Development and validation of an LC-MS/MS method for quantifying NAD+ and related metabolites in mice sciatic nerves and its application to a nerve injury animal model.

Recent studies highlight the pivotal roles of Nicotinamide adenine dinucleotide (NAD+) and its metabolites in aging and neurodegeneration. Accurate quantification of NAD+ and its metabolite levels in cells or tissues is crucial for advancing biochemical research and interventions targeting aging and neurodegenerative diseases. This study presents an accurate, precise, and rapid LC-MS/MS method using a surrogate matrix to quantify endogenous substances NAD+, nicotinamide mononucleotide (NMN), nicotinamide (NAM), adenosine diphosphate ribose (ADPR), and cyclic adenosine diphosphate ribose (cADPR) concentrations in mice sciatic nerves. Considering the properties of the phosphate groups in the analytes, the column and mobile phase were systematically optimized. These five polar analytes exhibited excellent analytical performance and baseline separation within 5 min on an Atlantis Premier BEH C18 AX column, with methylene phosphonic acid as a mobile phase additive. Enhanced sensitivity addressed the challenges posed by the small sample size of mice sciatic nerve and low NMN and cADPR detection. The method was fully validated, with linear correlation coefficients exceeding 0.992, precision (%relative standard deviation, RSD) values within 8.8%, and accuracy values between 92.2% and 107.3%, suggesting good reproducibility. Analytical recoveries in spiked and diluted matrix ranged from 87.8% to 104.7%, indicating the suitability of water as a surrogate matrix. Application of the method to quantify NAD+ and its metabolite levels in normal and injured mice sciatic nerve identified cADPR as a sensitive biomarker in the nerve injury model. This method is anticipated to deepen our understanding of the connections between NAD+ and its metabolites in health and disease, potentially improving diagnoses of various neurological disorders and aiding drug development for aging and neurodegenerative diseases.

Essential role of CD38 in platelet aggregation through the PKC-mediated internalization and activation.

Introduction: CD38 is a multifunctional enzyme with a potent Ca2+ mobilizing effect, cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). Here, we aimed to demonstrate the role of CD38 in platelets via protein kinase C (PKC)-mediated internalization and activation. Methods: Mouse platelets were used in this study. Thrombin, an agonist of platelet function, provoked a prompt and long-lasting increase in intracellular Ca2+ concentration ([Ca2+]i), resulting from an interplay of multifold Ca2+ mobilizing messengers.The signaling pathway was delineated using different inhibitors and techniques such as platelet aggregation assay, intracellular calcium measurements, immunoprecipitation, immunoblotting, and flow cytometry. Results: We observed a sequential formation of cADPR and NAADP through CD38 activation by PKC of non-muscle myosin heavy chain IIA (MHCIIA), resulting in phospholipase C (PLC) activation in the thrombin-stimulated platelets. These findings reveal that PKC is fundamental in activating CD38 and elicits a physiological response in the murine platelets. Conclusion: PKC is involved in many signaling pathways. Specifically, PKC is involved in the internalization of CD38 via MHCIIA in CD38+/+ wild-type (WT) and CD38-/- knockout mice (KO). CD38 generates calcium-mobilizing agents that act on specific receptors of the calcium stores. Calcium triggered platelet aggregation while serving as a secondary messenger.

Inhibition of CD38 enzymatic activity enhances CAR-T cell immune-therapeutic efficacy by repressing glycolytic metabolism.

