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.

Nicotinamide Mononucleotide Supplementation: Understanding Metabolic Variability and Clinical Implications

“…However, the slow processing time of this pathway, exceeding several hours in some studies, cannot account for rapid NMN absorption within the gut (2–3 min) and tissue uptake (10–30 min) observed in mice [ 33 ]. Additionally, robust NMN uptake in cells despite inhibition of CD73/ENT or NRK1 further suggests an alternative route exists [ 22 , 43 ]. The discovery of Slc12a8, a highly specific NMN transporter abundantly expressed in the gut, pancreas, liver, and white adipose tissues, provides a compelling mechanism for rapid NMN absorption and distribution.…”

“…Distinct NAD+ pools exist in the cytosol, mitochondria, and nucleus, each serving specific functions [ 19 ]. The identification of separate transporters for NAD+ (SLC25A51) and NMN (Slc25a45) in mitochondria suggests a fascinating level of compartmentalized regulation within cells [ 43 , 159 ]. This implies that cells can precisely control the influx of these molecules based on specific needs, potentially offering a new avenue for understanding and influencing cellular health.…”