Specific ablation of Nampt in adult neural stem cells recapitulates their functional defects during aging

Neural stem/progenitor cell (NSPC) proliferation and self‐renewal, as well as insult‐induced differentiation, decrease markedly with age. The molecular mechanisms responsible for these declines remain unclear. Here, we show that levels of NAD⁺ and nicotinamide phosphoribosyltransferase (Nampt), the rate‐limiting enzyme in mammalian NAD⁺ biosynthesis, decrease with age in the hippocampus. Ablation of Nampt in adult NSPCs reduced their pool and proliferation in vivo. The decrease in the NSPC pool during aging can be rescued by enhancing hippocampal NAD⁺ levels. Nampt is the main source of NSPC NAD⁺ levels and required for G1/S progression of the NSPC cell cycle. Nampt is also critical in oligodendrocytic lineage fate decisions through a mechanism mediated redundantly by Sirt1 and Sirt2. Ablation of Nampt in the adult NSPCs in vivo reduced NSPC‐mediated oligodendrogenesis upon insult. These phenotypes recapitulate defects in NSPCs during aging, giving rise to the possibility that Nampt‐mediated NAD⁺ biosynthesis is a mediator of age‐associated functional declines in NSPCs.     

SIRT2 induces the checkpoint kinase BubR1 to increase lifespan

Mice overexpressing the mitotic checkpoint kinase gene BubR1 live longer, whereas mice hypomorphic for BubR1 (BubR1^H/H) live shorter and show signs of accelerated aging. As wild‐type mice age, BubR1 levels decline in many tissues, a process that is proposed to underlie normal aging and age‐related diseases. Understanding why BubR1 declines with age and how to slow this process is therefore of considerable interest. The sirtuins (SIRT1‐7) are a family of NAD⁺‐dependent deacetylases that can delay age‐related diseases. Here, we show that the loss of BubR1 levels with age is due to a decline in NAD⁺ and the ability of SIRT2 to maintain lysine‐668 of BubR1 in a deacetylated state, which is counteracted by the acetyltransferase CBP. Overexpression of SIRT2 or treatment of mice with the NAD⁺ precursor nicotinamide mononucleotide (NMN) increases BubR1 abundance in vivo. Overexpression of SIRT2 in BubR1^H/H animals increases median lifespan, with a greater effect in male mice. Together, these data indicate that further exploration of the potential of SIRT2 and NAD⁺ to delay diseases of aging in mammals is warranted.     

Dietary restriction involves NAD+‐dependent mechanisms and a shift toward oxidative metabolism

Interventions that slow aging and prevent chronic disease may come from an understanding of how dietary restriction (DR) increases lifespan. Mechanisms proposed to mediate DR longevity include reduced mTOR signaling, activation of the NAD⁺‐dependent deacylases known as sirtuins, and increases in NAD⁺ that derive from higher levels of respiration. Here, we explored these hypotheses in Caenorhabditis elegans using a new liquid feeding protocol. DR lifespan extension depended upon a group of regulators that are involved in stress responses and mTOR signaling, and have been implicated in DR by some other regimens [DAF‐16 (FOXO), SKN‐1 (Nrf1/2/3), PHA‐4 (FOXA), AAK‐2 (AMPK)]. Complete DR lifespan extension required the sirtuin SIR‐2.1 (SIRT1), the involvement of which in DR has been debated. The nicotinamidase PNC‐1, a key NAD⁺ salvage pathway component, was largely required for DR to increase lifespan but not two healthspan indicators: movement and stress resistance. Independently of pnc‐1, DR increased the proportion of respiration that is coupled to ATP production but, surprisingly, reduced overall oxygen consumption. We conclude that stress response and NAD⁺‐dependent mechanisms are each critical for DR lifespan extension, although some healthspan benefits do not require NAD⁺ salvage. Under DR conditions, NAD⁺‐dependent processes may be supported by a DR‐induced shift toward oxidative metabolism rather than an increase in total respiration.     

