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

摘要 视网膜色素变性（RP）是一种渐进形式的视网膜变性，由70个基因中至少3,000个突变引起。 不同的基因导致了杆状细胞的死亡，接着是锥细胞的死亡，最终导致失明。虽然氧化 在RP中观察到应激和炎症，疾病进展的早期分子机制尚不清楚。 我们建议将RP，rd 10的遗传小鼠模型与野生型小鼠进行比较，以确定 提出了利用广谱抗视网膜变性新策略治疗视网膜变性的治疗靶点 与蛋白质组学和线粒体功能障碍分析相结合的代谢组学。rd 10鼠标有一个 磷酸二酯酶6（PDE 6）β亚基的突变，其响应于光而水解cGMP，作为 光反应。该基因的突变导致人类隐性RP。我们最近的非靶向代谢组学 一项研究发现，几种脱氧嘧啶和脱氧嘌呤的水平异常升高， 与野生型小鼠相比，rd 10中辅酶A合成途径中的代谢产物。这些变化 在P18时观察到，这是首次检测到细胞死亡和感光功能降低的时候。我们假设 在rd 10小鼠中观察到的代谢组的变化是由于相关的 蛋白质/酶。确定在光感受器功能降低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的广谱（非靶向）和P14和P18的靶向代谢组学。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。随后将有针对性地评估蛋白质， 与我们在SA 1中的代谢组学结果相关，使用计算机分析整合代谢组学和 蛋白质组学数据用于开发早期机械性疾病谱。具体目标3将评估线粒体 P14时的耗氧率（OCR），基于我们的初步数据，表明rd 10中的OCR降低 早在P16和P18，提示线粒体功能障碍是RP模型的早期事件。 我们还将通过对线粒体健康状况进行评估来验证这一假设，例如线粒体 动态（融合和裂变）和线粒体结构的完整性，使用连续块面扫描 电子显微镜（SBFSEM）。在这一目标中确定的异常线粒体结构或功能将被 与来自SA 1和SA 2的代谢组学和蛋白质组学信息整合。对于所有特定的目标，黑暗饲养 和光饲养的小鼠进行比较，因为已知光是由 rd 10突变。基于氧化应激发生在不同时间点且不同定位的报告， 在雄性和雌性rd 10小鼠中，我们还将性别作为变量。这项建议的最终目的是 在视网膜变性的早期关键通路中确定潜在的治疗靶点。

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.