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。对细胞模型中基因表达的分析导致了釉蛋白(AMTN)的诱导，这是一种钙/羟基磷灰石(HAP)矿化的蛋白质。在细胞培养中，AMTN负责HAP的沉积，下调AMTN的siRNA可抑制钙化。此外，我们还发现AMTN在患有干性AMD的人供眼中有表达，并且与HAP小球相关。 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，将人AMTN特异性地靶向视网膜色素上皮细胞。这在小鼠RPE中高效和特异地表达人AMTN，并导致RPE和Bruchs膜的形态变化，类似于AMD中看到的变化。 我们现在已经发现了一种实验性的创伤系统，可以在小鼠的RPE中诱导HAP/AMTN沉积。这正被用来测试AMTN的siRNA抑制的效果，以用于潜在的治疗用途。 我们给出了几种FDA批准的抑制AMTN功能的药物，并将作为减缓AMD进展的潜在疗法进行测试。我们还在合作确定这些药物对患者群体中AMD进展的任何影响。 我们已经启动了关于AMTM的协作性结构研究。这种蛋白质本质上是无序的，并为新的方法学方法提供了机会。我们的合作者已经为AMTN的结构和行为提供了新的见解，对类似蛋白质的研究具有重要意义。结合分子模拟，这给出了可能的作用机制的提示，

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. Analysis of gene expression in the cell model has led to the discovery of the induction of amelotin (AMTN), a protein of calcium/hydroxyapatite (HAP) mineralization. In cell culture AMTN is responsible for HAP deposition and siRNA knockdown of AMTN inhibits calcification. Furthermore, we showed that AMTN is expressed in human donor eyes with dry AMD and is associated with HAP spherules. We have also developed a transgenic mouse model which specifically targets expression of human AMTN to retinal pigment epithelium using a novel cassette based on the RPE65 gene. This efficiently and specifically expresses human AMTN in mouse RPE and causes morphological changes in RPE and Bruch's membrane similar to those seen in AMD. We have now found an experimental wound system that induces HAP/AMTN deposits in RPE in mice. This is being used to test the effects of siRNA inhibition of AMTN for potential therapeutic use. We gave identified several FDA approved drugs that inhibit AMTN function and will be tested as potential therapies to slow AMD progression. We are also collaborating to identify any effects of these drugs on AMD progression in patient populations. We have initiated collaborative structural studies on AMTM. The protein is intrinsically disordered and presents opportunities for new methodological approaches. Our collaborators have already provided new insights into the structure and behavior of AMTN with important implications for work on similar proteins. Combined with molecular modelling, this is giving hints of possible functional mechanisms,