Chimeric antigen receptor (CAR)-T therapy has shown superior efficacy against hematopoietic malignancies. However, many patients failed to achieve sustainable tumor control partially due to CAR-T cell exhaustion and limited persistence. In this study, by performing single-cell multi-omics data analysis on patient-derived CAR-T cells, we identify CD38 as a potential hallmark of exhausted CAR-T cells, which is positively correlated with exhaustion-related transcription factors and further confirmed with in vitro exhaustion models. Moreover, inhibiting CD38 activity reverses tonic signaling- or tumor antigen-induced exhaustion independent of single-chain variable fragment design or costimulatory domain, resulting in improved CAR-T cell cytotoxicity and antitumor response. Mechanistically, CD38 inhibition synergizes the downregulation of CD38-cADPR -Ca2+ signaling and activation of the CD38-NAD+-SIRT1 axis to suppress glycolysis. Collectively, our findings shed light on the role of CD38 in CAR-T cell exhaustion and suggest potential clinical applications of CD38 inhibition in enhancing the efficacy and persistence of CAR-T cell therapy.

SARM1 regulates NAD+-linked metabolism and select immune genes in macrophages.

Sterile alpha and HEAT/armadillo motif-containing protein (SARM1) was recently described as a NAD+-consuming enzyme and has previously been shown to regulate immune responses in macrophages. Neuronal SARM1 is known to contribute to axon degeneration due to its NADase activity. However, how SARM1 affects macrophage metabolism has not been explored. Here, we show that macrophages from Sarm1-/- mice display elevated NAD+ concentrations and lower cyclic ADP-ribose, a known product of SARM1-dependent NAD+ catabolism. Further, SARM1-deficient macrophages showed an increase in the reserve capacity of oxidative phosphorylation and glycolysis compared to WT cells. Stimulation of macrophages to a proinflammatory state by lipopolysaccharide (LPS) revealed that SARM1 restricts the ability of macrophages to upregulate glycolysis and limits the expression of the proinflammatory gene interleukin (Il) 1b, but boosts expression of anti-inflammatory Il10. In contrast, we show macrophages lacking SARM1 induced to an anti-inflammatory state by IL-4 stimulation display increased oxidative phosphorylation and glycolysis, and reduced expression of the anti-inflammatory gene, Fizz1. Overall, these data show that SARM1 fine-tunes immune gene transcription in macrophages via consumption of NAD+ and altered macrophage metabolism.

Biosynthesis and Function of VIP and Oxytocin: Mechanisms of C-terminal Amidation, Oxytocin Secretion and Transport.

In this review, we provide the status of research on vasoactive intestinal peptide (VIP) and oxytocin, typical C-terminal α-amidated peptide hormones, including their precursor protein structures, processing and C-terminal α-amidation, and the recently identified mechanisms of regulation of oxytocin secretion and its transportation through the blood brain barrier. More than half of neural and endocrine peptides, such as VIP and oxytocin, have the α-amide structure at their C-terminus, which is essential for biological activities. We have studied the synthesis and function of C-terminal α-amidated peptides, including VIP and oxytocin, since the 1980s. Human VIP mRNA encoded not only VIP but also another related C-terminal α-amidated peptide, PHM-27 (peptide having amino-terminal histidine, carboxy-terminal methionine amide, and 27 amino acid residues). The human VIP/PHM-27 gene is composed of 7 exons and regulated synergistically by cyclic AMP and protein kinase C pathways. VIP has an essential role in glycemic control using transgenic mouse technology. The peptide C-terminal α-amidation proceeded through a 2-step mechanism catalyzed by 2 different enzymes encoded in a single mRNA. In the oxytocin secretion from the hypothalamus/the posterior pituitary, the CD38-cyclic ADP-ribose signal system, which was first established in the insulin secretion from pancreatic ß cells of the islets of Langerhans, was found to be essential. A possible mechanism involving RAGE (receptor for advanced glycation end-products) of the oxytocin transportation from the blood stream into the brain through the blood-brain barrier has also been suggested.

Postnatal expression of CD38 in astrocytes regulates synapse formation and adult social memory.

