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The addiction of tumors to elevated nicotinamide adenine dinucleotide (NAD+) levels is a hallmark of cancer metabolism. Obstructing NAD+ biosynthesis in tumors is a new and promising antineoplastic strategy. Inhibitors developed against nicotinamide phosphoribosyltransferase (NAMPT), the main enzyme in NAD+ production from nicotinamide, elicited robust anticancer activity in preclinical models but not in patients, implying that other NAD+-biosynthetic pathways are also active in tumors and provide sufficient NAD+ amounts despite NAMPT obstruction. Recent studies show that NAD+ biosynthesis through the so-called "Preiss-Handler (PH) pathway", which utilizes nicotinate as a precursor, actively operates in many tumors and accounts for tumor resistance to NAMPT inhibitors. The PH pathway consists of three sequential enzymatic steps that are catalyzed by nicotinate phosphoribosyltransferase (NAPRT), nicotinamide mononucleotide adenylyltransferases (NMNATs), and NAD+ synthetase (NADSYN1). Here, we focus on these enzymes as emerging targets in cancer drug discovery, summarizing their reported inhibitors and describing their current or potential exploitation as anticancer agents. Finally, we also focus on additional NAD+-producing enzymes acting in alternative NAD+-producing routes that could also be relevant in tumors and thus become viable targets for drug discovery. [ABSTRACT FROM AUTHOR]

The past 20 years of research on axon degeneration has revealed fine details on how NAD biology controls axonal survival. Extensive data demonstrate that the NAD precursor NMN binds to and activates the pro-degenerative enzyme SARM1, so a failure to convert sufficient NMN into NAD leads to toxic NMN accumulation and axon degeneration. This involvement of NMN brings the axon degeneration field to an unexpected overlap with research into ageing and extending healthy lifespan. A decline in NAD levels throughout life, at least in some tissues, is believed to contribute to age-related functional decay and boosting NAD production with supplementation of NMN or other NAD precursors has gained attention as a potential anti-ageing therapy. Recent years have witnessed an influx of NMN-based products and related molecules on the market, sold as food supplements, with many people taking these supplements daily. While several clinical trials are ongoing to check the safety profiles and efficacy of NAD precursors, sufficient data to back their therapeutic use are still lacking. Here, we discuss NMN supplementation, SARM1 and anti-ageing strategies, with an important question in mind: considering that NMN accumulation can lead to axon degeneration, how is this compatible with its beneficial effect in ageing and are there circumstances in which NMN supplementation could become harmful? • Perturbed NAD metabolism is believed to contribute to age-related functional decay. • NMN and other NAD precursors are commercially available as anti-ageing supplements. • A toxic role of NMN in programmed axon death has recently been identified. • NMN accumulation activates SARM1 leading to NAD consumption and axon degeneration. • Can NMN and other NAD precursors cause neurotoxicity in humans through SARM1?. [ABSTRACT FROM AUTHOR]

The addiction of tumors to elevated nicotinamide adenine dinucleotide (NAD+) levels is a hallmark of cancer metabolism. Obstructing NAD+ biosynthesis in tumors is a new and promising antineoplastic strategy. Inhibitors developed against nicotinamide phosphoribosyltransferase (NAMPT), the main enzyme in NAD+ production from nicotinamide, elicited robust anticancer activity in preclinical models but not in patients, implying that other NAD+-biosynthetic pathways are also active in tumors and provide sufficient NAD+ amounts despite NAMPT obstruction. Recent studies show that NAD+ biosynthesis through the so-called “Preiss-Handler (PH) pathway”, which utilizes nicotinate as a precursor, actively operates in many tumors and accounts for tumor resistance to NAMPT inhibitors. The PH pathway consists of three sequential enzymatic steps that are catalyzed by nicotinate phosphoribosyltransferase (NAPRT), nicotinamide mononucleotide adenylyltransferases (NMNATs), and NAD+ synthetase (NADSYN1). Here, we focus on these enzymes as emerging targets in cancer drug discovery, summarizing their reported inhibitors and describing their current or potential exploitation as anticancer agents. Finally, we also focus on additional NAD+-producing enzymes acting in alternative NAD+-producing routes that could also be relevant in tumors and thus become viable targets for drug discovery.

The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the C. elegans mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD+) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration.

The intracellular parasite Leishmania braziliensis is the causal agent of cutaneous and mucocutaneous leishmaniasis, a group of endemic diseases in tropical regions, including Latin America. New therapeutic targets are required to inhibit the pathogen without affecting the host. The enzyme nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT; EC: 2.7.7.1/18) is a potential target, since it catalyzes the final step in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), which is an essential metabolite in multiple cellular processes. In this work, we produced and evaluated the catalytic activity of the recombinant protein 6HisΔ241-249LbNMNAT to study the functional relevance of the exclusive insertion present in the enzyme of L. braziliensis (LbNMNAT), but absent in the primary structure of human NMNATs. Our results indicate that the 241–249 insertion constitutes a structural element that connects the protein structure Rossmann topology with the carboxyl-terminal domain of the enzyme. The removal of this region drastically decreases the solubility, and enzymatic activity of the recombinant, causing its inactivation. Molecular dynamics simulations were carried out with the wild-type and truncated enzymes to verify additional changes in their stability, which indicated a better stability in the wild-type protein. These findings constitute an initial step to identify a new inhibition mechanism for the development of focused pharmacological strategies on exclusive insertions from the LbNMNAT protein.

The enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) catalyzes a reaction central to all known NAD biosynthetic routes. In mammals, three isoforms with distinct molecular and catalytic properties, different subcellular and tissue distribution have been characterized. Each isoform is essential for cell survival, with a critical role in modulating NAD levels in a compartment‐specific manner. Each isoform supplies NAD to specific NAD‐dependent enzymes, thus regulating their activity with impact on several biological processes, including DNA repair, proteostasis, cell differentiation, and neuronal maintenance. The nuclear NMNAT1 and the cytoplasmic NMNAT2 are also emerging as relevant targets in specific types of cancers and NMNAT2 has a key role in the activation of antineoplastic compounds. This review recapitulates the biochemical properties of the three isoforms and focuses on recent advances on their protective function, involvement in human diseases and role as druggable targets. [ABSTRACT FROM AUTHOR]

Significant advances have been made in recent years in identifying the genetic components of Wallerian degeneration, the process that brings the progressive destruction and removal of injured axons. It has now been accepted that Wallerian degeneration is an active and dynamic cellular process that is well regulated at molecular and cellular levels. In this review, we describe our current understanding of Wallerian degeneration, focusing on the molecular players and mechanisms that mediate the injury response, activate the degenerative program, transduce the death signal, execute the destruction order, and finally, clear away the debris. By highlighting the starring roles and sketching out the molecular script of Wallerian degeneration, we hope to provide a useful framework to understand Wallerian and Wallerian-like degeneration and to lay a foundation for developing new therapeutic strategies to treat axon degeneration in neural injury as well as in neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Summary: SARM1 is an inducible TIR-domain NAD+ hydrolase that mediates pathological axon degeneration. SARM1 is activated by an increased ratio of NMN to NAD+, which competes for binding to an allosteric activating site. When NMN binds, the TIR domain is released from autoinhibition, activating its NAD+ hydrolase activity. The discovery of this allosteric activating site led us to hypothesize that other NAD+-related metabolites might activate SARM1. Here, we show the nicotinamide analog 3-acetylpyridine (3-AP), first identified as a neurotoxin in the 1940s, is converted to 3-APMN, which activates SARM1 and induces SARM1-dependent NAD+ depletion, axon degeneration, and neuronal death. In mice, systemic treatment with 3-AP causes rapid SARM1-dependent death, while local application to the peripheral nerve induces SARM1-dependent axon degeneration. We identify 2-aminopyridine as another SARM1-dependent neurotoxin. These findings identify SARM1 as a candidate mediator of environmental neurotoxicity and suggest that SARM1 agonists could be developed into selective agents for neurolytic therapy.

Nicotinamide adenine dinucleotide (NAD) is an essential coenzyme that mediates various redox reactions. Particularly, mitochondrial NAD plays a critical role in energy production pathways, including the tricarboxylic acid (TCA) cycle, fatty acid oxidation, and oxidative phosphorylation. NAD also serves as a substrate for ADP-ribosylation and deacetylation by poly(ADP-ribose) polymerases (PARPs) and sirtuins, respectively. Thus, NAD regulates energy metabolism, DNA damage repair, gene expression, and stress response. Numerous studies have demonstrated the involvement of NAD metabolism in neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), and retinal degenerative diseases. Mitochondrial dysfunction is considered crucial pathogenesis for neurodegenerative diseases such as AD and PD. Maintaining appropriate NAD levels is important for mitochondrial function. Indeed, decreased NAD levels are observed in AD and PD, and supplementation of NAD precursors ameliorates disease phenotypes by activating mitochondrial functions. NAD metabolism also plays an important role in axonal degeneration, a characteristic feature of peripheral neuropathy and neurodegenerative diseases. In addition, dysregulated NAD metabolism is implicated in retinal degenerative diseases such as glaucoma and Leber congenital amaurosis, and NAD metabolism is considered a therapeutic target for these diseases. In this review, we summarize the involvement of NAD metabolism in axon degeneration and various neurodegenerative diseases and discuss perspectives of nutritional intervention using NAD precursors. [ABSTRACT FROM AUTHOR]

The intracellular parasite Leishmania braziliensis is the etiological agent of cutaneous leishmaniasis, an endemic disease in the tropics, whose pharmacological treatments are toxic and for which there is currently no vaccine. For this reason, the study of proteins related to the energy metabolism of the parasite is relevant given its importance for its survival. In this study, based on the first 18 residues of the N-terminal end of the nicotinamide/nicotinate mononucleotide adenylyl transferase protein from L. braziliensis (Lb-NMNAT) as a template, peptides were synthesized implementing the Fmoc/tert-Butyl strategy in a Rink amide MBHA resin. The peptides were purified by C18 column chromatography and characterized by RP-HPLC. The recombinant 6xHisLb-NMNAT protein was expressed in Escherichia coli M15 cells and partially purified using immobilized metal affinity chromatography. The enzymatic activity of the protein was confirmed through direct enzymatic assays analyzed by RPHPLC. The synthesized peptides were used to evaluate their effect on the enzymatic activity of the 6xHisLb-NMNAT protein, observing a differential modulation, which is promising for the design of chemotherapeutic tools based on the N-terminal sequence of the Lb-NMNAT protein. [ABSTRACT FROM AUTHOR]

Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimer’s disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD+ synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model. By combining NMR spectroscopy, crystallography, single-molecule and computational approaches, we revealed that NMNAT adopts its enzymatic pocket to specifically bind the phosphorylated sites of pTau, which can be competitively disrupted by the enzymatic substrates of NMNAT. Moreover, we found that NMNAT serves as a co-chaperone of Hsp90 for the specific recognition of pTau over Tau. Our work uncovers a dedicated chaperone of pTau and suggests NMNAT as a key node between NAD+ metabolism and Tau homeostasis in aging and neurodegeneration.

