Quantification of localized NAD+ changes reveals unique specificity of NAD+ regulation in the hypothalamus

Abstract Recently, it has become a consensus that systemic decreases in NAD+ are a critical trigger for age-associated functional decline in multiple tissues and organs. The hypothalamus, which contains several functionally distinct subregions called nuclei, functions as a high-order control center of aging in mammals. However, due to a technical difficulty, how NAD+ levels change locally in each hypothalamic nucleus during aging remains uninvestigated. We were able to establish a new combinatorial methodology, using laser-captured microdissection (LCM) and high-performance liquid chromatography (HPLC), to accurately measure NAD+ levels in small tissue samples. We applied this methodology to examine local NAD+ changes in hypothalamic nuclei and found that NAD+ levels were decreased significantly in the arcuate nucleus (ARC), ventromedial hypothalamus (VMH), and lateral hypothalamus (LH), but not in the dorsomedial hypothalamus (DMH) of 22-month-old mice, compared to those of 3-month-old mice. The administration of nicotinamide mononucleotide (NMN) significantly increased NAD+ levels in all these hypothalamic nuclei. Interestingly, the administration of extracellular nicotinamide phosphoribosyltransferase-containing extracellular vesicles (eNampt-EVs) purified from young mice increased NAD+ levels in the ARC and DMH. These results reveal the unique specificity of NAD+ regulation in the hypothalamus during aging.