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(A) Quantification of NAD in control or FKSG67-transfected HEK cells. The effect of NAD, nicotinamide (Nam), NMN, nicotinamide riboside (NR) or nicotinic acid (NA) on NAD contents of FKSG-transfected cells is shown (NAD and its precursors have been added to the incubating media at 1 mM for 48 hrs). Basal NAD content was 12.6±2 nmol/mg prot. (B) Western blotting evaluation of the effect of NAD, Nam, NMN, NR or nicotinic acid NA (1 mM/48 hrs) on depletion of mitochondrial PAR content induced by FKSG76 co-transfection in mitoPARP1cd-transfected cells. Tubulin is shown as loading control. (C) Densitometric analysis on the experiment shown in (B). (D) Effect of oligomycin (10 µM/30 min) and/or glucose deprivation (30 min) on cellular ATP contents. (E) Effects of exogenous NAD (1 mM/3 hrs) on mitochondrial PAR contents in mitoPARP1cd-transfected cells under control conditions or exposed to oligomycin (10 µM) in the presence or absence of glucose. (F) Densitometric analysis on the experiment shown in (E). Columns represent the mean ± SEM of 3 experiments. Western blotting is representative of 3 (E) and 4 (C) experiments.* p<0.05; ** p<0.01 vs control (Student's t test).
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Among the enzymes involved in NAD homeostasis, nicotinamide mononucleotide adenylyltransferases (NMNAT1-3) are central to intracellular NAD formation. Although NMNAT3 is postulated to be a mitochondrial enzyme contributing to NAD-dependent organelle functioning, information on endogenous proteins is lacking. We report that in human cells a single g...
Citations
... Yet no study has confirmed NAD + being converted from NMN in isolated mitochondria. 42,43 The discovery of mitochondrial NAD + transporters further supports the direct transport of mitochondrial NAD + from cytosol. However, the knockdown of NMNAT3 also decreases mitochondrial NAD + , 22 implying that NMN contributes to the mitochondrial NAD + pool. ...
Nicotinamide mononucleotide (NMN) is a major precursor for NAD + metabolism with promising effects in treating NAD +-and aging-related pathologies. However, measuring live cell NMN dynamics was not possible, leaving key questions in intracellular NMN uptake and regulation unanswered. Here we developed a genetically encoded bioluminescent sensor to quantify subcellular NMN in live cells by fusing engineered NMN-responsive binding domain to bioluminescent and fluorescent proteins from BRET pairs. The sensor dissected the multimechanistic uptake of extracellular NMN and precursors in live cells. We then captured the notably low mitochondrial NMN content and the thereafter vulnerable NMN/NAD + ratio and SARM1 activation in mitochondria, establishing NMN/NAD + ratio as an important parameter in evaluating NAD + boosting strategies. Moreover, we characterized the signature of major NAD + regulating enzymes on NMN and NMN/NAD + ratios, in which Slc25a45 was identified to be a potential mitochondrial NMN transporter for its unique fingerprint on mitochondrial NMN/NAD + ratio.
... NMNAT3 expression was greatest in rods and cones, followed by RGCs ( Fig. 2A-C), given that NMNAT3 mRNA has a mitochondrial targeting sequence this would appear to fit with the high density of mitochondria in these neurons. However, NMNAT3 is known to be translationally repressed due to an upstream open reading frame in the mRNA 5′UTR region and mature protein has only been identified in cells following over-expression through plasmid transfection [17]. Although rods, cones, and RGCs express NMNAT3 it is, therefore, unlikely to have a functional role at a protein level in these cells. ...
Glaucoma is the leading cause of irreversible blindness and is a major health and economic burden. Current treatments do not address the neurodegenerative component of glaucoma. In animal models of glaucoma, the capacity to maintain retinal nicotinamide adenine dinucleotide (NAD) pools declines early during disease pathogenesis. Treatment with nicotinamide, an NAD precursor through the NAD salvage pathway, robustly protects against neurodegeneration in a number of glaucoma models and improves vision in existing glaucoma patients. However, it remains unknown in humans what retinal cell types are able to process nicotinamide to NAD and how these are affected in glaucoma. To address this, we utilized publicly available RNA-sequencing data (bulk, single cell, and single nucleus) and antibody labelling in highly preserved enucleated human eyes to identify expression of NAD synthesizing enzyme machinery. This identifies that the neural retina favors expression of the NAD salvage pathway, and that retinal ganglion cells are particularly enriched for these enzymes. NMNAT2, a key terminal enzyme in the salvage pathway, is predominantly expressed in retinal ganglion cell relevant layers of the retina and declines in glaucoma. These findings suggest that human retinal ganglion cells can directly utilize nicotinamide and could maintain a capacity to do so in glaucoma, showing promise for ongoing clinical trials.
... NMNAT1 is a nuclear enzyme that is ubiquitously expressed [187,188]. In contrast, NMNAT2 is associated with the Golgi and acts in the cytoplasm [188,189], while NMNAT3 has been identified in both cytosolic and mitochondrial compartments, with cell/tissue-specific subcellular localization patterns [189][190][191]. The presence of NMNAT enzymes in different compartments suggest that salvage of NAD + precursors might occur in multiple cellular locations. ...
Alterations in cellular nicotinamide adenine dinucleotide (NAD⁺) levels have been observed in multiple lifestyle and age-related medical conditions. This has led to the hypothesis that dietary supplementation with NAD⁺ precursors, or vitamin B3s, could exert health benefits. Among the different molecules that can act as NAD⁺ precursors, Nicotinamide Riboside (NR) has gained most attention due to its success in alleviating and treating disease conditions at the pre-clinical level. However, the clinical outcomes for NR supplementation strategies have not yet met the expectations generated in mouse models. In this review we aim to provide a comprehensive view on NAD⁺ biology, what causes NAD⁺ deficits and the journey of NR from its discovery to its clinical development. We also discuss what are the current limitations in NR-based therapies and potential ways to overcome them. Overall, this review will not only provide tools to understand NAD⁺ biology and assess its changes in disease situations, but also to decide which NAD⁺ precursor could have the best therapeutic potential.