Characterization and possible redox regulation of the purified calmodulin‐dependent NAD+ kinase from Lycopersicon pimpinellifolium

The soluble and calmodulin (CaM)‐dependent NAD⁺ kinase from Lycopersicon pimpinellifolium was previously shown to be largely inactivated in isolated cells exposed to a short‐term NaCl stress (Delumeau, Morère‐Le Paven, Montrichard, Laval‐Martin (2000) Plant Cell & Environment 23, 329–336). Nevertheless, the activity could be restored by adding a high dithiothreitol concentration to the protein extract, suggesting that the salt stress triggers an oxidation of the enzyme which leads to its inactivation. It was then interesting to investigate the effect of thiol‐modifying reagents and disulphide reductants on the activity of L. pimpinellifolium NAD⁺ kinase. A three‐step purification procedure was then established and allowed isolation of the enzyme which exists under two forms: a monomer and a dimer of a 56 kDa subunit, characterized, respectively, by pIs of 6·8 and 7·1. Isolated NAD⁺ kinase had a high affinity for CaM, half saturation being obtained for 7 ng mL^?1 bovine CaM. The activity of NAD⁺ kinase was strongly inhibited by thiol‐modifying reagents and oxidized glutathione. NAD⁺ kinase was also found to be air‐inactivated, the residual activity being stimulated by disulphide reductants. The most efficient of them is reduced thioredoxin from Escherichia coli which induced a five‐fold increase in activity and restored 80% of the initial activity. These results which can be related to those previously observed in vivo suggest that the activity of the L. pimpinellifolium NAD⁺ kinase, besides its dependence on CaM, is also dependent on the reduction state of the protein which could be regulated by the thioredoxin h/NADP‐thioredoxin reductase system.     

Role of the global regulator Rex in control of NAD+‐regeneration in Clostridioides (Clostridium) difficile

For the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D‐proline reductase (PR), a Stickland reaction that regenerates NAD⁺ from NADH. Many Clostridium spp. use Stickland metabolism (co‐fermentation of pairs of amino acids) to generate ATP and NAD⁺. Synthesis of PR is activated by PrdR, a proline‐responsive regulatory protein. Here we report that PrdR, in the presence of proline, represses other NAD⁺‐generating pathways, such as the glycine reductase and succinate‐acetyl CoA utilization pathways leading to butyrate production, but does so indirectly by affecting the activity of Rex, a global redox‐sensing regulator that responds to the NAD⁺/NADH ratio. Our results indicate that PR activity is the favored mechanism for NAD⁺ regeneration and that both Rex and PrdR influence toxin production. Using the hamster model of C. difficile infection, we revealed the importance of PrdR‐regulated Stickland metabolism in the virulence of C. difficile.     

Augmentation of cellular NAD+ by NQO1 enzymatic action improves age‐related hearing impairment

Age‐related hearing loss (ARHL) is a major neurodegenerative disorder and the leading cause of communication deficit in the elderly population, which remains largely untreated. The development of ARHL is a multifactorial event that includes both intrinsic and extrinsic factors. Recent studies suggest that NAD⁺/NADH ratio may play a critical role in cellular senescence by regulating sirtuins, PARP‐1, and PGC‐1α. Nonetheless, the beneficial effect of direct modulation of cellular NAD⁺ levels on aging and age‐related diseases has not been studied, and the underlying mechanisms remain obscure. Herein, we investigated the effect of β‐lapachone (β‐lap), a known plant‐derived metabolite that modulates cellular NAD⁺ by conversion of NADH to NAD⁺ via the enzymatic action of NADH: quinone oxidoreductase 1 (NQO1) on ARHL in C57BL/6 mice. We elucidated that the reduction of cellular NAD⁺ during the aging process was an important contributor for ARHL; it facilitated oxidative stress and pro‐inflammatory responses in the cochlear tissue through regulating sirtuins that alter various signaling pathways, such as NF‐κB, p53, and IDH2. However, augmentation of NAD⁺ by β‐lap effectively prevented ARHL and accompanying deleterious effects through reducing inflammation and oxidative stress, sustaining mitochondrial function, and promoting mitochondrial biogenesis in rodents. These results suggest that direct regulation of cellular NAD⁺ levels by pharmacological agents may be a tangible therapeutic option for treating various age‐related diseases, including ARHL.     

Porcine CD38 exhibits prominent secondary NAD+ cyclase activity

Cyclic ADP‐ribose (cADPR) mobilizes intracellular Ca²⁺ stores and activates Ca²⁺ influx to regulate a wide range of physiological processes. It is one of the products produced from the catalysis of NAD⁺ by the multifunctional CD38/ADP‐ribosyl cyclase superfamily. After elimination of the nicotinamide ring by the enzyme, the reaction intermediate of NAD⁺ can either be hydrolyzed to form linear ADPR or cyclized to form cADPR. We have previously shown that human CD38 exhibits a higher preference towards the hydrolysis of NAD⁺ to form linear ADPR while Aplysia ADP‐ribosyl cyclase prefers cyclizing NAD⁺ to form cADPR. In this study, we characterized the enzymatic properties of porcine CD38 and revealed that it has a prominent secondary NAD⁺ cyclase activity producing cADPR. We also determined the X‐ray crystallographic structures of porcine CD38 and were able to observe conformational flexibility at the base of the active site of the enzyme which allow the NAD⁺ reaction intermediate to adopt conformations resulting in both hydrolysis and cyclization forming linear ADPR and cADPR respectively.     

