NAD Metabolism in Aging and Disease: Dysfunction and Intervention

摘要哺乳动物的衰老是复杂的，其特点包括干细胞自我更新的倾向降低， DNA修复缺陷，对生长刺激激素和营养物质的反应减少，代谢 破坏和增加了疾病发作的易感性。自我更新缺陷的根源 修复和代谢动态平衡是老龄化领域的中心问题。可以说，没有一个单一的因素可以 被确认为提供因果关系的。我们一直关注NAD+代谢缺陷，认为这可能是一种 多效性效应导致下游细胞、组织和生物体健康功能障碍。这样的过程 如衰老，更容易由遗传因素引起，如DNA修复基因的缺陷(如 Werner和Bloom综合征)为进一步研究NAD+的作用提供了一个有趣的机会 缺乏，因为转基因成纤维细胞可以作为这方面的潜在工具在商业上获得。 我们建议概括地描述衰老如何影响孕激素细胞和老龄小鼠的NAD+代谢。 具体地说，我们将表征NAD+的生物合成潜力以及NAD+的周转率。此外，我们 将评估细胞对增加NAD+生物合成的药物干预的反应，以便 确定这些干预措施是否减轻了这些成纤维细胞的年龄依赖性表型。这些研究将 更深入地了解NAD+如何在细胞和组织中发生下降，以及某些细胞和组织是否 比其他人更容易受到这种下降的影响，以及为什么。应用程序的第二部分侧重于发现 一种名为二氢烟酰胺核苷(NRH)的新型NAD+增强剂，它可以将NAD+浓度从3- 在哺乳动物细胞中是10倍。初步数据显示，NRH使用了一种新的作用机制，其中 转化为NMNH，独立于已知的激酶NRK1或NRK2，导致NAD+的生物合成。在小鼠身上 这种化合物使大多数组织中的NAD+浓度比对照增加了许多倍。此应用程序 研究其在成纤维细胞和小鼠体内的作用机制，以阐明一种新的NAD+生物合成途径 具有治疗疾病的翻译潜力。因此，在应用程序的后半部分，我们描述 NRH对衰老小鼠的影响，并确定它是否可以诱导线粒体的生物发生。我们提供研究，以 使用新陈代谢组学方法来表征其效果。最后，我们测试了NRH来治疗一个新陈代谢模型 并描述年龄对疾病和治疗结果的影响。这笔赠款的目标 通过三个特定的目标来实现：1.表征细胞和小鼠的NAD+内稳态作为一种功能 年龄。2.阐明NRH在成纤维细胞和小鼠体内的作用及代谢途径。3.确定 NRH对老龄小鼠NAD+代谢、线粒体生物发生的影响及对代谢模型的缓解作用 综合症。这些目标的实现将使我们对NAD+如何成为 衰老以及下一代NAD+前体细胞是否可以改变作为衰老标志的表型。

SUMMARY Aging in mammals is complex, with hallmarks including reduced propensity for stem cell self renewal, deficiencies in DNA repair, reduced responses to growth stimulating hormones and nutrients, metabolic disruption and increased susceptibilities to the onset of diseases. The origins of the deficits in self renewal, self repair, and metabolic homeostasis are central questions in the aging field. Arguably, no single factor can be identified that provides a causative effect. We have focused upon deficits in NAD+ metabolism as a potentially pleiotropic effector leading to downstream dysfunctions in cellular, tissue and organism health. Processes such as senescence, which can more readily arise from genetic factors such as defects in DNA repair genes (e.g. Werner and Bloom Syndromes) present an interesting opportunity to further investigate the role of NAD+ deficiency, given that genetically altered fibroblasts are commercially accessible as potential tools in this regard. We propose to generally characterize how aging affects NAD+ metabolism in progeroid cells, and in aged mice. Specifically, we will characterize NAD+ biosynthetic potential as well as rates of NAD+ turnover. Moreover we will assess how cells respond to pharmacologic interventions that increase NAD+ biosynthesis in order to determine if these interventions mitigate age-dependent phenotypes in these fibroblast cells. These studies will provide a deeper view of how NAD+ decline occurs in cells and tissues, and if some cells and tissues are more susceptible to this decline than others and why. A second part of the application focuses on the discovery of a novel NAD+ enhancer called dihydronicotinamide riboside (NRH), which can raise NAD+ concentrations from 3- 10 fold in mammalian cells. Preliminary data shows that NRH uses a novel mechanism of action,wherein it is converted to NMNH, independent of the known kinases Nrk1 or Nrk2, leading to biosynthesis of NAD+. In mice this compound increases NAD+ concentrations many-fold over control in most tissues. This application investigates its mechanism of action in fibroblasts and in mice to elucidate a novel biosynthetic pathway to NAD+ with translational potential for treatment of disease. Thus, in the latter part of the application, we characterize NRH effects in aged mice and ascertain if it can induce mitochondrial biogenesis. We provide studies to characterize its effects using metabolomics approaches. Finally we test NRH to treat a model of metabolic syndrome and to characterize the effect of age on disease and treatment outcomes. The objectives of the grant are accomplished via 3 specific aims: 1. To characterize NAD+ homeostasis in cells and mice as a function of age. 2. To elucidate effect and metabolic pathway of NRH in fibroblasts and mice. 3. To determine the effect of NRH on aged mice in altering NAD+ metabolism, mitochondrial biogenesis and mitigation of a model of metabolic syndrome. The accomplishment of the objectives will provide new understanding of how NAD+ is a key factor in aging and whether next generation NAD+ precursors can alter phenotypes that are hallmarks of aging.