Monitoring NAD+ levels in aging using a novel genetically-encoded biosensor

使用新型基因编码生物传感器监测衰老过程中的 NAD 水平

• 批准号: 9284238
• 负责人: RICHARD H. GOODMAN
• 金额: $ 38.5万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9284238
• 关键词: Affect; Aging; Anabolism; Animals; Beta Cell; Binding; Biology; Biosensor; Caloric Restriction; Cell Culture Techniques; Cell Death; Cell Nucleus; Cells; Consequentialism; Cytoplasm; Development; Diabetes Mellitus; Disease; Electrons; Exercise; Gene Expression; Genes; Genome Stability; Goals; Hand; Homeostasis; Impairment; Injury; Intramuscular Injections; Libraries; Link; LoxP-flanked allele; MM form creatine kinase; Measurement; Measures; Mediating; Methods; Mitochondria; Modeling; Monitor; Mus; Muscle; Muscle Cells; Muscle Fibers; Muscle Weakness; Muscle function; Mutation; NADH; Nature; Nicotinamide adenine dinucleotide; Nuclear; Nutrient; Oxidation-Reduction; Polymerase; Production; Proteins; Regulation; Ribose; Role; Sirtuins; Skeletal Muscle; Structure of beta Cell of islet; Testing; Tissues; Transgenic Mice; Transgenic Organisms; age effect; age related; aged; disorder prevention; experimental study; functional disability; improved; in vivo; insight; insulin secretion; nicotinamide phosphoribosyltransferase; nicotinamide-beta-riboside; novel; prevent; regenerative; satellite cell; sensor

Project Summary Nicotinamide adenine dinucleotide (NAD+) is the substrate for sirtuins and polyADP-ribose polymerases (PARPs), linking it to gene expression and genomic stability. These enzymatic activities also connect NAD+ to such aging-related conditions as diabetes and muscle weakness. NAD+ alterations also figure prominently in the relationship between calorie restriction (CR) and disease prevention, but the exact nature of this link remains unknown. One model is that CR elevates intracellular NAD+, which then controls activity of sirtuins that regulate fuel utilization and expression of nutrient-responsive genes. PARP activation can also influence cellular energy homeostasis by depleting NAD+, ultimately leading to cell death. It is important to remember that the contribution of NAD+ to sirtuins and PARPs depends entirely upon the free NAD+ concentration and not on redox, that is, the NAD+/NADH ratio. Although measuring the NAD+/NADH ratio is straightforward, monitoring NAD+ is not—our development of an NAD+ biosensor has provided the first glimpses into NAD+ regulation within subcellular compartments of intact cells. This is an important advance because previous studies could not distinguish free from bound NAD+ or monitor differences in NAD+ regulation across compartments. With this novel sensor in hand, we will determine how NAD+ levels in pancreatic beta cells and skeletal muscle are regulated during aging and whether age-related changes can be prevented by CR or augmentation of NAD+ production. Although we have gained significant insights into NAD+ biology using the current sensor, it would be of great value to extend our studies into intact animals. Thus, our first goal is to develop a conditional transgenic mouse line expressing the NAD+ biosensor. We have already made significant progress optimizing our sensor for in vivo dynamic measurements and describe strategies for increasing its sensitivity and dynamic range further. We will then test whether aging decreases, and CR or NAD+ precursor administration increases, NAD+ levels in pancreatic beta cells and skeletal muscle cells by generating tissue- specific sensor strains capable of monitoring NAD+ levels in the nucleus, cytoplasm, and mitochondria. NAD+ depletion is thought to mediate age-related decreases in insulin secretion. Similarly, age-dependent NAD+ decreases have been proposed to underlie muscle weakness and impairments in muscle regenerative capacity. Our biosensor provides an unprecedented opportunity to examine the effect of aging on NAD+ levels, the contribution of NAD+ to age-related disorders, and the efficacy of several proposed approaches to ameliorating these conditions.

项目摘要 烟酰胺腺嘌呤二核苷酸（NAD+）是沉默调节蛋白和聚ADP-核糖聚合酶的底物 （PARP），将其与基因表达和基因组稳定性联系起来。这些酶活性也将NAD+连接到 与衰老有关的疾病，如糖尿病和肌肉无力。NAD+的改变也在 热量限制（CR）和疾病预防之间的关系，但这种联系的确切性质仍然存在 未知一种模型是CR升高细胞内NAD+，然后控制沉默调节蛋白的活性， 调节燃料利用和营养反应基因的表达。PARP激活也可以影响 细胞能量稳态通过消耗NAD+，最终导致细胞死亡。重要的是要记住 NAD+对sirtuins和PARP的贡献完全取决于游离NAD+浓度， 不依赖于氧化还原，即NAD+/NADH比率。虽然测量NAD+/NADH比率很简单， 监测NAD+不是我们开发的NAD+生物传感器提供了对NAD+的第一次一瞥 在完整细胞的亚细胞区室内调节。这是一个重要的进步，因为以前 研究无法区分游离NAD+和结合NAD+，也无法监测NAD+调节的差异。 隔间有了这种新型传感器，我们将确定胰腺β细胞中的NAD+水平， 骨骼肌在衰老过程中受到调节，以及CR或 增加NAD+产量。虽然我们已经获得了显着的见解NAD+生物学使用 电流传感器，将我们的研究扩展到完整的动物将是非常有价值的。因此，我们的首要目标是 开发表达NAD+生物传感器的条件转基因小鼠系。我们已经取得了重大进展， 进展优化我们的传感器在体内动态测量和描述的战略，以增加其 灵敏度和动态范围。然后，我们将测试老化是否减少，CR或NAD+前体 给药增加了胰腺β细胞和骨骼肌细胞中的NAD+水平， 能够监测细胞核、细胞质和线粒体中NAD+水平的特异性传感器菌株。NAD+ 消耗被认为介导与年龄相关的胰岛素分泌减少。同样，年龄依赖性NAD+ 已经提出降低是肌肉无力和肌肉再生能力受损的基础。 我们的生物传感器提供了一个前所未有的机会来检查老化对NAD+水平的影响， NAD+对年龄相关疾病的贡献，以及几种改善老年痴呆症的方法的疗效。 了以下条件