NAD is a vital coenzyme participating in a multitude of metabolic reactions. Moreover, it serves as a signaling molecule to mediate fundamental cellular processes including DNA repair, cell cycle progression, transcriptional, epigenetic and metabolic regulation. In these processes, NAD is cleaved to liberate nicotinamide, and the ADP-ribosyl moiety is used to perform protein or nucleic acid modifications or to generate messenger molecules. To maintain cellular NAD levels, the released nicotinamide is recycled into NAD synthesis through the salvage pathway.
Here, we aimed to understand the potential interaction between different subcellular NAD pools with a main interest in mitochondria. These organelles represent a major pool with the highest concentration of the dinucleotide. Following targeted overexpression of an NAD consumer in a variety of subcellular compartments we measured NAD turnover using stable isotope-labeled precursors. Remarkably, turnover was hardly affected by the induced increase of NAD-consuming activity, irrespective of its subcellular expression. Accordingly, no upregulation of NAD synthesis was observed and therefore, NAD levels were chronically decreased to limit NAD consumption to the original value. We hypothesize that these observations might provide a mechanistic background for age-dependent cellular NAD decline.
The mitochondrial NAD pool is known to have a certain degree of autonomy, and this has been linked to the mitochondrial localization of NMNAT3, an enzyme catalyzing the reversible, final step of NAD formation from NMN and ATP. However, NMNAT3 is dispensable in mice, and the recent identification of SLC25A51, or MCART1, as a mitochondrial NAD+ transporter, seems to finally have settled the question regarding the establishment and maintenance of the mitochondrial NAD pool in mammals. Based on a large set of experiments including genetically modified cell lines, we propose a key role of NMNAT3 in the mechanism how mitochondria maintain a balanced NAD pool. We posit that the reversible cleavage of imported NAD into NMN and ATP establishes an equilibrium between NAD and NMN (and ATP) that can be shifted to either side upon demand. For example, high cytosolic NAD increases mitochondrial uptake and subsequent cleavage of NAD, whereas high NAD consumption activity would favor NAD synthesis. This mechanism would establish a buffer to compensate fluctuations in mitochondrial NAD concentrations. Moreover, in concert with reversible NAD exchange through SLC25A51, this buffer would be functional for NAD pools in other subcellular locations. Indeed, we demonstrate that the mitochondrial NAD pool is “tapped” when the NAD consumer is overexpressed outside mitochondria. We conclude that subcellular NAD pools are interconnected with a major role of mitochondria in maintaining the cellular homeostasis of this coenzyme.