Functional Analysis of Novel Genes in Eye Development and Vision

老年性黄斑变性（AMD）是视力丧失的主要原因。我们利用rpe来源的细胞建立了血清剥夺性AMD的细胞培养模型。在AMD中，Bruch膜和毛细血管床的改变可能会限制血清成分进入RPE。我们已经证明，血清中RPE细胞的缺失导致胆固醇合成和运输的显著上调以及RPE中胆固醇的积累。这与我们和其他人在人类AMD中看到的胆固醇RPE积累非常相似。此外，血清剥夺导致细胞内锌的消耗，而许多锌结合蛋白被诱导。对细胞模型中基因表达的分析导致发现了意想不到的基因，这些基因与AMD中Bruch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敲低可以抑制钙化。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基因的新型卡带特异性地将靶基因表达到视网膜色素上皮。该模型为测试抑制HAP形成的治疗策略提供了工具。

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.