Identification of novel contributors to retinitis pigmentosa using metabolic and proteomic approaches

使用代谢和蛋白质组学方法鉴定视网膜色素变性的新因素

• 批准号: 10298716
• 负责人: Ellen Ruth Weiss
• 金额: $ 57.74万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10298716
• 关键词: Affect; Anabolism; Blindness; Carnitine; Cell Death; Cell Nucleus; Cessation of life; Coenzyme A; Complete Blindness; Computer Analysis; Computer software; Cone; Critiques; Cyclic GMP; DNA copy number; Data; Data Analyses; Defect; Disease; Disease Progression; Enzymes; Evaluation; Event; Exhibits; Face; Female; Gender; Genes; Genetic; Genetic Diseases; Genetic Models; Goals; Health; Human; Impairment; In Situ Nick-End Labeling; Individual; Inflammation; Lead; Libraries; Light; Link; Mass Spectrum Analysis; Membrane; Metabolic; Metabolic dysfunction; Methods; Mitochondria; Mitochondrial DNA; Modeling; Molecular; Mus; Mutation; Nuclear; Other Genetics; Oxidative Stress; Oxygen Consumption; Paper; Pathway Analysis; Pathway interactions; Periodicity; Photoreceptors; Post-Translational Protein Processing; Process; Proteins; Proteomics; Publishing; Reactive Oxygen Species; Reagent; Reporting; Research; Resolution; Retina; Retinal Cone; Retinal Degeneration; Retinitis Pigmentosa; Rod; Scanning Electron Microscopy; Severity of illness; Stains; Structure; Testing; Therapeutic; Time; Western Blotting; Wild Type Mouse; base; dark rearing; experimental study; genomic locus; immunocytochemistry; male; metabolome; metabolomics; mitochondrial dysfunction; mouse model; mutant; new therapeutic target; non-genetic; novel; novel strategies; phosphodiesterase 6; phosphoric diester hydrolase; photoreceptor degeneration; protein expression; response; retinal rods; targeted treatment; therapeutic target

ABSTRACT Retinitis pigmentosa (RP) is a progressive form of retinal degeneration caused by at least 3,000 mutations in 70 different genes that result in the death of rods, followed by cones, and ultimately blindness. Although oxidative stress and inflammation are observed in RP, early molecular mechanisms in disease progression are unknown. We propose to compare a genetic mouse model of RP, rd10, with wild type mice to determine key pathways that suggest therapeutic targets for treating retinal degeneration utilizing a novel strategy of broad-spectrum metabolomics integrated with proteomics and mitochondrial dysfunction analysis. The rd10 mouse has a mutation in the β subunit of phosphodiesterase 6 (PDE6), which hydrolyzes cGMP in response to light as part of the photoresponse. Mutations in this gene cause recessive RP in humans. Our recent untargeted metabolomics study found abnormal increases in the levels of several deoxypyrimidines and deoxypurines, as well as a key metabolite in the Coenzyme A synthesis pathway, in rd10 compared to wild type mice. These changes were observed at P18, which is when cell death and reduced photoreceptor function are first detected. We hypothesize that the observed changes in the metabolome in rd10 mice are due to changes in the activities of the related proteins/enzymes. To identify changes in the metabolome that occur before reduced photoreceptor function and cell death are detected, we propose to analyze mice at the earlier timepoint, P14. In Specific Aim 1, we will use broad spectrum (untargeted) at P14 and targeted metabolomics at both P14 and P18. In Specific Aim 2, untargeted proteomics will be used to identify proteins/enzymes in membrane, soluble and mitochondrial fractions that may be linked to the disease process. This will be followed by targeted evaluation of proteins that are related to our metabolomic results in SA1 using computer analysis to integrate the metabolomic and proteomic data for developing an early mechanistic disease profile. Specific Aim 3 will evaluate mitochondrial oxygen consumption rate (OCR) at P14, based on our preliminary data demonstrating a reduced OCR in rd10 mice as early as P16 and P18, suggesting that mitochondrial dysfunction is an early event in this model of RP. We will also test this hypothesis by performing evaluations of mitochondrial health such as mitochondrial dynamics (fusion and fission) and the integrity of the mitochondrial structure using Serial Block Face Scanning Electron Microscopy (SBFSEM). Abnormal mitochondrial structure or function identified in this aim will be integrated with the metabolomic and proteomic information from SA1 and SA2. For all specific aims, dark-reared and light-reared mice will be compared, since light is known to be an exacerbating factor for RP caused by the rd10 mutation. Based on a report that oxidative stress occurs at different time points and is differentially localized in male and female rd10 mice, we will also include gender as a variable. The ultimate goal of this proposal is to identify potential therapeutic targets in key pathways early in retinal degeneration.

抽象的 色素性视网膜炎 (RP) 是一种进行性视网膜变性，由 70 种基因中至少 3,000 个突变引起 不同的基因导致视杆细胞死亡，然后是视锥细胞死亡，最终失明。虽然氧化 在 RP 中观察到应激和炎症，但疾病进展的早期分子机制尚不清楚。 我们建议将 RP 基因小鼠模型（rd10）与野生型小鼠进行比较，以确定关键途径 提出利用广谱新策略治疗视网膜变性的治疗靶点 代谢组学与蛋白质组学和线粒体功能障碍分析相结合。 rd10 鼠标有一个 磷酸二酯酶 6 (PDE6) 的 β 亚基发生突变，它会响应光而水解 cGMP，作为 光响应。该基因的突变会导致人类隐性 RP。我们最近的非靶向代谢组学 研究发现几种脱氧嘧啶和脱氧嘌呤的水平异常增加，以及一个关键的 与野生型小鼠相比，rd10 中辅酶 A 合成途径中的代谢物。这些变化是 在 P18 观察到，这是首次检测到细胞死亡和光感受器功能降低的时间。我们假设 观察到 rd10 小鼠代谢组的变化是由于相关相关活性的变化 蛋白质/酶。识别光感受器功能下降之前发生的代谢组变化 检测到细胞死亡，我们建议在较早的时间点 P14 分析小鼠。在具体目标 1 中，我们将使用 P14 为广谱（非靶向），P14 和 P18 为靶向代谢组学。在具体目标 2 中， 非靶向蛋白质组学将用于鉴定膜、可溶性和线粒体中的蛋白质/酶 可能与疾病过程有关的部分。随后将对蛋白质进行有针对性的评估 与我们在 SA1 中的代谢组学结果相关，使用计算机分析来整合代谢组学和 用于开发早期机械疾病概况的蛋白质组数据。具体目标 3 将评估线粒体 P14 的耗氧率 (OCR)，基于我们的初步数据，表明 rd10 中 OCR 降低 早在 P16 和 P18 小鼠中，表明线粒体功能障碍是该 RP 模型的早期事件。 我们还将通过评估线粒体健康状况（例如线粒体）来检验这一假设。 使用串行块面扫描分析线粒体结构的动力学（融合和裂变）和完整性 电子显微镜（SBFSEM）。在此目标中发现的异常线粒体结构或功能将被 与 SA1 和 SA2 的代谢组学和蛋白质组学信息整合。对于所有特定目标，黑暗饲养的 将与光饲养的小鼠进行比较，因为已知光是由光饲养的小鼠引起的 RP 的加剧因素。 rd10突变。基于氧化应激发生在不同时间点且局部性不同的报告 在雄性和雌性 rd10 小鼠中，我们还将性别作为变量。该提案的最终目标是 确定视网膜变性早期关键通路中的潜在治疗靶点。