Quantitative dynamics of intracellular NMN by genetically encoded biosensor

Abstract Nicotinamide mononucleotide (NMN) is a major precursor for NAD + metabolism with promising effects in treating NAD + - and aging-related pathologies. However, measuring live cell NMN dynamics was not possible, leaving key questions in intracellular NMN uptake and regulation unanswered. Here we developed a genetically encoded bioluminescent sensor to quantify subcellular NMN in live cells by fusing engineered NMN-responsive binding domain to bioluminescent and fluorescent proteins from BRET pairs. The sensor dissected the multimechanistic uptake of extracellular NMN and precursors in live cells. We then captured the notably low mitochondrial NMN content and the thereafter vulnerable NMN/NAD + ratio and SARM1 activation in mitochondria, establishing NMN/NAD + ratio as an important parameter in evaluating NAD + boosting strategies. Moreover, we characterized the signature of major NAD + regulating enzymes on NMN and NMN/NAD + ratios, in which Slc25a45 was identified to be a potential mitochondrial NMN transporter for its unique fingerprint on mitochondrial NMN/NAD + ratio.