that occurs at an around Bruch's membrane in AMD. We have shown that siRNA knockdown of target genes inhibits calcification. This has therapeutic potential.We have also developed a transgenic mouse model which specifically targets expression of target gene to retinal pigment epithelium using a novel cassette based on the RPE65 gene. This model provides a tool for testing therapeutic strategies to inhibit HAP formation. In the lens, we have shown that deletion of KLPH/g-klotho leads to complete loss of expression of Clic5 in the lens. In normal lens, Clic5 is localized to the cilium/centrosome complex at the apical tip of the lens fibers. Clic5 is known to be associated with a ciliopathy in the inner ear. We have now shown that loss of Clic5 also has a photoreceptor phenotype suggesting this has relevance as a model for Usher's syndromes. Retbindin is a novel protein of retinal photoreceptors. A knockout mouse model shows progressive deficits in visual response and age-related defects in the outer retina that have striking similarities to some forms of age-related macular degeneration. This includes formation of drusen, RPE dysfunction, deposition of lipids and calcium, activation of microglia and premature loss of light sensitive retinal ganglion cells.

与年龄相关的黄斑变性（AMD）是视力丧失的主要原因。我们已经开发了一种使用RPE衍生的细胞的细胞培养模型，用于血清剥夺AMD。在AMD中，BRUCH的膜和毛细管床的变化可能会限制血清成分进入RPE。我们已经表明，RPE细胞的血清剥夺导致胆固醇合成和转运的明显上调以及胆固醇在RPE中的积累。这强烈让人联想到我们和其他人在人类AMD中看到的胆固醇RPE的积累。此外，血清剥夺会导致细胞内锌的消耗，而许多锌结合蛋白是诱导的。细胞模型中基因表达的分析导致发现了与羟基磷灰石矿化有关的意外基因，该基因发生在AMD中的Bruch膜周围。我们已经表明，靶基因的siRNA敲低抑制钙化。我们还开发了一种转基因小鼠模型，该模型使用基于RPE65基因的新型盒式盒子将靶基因的表达靶向视网膜色素上皮。该模型为测试抑制HAP形成的治疗策略提供了一种工具。在镜头中，我们表明klph/g-klotho的缺失导致镜头中Clic5表达的完全丧失。在正常晶状体中，Clic5位于晶状体纤维顶端的纤毛/中心体络合物中。已知Clic5与内耳中的纤毛病有关。现在，我们已经表明，CLIC5的损失也具有感光光表型，这表明这是作为Usher综合征的模型的相关性。视毒素是视网膜感光体的一种新型蛋白质。敲除小鼠模型显示出视觉反应的渐进性和外部视网膜与年龄相关的缺陷，这些缺陷与某些形式的与年龄相关的黄斑变性具有惊人的相似性。这包括形成drusen，RPE功能障碍，脂质沉积和钙的沉积，小胶质细胞的激活以及光敏性视网膜神经节细胞的过早丧失。