Free flavin adenine dinucleotide (FAD) is metabolized to flavin mononucleotide (FMN) and adenine monophosphate (AMP) by hydrolases and to 4',5'-cyclic phosphoriboflavin (cFMN) and AMP by the triose kinase FMN cyclase (TKFC). Yet, the lack of analytical standards for cFMN might have resulted in the incidence of cFMN in biological specimens being underreported. To address this shortcoming, cFMN was synthesized from either FMN or FAD. The optimization of the FAD to cFMN reaction conditions revealed that an equimolar ratio of ZnSO4 and FAD yielded pure cFMN upon the precipitation of AMP-Zn salts. cFMN is stable to aqueous acidic and basic conditions and is readily extracted from biological samples for detection by liquid chromatography coupled with mass spectrometry. Although cFMN is hydrolyzed by liver tissue extracts to FMN and riboflavin, the mechanisms for this conversion remain elusive.

CtBP1-S/BARS (C-terminal binding protein 1-S/Brefeldin A ADP-Ribosylation Substrate) is a moonlighting protein with key roles in membrane trafficking and gene regulation. We show that CtBP1-S/BARS couples enzymatic lipid remodeling [lysophosphatidic acid (LPA)-to-phosphatidic acid (PA) conversion] with membrane deformation to drive fission and that this activity is directly controlled by metabolic ligands. CtBP1-S/BARS exists as a monomer or dimer. The monomeric, acyl-CoA-bound form drives membrane fission by coupling acyltransferase-dependent LPA-to-PA conversion with amphipathic helix insertion into PA-enriched membranes. Under metabolic stress, reduced nicotinamide adenine dinucleotide (NADH) binding triggers dimerization and structural rearrangements that disable fission, enabling the binding of transcription factors regulating apoptosis and energy metabolism. This NADH/acyl-CoA competition likely coordinates trafficking shutdown with gene expression programs through a single conformational change. In intact cells, increased NADH promotes nuclear accumulation of dimeric/tetrameric CtBP1-S/BARS, whereas elevated acyl-CoA favors the cytosolic, membrane-associated monomer, indicating that cofactor availability determines protein function under physiological and stress conditions. This work reveals the structural basis for integrating membrane transport with transcriptional control, demonstrating how evolution embeds distinct cellular functions into a unified molecular pathway.

Nicotinamide adenine dinucleotide (NAD+) precursor supplementation shows metabolic and functional benefits in rodent models of disease and is being explored as potential therapeutic strategy in humans. However, the wide range of processes that involve NAD+ in every cell and subcellular compartment make it difficult to narrow down the mechanisms of action. Here we show that the rate of liver regeneration is closely associated with the concentration of NAD+ in hepatocyte mitochondria. We find that the mitochondrial NAD+ concentration in hepatocytes of male mice is determined by the expression of the transporter SLC25A51 (MCART1). The heterozygous loss of SLC25A51 modestly decreases mitochondrial NAD+ content in multiple tissues and impairs liver regeneration, whereas the hepatocyte-specific overexpression of SLC25A51 is sufficient to enhance liver regeneration comparably to the effect of systemic NAD+ precursor supplements. This benefit is observed even though NAD+ levels are increased only in mitochondria. Thus, the hepatocyte mitochondrial NAD+ pool is a key determinant of the rate of liver regeneration.

