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.