Metabolic dysfunction from ECM remodeling in diseases of human RPE

人类 RPE 疾病中 ECM 重塑的代谢功能障碍

• 批准号: 10680561
• 负责人: Jennifer Rayming Chao
• 金额: $ 56.77万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680561
• 关键词: Acids; Affect; Age related macular degeneration; Autopsy; Biochemical; Biological Assay; Biology; Branched-Chain Amino Acids; Buffers; CRISPR/Cas technology; Cell Respiration; Cells; Clinical; Data; Degenerative Disorder; Deposition; Development; Disease; Drusen; Energy Metabolism; Exhibits; Extracellular Matrix; Extracellular Matrix Degradation; Generations; Genes; Glucose; Glutathione; Glutathione Metabolism Pathway; Goals; Human; Impairment; Lipids; Metabolic; Metabolic Pathway; Metabolic dysfunction; Metabolism; Mitochondria; Mutation; NADP; Nuclear RNA; Outcome; Oxidation-Reduction; Oxidative Stress; Pathologic; Patients; Production; Protein Biosynthesis; Proteomics; Research; Resources; Role; Sorsby' s fundus dystrophy; Stress; Structure of retinal pigment epithelium; TIMP3 gene; Testing; Thinness; comparison control; human disease; induced pluripotent stem cell; inherited retinal degeneration; ketogentic; lipid metabolism; metabolomics; novel strategies; nutrient metabolism; oxidation; oxidative damage; programs; protein degradation; stressor; sugar; transcriptome sequencing; treatment strategy

PROJECT SUMMARY/ABSTRACT The presence of lipid-rich deposits underneath the retinal pigment epithelium (RPE) is a pathologic feature of early age-related macular degeneration (AMD). Drusen development has been associated with RPE lipid metabolism, redox biology and extracellular matrix (ECM) degradation. Mutations in a gene affecting ECM degradation, tissue inhibitor of metalloproteinase 3 (TIMP3), results in a rare inherited retinal degeneration with similar clinical features to AMD, called Sorsby Fundus Dystrophy (SFD). The mechanism by which abnormal ECM turnover influences lipid metabolism and RPE redox resulting in the formation of sub-RPE deposits remains unknown. The goal of this proposal is to test the hypothesis that ECM degradation overloads the RPE with ECM-derived metabolites, resulting in the reprogramming of RPE towards lipid synthesis and mitochondrial oxidative metabolism. This in turn results in the deposition of excess lipids and reduced antioxidative capacity of the RPE. The proposed specific aims are: Aim 1. Determine the influence of ECM degradation on lipid metabolism. Our preliminary results show that increased ECM degradation in SFD RPE activates lipid synthesis and oxidation of branch-chain amino acids (BCAAs). BCAAs are ketogenic and abundant in the ECM. The goal of Aim 1 is to test the hypothesis that ECM degradation of protein-rich components reprograms RPE metabolism towards enhanced BCAA oxidation for lipid synthesis and lipid deposition. We will use quantitative proteomics, quantitative metabolomics, metabolic flux analysis, perifusion assays, CRISPR/Cas9 gene-editing, and single cell nuclear RNA-Seq of patient-derived iPSC RPE to comprehensively investigate the metabolic pathways in ECM remodeling and lipid deposition. Aim 2. Determine the influence of ECM degradation on redox metabolism. Our preliminary data show that both NADPH and glutathione are depleted in SFD RPE, and ECM-derived metabolites interfere with NADPH and glutathione metabolism. The goal of Aim 2 is to test the hypothesis that increased ECM turnover results in impaired NADPH and glutathione metabolism. We will quantify the metabolic flux of ECM degradation, determine the roles of ECM-related metabolites in NADPH production and glutathione synthesis, and restore cellular redox with different approaches to enhance antioxidative capacity. The proposed research will define the biochemical impacts of ECM turnover on RPE metabolism, including changes in lipid metabolism and oxidative stress, and identify the relationship between nutrient metabolism, protein synthesis and degradation, and redox biology in normal and disease-relevant RPE.

项目摘要/摘要 视网膜色素上皮（RPE）下面存在富含脂质的沉积物是一种病理特征 与年龄相关的黄斑变性（AMD）。 Drusen开发与RPE脂质有关 代谢，氧化还原生物学和细胞外基质（ECM）降解。影响ECM的基因突变 降解，金属蛋白酶3的组织抑制剂（Timp3），导致罕见的遗传性视网膜变性 与AMD相似的临床特征，称为SORSBY眼底营养不良（SFD）。异常的机制 ECM周转影响脂质代谢和RPE氧化还原，导致子RPE沉积物形成 仍然未知。 该提案的目的是检验以下假设：ECM降解与ECM衍生的RPE超载 代谢物，导致RPE对脂质合成和线粒体氧化的重新编程 代谢。反过来，这导致过量脂质的沉积和降低的抗氧化能力 RPE。拟议的具体目的是： 目标1。确定ECM降解对脂质代谢的影响。我们的初步结果显示 SFD RPE中的ECM降解增加了分支链氨基的脂质合成和氧化 酸（BCAA）。 BCAA在ECM中具有生酮且丰富。目标1的目的是检验假设 富含蛋白质的成分的ECM降解重编程RPE代谢对增强的BCAA 脂质合成和脂质沉积的氧化。我们将使用定量蛋白质组学，定量 代谢组学，代谢通量分析，促进测定，CRISPR/CAS9基因编辑和单细胞核 患者衍生的IPSC RPE的RNA-seq全面研究ECM中的代谢途径 重塑和脂质沉积。 目标2。确定ECM降解对氧化还原代谢的影响。我们的初步数据表明 NADPH和谷胱甘肽在SFD RPE中都耗尽，ECM衍生的代谢物干扰了NADPH 和谷胱甘肽代谢。目标2的目的是检验以下假设，即ECM周转率增加导致 NADPH和谷胱甘肽代谢受损。我们将量化ECM降解的代谢通量， 确定ECM相关代谢物在NADPH生产和谷胱甘肽合成中的作用，并恢复 细胞氧化还原，采用不同的方法来增强抗氧化能力。 拟议的研究将定义ECM营业额对RPE代谢的生化影响，包括 脂质代谢和氧化应激的变化，并确定营养代谢之间的关系， 蛋白质的合成和降解，以及与疾病相关的RPE中的氧化还原生物学。