Objecfive: Constructing the specific overexpression of Nmnat gene in the heart of fruit flies using UAS/ hand-gal4 system to study the effect of the overexpression of Nmnat gene in the heart combined with endurance exercise on lipid toxicity induced by high-fat diet, and to analyze the molecular mechanism. Methods: Male w1118 and Nmnat-UAS fruit flies were respectively hybridized with female hand-gal4. Collecting the normal expression of heart Nmnat (hand>w1118) and the overexpression (hand>Nmnat) of F1 generation female flies. After feeding for 15 days, they began to perform endurance exercise (E) and high-fat diet (HFD). They were divided into hand>w1118, hand>w1118+E, hand>w1118+HFD. hand>w1118+HFD+E, hand>Nmnat, hand>Nmnat +E, hand>Nmnat+HFD, hand>Nmnat+HFD+E, 410 flies in each group. The levels of cardiac TAG, NAD+, SIR2 protein deacetylation, SOD activity and MDA were detected by ELISA. The expression of cardiac related genes by RT-PCR; M-mode detects cardiac function. Results: the expression of Nmnat, dFoxo, bmm, NAD+ level, SIR2 deacetylation, SOD activity and shortened fraction of hand>w1118+HFD+E group were significantly higher than that of hand>w1118+HFD group (P<0.05 or P<0.01), but the expression of dFAS, Col4al, TAG and MDA level, heart rate and fibrillation were significantly lower than that of hand>w"18+HFD group (P<0.05 or P<0.01). There was no significant difference between the heart hand>Nmnat group and hand>Nmnat+HFD group (P>0.05). The expression of Nmnat, dFoxo, bmm, NAD+, SIR2 deacetylation, SOD activity and shortened heart fraction in the hand>Nmnat+HFD+E group were significantly higher than those in the hand>Nmnat+HFD group (P<0.05 or P<0.01), but the expression of dFAS, Col4al, TAG and MDA levels, heart rate and fibrillation were significantly lower than those in the hand>Nmnat+HFD group (P<0.05 or P<0.01). Conclusion: both overexpression of cardiac Nmnat and endurance exercise can prevent excessive accumulation of cardiac lipids, oxidative damage, decreased cardiac contractility and increased fibrillation induced by high-fat diet. The molecular mechanism is related to the fact that both of them can up-regulate the activity of cardiac Nm-nat/NAD+/SIR2/dFoxo pathway. The overexpression of cardiac Nmnat gene combined with endurance exercise can better reduce the occurrence of lipid toxic cardiomyopathy, which is related to the superposition effect of the combination on the up-regulation of cardiac Nmnat/NAD+/SIR2/dFoxo pathway activity. [ABSTRACT FROM AUTHOR]

Recombinant proteins have become useful tools in biochemistry research. During their production, however, inclusion bodies (IB) appear, on the one hand, due to the high expression rate from the recombinant plasmids, which have high efficiency promoters, and, on the other hand, intrinsic characteristics of the expressed protein. Furhtermore, the nicotinamide/nicotinate mononucleotide adenilyl transferase (NMNAT) is a central protein in NAD(H)+ biosynthesis, an essential cofactor in cell metabolism, and in protozoon parasite has been studied. To study the NMNAT protein of these parasites, their recombinant version in E. coli has been expressed, getting a great quantity of IB as a by-product. To increase the solubility of the protein, the coding sequence of the NMNAT enzyme of Plasmodium falciparum was cloned in different expression plasmids which were subsequently transformed into E. coli BL21(DE3) expression strain. The solubility of the recombinant proteins was assessed and the one with the highest presence in the soluble fraction was subsequently purified and its enzyme activity was determined. The recombinant protein with a MBP (maltose-binding protein) tag showed an increased solubility and purity.

BACKGROUND Nicotinamide adenine dinucleotide (NAD) plays a central role in energy metabolism and integrates cellular metabolism with signalling and gene expression. NAD biosynthesis depends on the enzyme nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT; EC: 2.7.7.1/18), in which converge the de novo and salvage pathways. OBJECTIVE The purpose of this study was to analyse the protein-protein interactions (PPI) of NMNAT of Leishmania braziliensis (LbNMNAT) in promastigotes. METHODS Transgenic lines of L. braziliensis promastigotes were established by transfection with the pSP72αneoαLbNMNAT-GFP vector. Soluble protein extracts were prepared, co-immunoprecipitation assays were performed, and the co-immunoprecipitates were analysed by mass spectrometry. Furthermore, bioinformatics tools such as network analysis were applied to generate a PPI network. FINDINGS Proteins involved in protein folding, redox homeostasis, and translation were found to interact with the LbNMNAT protein. The PPI network indicated enzymes of the nicotinate and nicotinamide metabolic routes, as well as RNA-binding proteins, the latter being the point of convergence between our experimental and computational results. MAIN CONCLUSION We constructed a model of PPI of LbNMNAT and showed its association with proteins involved in various functions such as protein folding, redox homeostasis, translation, and NAD synthesis.

Accumulative aggregation of mutant Huntingtin (Htt) is a primary neuropathological hallmark of Huntington's disease (HD). Currently, mechanistic understanding of the cytotoxicity of mutant Htt aggregates remains limited, and neuroprotective strategies combating mutant Htt-induced neurodegeneration are lacking. Here, we show that in Drosophila models of HD, neuronal compartment-specific accumulation of mutant Htt aggregates causes neurodegenerative phenotypes. In addition to the increase in the number and size, we discovered an age-dependent acquisition of thioflavin S+, amyloidlike adhesive properties of mutant Htt aggregates and a concomitant progressive clustering of aggregates with mitochondria and synaptic proteins, indicating that the amyloid-like adhesive property underlies the neurotoxicity of mutant Htt aggregation. Importantly, nicotinamide mononucleotide adenylyltransferase (NMNAT), an evolutionarily conserved nicotinamide adenine dinucleotide (NAD+) synthase and neuroprotective factor, significantly mitigates mutant Htt-induced neurodegeneration by reducing mutant Htt aggregation through promoting autophagic clearance. Additionally, Nmnat overexpression reduces progressive accumulation of amyloid-like Htt aggregates, neutralizes adhesiveness, and inhibits the clustering of mutant Htt with mitochondria and synaptic proteins, thereby restoring neuronal function. Conversely, partial loss of endogenous Nmnat exacerbates mutant Htt-induced neurodegeneration through enhancing mutant Htt aggregation and adhesive property. Finally, conditional expression of Nmnat after the onset of degenerative phenotypes significantly delays the progression of neurodegeneration, revealing the therapeutic potential of Nmnat-mediated neuroprotection at advanced stages of HD. Our study uncovers essential mechanistic insights to the neurotoxicity of mutant Htt aggregation and describes the molecular basis of Nmnat-mediated neuroprotection in HD. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD+) is an essential biomolecule involved in many critical processes. Its role as both a driver of energy production and a signaling molecule underscores its importance in health and disease. NAD+ signaling impacts multiple processes that are dysregulated in cancer, including DNA repair, cell proliferation, differentiation, redox regulation, and oxidative stress. Distribution of NAD+ is highly compartmentalized, with each subcellular NAD+ pool differentially regulated and preferentially involved in distinct NAD+-dependent signaling or metabolic events. Emerging evidence suggests that targeting NAD+ metabolism is likely to repress many specific mechanisms underlying tumor development and progression, including proliferation, survival, metabolic adaptations, invasive capabilities, heterotypic interactions with the tumor microenvironment, and stress response including notably DNA maintenance and repair. Here we provide a comprehensive overview of how compartmentalized NAD+ metabolism in mitochondria, nucleus, cytosol, and extracellular space impacts cancer formation and progression, along with a discussion of the therapeutic potential of NAD+-targeting drugs in cancer. [ABSTRACT FROM AUTHOR]

