TITLE OF PROJECT eNAMPT-mediated adipo-hypothalamic communication for NAD+ production and aging ABSTRACT In recent years, nicotinamide adenine dinucleotide (NAD+) metabolism has emerged as a central topic in the field of aging and longevity research. It has been established that NAD+ availability declines over age at a systemic level, triggering a variety of age-associated pathophysiological changes in diverse model organisms. In mammalian NAD+ biosynthesis, nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme that converts nicotinamide and 5?-phosphoribosyl-pyrophosphate to nicotinamide mononucleotide (NMN), an important NAD+ intermediate. Interestingly, there are two distinct forms of NAMPT in mammals: intra- and extracellular NAMPT (iNAMPT and eNAMPT, respectively). We have previously demonstrated that eNAMPT mediates a novel intertissue communication system between adipose tissue and the hypothalamus, regulating hypothalamic NAD+ levels and functions. We have now found that eNAMPT is carried in exosomes through the circulation in mice and humans. Exosomal eNAMPT is internalized into primary hypothalamic neurons and enhances NAD+ biosynthesis intracellularly. The genetically engineered mice that can maintain higher levels of exosomal eNAMPT at old ages exhibit a variety of anti-aging phenotypes and a significant extension of healthspan. These new findings demonstrate a novel systemic mechanism that regulates the process of aging and determines healthspan/lifespan, driven by an exosome-mediated delivery of eNAMPT. Thus, we hypothesize that exosomal eNAMPT is delivered to specific tissues through the interaction with a specific receptor-like protein and regulates various tissue functions, including the hippocampus-dependent cognitive functions. The K53R mutant of NAMPT, whose secretion is enhanced, could be used as a genetically engineered biologic that mitigates age-associated functional decline in mice. We will address this hypothesis by the following specific aims: 1) To elucidate the mechanism of exosomal eNAMPT targeting, we will examine the requirement of the NAMPT protein structure and also test a potential receptor candidate for exosomal eNAMPT to be internalized, 2) to further analyze the effects of supplementing eNAMPT-containing exosomes in aged mice, we will analyze their effect on the hippocampus-mediated cognitive functions, and 3) to test eNAMPT as an anti-aging biologic, we will examine the effects of eNAMPT mutants encapsulated into exosomes on cognitive, behavioral, and other tissue functions in aged mice. Thus, the anticipated outcome of the proposed research will open a new avenue to understand how systemic NAD+ homeostasis regulates aging and longevity and develop a novel anti-aging intervention by using exosomal eNAMPT as a biologic.