Social behavior is essential for health, survival, and reproduction of animals; however, the role of astrocytes in social behavior remains largely unknown. The transmembrane protein CD38, which acts both as a receptor and ADP-ribosyl cyclase to produce cyclic ADP-ribose (cADPR) regulates social behaviors by promoting oxytocin release from hypothalamic neurons. CD38 is also abundantly expressed in astrocytes in the postnatal brain and is important for astroglial development. Here, we demonstrate that the astroglial-expressed CD38 plays an important role in social behavior during development. Selective deletion of CD38 in postnatal astrocytes, but not in adult astrocytes, impairs social memory without any other behavioral abnormalities. Morphological analysis shows that depletion of astroglial CD38 in the postnatal brain interferes with synapse formation in the medial prefrontal cortex (mPFC) and hippocampus. Moreover, astroglial CD38 expression promotes synaptogenesis of excitatory neurons by increasing the level of extracellular SPARCL1 (also known as Hevin), a synaptogenic protein. The release of SPARCL1 from astrocytes is regulated by CD38/cADPR/calcium signaling. These data demonstrate a novel developmental role of astrocytes in neural circuit formation and regulation of social behavior in adults.

NAD+ Consuming Enzymes: Involvement in Therapies and Prevention of Human Diseases.

Neuroprotection is one of the hot topics in medicine. Alzheimer's disease, amyotrophic lateral sclerosis, retinal pigment epithelial (RPE) degeneration, and axonal degeneration have been studied for the involvement of NAD depletion. Localized NAD+ depletion could lead to overactivation and crowding of local NAD+ salvage pathways. It has been stated that NAD+ depletion caused by PARPs and PAR cycling has been related to metabolic diseases and cancer. Additionally, it is now acknowledged that SARM1 dependent NAD+ depletion causes axon degeneration. New targeted therapeutics, such as SARM1 inhibitors, and NAD+ salvage drugs will help alleviate the dysfunctions affecting cell life and death in neurodegeneration as well as in metabolic diseases and cancer.

Enzymology of Ca2+-Mobilizing Second Messengers Derived from NAD: From NAD Glycohydrolases to (Dual) NADPH Oxidases.

Nicotinamide adenine dinucleotide (NAD) and its 2'-phosphorylated cousin NADP are precursors for the enzymatic formation of the Ca2+-mobilizing second messengers adenosine diphosphoribose (ADPR), 2'-deoxy-ADPR, cyclic ADPR, and nicotinic acid adenine dinucleotide phosphate (NAADP). The enzymes involved are either NAD glycohydrolases CD38 or sterile alpha toll/interleukin receptor motif containing-1 (SARM1), or (dual) NADPH oxidases (NOX/DUOX). Enzymatic function(s) are reviewed and physiological role(s) in selected cell systems are discussed.

Dual amplification strategy turns TRPM2 channels into supersensitive central heat detectors.

The Ca2+ and ADP ribose (ADPR)-activated cation channel TRPM2 is the closest homolog of the cold sensor TRPM8 but serves as a deep-brain warmth sensor. To unravel the molecular mechanism of heat sensing by the TRPM2 protein, we study here temperature dependence of TRPM2 currents in cell-free membrane patches across ranges of agonist concentrations. We find that channel gating remains strictly agonist-dependent even at 40°C: heating alone or in combination with just Ca2+, just ADPR, Ca2+ + cyclic ADPR, or H2O2 pretreatment only marginally activates TRPM2. For fully liganded TRPM2, pore opening is intrinsically endothermic, due to ~10-fold larger activation enthalpy for opening (~200 kJ/mol) than for closure (~20 kJ/mol). However, the temperature threshold is too high (>40°C) for unliganded but too low (<15°C) for fully liganded channels. Thus, warmth sensitivity around 37°C is restricted to narrow ranges of agonist concentrations. For ADPR, that range matches, but for Ca2+, it exceeds bulk cytosolic values. The supraphysiological [Ca2+] needed for TRPM2 warmth sensitivity is provided by Ca2+ entering through the channel's pore. That positive feedback provides further strong amplification to the TRPM2 temperature response (Q10 ~ 1,000), enabling the TRPM2 protein to autonomously respond to tiny temperature fluctuations around 37°C. These functional data together with published structures suggest a molecular mechanism for opposite temperature dependences of two closely related channel proteins.

