Functional Analysis of Novel Genes in Eye Development and Vision

视网膜相关性黄斑变性（AMD）是视力丧失的主要原因。我们已经开发了一个细胞培养模型，用于血清剥夺AMD使用RPE衍生的细胞。在AMD中，布鲁赫膜和毛细血管床的变化可能限制血清组分进入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显著上调以及胆固醇在RPE中的积累。这强烈地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，血清剥夺导致细胞内锌耗尽，同时诱导许多锌结合蛋白。对细胞模型中基因表达的分析已经导致发现了意想不到的基因，这些基因与发生在AMD中布鲁赫膜周围的羟基磷灰石矿化有关。我们已经证明，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RPE 65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。该模型提供了用于测试抑制HAP形成的治疗策略的工具。 在透镜中，我们已经表明KLPH/g-klotho的缺失导致透镜中Clic 5表达的完全丧失。在正常透镜中，Clic 5定位于透镜纤维顶端的纤毛/中心体复合体。已知Clic 5与内耳纤毛病变有关。我们现在已经表明Clic 5的缺失也具有光感受器表型，表明这与Usher综合征的模型相关。 Retbindin是一种新的视网膜光感受器蛋白。基因敲除小鼠模型显示视觉反应的进行性缺陷和外层视网膜中与年龄相关的缺陷，这些缺陷与某些形式的年龄相关性黄斑变性具有惊人的相似性。这包括玻璃疣的形成、RPE功能障碍、脂质和钙的沉积、小胶质细胞的活化和光敏视网膜神经节细胞的过早丧失。

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.