IntroductionCD38, a regulator of intracellular calcium signalling, is highly expressed in immune cells. Mice lacking CD38 are very susceptible to acute bacterial infections, implicating CD38 in innate immune responses. The effects of CD38 inhibition on NLRP3 inflammasome activation in human primary monocytes and monocyte-derived macrophages have not been investigated. Apigenin is a naturally occurring flavonoid known to inhibit CD38. However, apigenin has also been proposed to inhibit the extracellular ATP receptor P2XR7, an upstream activator of NLRP3. In this study we aimed to investigate whether apigenin attenuates NLRP3 inflammasome activation in human monocytes and monocyte-derived macrophages through CD38 inhibition. MethodsLPS-primed human monocytes and monocyte-derived macrophages were treated with apigenin, the CD38 inhibitor 78c, antagonists of CD38 second messengers (8-br-ADPR and 8-br-cADPR) or the ATP hydrolase, apyrase, prior to NLRP3 activation with ATP, monosodium urate crystals (MSU) or nigericin. IL-1β and TNF secretion and mRNA expression, as well as N-terminal gasdermin-D formation were quantified. Ca2+ mobilization was determined by live confocal microscopy. NLRP3 activity was also compared in WT and CD38-/- mouse bone marrow-derived macrophages (BMDMs) with and without CD38 inhibitors.ResultsApigenin significantly inhibited IL-1β release from LPS-primed monocytes and macrophages activated with ATP, MSU, or nigericin. CD38 inhibition with 78c also attenuated NLRP3-dependent IL-1β release. Apigenin was a potent inhibitor of Ca2+ flux from the endoplasmic reticulum to the cytosol in human monocyte-derived macrophages. Apyrase attenuated IL-1β release induced by ATP or MSU, but not by nigericin. However, the NLRP3 inflammasome is not compromised in CD38-/- bone marrow-derived macrophages compared to corresponding WT cells, and apigenin moderated IL-1β release in both genotypes. DiscussionOur data support that apigenin attenuates NLRP3 activation independently of CD38. Our results also suggest that MSU crystals activate NLRP3 through autocrine or paracrine ATP signalling.

Beyond their classical functions as redox cofactors, recent fundamental and clinical research has expanded our understanding of the diverse roles of nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) in signaling pathways, epigenetic regulation and energy homeostasis. Moreover, NAD and NADP influence numerous diseases as well as the processes of aging, and are emerging as targets for clinical intervention. Here, we summarize safety, bioavailability and efficacy data from NAD+-related clinical trials, focusing on aging and neurodegenerative diseases. We discuss the established NAD+ precursors nicotinic acid and nicotinamide, newer compounds such as nicotinamide riboside and nicotinamide mononucleotide, and emerging precursors. We also discuss technological advances including in industrial-scale production and real-time detection, which are facilitating NAD+ research and clinical translation. Finally, we emphasize the need for further large-scale studies to determine optimal dose, administration routes and frequency, as well as long-term safety and interindividual variability in response. Levels of the metabolic coenzyme NAD+ decline during aging, which is linked to many age-related diseases. Zhang et al. review recent clinical and translational evidence testing NAD+ supplementation in age-related diseases, highlighting therapeutic challenges and opportunities.

The interpretation of multi-omics datasets obtained from high-throughput approaches is important to understand disease-related physiological changes and to predict biomarkers in body fluids. We present a new metabolite-centred genome-scale metabolic modelling algorithm, the Gene Expression-based Metabolite Centrality Analysis Tool (GEMCAT). GEMCAT enables integration of transcriptomics or proteomics data to predict changes in metabolite concentrations, which can be verified by targeted metabolomics. In addition, GEMCAT allows to trace measured and predicted metabolic changes back to the underlying alterations in gene expression or proteomics and thus enables functional interpretation and integration of multi-omics data. We demonstrate the predictive capacity of GEMCAT on three datasets and genome-scale metabolic networks from two different organisms: (i) we integrated transcriptomics and metabolomics data from an engineered human cell line with a functional deletion of the mitochondrial NAD transporter; (ii) we used a large multi-tissue multi-omics dataset from rats for transcriptome- and proteome-based prediction and verification of training-induced metabolic changes and achieved an average prediction accuracy of 70%; and (iii) we used proteomics measurements from patients with inflammatory bowel disease and verified the predicted changes using metabolomics data from the same patients. For this dataset, the prediction accuracy achieved by GEMCAT was 79%.