Gut motility malfunction and pathological changes in the enteric nervous system (ENS) are observed in the early stages of Parkinson’s disease (PD). In many cases disturbances in the autonomous functions such as gut motility precedes the observed loss of central motor functions in PD. However, the mechanism by which ENS degeneration occurs in PD is unknown. We show that parkinsonian mimetics rotenone and MPP+ induce neurite degeneration that precedes cell death in primary enteric neurons cultured in vitro. If the neuronal death signals originate from degenerating neurites, neuronal death should be prevented by inhibiting neurite degeneration. Our data demonstrate that overexpression of cytNmnat1, an axon protector, maintains healthy neurites in enteric neurons treated with either of the parkinsonian mimetics, but cannot protect the soma. We also demonstrate that neurite protection via cytNmnat1 is independent of mitochondrial dynamics or ATP levels. Overexpression of Bcl-xl, an anti-apoptotic factor, protects both the neuronal cell body and the neurites in both rotenone and MPP+ treated enteric neurons. Our data reveals that Bcl-xl and cytNmnat1 act through separate mechanisms to protect enteric neurites. Our findings suggest that neurite protection alone is not sufficient to inhibit enteric neuronal degeneration in rotenone or MPP+ toxicity, and enteric neurodegeneration in PD may be occurring through independent somatic and neuritic mechanisms. Thus, therapies targeting both axonal and somal protection can be important in finding interventions for enteric symptoms in PD. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor that mediates numerous biological processes in all living cells. Multiple NAD+ biosynthetic enzymes and NAD+-consuming enzymes are involved in neuroprotection and axon regeneration. The nematode Caenorhabditis elegans has served as a model to study the neuronal role of NAD+ because many molecular components regulating NAD+ are highly conserved. This review focuses on recent findings using C. elegans models of neuronal damage pertaining to the neuronal functions of NAD+ and its precursors, including a neuroprotective role against excitotoxicity and axon degeneration as well as an inhibitory role in axon regeneration. The regulation of NAD+ levels could be a promising therapeutic strategy to counter many neurodegenerative diseases, as well as neurotoxin-induced and traumatic neuronal damage.

The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the C. elegans mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD+) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration.

The biochemical pathways involved in nicotinamide adenine dinucleotide (NAD) biosynthesis converge at the enzymatic step catalysed by nicotinamide mononucleotide adenylyltransferase (NMNAT, EC: 2.7.7.1). The majority of NMNATs are assembled into homo-oligomeric states that comprise 2-6 subunits. Recently, the NMNAT of Plasmodium falciparum (PfNMNAT) has been identified as a pharmacological target. The enzymatic characterisation, cellular location, and tertiary structure of the PfNMNAT protein have been reported. Nonetheless, its quaternary structure remains to be explored. The present study describes the oligomeric assembly of the 6 x His-PfNMNAT recombinant protein using immobilised metal affinity chromatography coupled with size exclusion chromatography (SEC) and native protein electrophoresis combined with Ferguson plot graphing. These chromatographic approaches resulted in the elution of an active monomer from the SEC column, whereas the Ferguson plot indicated a dimeric assembly of the 6 x His-PfNMNAT protein.

Chemotherapy-induced peripheral neuropathy (CIPN) is the major dose-limiting side effect of many commonly used chemotherapeutic agents, including paclitaxel. Currently, there are no neuroprotective or effective symptomatic treatments for CIPN. Lack of understanding of the in vivo mechanisms of CIPN has greatly impeded the identification of therapeutic targets. Here, we optimized a model of paclitaxel-induced peripheral neuropathy using Drosophila larvae that recapitulates aspects of chemotherapy-induced sensory dysfunction. We showed that nociceptive sensitivity is associated with disrupted organization of microtubule-associated MAP1B/Futsch and aberrant stabilization of peripheral sensory dendrites. These findings establish a robust and amenable model for studying peripheral mechanisms of CIPN. Using this model, we uncovered a critical role for nicotinamide mononucleotide adenylyltransferase (Nmnat) in maintaining the integrity and function of peripheral sensory neurons and uncovered Nmnat's therapeutic potential against diverse sensory symptoms of CIPN.

