Subcellular NAMPT‐mediated NAD⁺ salvage pathways and their roles in bioenergetics and neuronal protection after ischemic injury

Abstract

NAD+ is a cofactor required for glycolysis, tricarboxylic acid cycle, and complex I enzymatic reaction. In mammalian cells, NAD+ is predominantly synthesized through the salvage pathway, where nicotinamide phosphoribosyltransferase (NAMPT) is the rate‐limiting enzyme. Previously, we demonstrated that NAMPT exerts a neuroprotective effect in ischemia through the suppression of mitochondrial dysfunction. Mammalian cells maintain distinct NAD+ pools in the cytosol, mitochondria, and nuclei. However, it is unknown whether mitochondria have an intact machinery for NAD+ salvage, and if so, whether it plays a dominant role in bioenergetics, mitochondrial function, and neuronal protection after ischemia. Here, using mouse primary cortical neuron and cortical tissue preparations, and multiple technologies including cytosolic and mitochondrial subfractionation, viral over‐expression of transgenes, molecular biology, and confocal microscopy, we provided compelling evidence that neuronal mitochondria possess an intact machinery of NAMPT‐mediated NAD+ salvage pathway, and that NAMPT and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3) are localized in the mitochondrial matrix. By knocking down NMNAT1‐3 and NAMPT with siRNA, we found that NMNAT3 has a larger effect on basal and ATP production‐related mitochondrial respiration than NMNAT1‐2 in primary cultured neurons, while NMNAT1‐2 have a larger effect on glycolytic flux than NMNAT3. Using an oxygen glucose deprivation model, we found that mitochondrial, cytoplasmic, and non‐subcellular compartmental over‐expressions of NAMPT have a comparable effect on neuronal protection and suppression of apoptosis‐inducing factor translocation. The current study provides novel insights into the roles of subcellular compartmental NAD+ salvage pathways in NAD+ homeostasis, bioenergetics, and neuronal protection in ischemic conditions.

NAD+ metabolism, stemness, the immune response, and cancer

“…Eliminating cytoplasmic NAD + blocks glycolysis and leads to cell death. 3 , 50 – 54 However, many works have suggested the NAD + biosynthetic machinery inside the mitochondria maintain the mitochondrial NAD + pool in response to environmental and nutritional stresses. 3 , 17 …”

Notoginseng Leaf Triterpenes Ameliorates OGD/R-Induced Neuronal Injury via SIRT1/2/3-Foxo3a-MnSOD/PGC-1α Signaling Pathways Mediated by the NAMPT-NAD Pathway

“…NAMPT, as a rate-limiting enzyme of NAD biosynthesis in the salvage pathway, positively regulates the activity of sirtuins and exerts protective effects in many cell types [ 46 , 47 ]. To further explore the relationship between the PNGL and the NAMPT-NAD-mediated SIRT1/2/3 pathways in vitro , we also used FK866 to investigate in vitro , which are involved in neuronal energy metabolism and mitochondrial functions following neuronal oxidative stress.…”

Mono(ADP-ribosyl)ation Enzymes and NAD+ Metabolism: A Focus on Diseases and Therapeutic Perspectives

“…Previous results have shown the coordinated requirement of NAMPT and NMNAT2 in NAD + synthesis and how their inhibition could lead to derangement of the NAD + pool. There are structural and functional similarities between human NAMPT and nicotinate phosphoribosyltransferase (NAPRT), which uses nicotinic acid (NA) to produce nicotinic acid mononucleotide (NaMN) [ 38 ]. Using FK866 to inhibit NAMPT, A375 cells were treated with nicotinamide, nicotinic acid, nicotinamide riboside, kynurenine and quinolinic acid as precursors of NAD + , showing that each substrate has organelle-specific ability to rescue from NAMPT block [ 38 ].…”

“…There are structural and functional similarities between human NAMPT and nicotinate phosphoribosyltransferase (NAPRT), which uses nicotinic acid (NA) to produce nicotinic acid mononucleotide (NaMN) [ 38 ]. Using FK866 to inhibit NAMPT, A375 cells were treated with nicotinamide, nicotinic acid, nicotinamide riboside, kynurenine and quinolinic acid as precursors of NAD + , showing that each substrate has organelle-specific ability to rescue from NAMPT block [ 38 ]. The authors showed that cytosolic NAD + content decreased, mitochondrial NAD + did not change, so that NAMPT was not affected in the mitochondrial compartment [ 38 ], whereas nicotinamide riboside kinase (NRK) was found active in nuclei and in mitochondria, and NAPRT was predominant in cytosol and mitochondria [ 39 ].…”

“…Using FK866 to inhibit NAMPT, A375 cells were treated with nicotinamide, nicotinic acid, nicotinamide riboside, kynurenine and quinolinic acid as precursors of NAD + , showing that each substrate has organelle-specific ability to rescue from NAMPT block [ 38 ]. The authors showed that cytosolic NAD + content decreased, mitochondrial NAD + did not change, so that NAMPT was not affected in the mitochondrial compartment [ 38 ], whereas nicotinamide riboside kinase (NRK) was found active in nuclei and in mitochondria, and NAPRT was predominant in cytosol and mitochondria [ 39 ]. NAMPT and NAPRT can be secreted extracellularly (eNAMPT, eNAPRT) as cytokines and damage-associated molecular patterns (DAMPs): eNAMPT is known as pre-B cell colony-enhancing factor or visfatin [ 40 ], acting through the activation of Toll-like receptor 4 (TLR4) [ 41 ].…”