CD38-Cyclic ADP-Ribose Signal System in Physiology, Biochemistry, and Pathophysiology.

Calcium (Ca2+) is a ubiquitous and fundamental signaling component that is utilized by cells to regulate a diverse range of cellular functions, such as insulin secretion from pancreatic ß-cells of the islets of Langerhans. Cyclic ADP-ribose (cADPR), synthesized from NAD+ by ADP-ribosyl cyclase family proteins, such as the mammalian cluster of differentiation 38 (CD38), is important for intracellular Ca2+ mobilization for cell functioning. cADPR induces Ca2+ release from endoplasmic reticulum via the ryanodine receptor intracellular Ca2+ channel complex, in which the FK506-binding protein 12.6 works as a cADPR-binding regulatory protein. Recently, involvements of the CD38-cADPR signal system in several human diseases and animal models have been reported. This review describes the biochemical and molecular biological basis of the CD38-cADPR signal system and the diseases caused by its abnormalities.

A TIReless battle: TIR domains in plant-pathogen interactions.

Toll/interleukin-1 receptor (TIR) domain-containing proteins are conserved across kingdoms, and their mechanistic understanding holds promise for basic plant biology and agriculture. Here, we discuss the novel enzymatic TIR domain functions of nucleotide-binding leucine-rich repeat receptors (NLRs) in cell death, and posit how TIR domain-containing effectors mechanistically subvert host immune systems.

A phytobacterial TIR domain effector manipulates NAD+ to promote virulence.

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.

The calcium signaling enzyme CD38 - a paradigm for membrane topology defining distinct protein functions.

CD38 is a single-pass transmembrane enzyme catalyzing the synthesis of two nucleotide second messengers, cyclic ADP-ribose (cADPR) from NAD and nicotinic acid adenine dinucleotide phosphate (NAADP) from NADP. The former mediates the mobilization of the endoplasmic Ca2+-stores in response to a wide range of stimuli, while NAADP targets the endo-lysosomal stores. CD38 not only possesses multiple enzymatic activities, it also exists in two opposite membrane orientations. Type III CD38 has the catalytic domain facing the cytosol and is responsible for producing cellular cADPR. The type II CD38 has an opposite orientation and is serving as a surface receptor mediating extracellular functions such as cell adhesion and lymphocyte activation. Its ecto-NADase activity also contributes to the recycling of external NAD released by apoptosis. Endocytosis can deliver surface type II CD38 to endo-lysosomes, which acidic environment favors the production of NAADP. This article reviews the rationale and evidence that have led to CD38 as a paradigm for membrane topology defining distinct functions of proteins. Also described is the recent discovery of a hitherto unknown cADPR-synthesizing enzyme, SARM1, ushering in a new frontier in cADPR-mediated Ca2+-signaling.

Small Molecule CD38 Inhibitors: Synthesis of 8-Amino-N1-inosine 5'-monophosphate, Analogues and Early Structure-Activity Relationship.

Although a monoclonal antibody targeting the multifunctional ectoenzyme CD38 is an FDA-approved drug, few small molecule inhibitors exist for this enzyme that catalyzes inter alia the formation and metabolism of the N1-ribosylated, Ca2+-mobilizing, second messenger cyclic adenosine 5'-diphosphoribose (cADPR). N1-Inosine 5'-monophosphate (N1-IMP) is a fragment directly related to cADPR. 8-Substituted-N1-IMP derivatives, prepared by degradation of cyclic parent compounds, inhibit CD38-mediated cADPR hydrolysis more efficiently than related cyclic analogues, making them attractive for inhibitor development. We report a total synthesis of the N1-IMP scaffold from adenine and a small initial compound series that facilitated early delineation of structure-activity parameters, with analogues evaluated for inhibition of CD38-mediated hydrolysis of cADPR. The 5'-phosphate group proved essential for useful activity, but substitution of this group by a sulfonamide bioisostere was not fruitful. 8-NH2-N1-IMP is the most potent inhibitor (IC50 = 7.6 µM) and importantly HPLC studies showed this ligand to be cleaved at high CD38 concentrations, confirming its access to the CD38 catalytic machinery and demonstrating the potential of our fragment approach.