Highlights • NAD levels decline with age due to an imbalance between its synthesis and destruction. • Decreased NAD metabolism is associated with various physiological aging processes. • NAD precursors protect against various aging-associated diseases. • Boosting NAD metabolism can extend the lifespan of diverse organisms. Abstract Nicotinamide adenine dinucleotide (NAD) is an important co-factor involved in numerous physiological processes, including metabolism, post-translational protein modification, and DNA repair. In living organisms, a careful balance between NAD production and degradation serves to regulate NAD levels. Recently, a number of studies have demonstrated that NAD levels decrease with age, and the deterioration of NAD metabolism promotes several aging-associated diseases, including metabolic and neurodegenerative diseases and various cancers. Conversely, the upregulation of NAD metabolism, including dietary supplementation with NAD precursors, has been shown to prevent the decline of NAD and exhibits beneficial effects against aging and aging-associated diseases. In addition, many studies have demonstrated that genetic and/or nutritional activation of NAD metabolism can extend the lifespan of diverse organisms. Collectively, it is clear that NAD metabolism plays important roles in aging and longevity. In this review, we summarize the basic functions of the enzymes involved in NAD synthesis and degradation, as well as the outcomes of their dysregulation in various aging processes. In addition, a particular focus is given on the role of NAD metabolism in the longevity of various organisms, with a discussion of the remaining obstacles in this research field. [ABSTRACT FROM AUTHOR]

Nmnat is a stress response protein which has been involved in a variety of biological processes. However, the effects of Nmnat on aging have not yet been investigated. The present study revealed the effects of Nmnat on aging of Drosophila and uncovered its underlying mechanism. Therefore, the overexpression of Nmnat was established by arm/Gal4 system in Drosophila with an aim to determine the functions of Nmnat during aging process. In this study, our results showed Nmnat was a positive factor on lifespan and movement capacity, which was consistent on d -galactose induced aging acceleration. Further investigation showed that oxidative stress biomarkers, longevity gene, mitochondria related genes and ATP levels were significantly improved in the Nmnat overexpression Drosophila , which suggested that the underlying mechanism of Nmnat on aging process and movement capacity was partly due to its anti-oxidative stress and mitochondrial-protection function. In addition, on H 2 O 2 challenge tests, Nmnat overexpression was sufficient to increase the survival time and movement capacity of Drosophila , which was probably due to its protection against oxidative stress. On rotenone induced mitochondrial dysfunction, Nmnat overexpression also showed better health span and movement capacity than the control group. Based on these data, Nmnat may be a new molecular target to improve health span by enhancing stress resistance and locomotor activity in aging process. [ABSTRACT FROM AUTHOR]

Summary Axons require the axonal NAD-synthesizing enzyme NMNAT2 to survive. Injury or genetically induced depletion of NMNAT2 triggers axonal degeneration or defective axon growth. We have previously proposed that axonal NMNAT2 primarily promotes axon survival by maintaining low levels of its substrate NMN rather than generating NAD; however, this is still debated. NMN deamidase, a bacterial enzyme, shares NMN-consuming activity with NMNAT2, but not NAD-synthesizing activity, and it delays axon degeneration in primary neuronal cultures. Here we show that NMN deamidase can also delay axon degeneration in zebrafish larvae and in transgenic mice. Like overexpressed NMNATs, NMN deamidase reduces NMN accumulation in injured mouse sciatic nerves and preserves some axons for up to three weeks, even when expressed at a low level. Remarkably, NMN deamidase also rescues axonal outgrowth and perinatal lethality in a dose-dependent manner in mice lacking NMNAT2. These data further support a pro-degenerative effect of accumulating NMN in axons in vivo. The NMN deamidase mouse will be an important tool to further probe the mechanisms underlying Wallerian degeneration and its prevention. [ABSTRACT FROM AUTHOR]

Protein misfolding is implicated in numerous neurodegenerative disorders including amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease. A unifying feature of patients with these disorders is the accumulation of deposits comprised of misfolded protein. Aberrant protein folding can cause toxicity through a loss or gain of protein function, or both. An intriguing therapeutic approach to counter these disorders is the application of protein-remodeling factors to resolve these misfolded conformers and return the proteins to their native fold and function. Here, we describe the application of protein-remodeling factors to alleviate protein misfolding in neurodegenerative disease. We focus on Hsp104, Hsp110/Hsp70/Hsp40, NMNAT, and HtrA1, which can prevent and reverse protein aggregation. While many of these protein-remodeling systems are highly promising, their activity can be limited. Thus, engineering protein-remodeling factors to enhance their activity could be therapeutically valuable. Indeed, engineered Hsp104 variants suppress neurodegeneration in animal models, which opens the way to novel therapeutics and mechanistic probes to help understand neurodegenerative disease. [ABSTRACT FROM AUTHOR]

WldS mutation protects axons from degeneration in diverse experimental models of neurological disorders, suggesting that the mutation might act on a key step shared by different axon degeneration pathways. Here we test the hypothesis that WldS protects axons by preventing energy deficiency commonly encountered in many diseases. We subjected compartmentally cultured, mouse cortical axons to energy deprivation with 6 mM azide and zero glucose. In wild-type (WT) culture, the treatment, which reduced axon ATP level ([ATP]axon) by 65%, caused immediate axon depolarization followed by gradual free calcium accumulation and subsequent irreversible axon damage. The calcium accumulation resulted from calcium influx partially via L-type voltage-gated calcium channel (L-VGCC). Blocking L-VGCC with nimodipine reduced calcium accumulation and protected axons. Without altering baseline [ATP]axon, the presence of WldS mutation significantly reduced the axon ATP loss and depolarization, restrained the subsequent calcium accumulation, and protected axons against energy deprivation. WldS neurons possessed higher than normal nicotinamide mononucleotide adenylyltransferase (NMNAT) activity. The intrinsic WldS NMNAT activity was required for the WldS-mediated energy preservation and axon protection during but not prior to energy deprivation. NMNAT catalyzes the reversible reaction that produces nicotinamide adenine dinucleotide (NAD) from nicotinamide mononucleotide (NMN). Interestingly, preventing the production of NAD from NMN with FK866 increased [ATP]axon and protected axons from energy deprivation. These results indicate that the WldS mutation depends on its intrinsic WldS NMNAT activity and the subsequent increase in axon ATP but not NAD to protect axons, implicating a novel role of WldS NMNAT in axon bioenergetics and protection.

