Altered RPE matrix in Sorsby Fundus Dystrophy leads to metabolic dysfunction

项目摘要/摘要 老年性黄斑变性(AMD)是50岁以上成年人失明的主要原因，没有有效的治疗方法 提供治疗方法。AMD是由多种遗传和环境因素引起的，因此很难复制 在实验室里学习。相比之下，索比氏眼底营养不良(SFD)是一种罕见的单基因疾病，由 通过金属蛋白酶组织抑制物3(TIMP3)的突变，其表型与 AMD。利用SFD的动物和细胞培养模型，我希望了解SFD的潜在致病和 SFD和AMD中RPE/视网膜变性的分子机制。视网膜有一种独特的高 新陈代谢需求和为视网膜提供能量所需的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代谢才能获得足够的营养。视网膜利用 有氧糖酵解产生三磷酸腺苷，因此主要利用葡萄糖作为能量底物。基于 来自Anand-Apte实验室的初步数据，我假设RPE中异常的能量代谢 导致视网膜营养缺乏，是SFD病理的重要因素。要解决这个问题 假设，我提出了两个具体目标：(I)确定SFD RPE细胞是否有能力缺陷 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ARPE19细胞培养模型中。我要用气相色谱-质谱仪 (GC/MS)以量化体内和体外RPE中的营养物质转运。TIMP3突变与TIMP3基因突变的关系 代谢紊乱尚不清楚，特别是考虑到TIMP3具有细胞外定位，而 新陈代谢是一个细胞内的过程。(Ii)调查细胞外基质是否发生变化和HA 过量生产是SFD RPE能量代谢改变的原因。最近，Anand-Apte 实验室报道，表达突变体TIMP3 S179C的SFD RPE会产生过量的透明质酸(HA)，a 细胞外基质(ECM)中高度丰富的氨基葡萄糖。我假设房委会在年生产过剩 SFD RPE细胞是我们观察到的代谢表型的原因。由于透明质酸是由 糖酵解中间体HA可通过多种机制影响能量代谢。了解 黄斑变性的细节是发现有效预防措施的第一步，并将有助于 新疗法的发展。

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.