The preparation of custom-made media offers precise control over nutrient composition, enabling detailed studies of cellular metabolism. We demonstrate how self-made media formulations enable diverse assay designs and readouts to assess cancer metabolism. Self-made media can be used in Seahorse assays to measure mitochondrial respiration under defined conditions. In nutrient deprivation experiments, amino acid or vitamin removal can uncover how cancer cells adapt to metabolic stress. Using labeled amino acids enables analysis of nascent protein synthesis and translational regulation, while stable-isotope tracing reveals metabolic fluxes through key pathways. This guide presents a suite of metabolic assays using custom-made media, covering experimental design, the selection of controls, sample preparation, data acquisition, and interpretation. The accompanying online media calculator "Media Minds" streamlines the creation of custom media formulations, ensuring accuracy and reproducibility.

Werner syndrome (WS), caused by mutations in the RecQ helicase WERNER (WRN) gene, is a classical accelerated aging disease with patients suffering from several metabolic dysfunctions without a cure. While, as we previously reported, depleted NAD+ causes accumulation of damaged mitochondria, leading to compromised metabolism, how mitochondrial NAD+ changes in WS and the impact on WS pathologies were unknown. We show that loss of WRN increases senescence in mesenchymal stem cells (MSCs) likely related to dysregulation of metabolic and aging pathways. In line with this, NAD+ augmentation, via supplementation with nicotinamide riboside, reduces senescence and improves mitochondrial metabolic profiles in MSCs with WRN knockout (WRN-/-) and in primary fibroblasts derived from WS patients compared to controls. Moreover, WRN deficiency results in decreased mitochondrial NAD+ (measured indirectly via mitochondrially-expressed PARP activity), and altered expression of key salvage pathway enzymes, including NMNAT1 and NAMPT; ChIP-seq data analysis unveils a potential co-regulatory axis between WRN and the NMNATs, likely important for chromatin stability and DNA metabolism. However, restoration of mitochondrial or cellular NAD+ is not sufficient to reinstall cellular proliferation in immortalized cells with siRNA-mediated knockdown of WRN, highlighting an indispensable role of WRN in proliferation even in an NAD+ affluent environment. Further cell and animal studies are needed to deepen our understanding of the underlying mechanisms, facilitating related drug development.

Tuberculosis necrotizing toxin (TNT) is a protein domain discovered on the outer membrane of Mycobacterium tuberculosis (Mtb), and the fungal pathogen Aspergillus fumigatus. TNT domains have pure NAD(P) hydrolytic activity, setting them apart from other NAD-cleaving domains such as ADP-ribosyl cyclase and Toll/interleukin-1 receptor homology (TIR) domains which form a wider set of products. Importantly, the Mtb TNT domain has been shown to be involved in immune evasion via depletion of the intracellular NAD pool of macrophages. Therefore, an intriguing hypothesis is that TNT domains act as "NAD killers" in host cells facilitating pathogenesis. Here, we explore the phylogenetic distribution of TNT domains and detect their presence solely in bacteria and fungi. Within fungi, we discerned six TNT clades. In addition, X-ray crystallography and AlphaFold2 modeling unveiled clade-specific strategies to promote homodimer stabilization of the fungal enzymes, namely, Ca2+ binding, disulfide bonds, or hydrogen bonds. We show that dimer stabilization is a requirement for NADase activity and that the group-specific strategies affect the active site conformation, thereby modulating enzyme activity. Together, these findings reveal the evolutionary lineage of fungal TNT enzymes, corroborating the hypothesis of them being pure extracellular NAD (eNAD) cleavers, with possible involvement in microbial warfare and host immune evasion.

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.