Probing the Ca2+ mobilizing properties on primary cortical neurons of a new stable cADPR mimic.

Cyclic adenosine diphosphate ribose (cADPR) is a second messenger involved in the Ca2+ homeostasis. Its chemical instability prompted researchers to tune point by point its structure, obtaining stable analogues featuring interesting biological properties. One of the most challenging derivatives is the cyclic inosine diphosphate ribose (cIDPR), in which the hypoxanthine isosterically replaces the adenine. As our research focuses on the synthesis of N1 substituted inosines, in the last few years we have produced new flexible cIDPR analogues, where the northern ribose has been replaced by alkyl chains. Interestingly, some of them mobilized Ca2+ ions in PC12 cells. To extend our SAR studies, herein we report on the synthesis of a new stable cIDPR derivative which contains the 2″S,3″R dihydroxypentyl chain instead of the northern ribose. Interestingly, the new cyclic derivative and its open precursor induced an increase in intracellular calcium concentration ([Ca2+]i) with the same efficacy of the endogenous cADPR in rat primary cortical neurons.

NAD+ Degrading Enzymes, Evidence for Roles During Infection.

Declines in cellular nicotinamide adenine dinucleotide (NAD) contribute to metabolic dysfunction, increase susceptibility to disease, and occur as a result of pathogenic infection. The enzymatic cleavage of NAD+ transfers ADP-ribose (ADPr) to substrate proteins generating mono-ADP-ribose (MAR), poly-ADP-ribose (PAR) or O-acetyl-ADP-ribose (OAADPr). These important post-translational modifications have roles in both immune response activation and the advancement of infection. In particular, emergent data show viral infection stimulates activation of poly (ADP-ribose) polymerase (PARP) mediated NAD+ depletion and stimulates hydrolysis of existing ADP-ribosylation modifications. These studies are important for us to better understand the value of NAD+ maintenance upon the biology of infection. This review focuses specifically upon the NAD+ utilising enzymes, discusses existing knowledge surrounding their roles in infection, their NAD+ depletion capability and their influence within pathogenic infection.

Permeant fluorescent probes visualize the activation of SARM1 and uncover an anti-neurodegenerative drug candidate.

SARM1 regulates axonal degeneration through its NAD-metabolizing activity and is a drug target for neurodegenerative disorders. We designed and synthesized fluorescent conjugates of styryl derivative with pyridine to serve as substrates of SARM1, which exhibited large red shifts after conversion. With the conjugates, SARM1 activation was visualized in live cells following elevation of endogenous NMN or treatment with a cell-permeant NMN-analog. In neurons, imaging documented mouse SARM1 activation preceded vincristine-induced axonal degeneration by hours. Library screening identified a derivative of nisoldipine (NSDP) as a covalent inhibitor of SARM1 that reacted with the cysteines, especially Cys311 in its ARM domain and blocked its NMN-activation, protecting axons from degeneration. The Cryo-EM structure showed that SARM1 was locked into an inactive conformation by the inhibitor, uncovering a potential neuroprotective mechanism of dihydropyridines.

CD38: A Potential Therapeutic Target in Cardiovascular Disease.