Heterogeneity of active sites in recombinant betaine aldehyde dehydrogenase is modulated by potassium

Betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine using NAD⁺ as a coenzyme. Porcine kidney BADH (pkBADH) follows a bi‐bi ordered mechanism in which NAD⁺ binds to the enzyme before the aldehyde. Previous studies showed that NAD⁺ induces complex and unusual conformational changes on pkBADH and that potassium is required to maintain its quaternary structure. The aim of this work was to analyze the structural changes in pkBADH caused by NAD⁺ binding and the role played by potassium in those changes. The pkBADH cDNA was cloned and overexpressed in Escherichia coli, and the protein was purified by affinity chromatography using a chitin matrix. The pkBADH/NAD⁺ interaction was analyzed by circular dichroism (CD) and by isothermal titration calorimetry (ITC) by titrating the enzyme with NAD⁺. The cDNA has an open reading frame of 1485 bp and encodes a protein of 494 amino acids, with a predicted molecular mass of 53.9 kDa. CD data showed that the binding of NAD⁺ to the enzyme caused changes in its secondary structure, whereas the presence of K⁺ helps maintain its α‐helix content. K⁺ increased the thermal stability of the pkBADH‐NAD⁺ complex by 5.3°C. ITC data showed that NAD⁺ binding occurs with different association constants for each active site between 37.5 and 8.6 μM. All the results support previous data in which the enzyme incubation with NAD⁺ provoked changes in reactivity, which is an indication of slow conformational rearrangements of the active site.     

Structure and activity of the NAD(P)+‐dependent succinate semialdehyde dehydrogenase YneI from Salmonella typhimurium

Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)⁺‐dependent succinate semialdehyde dehydrogenase YneI from S. typhimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD⁺ and NADP⁺ as cofactors, although affinity to NAD⁺ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD⁺ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD⁺ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site‐directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD⁺. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. © 2012 Wiley Periodicals, Inc.     

Steric hindrance controls pyridine nucleotide specificity of a flavin‐dependent NADH:quinone oxidoreductase

The crystal structure of the NADH:quinone oxidoreductase PA1024 has been solved in complex with NAD⁺ to 2.2 Å resolution. The nicotinamide C4 is 3.6 Å from the FMN N5 atom, with a suitable orientation for facile hydride transfer. NAD⁺ binds in a folded conformation at the interface of the TIM‐barrel domain and the extended domain of the enzyme. Comparison of the enzyme‐NAD⁺ structure with that of the ligand‐free enzyme revealed a different conformation of a short loop (75–86) that is part of the NAD⁺‐binding pocket. P78, P82, and P84 provide internal rigidity to the loop, whereas Q80 serves as an active site latch that secures the NAD⁺ within the binding pocket. An interrupted helix consisting of two α‐helices connected by a small three‐residue loop binds the pyrophosphate moiety of NAD⁺. The adenine moiety of NAD⁺ appears to π–π stack with Y261. Steric constraints between the adenosine ribose of NAD⁺, P78, and Q80, control the strict specificity of the enzyme for NADH. Charged residues do not play a role in the specificity of PA1024 for the NADH substrate.     

A single‐point mutation enables lactate dehydrogenase from Bacillus subtilis to utilize NAD+ and NADP+ as cofactor

The NAD⁺‐dependent lactate dehydrogenase from Bacillus subtilis (BsLDH) catalyzes the enantioselective reduction of pyruvate to lactate. BsLDH is highly specific to NAD⁺ and exhibits only a low activity with NADP⁺ as cofactor. Based on the high activity and good stability of LDHs, these enzymes have been frequently used for the regeneration of NAD⁺. While an application in the regeneration of NADP⁺ is not sufficient due to the cofactor preference of the BsLDH. In addition, NADP⁺‐dependent LDHs have not yet been found in nature. Therefore, a structure‐based approach was performed to predict amino acids involved in the cofactor specificity. Methods of site‐saturation mutagenesis were applied to vary these amino acids, with the aim to alter the cofactor specificity of the BsLDH. Five constructed libraries were screened for improved NADP⁺ acceptance. The mutant V39R was identified to have increased activity with NADP⁺ relative to the wild type. V39R was purified and biochemically characterized. V39R showed excellent kinetic properties with NADP(H) and NAD(H), for instance the maximal specific activity with NADPH was enhanced 100‐fold to 90.8 U/mg. Furthermore, a 249‐fold increased catalytic efficiency was observed. Surprisingly, the activity with NADH was also significantly improved. Overall, we were able to successfully apply V39R in the regeneration of NADP⁺ in an enzyme‐coupled approach combined with the NADP⁺‐dependent alcohol dehydrogenase from Lactobacillus kefir. We demonstrate for the first time an application of an LDH in the regeneration of NADP⁺.     

