NAD Metabolism in Aging and Disease: Dysfunction and Intervention

哺乳动物的衰老是复杂的，其特征包括干细胞自我更新倾向降低， DNA修复缺陷，对生长刺激激素和营养素的反应降低，代谢 破坏和增加疾病发作的易感性。自我更新缺陷的起源， 修复和代谢稳态是衰老领域的中心问题。可以说，没有一个单一的因素可以 被鉴定为提供了一种因果关系。我们关注NAD+代谢的缺陷， 多效性效应子导致细胞、组织和生物体健康的下游功能障碍。过程 衰老，这可能更容易由遗传因素引起，如DNA修复基因的缺陷（例如， Werner和Bloom Syndrome）为进一步研究NAD+的作用提供了有趣的机会 然而，由于遗传改变的成纤维细胞作为这方面的潜在工具在商业上是可获得的，因此存在缺陷。 我们建议一般的特点如何老化影响NAD+代谢的早衰细胞，并在老年小鼠。 具体而言，我们将表征NAD+生物合成潜力以及NAD+周转率。并且我们在 将评估细胞如何对增加NAD+生物合成的药理干预作出反应， 确定这些干预措施是否减轻了这些成纤维细胞中的年龄依赖性表型。这些研究将 更深入地了解NAD+在细胞和组织中是如何下降的，以及如果一些细胞和组织比其他细胞和组织更 比其他人更容易受到这种衰退的影响，以及为什么。该应用程序的第二部分侧重于发现 一种新的NAD+增强剂，称为二氢烟酰胺核苷（NRH），可以将NAD+浓度从3- 10倍于哺乳动物细胞。初步数据显示，NRH使用一种新的作用机制，其中它是 转化为NMNH，独立于已知的激酶Nrk 1或Nrk 2，导致NAD+的生物合成。小鼠 这种化合物使大多数组织中的NAD+浓度比对照组增加了许多倍。本申请 研究其在成纤维细胞和小鼠中的作用机制，以阐明NAD+的新生物合成途径 具有治疗疾病的转化潜力。因此，在本申请的后半部分，我们表征 NRH对老年小鼠线粒体生物合成的影响。我们提供研究， 使用代谢组学方法描述其作用。最后，我们测试了NRH治疗代谢性 综合征，并描述年龄对疾病和治疗结果的影响。赠款的目标 通过三个具体目标来实现：1。为了表征细胞和小鼠中NAD+稳态作为 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.以确定影响 NRH对老年小鼠NAD+代谢、线粒体生物合成和代谢紊乱模型的缓解作用 综合征这些目标的实现将使人们对NAD+如何成为 老化以及下一代NAD+前体是否可以改变作为老化标志的表型。

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.