Substantial research has demonstrated the association between cardiovascular disease and the dysregulation of intracellular calcium, ageing, reduction in nicotinamide adenine dinucleotide NAD+ content, and decrease in sirtuin activity. CD38, which comprises the soluble type, type II, and type III, is the main NADase in mammals. This molecule catalyses the production of cyclic adenosine diphosphate ribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), and adenosine diphosphate ribose (ADPR), which stimulate the release of Ca2+, accompanied by NAD+ consumption and decreased sirtuin activity. Therefore, the relationship between cardiovascular disease and CD38 has been attracting increased attention. In this review, we summarize the structure, regulation, function, targeted drug development, and current research on CD38 in the cardiac context. More importantly, we provide original views about the as yet elusive mechanisms of CD38 action in certain cardiovascular disease models. Based on our review, we predict that CD38 may serve as a novel therapeutic target in cardiovascular disease in the future.

CD38: An Immunomodulatory Molecule in Inflammation and Autoimmunity.

CD38 is a molecule that can act as an enzyme, with NAD-depleting and intracellular signaling activity, or as a receptor with adhesive functions. CD38 can be found expressed either on the cell surface, where it may face the extracellular milieu or the cytosol, or in intracellular compartments, such as endoplasmic reticulum, nuclear membrane, and mitochondria. The main expression of CD38 is observed in hematopoietic cells, with some cell-type specific differences between mouse and human. The role of CD38 in immune cells ranges from modulating cell differentiation to effector functions during inflammation, where CD38 may regulate cell recruitment, cytokine release, and NAD availability. In line with a role in inflammation, CD38 appears to also play a critical role in inflammatory processes during autoimmunity, although whether CD38 has pathogenic or regulatory effects varies depending on the disease, immune cell, or animal model analyzed. Given the complexity of the physiology of CD38 it has been difficult to completely understand the biology of this molecule during autoimmune inflammation. In this review, we analyze current knowledge and controversies regarding the role of CD38 during inflammation and autoimmunity and novel molecular tools that may clarify current gaps in the field.

On a Magical Mystery Tour with 8-Bromo-Cyclic ADP-Ribose: From All-or-None Block to Nanojunctions and the Cell-Wide Web.

A plethora of cellular functions are controlled by calcium signals, that are greatly coordinated by calcium release from intracellular stores, the principal component of which is the sarco/endooplasmic reticulum (S/ER). In 1997 it was generally accepted that activation of various G protein-coupled receptors facilitated inositol-1,4,5-trisphosphate (IP3) production, activation of IP3 receptors and thus calcium release from S/ER. Adding to this, it was evident that S/ER resident ryanodine receptors (RyRs) could support two opposing cellular functions by delivering either highly localised calcium signals, such as calcium sparks, or by carrying propagating, global calcium waves. Coincidentally, it was reported that RyRs in mammalian cardiac myocytes might be regulated by a novel calcium mobilising messenger, cyclic adenosine diphosphate-ribose (cADPR), that had recently been discovered by HC Lee in sea urchin eggs. A reputedly selective and competitive cADPR antagonist, 8-bromo-cADPR, had been developed and was made available to us. We used 8-bromo-cADPR to further explore our observation that S/ER calcium release via RyRs could mediate two opposing functions, namely pulmonary artery dilation and constriction, in a manner seemingly independent of IP3Rs or calcium influx pathways. Importantly, the work of others had shown that, unlike skeletal and cardiac muscles, smooth muscles might express all three RyR subtypes. If this were the case in our experimental system and cADPR played a role, then 8-bromo-cADPR would surely block one of the opposing RyR-dependent functions identified, or the other, but certainly not both. The latter seemingly implausible scenario was confirmed. How could this be, do cells hold multiple, segregated SR stores that incorporate different RyR subtypes in receipt of spatially segregated signals carried by cADPR? The pharmacological profile of 8-bromo-cADPR action supported not only this, but also indicated that intracellular calcium signals were delivered across intracellular junctions formed by the S/ER. Not just one, at least two. This article retraces the steps along this journey, from the curious pharmacological profile of 8-bromo-cADPR to the discovery of the cell-wide web, a diverse network of cytoplasmic nanocourses demarcated by S/ER nanojunctions, which direct site-specific calcium flux and may thus coordinate the full panoply of cellular processes.