Functional Analysis of Novel Genes in Eye Development and Vision

老年性黄斑变性(AMD)是导致视力丧失的主要原因。我们利用RPE来源的细胞建立了一种血清剥夺AMD的细胞培养模型。在AMD中，Bruch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，剥夺RPE细胞的血清会显著上调胆固醇的合成和运输，并导致RPE中胆固醇的积累。这强烈地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，血清剥夺导致细胞内锌的耗竭，同时诱导了许多锌结合蛋白。对细胞模型中基因表达的分析导致了与AMD患者Bruch膜周围的羟基磷灰石矿化有关的意外基因的发现。我们已经证明，靶基因的siRNA敲除可以抑制钙化。我们还开发了一种转基因小鼠模型，利用基于RPE65基因的新型盒，将靶基因特异性地表达到视网膜色素上皮细胞。该模型为测试抑制HAP形成的治疗策略提供了一种工具。 在晶状体中，我们已经证明KLPH/g-klotho的缺失会导致Clic5在晶状体中的表达完全丧失。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与内耳的纤毛疾病有关。我们现在已经表明，Clic5的缺失也具有光感受器表型，这表明这与Usher综合征的模型有关。 视黄醇结合蛋白是一种新的视网膜光感受器蛋白。基因敲除的小鼠模型显示出视觉反应的进行性缺陷和外层视网膜的年龄相关缺陷，这些缺陷与某些形式的年龄相关黄斑变性有着惊人的相似之处。这包括玻璃体形成、RPE功能障碍、脂质和钙沉积、小胶质细胞激活和光敏性视网膜神经节细胞过早丧失。

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.