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

• 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
• 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' 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

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.

年龄相关性黄斑变性（AMD）是视力丧失的主要原因。我们使用 RPE 衍生细胞开发了一种用于血清剥夺 AMD 的细胞培养模型。在 AMD 中，布鲁赫膜和毛细血管床的变化可能会限制血清成分进入 RPE。我们已经证明，RPE 细胞的血清剥夺会导致胆固醇合成和运输的显着上调以及 RPE 中胆固醇的积累。这强烈让人想起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，同时诱导许多锌结合蛋白的产生。对细胞模型中基因表达的分析发现了意想不到的基因，这些基因与 AMD 中布鲁赫膜周围发生的羟基磷灰石矿化有关。我们已经证明，靶基因的 siRNA 敲除可抑制钙化。这具有治疗潜力。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，将靶基因的表达特异性靶向视网膜色素上皮。该模型提供了测试抑制 HAP 形成的治疗策略的工具。 在晶状体中，我们已经证明，KLPH/g-klotho 的缺失会导致晶状体中 Clic5 的表达完全丧失。在正常晶状体中，Clic5 定位于晶状体纤维顶端的纤毛/中心体复合体。已知 Clic5 与内耳纤毛病有关。我们现在已经证明，Clic5 的缺失也具有光感受器表型，表明这与亚瑟综合征的模型相关。 Retbindin 是一种新型的视网膜感光蛋白。基因敲除小鼠模型显示出视觉反应的渐进性缺陷和外层视网膜的与年龄相关的缺陷，这与某些形式的与年龄相关的黄斑变性具有惊人的相似性。这包括玻璃膜疣的形成、RPE功能障碍、脂质和钙的沉积、小胶质细胞的激活以及光敏感的视网膜神经节细胞的过早丧失。