Subcellular NAD Pools Are Interconnected and Buffered by Mitochondrial NAD

The coenzyme NAD is consumed by signalling enzymes, including poly-ADP-ribosyltransferases (PARPs) and sirtuins. Ageing 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 overconsumption 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 modelling 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 nicotinamide mononucleotide and ATP when NMNAT3 is present. Thus, these organelles can maintain an additional, virtual NAD pool. Our results are consistent with a well-tolerated ageing-related NAD decline as long as the vulnerable mitochondrial pool is not directly affected.