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Nicotinamide adenine dinucleotide (NAD.sup.+) is an essential co-factor for cellular metabolism and serves as a substrate in enzymatic processes. NAD.sup.+ is produced by de novo synthesis or salvage pathways in nearly all bacterial species. Haemophilus influenzae lacks the capacity for de novo synthesis, so it is dependent on import of NAD.sup.+ from the external environment or salvage biosynthetic pathways for recycling of NAD.sup.+ precursors and breakdown products. However, the actual sources of NAD.sup.+ utilized by H. influenzae in the respiratory tract are not well defined. In this study, we found that a variety of bacteria, including species found in the upper airway of humans, released NAD.sup.+ that was readily detectable in extracellular culture fluid, and which supported growth of H. influenzae in vitro. By contrast, certain strains of Streptococcus pyogenes (group A streptococcus or GAS) inhibited growth of H. influenzae in vitro by secreting NAD.sup.+ -glycohydrolase (NADase), which degraded extracellular NAD.sup.+ . Conversely, GAS strains that lacked enzymatically active NADase released extracellular NAD.sup.+, which could support H. influenzae growth. Our results suggest that many bacterial species, including normal flora of the upper airway, release NAD.sup.+ into the environment. GAS is distinctive in its ability to both release and degrade NAD.sup.+ . Thus, colonization of the airway with H. influenzae may be promoted or restricted by co-colonization with GAS in a strain-specific manner that depends, respectively, on release of NAD.sup.+ or secretion of active NADase. We suggest that, in addition to its role as a cytotoxin for host cells, NADase may serve a separate function by restricting growth of H. influenzae in the human respiratory tract.

Mitochondrial disorders represent a large collection of rare syndromes that are difficult to manage both because we do not fully understand biochemical pathogenesis and because we currently lack facile markers of severity. The m.3243A>G variant is the most common heteroplasmic mitochondrial DNA mutation and underlies a spectrum of diseases, notably mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS). To identify robust circulating markers of m.3243A>G disease, we first performed discovery proteomics, targeted metabolomics, and untargeted metabolomics on plasma from a deeply phenotyped cohort (102 patients, 32 controls). In a validation phase, we measured concentrations of prioritized metabolites in an independent cohort using distinct methods. We validated 20 analytes (1 protein, 19 metabolites) that distinguish patients with MELAS from controls. The collection includes classic (lactate, alanine) and more recently identified (GDF-15, α-hydroxybutyrate) mitochondrial markers. By mining untargeted mass-spectra we uncovered 3 less well-studied metabolite families: N-lactoyl-amino acids, β-hydroxy acylcarnitines, and β-hydroxy fatty acids. Many of these 20 analytes correlate strongly with established measures of severity, including Karnofsky status, and mechanistically, nearly all markers are attributable to an elevated NADH/NAD+ ratio, or NADH-reductive stress. Our work defines a panel of organelle function tests related to NADH-reductive stress that should enable classification and monitoring of mitochondrial disease.

NAD is an essential metabolite that exists in NAD ⁺ or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD ⁺/NADH redox state and modulating cellular signaling processes through the activity of the NAD ⁺-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD ⁺ and NADH contents and the NAD ⁺/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD ⁺, total NAD contents, and NAD ⁺/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ. Significance Decline in NAD ⁺ availability and abnormal NAD ⁺/NADH redox state are tightly linked to age-related metabolic diseases and neurodegenerative disorders. To better understand the roles of NAD metabolism and redox state in health and disease, it is important to assess the intracellular NAD and redox state in situ. We report herein the first in vivo NAD assay, to our knowledge, that is capable of noninvasively and simultaneously measuring intracellular NAD ⁺ and NADH concentrations and NAD ⁺/NADH ratio in the human brain and detecting the age-dependent changes in NAD contents and redox state associated with the normal aging. This method can potentially be applied to study various metabolic and neurodegenerative disorders by monitoring the NAD and redox state changes associated with disease progression or treatment in human patients.

