Altered RPE matrix in Sorsby Fundus Dystrophy leads to metabolic dysfunction

项目总结/摘要 视网膜相关性黄斑变性（AMD）是50岁以上成年人失明的主要原因， 可用的治疗。AMD是由多种遗传和环境因素引起的，难以复制 在实验室里研究。相比之下，Sorby眼底营养不良（SFD）是一种罕见的单基因疾病， 金属蛋白酶组织抑制剂3（TIMP 3）的突变，与 AMD.使用SFD的动物和细胞培养模型，我希望了解潜在的致病性和 SFD和可能的AMD中RPE/视网膜变性的分子机制。视网膜有一个独特的高 为视网膜提供动力所需的代谢需求和所有营养物质必须通过RPE运输或由RPE产生。 此外，视网膜需要适当的RPE代谢以获得足够的营养。视网膜利用 有氧糖酵解产生ATP，因此主要利用葡萄糖作为其能量底物。基于 根据Anand-Apte实验室的初步数据，我假设RPE中异常的能量代谢 导致视网膜中的营养缺乏，并且是SFD病理学的重要贡献者。为了解决这个 假设，我提出了2个具体的目标;（i）确定SFD RPE细胞是否缺乏其能力， 产生和/或运输必需的代谢物到视网膜。利用代谢组学和稳定同位素示踪 在SFD小鼠和人ARPE 19细胞培养模型中。我会用气相色谱质谱 (GC/MS）来量化体内和体外穿过RPE的营养转运。TIMP 3突变与 尤其是考虑到TIMP 3具有细胞外定位， 代谢是细胞内的过程。(ii)研究细胞外基质是否发生变化， 过量生产是SFD RPE中能量代谢改变的原因。最近，Anand-Apte 实验室报告称，表达突变体TIMP 3 S179 C的SFD RPE产生过量的透明质酸（HA），一种 在细胞外基质（ECM）中高度丰富的葡糖胺。我推测， SFD RPE细胞负责我们观察到的代谢表型。由于HA是由 糖酵解中间产物HA可通过多种机制影响能量代谢。了解 黄斑变性的详细信息是发现有效治疗药物的第一步， 开发新的治疗方法。

Project Summary/Abstract Age-related macular degeneration (AMD), is the leading cause of blindness in adults over 50 with no effective treatments available. AMD is caused by multiple genetic and environmental factors making it difficult to replicate and study in the laboratory. In contrast, Sorby’s Fundus Dystrophy (SFD) is a rare monogenetic disease caused by mutations in Tissue inhibitor of metalloproteinase 3 (TIMP3) that has significant phenotypic similarities to AMD. Using animal and cell culture models of SFD, I hope to understand the underlying pathogenic and molecular mechanisms of RPE/retinal degeneration in SFD and possibly AMD. The retina has a uniquely high metabolic demand and all nutrients needed to power the retina must be transported across or generated by RPE. Furthermore, proper RPE metabolism is required for the retina to receive adequate nutrients. Retina utilize aerobic glycolysis to generate ATP and therefore primarily utilize glucose as their energy substrate. Based on preliminary data from the Anand-Apte laboratory, I hypothesize that aberrant energy metabolism in the RPE results in nutrient deprivation in the retina and is a significant contributor to SFD pathology. To address this hypothesis, I propose 2 specific aims; (i) to determine if SFD RPE cells are deficient in their ability to produce and/or transport essential metabolites to the retina. Using metabolomics and stable isotopic tracing within SFD mouse and human ARPE19 cell culture models. I will use gas chromatography mass spectrometry (GC/MS) to quantify nutrient transport across RPE in vivo and in vitro. The link between TIMP3 mutations and metabolism perturbations are unclear especially considering that TIMP3 has extracellular localization while metabolism is an intracellular process. (ii) to investigate if extracellular matrix changes and HA overproduction are responsible for altered energy metabolism in SFD RPE. Recently, the Anand-Apte laboratory has reported that SFD RPE expressing mutant TIMP3 S179C produce excess hyaluronan (HA), a glucosamine that is highly abundant in the extracellular matrix (ECM). I hypothesize that HA overproduction in SFD RPE cells is responsible for the metabolic phenotype we have observed. Since HA is synthesized from glycolytic intermediates, HA could impact energy metabolism through multiple mechanisms. Understanding the details of macular degeneration is the first step towards discovering effective prophylactics and will aid in the development of new therapeutics.