Subcellular NAD+ pools are interconnected and buffered by mitochondrial NAD+

Summary

The coenzyme NAD+ is consumed by signaling enzymes, including poly-ADP-ribosyltransferases (PARPs) and sirtuins. Aging is associated with a decrease in cellular NAD+ levels but how cells cope with persistently decreased NAD+ concentrations is unclear. Here, we show that subcellular NAD+ pools are interconnected, with mitochondria acting as a rheostat to maintain NAD+ levels upon excessive consumption. To evoke chronic, compartment-specific over-consumption of NAD+, we engineered cell lines stably expressing PARP activity in mitochondria, the cytosol, endoplasmic reticulum, or peroxisomes, resulting in a decline of cellular NAD+ concentrations by up to 50%. Isotope-tracer flux measurements and mathematical modeling show that the lowered NAD+ concentration kinetically restricts NAD+ consumption to maintain a balance with the NAD+ biosynthesis rate, which remains unchanged. Chronic NAD+ deficiency is well tolerated unless mitochondria are directly targeted. Mitochondria maintain NAD+ by import through SLC25A51, and reversibly cleave NAD+ to NMN and ATP, when NMNAT3 is present. Thereby, these organelles can maintain an additional, virtual NAD+ pool. Our results are consistent with a well-tolerated aging-related NAD+ decline as long as the vulnerable mitochondrial pool is not directly affected.