Crystallization of NAD+ synthetase from Bacillus subtilis

NAD⁺-synthetase is a ubiquitous enzyme catalyzing the last step in the biosynthesis of NAD⁺. Mutants of NAD⁺ synthetase with impaired cellular functions have been isolated, indicating a key role for this enzyme in cellular metabolism. Crystals of the enzyme from Bacillus subtilis suitable for x-ray crystallographic investigation have been grown from polyethylene glycol solutions. Investigation on the structural organization of NAD⁺ synthetase, an enzyme fundamental for NAD⁺ biosynthesis, and belonging to the recently characterized amidotransferase enzymatic family, will provide more insight into the catalytic mechanism of deamido-NAD⁺ → NAD⁺ conversion, a biosynthetic process that is a potential target for the development of antibiotic compounds against Bacillus sp. and related bacteria. © 1996 Wiley-Liss, Inc.     

Oral nicotinamide riboside raises NAD+ and lowers biomarkers of neurodegenerative pathology in plasma extracellular vesicles enriched for neuronal origin

Declining nicotinamide adenine dinucleotide (NAD⁺) concentration in the brain during aging contributes to metabolic and cellular dysfunction and is implicated in the pathogenesis of aging-associated neurological disorders. Experimental therapies aimed at boosting brain NAD⁺ levels normalize several neurodegenerative phenotypes in animal models, motivating their clinical translation. Dietary intake of NAD⁺ precursors, such as nicotinamide riboside (NR), is a safe and effective avenue for augmenting NAD⁺ levels in peripheral tissues in humans, yet evidence supporting their ability to raise NAD⁺ levels in the brain or engage neurodegenerative disease pathways is lacking. Here, we studied biomarkers in plasma extracellular vesicles enriched for neuronal origin (NEVs) from 22 healthy older adults who participated in a randomized, placebo-controlled crossover trial (NCT02921659) of oral NR supplementation (500 mg, 2x /day, 6 weeks). We demonstrate that oral NR supplementation increases NAD⁺ levels in NEVs and decreases NEV levels of Aβ42, pJNK, and pERK1/2 (kinases involved in insulin resistance and neuroinflammatory pathways). In addition, changes in NAD(H) correlated with changes in canonical insulin–Akt signaling proteins and changes in pERK1/2 and pJNK. These findings support the ability of orally administered NR to augment neuronal NAD⁺ levels and modify biomarkers related to neurodegenerative pathology in humans. Furthermore, NEVs offer a new blood-based window into monitoring the physiologic response of NR in the brain.     

Fingerstick blood assay maps real-world NAD+ disparity across gender and age

Nicotinamide adenine dinucleotide (NAD⁺) level has been associated with various age-related diseases and its pharmacological modulation emerges as a potential approach for aging intervention. But human NAD⁺ landscape exhibits large heterogeneity. The lack of rapid, low-cost assays limits the establishment of whole-blood NAD⁺ baseline and the development of personalized therapies, especially for those with poor responses towards conventional NAD⁺ supplementations. Here, we developed an automated NAD⁺ analyzer for the rapid measurement of NAD⁺ with 5 μL of capillary blood using recombinant bioluminescent sensor protein and automated optical reader. The minimal invasiveness of the assay allowed a frequent and decentralized mapping of real-world NAD⁺ dynamics. We showed that aerobic sport and NMN supplementation increased whole-blood NAD⁺ and that male on average has higher NAD⁺ than female before the age of 50. We further revealed the long-term stability of human NAD⁺ baseline over 100 days and identified major real-world NAD⁺-modulating behaviors.     