Mitochondrial diseases are associated with neuronal death and mtDNA depletion. Astrocytes respond to injury or stimuli and damage to the central nervous system. Neurodegeneration can cause astrocytes to activate and acquire toxic functions that induce neuronal death. However, astrocyte activation and its impact on neuronal homeostasis in mitochondrial disease remain to be explored. Using patient cells carrying POLG mutations, we generated iPSCs and then differentiated these into astrocytes. POLG astrocytes exhibited mitochondrial dysfunction including loss of mitochondrial membrane potential, energy failure, loss of complex I and IV, disturbed NAD+/NADH metabolism, and mtDNA depletion. Further, POLG derived astrocytes presented an A1-like reactive phenotype with increased proliferation, invasion, upregulation of pathways involved in response to stimulus, immune system process, cell proliferation and cell killing. Under direct and indirect co-culture with neurons, POLG astrocytes manifested a toxic effect leading to the death of neurons. We demonstrate that mitochondrial dysfunction caused by POLG mutations leads not only to intrinsic defects in energy metabolism affecting both neurons and astrocytes, but also to neurotoxic damage driven by astrocytes. These findings reveal a novel role for dysfunctional astrocytes that contribute to the pathogenesis of POLG diseases.

Nicotinamide adenine dinucleotide, in its oxidized (NAD+) and reduced (NADH) forms, is a reduction-oxidation (redox) co-factor and substrate for signalling enzymes that have essential roles in metabolism. The recognition that NAD+ levels fall in response to stress and can be readily replenished through supplementation has fostered great interest in the potential benefits of increasing or restoring NAD+ levels in humans to prevent or delay diseases and degenerative processes. However, much about the biology of NAD+ and related molecules remains poorly understood. In this Review, we discuss the current knowledge of NAD+ metabolism, including limitations of, assumptions about and unappreciated factors that might influence the success or contribute to risks of NAD+ supplementation. We highlight several ongoing controversies in the field, and discuss the role of the microbiome in modulating the availability of NAD+ precursors such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), the presence of multiple cellular compartments that have distinct pools of NAD+ and NADH, and non-canonical NAD+ and NADH degradation pathways. We conclude that a substantial investment in understanding the fundamental biology of NAD+, its detection and its metabolites in specific cells and cellular compartments is needed to support current translational efforts to safely boost NAD+ levels in humans.

Nicotinamide adenine dinucleotide (NAD) is an essential molecule in all kingdoms of life, mediating energy metabolism and cellular signaling. Recently, a new class of highly active fungal surface NADases was discovered. The enzyme from the opportunistic human pathogen Aspergillus fumigatus was thoroughly characterized. It harbors a catalytic domain that resembles that of the tuberculosis necrotizing toxin from Mycobacterium tuberculosis, which efficiently cleaves NAD+ to nicotinamide and ADP-ribose, thereby depleting the dinucleotide pool. Of note, the A. fumigatus NADase has an additional Ca2+-binding motif at the C-terminus of the protein. Despite the presence of NADases in several fungal divisions, the Ca2+-binding motif is uniquely found in the Eurotiales order, which contains species that have immense health and economic impacts on humans. To identify the potential roles of the metal ion-binding site in catalysis or protein stability, we generated and characterized A. fumigatus NADase variants lacking the ability to bind calcium. X-ray crystallographic analyses revealed that the mutation causes a drastic and dynamic structural rearrangement of the homodimer, resulting in decreased thermal stability. Even though the calcium-binding site is at a long distance from the catalytic center, the structural reorganization upon the loss of calcium binding allosterically alters the active site, thereby negatively affecting NAD-glycohydrolase activity. Together, these findings reveal that this unique calcium-binding site affects the protein fold, stabilizing the dimeric structure, but also mediates long-range effects resulting in an increased catalytic rate.

