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RPE Energy Metabolism and Cell Phenotype

RPE 能量代谢和细胞表型

基本信息

批准号：
10382919
负责人：
Douglas E. Vollrath
金额：
$ 8.05万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-01 至 2022-08-31
项目状态：
已结题

项目摘要

The retinal pigment epithelium (RPE) is a highly differentiated, post-mitotic cell layer that performs a host of functions critical to retinal homeostasis. To discharge its many functions, the RPE requires ample energy. Studies of cultured RPE cells show that they can derive energy from glucose by either aerobic glycolysis or oxidative phosphorylation (OXPHOS), depending upon culture conditions. However, the balance between these two primary modes of RPE glucose metabolism in vivo is unknown, and it is unclear whether alterations in this balance occur under normal and/or disease conditions. Abundant evidence links changes in cellular energy metabolism with alterations in cell phenotype in a variety of fields including cancer, development, stem cell differentiation and aging. In the outer retina, mutations in mitochondrial DNA that compromise OXPHOS cause macular retinopathy. Moreover, disproportionate damage to mitochondrial DNA has been documented in the RPE of individuals with age-related macular degeneration (AMD), suggesting a causal link. We previously showed that postnatal loss of mitochondrial DNA and OXPHOS capacity in the murine RPE in vivo has surprising effects on cell phenotype, causing activation of cell growth pathways, increased glycolytic flux, and loss of epithelial functions and integrity. Our findings demonstrate that enforced changes in cellular energy metabolism in vivo can drive dedifferentiation and transdifferentiation of the RPE, and support a causal connection between diminished RPE OXPHOS capacity and AMD. However, our results raise new questions about how particular aspects of altered cellular energy metabolism read out as changes in cell phenotype. Can increased aerobic glycolysis alone, in the presence of intact OXPHOS, activate cell growth pathways, cause dedifferentiation/transdifferentiation and loss of epithelial integrity? Are features of the RPE glycolytic phenotype reversible through rebalancing of metabolism? What aspects of the altered phenotype of OXPHOS- deficient RPE result from lack of ATP production via OXPHOS versus loss of electron transport to oxygen? Does diurnal variation in energy metabolism affect the capacity of the RPE to phagocytize outer segment tips? We propose an ensemble of experiments to address these questions. Specifically we will modulate RPE aerobic glycolysis in vivo in the context of intact OXPHOS, restore respiration without ATP generation to OXPHOS-deficient RPE in vivo, and probe the relationship between RPE energy metabolism and diurnal phagocytic capacity. The requested equipment will allow us to continue our detailed characterization and quantification of the alterations in RPE cell phenotype caused by these in vivo metabolic changes, and thereby uncover mechanistic connections between energy metabolism and RPE cell phenotype. Because we will work in vivo, the impact of RPE metabolic modulation on photoreceptors will be apparent. Thus, success of this project will not only provide foundational knowledge of in vivo RPE metabolism and its relationship to cell phenotype, it will also inform studies of abnormal RPE metabolism in human retinal disease.

视网膜色素上皮(RPE)是一种高度分化的有丝分裂后细胞层，它执行一系列对视网膜动态平衡至关重要的功能。要发挥其众多功能，RPE需要充足的能量。对培养的RPE细胞的研究表明，它们可以通过有氧酵解或从葡萄糖中获得能量氧化磷酸化(OXPHOS)，取决于培养条件。然而，两者之间的平衡 RPE体内葡萄糖代谢的这两种主要模式尚不清楚，也不清楚是否发生了变化这种平衡发生在正常和/或疾病条件下。大量证据表明细胞内的变化能量代谢与癌症、发育、干细胞等不同领域细胞表型的变化细胞分化和衰老。在外视网膜，线粒体DNA的突变损害了OXPHOS 导致黄斑视网膜病变。此外，对线粒体DNA的不成比例的损害已经被记录在年龄相关性黄斑变性(AMD)患者的RPE，提示两者之间存在因果联系。我们之前结果表明，出生后小鼠RPE体内线粒体DNA和OXPHOS能力的丧失对细胞表型的惊人影响，导致细胞生长途径的激活，糖酵解通量增加，以及丧失上皮功能和完整性。我们的发现表明，细胞能量的强制变化体内代谢可以驱动RPE的去分化和转分化，并支持一种病因 RPE OXPHOS容量减少与AMD之间的联系。然而，我们的结果提出了新的问题关于细胞能量代谢变化的特定方面如何读出为细胞表型的变化。能在完整的OXPHOS存在的情况下，单独增加有氧糖酵解，激活细胞生长途径，导致去分化/转分化和上皮完整性丧失？是RPE糖酵解的特征通过新陈代谢的重新平衡，表型可逆？OXPHOS表型改变的哪些方面- RPE缺陷是由于缺乏通过OXPHOS产生的ATP，而不是失去向氧的电子传输？能量代谢的日变化是否影响RPE吞噬外节末端的能力？我们提出了一系列实验来解决这些问题。具体来说，我们将调节RPE 体内有氧糖酵解在完整OXPHOS的背景下，恢复呼吸而不产生ATP 体内OXPHOS缺陷型RPE，并探讨RPE能量代谢与昼夜节律的关系吞噬能力。所要求的设备将使我们能够继续我们的详细描述和量化由这些体内代谢变化引起的RPE细胞表型的变化，从而揭示能量代谢和RPE细胞表型之间的机制联系。因为我们会努力工作在体内，RPE代谢调节对光感受器的影响将是显而易见的。因此，这件事的成功该项目不仅将提供体内RPE代谢及其与细胞关系的基础知识表型，它还将为人类视网膜疾病中RPE代谢异常的研究提供信息。