Key Points NAD is an important redox factor and substrate in various signalling processes, in which it is irreversibly degraded to form molecules that are of key relevance to cellular homeostasis. Both NAD + -dependent metabolic and signalling pathways are altered in cancer cells, providing a number of potential drug targets. Permanent synthesis of NAD is essential to fuel bioenergetic processes and maintain balanced cell regulation. NAD + is synthesized from vitamin B 3 (niacin, including both nicotinamide and nicotinic acid) and the corresponding nucleosides. However, the predominant source to maintain NAD levels is nicotinamide (Nam), which arises endogenously from NAD + -dependent signalling processes. Therefore, nicotinamide phosphoribosyltransferase (NamPRT) is of outstanding importance, as it is the only human enzyme that salvages Nam into NAD + synthesis. NamPRT inhibitors are currently under scrutiny to evaluate their potential in cancer therapy based on NAD + depletion. Likewise, inhibitors of nicotinamide mononucleotide adenylyltransferases (NMNATs) have the potential to affect NAD levels, as these enzymes are required in all pathways of NAD + generation. Moreover, the expression of the three human isoforms is tissue- and cell compartment-specific, suggesting the possibility of more specific therapeutic approaches. However, so far, specific and potent inhibitors are not available. Several NAD-dependent signalling pathways are involved in the control of cell cycle progression, transcriptional regulation and DNA repair and have therefore been identified as promising targets in cancer therapy. The NAD + -dependent protein deacetylases (Sirtuins) SIRT1, SIRT3, SIRT6 and SIRT7 are also now of interest in the development of new cancer therapies. Inhibitors of polyADP ribose polymerases (PARPs) have a demonstrated potential in cancer therapy and have recently reached the clinical arena. Major current challenges in their use are selectivity towards specific PARP isoforms, potential impairment of DNA repair in healthy tissues and development of drug resistance. NAD is a vital molecule in all organisms and is a key component of both energy and signal transduction — processes that undergo crucial changes in cancer cells. NAD + -dependent signalling reactions involve the degradation of the molecule, so permanent nucleotide resynthesis through different biosynthetic pathways is crucial for incessant cancer cell proliferation. Is targeting of NAD metabolism a new therapeutic strategy for cancer treatment? NAD is a vital molecule in all organisms. It is a key component of both energy and signal transduction — processes that undergo crucial changes in cancer cells. NAD + -dependent signalling pathways are many and varied, and they regulate fundamental events such as transcription, DNA repair, cell cycle progression, apoptosis and metabolism. Many of these processes have been linked to cancer development. Given that NAD + -dependent signalling reactions involve the degradation of the molecule, permanent nucleotide resynthesis through different biosynthetic pathways is crucial for incessant cancer cell proliferation. This necessity supports the targeting of NAD metabolism as a new therapeutic concept for cancer treatment.

• The Pseudomonas syringae DC3000 type III effector HopAM1 suppresses plant immunity and contains a Toll/interleukin-1 receptor (TIR) domain homologous to immunity-related TIR domains of plant nucleotide-binding leucine-rich repeat receptors that hydrolyze nicotinamide adenine dinucleotide (NAD⁺) and activate immunity. In vitro and in vivo assays were conducted to determine if HopAM1 hydrolyzes NAD⁺ and if the activity is essential for HopAM1’s suppression of plant immunity and contribution to virulence. • HPLC and LC-MS were utilized to analyze metabolites produced from NAD⁺ by HopAM1 in vitro and in both yeast and plants. Agrobacterium-mediated transient expression and in planta inoculation assays were performed to determine HopAM1’s intrinsic enzymatic activity and virulence contribution. • HopAM1 is catalytically active and hydrolyzes NAD⁺ to produce nicotinamide and a novel cADPR variant (v2-cADPR). Expression of HopAM1 triggers cell death in yeast and plants dependent on the putative catalytic residue glutamic acid 191 (E191) within the TIR domain. Furthermore, HopAM1’s E191 residue is required to suppress both pattern-triggered immunity and effector-triggered immunity and promote P. syringae virulence. • HopAM1 manipulates endogenous NAD⁺ to produce v2-cADPR and promote pathogenesis. This work suggests that HopAM1’s TIR domain possesses different catalytic specificity than other TIR domain-containing NAD⁺ hydrolases and that pathogens exploit this activity to sabotage NAD⁺ metabolism for immune suppression and virulence.