Diseases caused by POLG mutations are the most common form of mitochondrial diseases and associated with phenotypes of varying severity. Clinical studies have shown that patients with compound heterozygous POLG mutations have a lower survival rate than patients with homozygous mutations, but the molecular mechanisms behind this remain unexplored. Using an induced pluripotent stem cell (iPSC) model, we investigate differences between homozygous and compound heterozygous genotypes in different cell types, including patient-specific fibroblasts, iPSCs, and iPSC-derived neural stem cells (NSCs) and astrocytes. We found that compound hetero-zygous lines exhibited greater impairment of mitochondrial function in NSCs than homozygous NSCs, but not in fibroblasts, iPSCs, or astrocytes. Compared with homozygous NSCs, compound heterozygous NSCs exhibited more severe functional defects, including reduced ATP production, loss of mitochondrial DNA (mtDNA) copy number and complex I expression, disturbance of NAD+ metabolism, and higher ROS levels, which further led to cellular senescence and activation of mitophagy. RNA sequencing analysis revealed greater downregulation of mitochondrial and metabolic pathways, including the citric acid cycle and oxidative phosphorylation, in com-pound heterozygous NSCs. Our iPSC-based disease model can be widely used to understand the genotype -phenotype relationship of affected brain cells in mitochondrial diseases, and further drug discovery applications.

NAD homeostasis in mammals requires the salvage of nicotinamide (Nam), which is cleaved from NAD+ by sirtuins, PARPs, and other NAD+-dependent signaling enzymes. Nam phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in vitamin B3 salvage, whereby Nam reacts with phosphoribosyl pyrophosphate (PRPP) to form nicotinamide mononucleotide. NAMPT has a high affinity towards Nam, which is further enhanced by autophosphorylation of His247. The mechanism of this enhancement has remained unknown. Here, we present high-resolution crystal structures and biochemical data that provide reasoning for the increased affinity of the phosphorylated NAMPT for its substrate. Structural and kinetic analyses suggest a mechanism that includes Mg2+ coordination by phospho-His247, such that PRPP is stabilized in a position highly favorable for catalysis. Under these conditions, nicotinic acid (NA) can serve as a substrate. Moreover, we demonstrate that a stretch of 10 amino acids, present only in NAMPTs from deuterostomes, facilitates conformational plasticity and stabilizes the chemically unstable phosphorylation of His247. Thereby the apparent substrate affinity is considerably enhanced compared to prokaryotic NAMPTs. Collectively, our study provides a structural basis for the important function of NAMPT to recycle Nam into NAD biosynthesis with high affinity.

SLC25A51 is a member of the mitochondrial carrier family (MCF) but lacks key residues that contribute to the mechanism of other nucleotide MCF transporters. Thus, how SLC25A51 transports NAD+ across the inner mitochondrial membrane remains unclear. To elucidate its mechanism, we use Molecular Dynamics simulations to reconstitute SLC25A51 homology models into lipid bilayers and to generate hypotheses to test. We observe spontaneous binding of cardiolipin phospholipids to three distinct sites on the exterior of SLC25A51's central pore and find that mutation of these sites impairs cardiolipin binding and transporter activity. We also observe that stable formation of the required matrix gate is controlled by a single salt bridge. We identify binding sites in SLC25A51 for NAD+ and show that its selectivity for NAD+ is guided by an electrostatic interaction between the charged nicotinamide ring in the ligand and a negatively charged patch in the pore. In turn, interaction of NAD+ with interior residue E132 guides the ligand to dynamically engage and weaken the salt bridge gate, representing a ligand-induced initiation of transport.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have distinct origins: ESCs are derived from pre-implanted embryos while iPSCs are reprogrammed somatic cells. Both have their own characteristics and lineage specificity, and both are valuable tools for studying human neurological development and disease. Thus far, few studies have analyzed how differences between stem cell types influence mitochondrial function and mitochondrial DNA (mtDNA) homeostasis during differentiation into neural and glial lineages. In this study, we compared mitochondrial function and mtDNA replication in human ESCs and iPSCs at three different stages − pluripotent, neural progenitor and astrocyte. We found that while ESCs and iPSCs have a similar mitochondrial signature, neural and astrocyte derivations manifested differences. At the neural stem cell (NSC) stage, iPSC-NSCs displayed decreased ATP production and a reduction in mitochondrial respiratory chain (MRC) complex IV expression compared to ESC-NSCs. IPSC-astrocytes showed increased mitochondrial activity including elevated ATP production, MRC complex IV expression, mtDNA copy number and mitochondrial biogenesis relative to those derived from ESCs. These findings show that while ESCs and iPSCs are similar at the pluripotent stage, differences in mitochondrial function may develop during differentiation and must be taken into account when extrapolating results from different cell types.Abbreviation: BSA: Bovine serum albumin; DCFDA: 2′,7′‐dichlorodihydrofluorescein diacetate; DCX: Doublecortin; EAAT-1: Excitatory amino acid transporter 1; ESCs: Embryonic stem cells; GFAP: Glial fibrillary acidic protein; GS: Glutamine synthetase; iPSCs: Induced pluripotent stem cells; LC3B: Microtubule-associated protein 1 light chain 3β; LC-MS: Liquid chromatography-mass spectrometry; mito-ROS: Mitochondrial ROS; MMP: Mitochondrial membrane potential; MRC: Mitochondrial respiratory chain; mtDNA: Mitochondrial DNA; MTDR: MitoTracker Deep Red; MTG: MitoTracker Green; NSCs: Neural stem cells; PDL: Poly-D-lysine; PFA: Paraformaldehyde; PGC-1α: PPAR-γ coactivator-1 alpha; PPAR-γ: Peroxisome proliferator-activated receptor-gamma; p-SIRT1: Phosphorylated sirtuin 1; p-ULK1: Phosphorylated unc-51 like autophagy activating kinase 1; qPCR: Quantitative PCR; RT: Room temperature; RT-qPCR: Quantitative reverse transcription PCR; SEM: Standard error of the mean; TFAM: Mitochondrial transcription factor A; TMRE: Tetramethylrhodamine ethyl ester; TOMM20: Translocase of outer mitochondrial membrane 20