The use of hens for the production of polyclonal antibodies reduces animal intervention and moreover yields a higher quantity of antibodies than other animal models. The phylogenetic distance between bird and mammal antigens, often leads to more specific avian antibodies than their mammalian counterparts.Since a large amount of antigen is required for avian antibody production, the use of recombinant proteins for this procedure has been growing faster over the last years. Nevertheless, recombinant protein production through heterologous systems frequently prompts the protein to precipitate, forming insoluble aggregates of limited utility (inclusion bodies). A methodology for the production of avian polyclonal antibodies, using recombinant protein from inclusion bodies is presented in this article.In order to produce the antigen, a recombinant Nicotinamide mononucleotide adenylyltransferase from Giardia intestinalis (His-GiNMNAT) was expressed in Escherichia coli. The protein was purified through solubilization from inclusion bodies prior to its renaturalization. Antibodies were purified from egg yolk of immunized hens by water dilution, followed by ammonium sulfate precipitation and thiophilic affinity chromatography.The purified antibodies were tested against His-GiNMNAT protein in Western blot essays. From one egg yolk, 14.4 mg of highly pure IgY were obtained; this antibody was able to detect 15ng of His-GiNMNAT. IgY specificity was improved by means of antigen affinity purification, allowing its use for parasite protein recognition.

Overexpression of the NAD+ biosynthetic enzyme NMNAT1 leads to preservation of injured axons. While increased NAD+ or decreased NMN levels are thought to be critical to this process, the mechanism(s) of this axon protection remain obscure. Using steady-state and flux analysis of NAD+ metabolites in healthy and injured mouse dorsal root ganglion axons, we find that rather than altering NAD+ synthesis, NMNAT1 instead blocks the injury-induced, SARM1-dependent NAD+ consumption that is central to axon degeneration.

Axon degeneration is a prominent feature of various neurodegenerative diseases, such as Parkinson’s and Alzheimer’s, and is often characterized by aberrant mitochondrial dynamics. Mitochondrial fission, fusion, and motility have been shown to be particularly important in progressive neurodegeneration. Thus we investigated these imperative dynamics, as well as mitochondrial fragmentation in vincristine induced axon degradation in cultured DRG neurons. CytNmnat1 inhibits axon degeneration in various paradigms including vincristine toxicity. The mechanism of its protection is not yet fully understood; therefore, we also investigated the effect of cytNmnat1 on mitochondrial dynamics in vincristine treated neurons. We observed that vincristine treatment decreases the rate of mitochondrial fission, fusion and motility and induces mitochondrial fragmentation. These mitochondrial events precede visible axon degeneration. Overexpression of cytNmnat1 inhibits axon degeneration and preserves the normal mitochondrial dynamics and motility in vincristine treated neurons. We suggest the alterations in mitochondrial structure and dynamics are early events which lead to axon degeneration and cytNmnat1 blocks axon degeneration by halting the vincristine induced changes to mitochondrial structure and dynamics.

Axon degeneration is a prominent feature of various neurodegenerative diseases, such as Parkinson's and Alzheimer's, and is often characterized by aberrant mitochondrial dynamics. Mitochondrial fission, fusion, and motility have been shown to be particularly important in progressive neurodegeneration. Thus we investigated these imperative dynamics, as well as mitochondrial fragmentation in vincristine induced axon degradation in cultured dorsal root ganglia (DRG) neurons. CytNmnat1 inhibits axon degeneration in various paradigms including vincristine toxicity. The mechanism of its protection is not yet fully understood; therefore, we also investigated the effect of cytNmnat1 on mitochondrial dynamics in vincristine treated neurons. We observed that vincristine treatment decreases the rate of mitochondrial fission, fusion and motility and induces mitochondrial fragmentation. These mitochondrial events precede visible axon degeneration. Overexpression of cytNmnat1 inhibits axon degeneration and preserves the normal mitochondrial dynamics and motility in vincristine treated neurons. We suggest the alterations in mitochondrial structure and dynamics are early events which lead to axon degeneration and cytNmnat1 blocks axon degeneration by halting the vincristine induced changes to mitochondrial structure and dynamics. [ABSTRACT FROM AUTHOR]

Nicotinic acid phosphoribosyltransferase (EC 2.4.2.11) (NaPRTase) is the rate-limiting enzyme in the three-step Preiss–Handler pathway for the biosynthesis of NAD. The enzyme catalyzes the conversion of nicotinic acid (Na) and 5-phosphoribosyl-1-pyrophosphate (PRPP) to nicotinic acid mononucleotide (NaMN) and pyrophosphate (PPi). Several studies have underlined the importance of NaPRTase for NAD homeostasis in mammals, but no crystallographic data are available for this enzyme from higher eukaryotes. Here, we report the crystal structure of human NaPRTase that was solved by molecular replacement at a resolution of 2.9 Å in its ligand-free form. Our structural data allow the assignment of human NaPRTase to the type II phosphoribosyltransferase subfamily and reveal that the enzyme consists of two domains and functions as a dimer with the active site located at the interface of the monomers. The substrate-binding mode was analyzed by molecular docking simulation and provides hints into the catalytic mechanism. Moreover, structural comparison of human NaPRTase with the other two human type II phosphoribosyltransferases involved in NAD biosynthesis, quinolinate phosphoribosyltransferase and nicotinamide phosphoribosyltransferase, reveals that while the three enzymes share a conserved overall structure, a few distinctive structural traits can be identified. In particular, we show that NaPRTase lacks a tunnel that, in nicotinamide phosphoribosiltransferase, represents the binding site of its potent and selective inhibitor FK866, currently used in clinical trials as an antitumoral agent. [ABSTRACT FROM AUTHOR]