PGC1α protects against kidney injury by upregulating enzymes that enhance nicotinamide adenine dinucleotide (NAD) and driving local accumulation of the fatty acid breakdown product β-hydroxybutyrate, which leads to increased production of the renoprotective prostaglandin E 2 . Kidney protection by PGC1α Samir Parikh and colleagues show that the mitochondrial biogenesis regulator PGC1α protects against kidney injury by regulating NAD biosynthesis. In a mouse model, PGC1α upregulates NAMPT, an enzyme required form for NAD + biosynthesis, and drives local accumulation of the fatty acid breakdown product β-hydroxybutyrate. This, in turn, leads to increased production of the renoprotective prostaglandin PGE 2 . The authors further show that treatment with the NAD precursor nicotinamide (NAM) can reverse established ischaemic kidney injury. The energetic burden of continuously concentrating solutes against gradients along the tubule may render the kidney especially vulnerable to ischaemia. Acute kidney injury (AKI) affects 3% of all hospitalized patients 1 , 2 . Here we show that the mitochondrial biogenesis regulator, PGC1α 3 , 4 , is a pivotal determinant of renal recovery from injury by regulating nicotinamide adenine dinucleotide (NAD) biosynthesis. Following renal ischaemia, Pgc1α −/− (also known as Ppargc1a −/− ) mice develop local deficiency of the NAD precursor niacinamide (NAM, also known as nicotinamide), marked fat accumulation, and failure to re-establish normal function. Notably, exogenous NAM improves local NAD levels, fat accumulation, and renal function in post-ischaemic Pgc1α −/− mice. Inducible tubular transgenic mice (iNephPGC1α) recapitulate the effects of NAM supplementation, including more local NAD and less fat accumulation with better renal function after ischaemia. PGC1α coordinately upregulates the enzymes that synthesize NAD de novo from amino acids whereas PGC1α deficiency or AKI attenuates the de novo pathway. NAM enhances NAD via the enzyme NAMPT and augments production of the fat breakdown product β-hydroxybutyrate, leading to increased production of prostaglandin PGE 2 (ref. 5 ), a secreted autacoid that maintains renal function. NAM treatment reverses established ischaemic AKI and also prevented AKI in an unrelated toxic model. Inhibition of β-hydroxybutyrate signalling or prostaglandin production similarly abolishes PGC1α-dependent renoprotection. Given the importance of mitochondrial health in ageing and the function of metabolically active organs, the results implicate NAM and NAD as key effectors for achieving PGC1α-dependent stress resistance.

Pathological degeneration of axons disrupts neural circuits and represents one of the hallmarks of neurodegeneration.sup.1-4. Sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) is a central regulator of this neurodegenerative process.sup.5-8, and its Toll/interleukin-1 receptor (TIR) domain exerts its pro-neurodegenerative action through NADase activity.sup.9,10. However, the mechanisms by which the activation of SARM1 is stringently controlled are unclear. Here we report the cryo-electron microscopy structures of full-length SARM1 proteins. We show that NAD.sup.+ is an unexpected ligand of the armadillo/heat repeat motifs (ARM) domain of SARM1. This binding of NAD.sup.+ to the ARM domain facilitated the inhibition of the TIR-domain NADase through the domain interface. Disruption of the NAD.sup.+-binding site or the ARM-TIR interaction caused constitutive activation of SARM1 and thereby led to axonal degeneration. These findings suggest that NAD.sup.+ mediates self-inhibition of this central pro-neurodegenerative protein.

