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: 10537228
• 负责人: Jennifer Rayming Chao
• 金额: $ 59.92万
• 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/10537228
• 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; human disease; induced pluripotent stem cell; inherited retinal degeneration; ketogentic; lipid metabolism; metabolomics; novel strategies; nutrient metabolism; oxidation; oxidative damage; protein degradation; retinal progenitor cell; 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.

项目总结/摘要 视网膜色素上皮（RPE）下富含脂质沉积物的存在是视网膜色素变性的病理特征。 早期年龄相关性黄斑变性（AMD）。玻璃疣的发展与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 金属蛋白酶组织抑制剂3（TIMP 3）的降解导致罕见的遗传性视网膜变性， 类似于AMD的临床特征，称为Sorsby眼底营养不良（SFD）。不正常的 ECM周转影响脂质代谢和RPE氧化还原，导致亚RPE沉积物的形成 仍然未知。 本提案的目的是检验ECM降解使RPE过载ECM衍生的RPE的假设。 代谢物，导致RPE向脂质合成和线粒体氧化重编程 新陈代谢.这反过来又导致过量脂质的沉积和细胞的抗氧化能力降低。 RPE。拟议的具体目标是： 目标1。确定ECM降解对脂质代谢的影响。我们的初步结果表明 SFD RPE中ECM降解的增加激活了脂质合成和支链氨基的氧化， 酸（支链氨基酸）。支链氨基酸是生酮的，在ECM中含量丰富。目标1的目的是检验假设 ECM降解富含蛋白质的成分， 氧化用于脂质合成和脂质沉积。我们将使用定量蛋白质组学， 代谢组学、代谢通量分析、灌流测定、CRISPR/Cas9基因编辑和单细胞核 患者来源的iPSC RPE的RNA-Seq，以全面研究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。初步数据显示， NADPH和谷胱甘肽在SFD RPE中耗尽，ECM衍生的代谢物干扰NADPH 和谷胱甘肽代谢。目标2的目的是检验ECM周转率增加导致 受损的NADPH和谷胱甘肽代谢。我们将量化ECM降解的代谢通量， 确定ECM相关代谢物在NADPH产生和谷胱甘肽合成中的作用，并恢复 细胞氧化还原与不同的方法，以提高抗氧化能力。 拟议的研究将确定ECM周转对RPE代谢的生化影响，包括 脂质代谢和氧化应激的变化，并找出营养代谢之间的关系， 蛋白质合成和降解，以及正常和疾病相关RPE中的氧化还原生物学。