The intracellular parasite Leishmania braziliensis is the causal agent of cutaneous and mucocutaneous leishmaniasis, a group of endemic diseases in tropical regions, including Latin America. New therapeutic targets are required to inhibit the pathogen without affecting the host. The enzyme nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT; EC: 2.7.7.1/18) is a potential target, since it catalyzes the final step in the biosynthesis of nicotinamide adenine dinucleotide (NAD + ), which is an essential metabolite in multiple cellular processes. In this work, we produced and evaluated the catalytic activity of the recombinant protein 6HisΔ 241-249 LbNMNAT to study the functional relevance of the exclusive insertion present in the enzyme of L. braziliensis (LbNMNAT), but absent in the primary structure of human NMNATs. Our results indicate that the 241-249 insertion constitutes a structural element that connects the protein structure Rossmann topology with the carboxyl-terminal domain of the enzyme. The removal of this region drastically decreases the solubility, and enzymatic activity of the recombinant, causing its inactivation. Molecular dynamics simulations were carried out with the wild-type and truncated enzymes to verify additional changes in their stability, which indicated a better stability in the wild-type protein. These findings constitute an initial step to identify a new inhibition mechanism for the development of focused pharmacological strategies on exclusive insertions from the LbNMNAT protein., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

The Wallenda (Wnd)/dual leucine zipper kinase (DLK)-Jnk pathway is an evolutionarily conserved MAPK signaling pathway that functions during neuronal development and following axonal injury. Improper pathway activation causes defects in axonal guidance and synaptic growth, whereas loss-of-function mutations in pathway components impairs axonal regeneration and degeneration after injury. Regulation of this pathway is in part through the E3 ubiquitin ligase Highwire (Hiw), which targets Wnd/DLK for degradation to limit MAPK signaling. To explore mechanisms controlling Wnd/DLK signaling, we performed a large-scale genetic screen in Drosophila to identify negative regulators of the pathway. Here we describe the identification and characterization of SkpA, a core component of SCF E3 ubiquitin ligases. Mutants in SkpA display synaptic overgrowth and an increase in Jnk signaling, similar to hiw mutants. The combination of hypomorphic alleles of SkpA and hiw leads to enhanced synaptic growth. Mutants in the Wnd-Jnk pathway suppress the overgrowth of SkpA mutants demonstrating that the synaptic overgrowth is due to increased Jnk signaling. These findings support the model that SkpA and the E3 ligase Hiw function as part of an SCF-like complex that attenuates Wnd/DLK signaling. In addition, SkpA, like Hiw, is required for synaptic and axonal responses to injury. Synapses in SkpA mutants are more stable following genetic or traumatic axonal injury, and axon loss is delayed in SkpA mutants after nerve crush. As in highwire mutants, this axonal protection requires Nmnat. Hence, SkpA is a novel negative regulator of the Wnd-Jnk pathway that functions with Hiw to regulate both synaptic development and axonal maintenance. [ABSTRACT FROM AUTHOR]

Abstract: Peripheral axons can re-extend robustly after nerve injury. Soon after a nerve crush regenerating axons grow through the nerve segment distal to the lesion in close proximity to distal axons that are still morphologically and molecularly preserved. Hence, following the progress of regenerating axons necessitates markers that can distinguish between regenerating and degenerating axons. Here, we show that axonal levels of superior cervical ganglion 10 (SCG10) are dynamically regulated after axonal injury and provide an efficient method to label regenerating axons. In contrast to the rapid loss of SCG10 in distal axons (Shin et al., 2012b), we report that SCG10 accumulates in the proximal axons within an hour after injury, leading to a rapid identification of the lesion site. The increase in SCG10 levels is maintained during axon regeneration after nerve crush or nerve repair and allows for more selective labeling of regenerating axons than the commonly used markers growth-associated protein 43 (GAP43) and YFP. SCG10 is preferentially expressed in regenerating sensory axons rather than motor axons in the sciatic nerve. In a mouse model of slow Wallerian degeneration, SCG10 labeling remains selective for regenerating axons and allows for a quantitative analysis of delayed regeneration in this mutant. Taken together, these data demonstrate the utility of SCG10 as an efficient and selective marker of sensory axon regeneration. [Copyright &y& Elsevier]

Highlights: [•] Origin of degeneration signal in amyloid β(Aβ) toxicity in hippocampal neurons was investigated. [•] Aβ1–42 toxicity-induced axon degeneration precedes cell death in hippocampal neurons. [•] Bcl-xl inhibited both axonal and cell body degeneration in the Aβ-42 treated neurons. [•] Inhibition of axon degeneration in Aβ1–42 treated neurons prevented neuronal cell death. [•] Conclusion: Axon degeneration is the key component of Aβ1–42 induced neuronal degeneration. [ABSTRACT FROM AUTHOR]

Highlights: [•] Neurons require a ‘maintenance plan’ that enables them to endure daily wear and tear. [•] NMNATs function as NAD synthesizing enzymes and chaperones. [•] Loss of NMNAT causes rapid and severe neurodegeneration. [•] NMNATs have the broadest and most potent neuroprotective effects. [•] Alzheimer's Disease, Parkinson's Disease, and other neurodegenerative disorders are accompanied by downregulation of NMNAT. [ABSTRACT FROM AUTHOR]

Abstract: WldS mutation protects axons from degeneration in diverse experimental models of neurological disorders, suggesting that the mutation might act on a key step shared by different axon degeneration pathways. Here we test the hypothesis that WldS protects axons by preventing energy deficiency commonly encountered in many diseases. We subjected compartmentally cultured, mouse cortical axons to energy deprivation with 6mM azide and zero glucose. In wild-type (WT) culture, the treatment, which reduced axon ATP level ([ATP]axon) by 65%, caused immediate axon depolarization followed by gradual free calcium accumulation and subsequent irreversible axon damage. The calcium accumulation resulted from calcium influx partially via L-type voltage-gated calcium channel (L-VGCC). Blocking L-VGCC with nimodipine reduced calcium accumulation and protected axons. Without altering baseline [ATP]axon, the presence of WldS mutation significantly reduced the axon ATP loss and depolarization, restrained the subsequent calcium accumulation, and protected axons against energy deprivation. WldS neurons possessed higher than normal nicotinamide mononucleotide adenylyltransferase (NMNAT) activity. The intrinsic WldS NMNAT activity was required for the WldS -mediated energy preservation and axon protection during but not prior to energy deprivation. NMNAT catalyzes the reversible reaction that produces nicotinamide adenine dinucleotide (NAD) from nicotinamide mononucleotide (NMN). Interestingly, preventing the production of NAD from NMN with FK866 increased [ATP]axon and protected axons from energy deprivation. These results indicate that the WldS mutation depends on its intrinsic WldS NMNAT activity and the subsequent increase in axon ATP but not NAD to protect axons, implicating a novel role of WldS NMNAT in axon bioenergetics and protection. [Copyright &y& Elsevier]

