Evolving concepts in NAD+ metabolism

Abstract

NAD(H) and NADP(H) have traditionally been viewed as co-factors (or co-enzymes) involved in a myriad of oxidation-reduction reactions including the electron transport in the mitochondria. However, NAD pathway metabolites have many other important functions, including roles in signaling pathways, post-translational modifications, epigenetic changes, and regulation of RNA stability and function via NAD-capping of RNA. Non-oxidative reactions ultimately lead to the net catabolism of these nucleotides, indicating that NAD metabolism is an extremely dynamic process. In fact, recent studies have clearly demonstrated that NAD has a half-life in the order of minutes in some tissues. Several evolving concepts on the metabolism, transport, and roles of these NAD pathway metabolites in disease states such as cancer, neurodegeneration, and aging have emerged in just the last few years. In this perspective, we discuss key recent discoveries and changing concepts in NAD metabolism and biology that are reshaping the field. In addition, we will pose some open questions in NAD biology, including why NAD metabolism is so fast and dynamic in some tissues, how NAD and its precursors are transported to cells and organelles, and how NAD metabolism is integrated with inflammation and senescence. Resolving these questions will lead to significant advancements in the field.

Keywords: NAD pathway metabolites; NAD(+); aging; disease; humans; mitochondria; transport; vitamin B3.

Figure 1.. An overview of the NAD metabolome and metabolic pathways.

This figure is an integrative view of the networks related to NAD metabolism, including synthesis (de novo, Preiss-Handler, and salvage pathways), degradation, excretion, and repair pathways. The structure of NAD, NMN, and NR is shown (upper right). The structure of S-NADHX is shown (repair pathway - green) and the hydroxy group located at position 6 of the nicotinyl ring is highlighted. In the R-configuration the hydroxy group of the nicotinyl ring is located at position 2 (not shown). Cyclic forms of both S- and R-NAD(P)HX (not shown) are also toxic metabolites. 2-PY= N-methyl-2-pyridone-5-carboxamide; 4PY= N-methyl-4-pyridone-3-carboxamide; ACMS= 2-Amino-3-carboxymuconic acid semialdehyde; AFMID= Arylformamidase; AOX= aldehyde oxidase; ARTs= ADP-ribosyl-transferases; CYP2E1= Cytochrome P450 2E1; GAPDH= Glyceraldehyde-3-phosphate dehydrogenase; HAAO= 3-hydroxyanthranilate 3,4-dioxygenase; IDO= indoleamine 2,3-dioxygenase; KMO= kynurenine 3-monooxygenase; KYNU= kynureninase; M-NAM= methyl nicotinamide; NA= nicotinic acid; NaAD= nicotinic acid adenine dinucleotide; NAD= nicotinamide adenine dinucleotide; NAM= nicotinamide; NaMN= nicotinic acid mononucleotide; NAMPT= nicotinamide phosphoribosyltransferase; NaPRT1= nicotinic acid phosphoribosyltransferase 1; NAXD= NAD(P)HX Dehydratase; NAXE= NAD(P)H-hydrate epimerase; NMN= nicotinamide mononucleotide; NMNAT1= nicotinamide/nicotinic acid mononucleotide adenylyltransferase 1; NNMT= nicotinamide N-methyltranferase; NR= nicotinamide riboside; PARP= poly (ADP-ribose) polymerase; PRPP= phosphoribosyl pyrophosphate; QPRT= quinolinate phosphoribosyl transferase; SARM1= short for sterile alpha and Toll/interleukin receptor (TIR) motif-containing protein 1; TPO= tryptophan 2,3-dioxygenase;

Figure 2.. Potential mechanisms involved in Sarm1 activation.

SARM1 homo-octamer assumes a packed inactive conformation which is stabilized by NAD binding to allosteric sites located in the ARM domains. NAD decline leads to the disassembly of SARM1’s peripheral ARM ring, allowing the formation of TIR dimers, which are responsible for SARM1 NADase activity. It has been postulated that NMN may promote NAD displacement from SARM1 inhibitory allosteric sites, resulting in SARM1 NADase activation. A) In normal physiological context NAD is bound to allosteric sites in SARM1 oligomers, far from its catalytic sites. CD38 present in the cellular plasma membrane would lead to the degradation of extracellular NMN, preventing the increase of intracellular NMN levels. B) In a condition where extracellular CD38 activity is blocked, the consequent increase in intracellular NMN levels could lead to the displacement of NAD from SARM1’s allosteric inhibitory sites leading to SARM1 activation. C) Increased expression/activity of intracellular NADases such as PARPs or intracellular CD38 (iCD38) can lead to a decrease in intracellular NAD levels and consequent activation of SARM1 activity. Further decrease in NAD levels could lead to metabolic collapse and cell death. SAM= sterile alpha motif; TIR= toll/interleukin-1 receptor (TIR) homology domain; ARM= armadillo repeat domain.

Figure 3.. Nicotinamide nucleotides transport into cells and organelles.

It is believed that extracellular NAD and NMN are degraded into ADPR and NAM or NR by exonucleotidases such as CD38 or CD73, respectively, before being transported into the cells. The transport of NR is mediated by equilibrative nucleoside transporters (ENT). Whether NMN can be taken up into cells through the transporter Slc12a8 or any other transporter is still under debate. NAM is known to enter into the cells, but the protein that mediates its transport is still unknown. The CD38 product ADPR can be used as substrate by other nucleotidases such as the nucleotide pyrophosphatase CD203a, which generates AMP. AMP is the main substrate of CD73, which generates adenosine, an immunosuppressive metabolite. Slc25a51, a member of the solute carrier transporter family, has recently been identified as the NAD transporter in the mitochondrial inner membrane. NAM= nicotinamide; NMN= nicotinamide mononucleotide; NR= nicotinamide riboside; ADPR= adenosine diphosphate ribose; AMP= adenosine mononucleotide; ADO= adenosine.

Figure 4.. NAD, inflammation, and senescence: double edged sword?

In oncogene-induced senescence (OIS), high mobility group A protein (HMGA) upregulates the expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate liming enzyme for the NAD salvage pathway. The HMGA-mediated NAMPT expression induces NAD synthesis, leading to higher NAD⁺/NADH ratios. This metabolic change can induce a higher pro-inflammatory SASP, which accelerates cancer progression in surrounding cells. During aging-related senescence (ARS), the chronic SASP causes a low-level pro-inflammatory environment, leading to inflammaging and upregulation of CD38 expression in M1 macrophages and other immune cells. Induction of CD38 expression in turn causes a reduction in NAD and NMN levels in surrounding tissues. Pro-inflammatory immune cells also have reduced NAD levels, but this reduction depends on other enzymes in addition to CD38, like PARP1. NAD boosting-therapies are attractive therapeutical approaches, but it is crucial to consider them carefully. NAD= nicotinamide adenine dinucleotide; NMN= nicotinamide mononucleotide; SASP= senescence associated secretory phenotype; OIS= oncogene-induced senescence