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）是一种渐进的视网膜变性形式，由至少3,000个突变引起 导致杆死亡的不同基因，其次是锥体，最终导致失明。虽然氧化 在RP中观察到应力和炎症，疾病进展的早期分子机制尚不清楚。 我们建议将RP，RD10的遗传小鼠模型与野生型小鼠进行比较，以确定关键途径 建议使用广谱的新策略来治疗视网膜变性的治疗靶标 与蛋白质组学和线粒体功能障碍分析相结合的代谢组学。 RD10鼠标有一个 磷酸二酯酶6（PDE6）的β亚基中的突变，该突变是响应于光的CGMP的一部分 光反应。该基因的突变导致人类隐性RP。我们最近的不靶向代谢组学 研究发现，几种脱氧酰亚胺和脱氧嘌呤的水平异常增加以及关键 与野生型小鼠相比，RD10中的辅酶A合成途径中的代谢产物。这些变化是 在P18上观察到，这是首先检测到细胞死亡和降低的感光受体功能的时间。我们假设 RD10小鼠中观察到的代谢组的变化是由于相关活动的变化 蛋白质/酶。确定在降低感光函数和 检测到细胞死亡，我们建议在较早的时间点P14上分析小鼠。在特定目标1中，我们将使用 p14处的宽光谱（未靶向），并在p14和p18处靶向代谢组学。在特定的目标2中， 未靶向的蛋白质组学将用于识别膜，可溶性和线粒体中的蛋白质/酶 可能与疾病过程有关的分数。接下来是对蛋白质的有针对性评估 使用计算机分析与我们在SA1中的代谢组结果有关，以整合代谢组和 用于发展早期机械疾病特征的蛋白质组学数据。特定目标3将评估线粒体 基于我们的初步数据，p14处的氧气消耗率（OCR）证明了RD10的OCR降低 早在P16和P18时，小鼠表明线粒体功能障碍是这种RP模型的早期事件。 我们还将通过对线粒体健康（例如线粒体）进行评估来检验这一假设 动力学（融合和裂变）以及使用串行块面扫描线粒体结构的完整性 电子显微镜（SBFSEM）。在此目标中确定的线粒体结构或功能异常将是 与来自SA1和SA2的代谢组和蛋白质组学信息集成。对于所有特定目标，深色 将比较浅毛的小鼠，因为已知光是由RP引起的恶化因素 RD10突变。基于一份报道，氧化应激发生在不同的时间点，并且局部不同 在雄性和雌性RD10小鼠中，我们还将将性别作为变量。该提议的最终目标是 在永久性变性早期确定关键途径中的潜在治疗靶标。