Dual treatment with kynurenine pathway inhibitors and NAD+ precursors synergistically extends life span in Drosophila

Tryptophan catabolism is highly conserved and generates important bioactive metabolites, including kynurenines, and in some animals, NAD⁺. Aging and inflammation are associated with increased levels of kynurenine pathway (KP) metabolites and depleted NAD⁺, factors which are implicated as contributors to frailty and morbidity. Contrastingly, KP suppression and NAD⁺ supplementation are associated with increased life span in some animals. Here, we used DGRP_229 Drosophila to elucidate the effects of KP elevation, KP suppression, and NAD⁺ supplementation on physical performance and survivorship. Flies were chronically fed kynurenines, KP inhibitors, NAD⁺ precursors, or a combination of KP inhibitors with NAD⁺ precursors. Flies with elevated kynurenines had reduced climbing speed, endurance, and life span. Treatment with a combination of KP inhibitors and NAD⁺ precursors preserved physical function and synergistically increased maximum life span. We conclude that KP flux can regulate health span and life span in Drosophila and that targeting KP and NAD⁺ metabolism can synergistically increase life span.     

Metabolism of NAD+ in Nuclei of Saccharomyces cerevisiae during Stimulation of Its Biosynthesis by Nicotinamide

The activities of nuclear enzymes involved in NAD⁺ metabolism in Saccharomyces cerevisiae strain 913a-1 and its mutant 110 previously selected as an NAD⁺ producer were investigated. The presence of extracellular nicotinamide increased the total NAD⁺ pool in the cells and increased [³H]nicotinic acid incorporation; however, NAD⁺ concentration in isolated nuclei decreased slightly. The stimulating effect of nicotinamide on intracellular synthesis of NAD⁺ correlated with increases in ADP-ribosyl transferase, NAD⁺-pyrophosphorylase, and NAD⁺ ase activities.     

The Streptococcus pyogenes NAD+ glycohydrolase modulates epithelial cell PARylation and HMGB1 release

Streptococcus pyogenes uses the cytolysin streptolysin O (SLO) to translocate an enzyme, the S. pyogenes NAD⁺ glycohydrolase (SPN), into the host cell cytosol. However, the function of SPN in this compartment is not known. As a complication, many S. pyogenes strains express a SPN variant lacking NAD⁺ glycohydrolase (NADase) activity. Here, we show that SPN modifies several SLO‐ and NAD⁺‐dependent host cell responses in patterns that correlate with NADase activity. SLO pore formation results in hyperactivation of the cellular enzyme poly‐ADP‐ribose polymerase‐1 (PARP‐1) and production of polymers of poly‐ADP‐ribose (PAR). However, while SPN NADase activity moderates PARP‐1 activation and blocks accumulation of PAR, these processes continued unabated in the presence of NADase‐inactive SPN. Temporal analyses revealed that while PAR production is initially independent of NADase activity, PAR rapidly disappears in the presence of NADase‐active SPN, host cell ATP is depleted and the pro‐inflammatory mediator high‐mobility group box‐1 (HMGB1) protein is released from the nucleus by a PARP‐1‐dependent mechanism. In contrast, HMGB1 is not released in response to NADase‐inactive SPN and instead the cells release elevated levels of interleukin‐8 and tumour necrosis factor‐α. Thus, SPN and SLO combine to induce cellular responses subsequently influenced by the presence or absence of NADase activity.     

Biochemical characterization of isocitrate dehydrogenase from Methylococcus capsulatus reveals a unique NAD+-dependent homotetrameric enzyme

The gene encoding isocitrate dehydrogenase (IDH) of Methylococcus capsulatus (McIDH) was cloned and overexpressed in Escherichia coli. The purified enzyme was NAD⁺-dependent with a thermal optimum for activity at 55–60°C and an apparent midpoint melting temperature (T _m) of 70°C. Analytical ultracentrifugation (AUC) revealed a homotetrameric state, and McIDH thus represents the first homotetrameric NAD⁺-dependent IDH that has been characterized. Based on a structural alignment of McIDH and homotetrameric homoisocitrate dehydrogenase (HDH) from Thermus thermophilus (TtHDH), we identified the clasp-like domain of McIDH as a likely site for tetramerization. McIDH showed moreover, higher sequence identity (48%) to TtHDH than to previously characterized IDHs. Putative NAD⁺-IDHs with high sequence identity (48–57%) to McIDH were however identified in a variety of bacteria showing that NAD⁺-dependent IDHs are indeed widespread within the domain, Bacteria. Phylogenetic analysis including these new sequences revealed a close relationship with eukaryal allosterically regulated NAD⁺-IDH and the subfamily III of IDH was redefined to include bacterial NAD⁺- and NADP⁺-dependent IDHs. This apparent relationship suggests that the mitochondrial genes encoding NAD⁺-IDH are derived from the McIDH-like IDHs.