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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要烟酰胺腺嘌呤二核苷酸 (NAD+) 是去乙酰化酶和聚 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+ 与 NAD+ 联系起来。诸如糖尿病和肌肉无力等与衰老相关的疾病也在该疾病中占据显着地位。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响应基因的表达也会影响 PARP 激活。通过消耗 NAD+ 来维持细胞能量稳态，最终导致细胞死亡，这一点很重要。 NAD+ 对 Sirtuins 和 PARP 的贡献完全取决于游离 NAD+ 浓度，不是氧化还原，即 NAD+/NADH 比率虽然测量 NAD+/NADH 比率很简单，监测 NAD+ 并非如此——我们开发的 NAD+ 生物传感器让人们首次了解 NAD+ 这是一个重要的进步，因为之前的研究。研究无法区分游离 NAD+ 和结合 NAD+ 或监测 NAD+ 调节的差异有了这种新型传感器，我们将确定胰腺 β 细胞和细胞中的 NAD+ 水平。骨骼肌在衰老过程中受到调节，CR 或 CR 是否可以预防与年龄相关的变化尽管我们使用 NAD+ 生物学获得了重要的见解。目前的传感器，将我们的研究扩展到完整的动物将具有很大的价值。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。优化我们的传感器用于体内动态测量的进展，并描述提高其性能的策略然后我们将进一步测试老化是否会降低以及 CR 或 NAD+ 前体的灵敏度和动态范围。施用通过产生组织-增加胰腺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+ 水平的特定传感器菌株。消耗被认为会介导与年龄相关的胰岛素分泌减少，同样，年龄依赖性 NAD+ 也会减少。已提出减少是肌肉无力和肌肉再生能力受损的原因。我们的生物传感器提供了前所未有的机会来检查衰老对 NAD+ 水平的影响，即 NAD+ 对年龄相关疾病的贡献，以及几种提议的改善方法的功效这些条件。