Bacteria use two alternative pathways to synthesize nicotinamide adenine dinucleotide (NAD) from nicotinamide (Nam). A short, two-step route proceeds through nicotinamide mononucleotide (NMN) formation, whereas the other pathway, a four-step route, includes the deamidation of Nam and the reamidation of nicotinic acid adenine dinucleotide (NAAD) to NAD. In addition to having twice as many enzymatic steps, the four-step route appears energetically unfavourable, because the amidation of NAAD includes the cleavage of ATP to AMP. Therefore, it is surprising that this pathway is prevalent not only in bacteria but also in yeast and plants. Here, we demonstrate that the considerably higher chemical stability of the deamidated intermediates, compared with their amidated counterparts, might compensate for the additional energy expenditure, at least at elevated temperatures. Moreover, comprehensive bioinformatics analyses of the available >6000 bacterial genomes indicate that an early selection of one or the other pathway occurred. The mathematical modelling of the NAD pathway dynamics supports this hypothesis, as there appear to be no advantages in having both pathways.

We conducted a double-blinded phase I clinical trial to establish whether nicotinamide adenine dinucleotide (NAD) replenishment therapy, via oral intake of nicotinamide riboside (NR), is safe, augments cerebral NAD levels, and impacts cerebral metabolism in Parkinson's disease (PD). Thirty newly diagnosed, treatment-naive patients received 1,000 mg NR or placebo for 30 days. NR treatment was well tolerated and led to a significant, but variable, increase in cerebral NAD levels-measured by (31)phosphorous magnetic resonance spectroscopy-and related metabolites in the cerebrospinal fluid. NR recipients showing increased brain NAD levels exhibited altered cerebral metabolism, measured by (18)fluoro-deoxyglucose positron emission tomography, and this was associated with mild clinical improvement. NR augmented the NAD metabolome and induced transcriptional upregulation of processes related to mitochondrial, lysosomal, and proteasomal function in blood cells and/or skeletal muscle. Furthermore, NR decreased the levels of inflammatory cytokines in serum and cerebrospinal fluid. Our findings nominate NR as a potential neuroprotective therapy for PD, warranting further investigation in larger trials.

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