NAD Metabolism in Aging and Disease: Dysfunction and Intervention

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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.