Over 100 years after its first discovery, several new aspects of the biology of the redox co-factor NAD are rapidly emerging. NAD, as well as its precursors, its derivatives, and its metabolic enzymes, have been recently shown to play a determinant role in a variety of biological functions, from the classical role in oxidative phosphorylation and redox reactions to a role in regulation of gene transcription, lifespan and cell death, from a role in neurotransmission to a role in axon degeneration, and from a function in regulation of glucose homeostasis to that of control of circadian rhythm. It is also becoming clear that this variety of specialized functions is regulated by the fine subcellular localization of NAD, its related nucleotides and its metabolic enzymatic machinery. Here we describe the known NAD biosynthetic and catabolic pathways, and review evidence supporting a specialized role for NAD metabolism in a subcellular compartment-dependent manner. [ABSTRACT FROM AUTHOR]

Axon degeneration is observed at the early stages of many neurodegenerative conditions and this often leads to subsequent neuronal loss. We previously showed that inactivating the c-Jun N-terminal kinase (JNK) pathway leads to axon degeneration in Drosophila mushroom body (MB) neurons. To understand this process, we screened candidate suppressor genes and found that the Wallerian degeneration slow (WldS) protein blocked JNK axonal degeneration. Although the nicotinamide mononucleotide adenylyltransferase (Nmnat1) portion of WldS is required, we found that its nicotinamide adenine dinucleotide (NAD+) enzyme activity and the WldS N-terminus (N70) are dispensable, unlike axotomy models of neurodegeneration. We suggest that WldS-Nmnat protects against axonal degeneration through chaperone activity. Furthermore, ectopically expressed heat shock proteins (Hsp26 and Hsp70) also protected against JNK and Nmnat degeneration phenotypes. These results suggest that molecular chaperones are key in JNK- and Nmnat-regulated axonal protective functions. [ABSTRACT FROM AUTHOR]

Abstract: The selective degeneration of dendrites precedes neuronal cell death in hypoxia–ischemia (HI) and is a neuropathological hallmark of stroke. While it is clear that a number of different molecular pathways likely contribute to neuronal cell death in HI, the mechanisms that govern HI-induced dendrite degeneration are largely unknown. Here, we show that the NAD synthase nicotinamide mononucleotide adenylyltransferase (Nmnat) functions endogenously to protect Drosophila class IV dendritic arborization (da) sensory neurons against hypoxia-induced dendritic damage. Whereas dendrites of wild-type class IV neurons are largely resistant to morphological changes during prolonged periods of hypoxia (<1.0% O2), class IV neurons of nmnat heterozygous mutants exhibit significant dendrite loss and extensive fragmentation of the dendritic arbor under the same hypoxic conditions. Although basal levels of autophagy are required for neuronal survival, we demonstrate that autophagy is dispensable for maintaining the dendritic integrity of class IV neurons. However, we find that genetically blocking autophagy can suppress hypoxia-induced dendrite degeneration of nmnat heterozygous mutants in a cell-autonomous manner, suggestive of a self-destructive role for autophagy in this context. We further show that inducing autophagy by overexpression of the autophagy-specific kinase Atg1 is sufficient to cause dendrite degeneration of class IV neurons under hypoxia and that overexpression of Nmnat fails to protect class IV dendrites from the effects of Atg1 overexpression. Our studies reveal an essential neuroprotective role for endogenous Nmnat in hypoxia and demonstrate that Nmnat functions upstream of autophagy to mitigate the damage incurred by dendrites in neurons under hypoxic stress. [Copyright &y& Elsevier]

Although the nicotinamide nucleotides NAD(H) and NADP(H) are essential for various metabolic reactions that play major roles in maintenance of cellular homeostasis, the significance of NAD biosynthesis is not well understood. Here, we investigated the dynamics of pollen nicotinamide nucleotides in response to imbibition, a representative germination cue. Metabolic analysis with capillary electrophoresis electrospray ionization mass spectrometry revealed that excess amount of NAD+ is accumulated in freshly harvested dry pollen, whereas it dramatically decreased immediately after contact with water. Importantly, excess of NAD+ impaired pollen tube growth. Moreover, NAD+ accumulation was retained after pollen was imbibed in the presence of NAD+-consuming reaction inhibitors and pollen germination was greatly retarded. Pollen deficient in the nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) gene, encoding a key enzyme in NAD biosynthesis, and a lack of NAD+ accumulation in the gametophyte, showed precocious pollen tube germination inside the anther locule and vigorous tube growth under high-humidity conditions. Hence, the accumulation of excess NAD+ is not essential for pollen germination, but instead participates in regulating the timing of germination onset. These results indicate that NAD+ accumulation acts to negatively regulate germination and a decrease in NAD+ plays an important role in metabolic state transition. [ABSTRACT FROM AUTHOR]

Active zones are specialized presynaptic structures critical for neurotransmission. We show that a neuronal maintenance factor, nicotinamide mononucleotide adenylyltransferase (NMNAT), is required for maintaining active zone structural integrity in Drosophila by interacting with the active zone protein, Bruchpilot (BRP), and shielding it from activity-induced ubiquitin-proteasome-mediated degradation. NMNAT localizes to the peri-active zone and interacts biochemically with BRP in an activity-dependent manner. Loss of NMNAT results in ubiquitination, mislocalization and aggregation of BRP, and subsequent active zone degeneration. We propose that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein-protein interaction. [ABSTRACT FROM AUTHOR]