Biological clocks allow organisms to anticipate cycles of feeding, activity, and rest so that metabolic enzymes in mitochondria are ready when needed. Peek et al. ( 10.1126/science.1243417 , published online 19 September; see the Perspective by Rey and Reddy ) describe a mechanism by which the biochemical elements of the circadian clock are linked to such control of mitochondrial metabolism. The clock controls rhythmic transcription of the gene encoding the rate-limiting enzyme required for synthesis of nicotinamide adenine dinucleotide (NAD + ). The concentration of NAD + in mitochondria determines the activity of the deacetylase SIRT3, which then controls acetylation and activity of key metabolic enzymes. NAD+ also influences clock function, and thus appears to be a versatile point at which regulation of oxidative metabolism is coordinated with the daily cycles of energy consumption. The coenzyme nicotinamide adenine dinucleotide mechanistically links the circadian clock to control of energy production by mitochondria. [Also see Perspective by Rey and Reddy ] Circadian clocks are self-sustained cellular oscillators that synchronize oxidative and reductive cycles in anticipation of the solar cycle. We found that the clock transcription feedback loop produces cycles of nicotinamide adenine dinucleotide (NAD + ) biosynthesis, adenosine triphosphate production, and mitochondrial respiration through modulation of mitochondrial protein acetylation to synchronize oxidative metabolic pathways with the 24-hour fasting and feeding cycle. Circadian control of the activity of the NAD + -dependent deacetylase sirtuin 3 (SIRT3) generated rhythms in the acetylation and activity of oxidative enzymes and respiration in isolated mitochondria, and NAD + supplementation restored protein deacetylation and enhanced oxygen consumption in circadian mutant mice. Thus, circadian control of NAD + bioavailability modulates mitochondrial oxidative function and organismal metabolism across the daily cycles of fasting and feeding.

The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) protects against redox stress by providing reducing equivalents to antioxidants such as glutathione and thioredoxin. NADPH levels decline with aging in several tissues, but whether this is a major driving force for the aging process has not been well established. Global or neural overexpression of several cytoplasmic enzymes that synthesize NADPH have been shown to extend lifespan in model organisms such as Drosophila suggesting a positive relationship between cytoplasmic NADPH levels and longevity. Mitochondrial NADPH plays an important role in the protection against redox stress and cell death and mitochondrial NADPH-utilizing thioredoxin reductase 2 levels correlate with species longevity in cells from rodents and primates. Mitochondrial NADPH shuttles allow for some NADPH flux between the cytoplasm and mitochondria. Since a decline of nicotinamide adenine dinucleotide (NAD+) is linked with aging and because NADP+ is exclusively synthesized from NAD+ by cytoplasmic and mitochondrial NAD+ kinases, a decline in the cytoplasmic or mitochondrial NADPH pool may also contribute to the aging process. Therefore pro-longevity therapies should aim to maintain the levels of both NAD+ and NADPH in aging tissues.

Excessive poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation kills cells via a cell-death process designated “parthanatos” in which PAR induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor to initiate chromatinolysis and cell death. Accompanying the formation of PAR are the reduction of cellular NAD ⁺ and energetic collapse, which have been thought to be caused by the consumption of cellular NAD ⁺ by PARP-1. Here we show that the bioenergetic collapse following PARP-1 activation is not dependent on NAD ⁺ depletion. Instead PARP-1 activation initiates glycolytic defects via PAR-dependent inhibition of hexokinase, which precedes the NAD ⁺ depletion in N -methyl- N -nitroso- N -nitroguanidine (MNNG)-treated cortical neurons. Mitochondrial defects are observed shortly after PARP-1 activation and are mediated largely through defective glycolysis, because supplementation of the mitochondrial substrates pyruvate and glutamine reverse the PARP-1–mediated mitochondrial dysfunction. Depleting neurons of NAD ⁺ with FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, does not alter glycolysis or mitochondrial function. Hexokinase, the first regulatory enzyme to initiate glycolysis by converting glucose to glucose-6-phosphate, contains a strong PAR-binding motif. PAR binds to hexokinase and inhibits hexokinase activity in MNNG-treated cortical neurons. Preventing PAR formation with PAR glycohydrolase prevents the PAR-dependent inhibition of hexokinase. These results indicate that bioenergetic collapse induced by overactivation of PARP-1 is caused by PAR-dependent inhibition of glycolysis through inhibition of hexokinase.