... The regulation of NAD + synthesis involves many enzymes, including plays an essential role in the synthesis of NAD + in the mitochondria [30][31][32] . ...
... NMNAT3 is a key enzyme in the synthesis of NAD + in mitochondria, which plays an essential role in the regulation of NAD + homeostasis [30][31][32][33][34] . ...
Oxidative stress damage is a common problem in bone marrow mesenchymal stem cell (BMSC) transplantation. Under stress conditions, the mitochondrial function of BMSCs is disrupted, which accelerates senescence and apoptosis of BMSCs, ultimately leading to poor efficacy. Therefore, improving mitochondrial function and enhancing the anti-oxidative stress capacity of BMSCs may be an effective way of improving the survival rate and curative effect of BMSCs. In this study, we have confirmed that overexpression of nicotinamide mononucleotide adenylyl transferase 3 (NMNAT3) improves mitochondrial function and resistance to stress-induced apoptosis in BMSCs. We further revealed the mechanism of NMNAT3-mediated resistance to stress-induced apoptosis in BMSCs. We increased the level of nicotinamide adenine dinucleotide (NAD+) by overexpressing NMNAT3 in BMSCs and found that it could significantly increase the activity of silent mating type information regulation 2 homolog 3 (Sirt3) and significantly decrease the acetylation levels of Sirt3-dependent deacetylation-related proteins isocitrate dehydrogenase 2 (Idh2) and Forkhead-box protein O3a (FOXO3a). These findings show that NMNAT3 may increase the activity of Sirt3 by increasing NAD+ levels. Our results confirm that the NMNAT3-NAD+-Sirt3 axis is a potential mechanism for improving mitochondrial function and enhancing anti-oxidative stress of BMSCs. In this study, we take advantage of the role of NMNAT3 in inhibiting stress-induced apoptosis of BMSCs and provide new methods and ideas for breaking through the bottleneck of transplantation efficacy of BMSCs in the clinic.
... 50 However, its contribution to NAD biosynthesis in mitochondria where NAD concentration ranges from 300 to 500 μM 54,57 is matter of debate. Some authors showed that NMNAT3 is dispensable for mitochondrial NAD maintenance, 58,59 whereas others evidenced an important role in regulating mitochondrial NAD levels 15,29 and mitochondrial mono ADP-ribosylation. 14 A recent study showed that inside endolysosomes, NMNAT3 is responsible, together with CD38, of the production of NAADP, a potent Ca +2 -mobilizing second messenger. ...
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.
... The salvage synthesis of NMN from NAM is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT), whereas the synthesis of NMN from NR is catalyzed by intracellular NR kinases (NRK1 and NRK2) [43]. Finally, NMN may be subsequently transformed into NAD+ by different subcellular forms of adenylyltransferases (NMNAT1-3) [44]. ...
Cardiovascular diseases are the leading cause of death worldwide. Aging and/or metabolic stress directly impact the cardiovascular system. Over the last few years, the contributions of altered nicotinamide adenine dinucleotide (NAD+) metabolism to aging and other pathological conditions closely related to cardiovascular diseases have been intensively investigated. NAD+ bioavailability decreases with age and cardiometabolic conditions in several mammalian tissues. Compelling data suggest that declining tissue NAD+ is commonly related to mitochondrial dysfunction and might be considered as a therapeutic target. Thus, NAD+ replenishment by either genetic or natural dietary NAD+-increasing strategies has been recently demonstrated to be effective for improving the pathophysiology of cardiac and vascular health in different experimental models as well as human health to a lesser extent. Here, we review and discuss recent experimental evidence illustrating that increasing NAD+ bioavailability, particularly by the use of natural NAD+ precursors, may offer hope for new therapeutic strategies to prevent and treat cardiovascular diseases.
... Another study suggests the existence of an unrecognized mammalian NAD + (or NADH) transporter in mitochondria (Davila et al, 2018), though the mitochondrial inner membrane is considered impermeable to pyridine nucleotides, raising the possibility that Nmnat3 in the cytosol can also regulate the mitochondrial NAD + levels. Conversely, it has been suggested that cytosolic NAD + maintains the mitochondrial NAD + pool via cytosolic Nmnat3 (Felici et al, 2013). ...
Mitochondrial translation dysfunction is associated with neurodegenerative and cardiovascular diseases. Cells eliminate defective mitochondria by the lysosomal machinery via autophagy. The relationship between mitochondrial translation and lysosomal function is unknown. In this study, mitochondrial translation-deficient hearts from p32-knockout mice were found to exhibit enlarged lysosomes containing lipofuscin, suggesting impaired lysosome and autolysosome function. These mice also displayed autophagic abnormalities, such as p62 accumulation and LC3 localization around broken mitochondria. The expression of genes encoding for nicotinamide adenine dinucleotide (NAD+ ) biosynthetic enzymes-Nmnat3 and Nampt-and NAD+ levels were decreased, suggesting that NAD+ is essential for maintaining lysosomal acidification. Conversely, nicotinamide mononucleotide (NMN) administration or Nmnat3 overexpression rescued lysosomal acidification. Nmnat3 gene expression is suppressed by HIF1α, a transcription factor that is stabilized by mitochondrial translation dysfunction, suggesting that HIF1α-Nmnat3-mediated NAD+ production is important for lysosomal function. The glycolytic enzymes GAPDH and PGK1 were found associated with lysosomal vesicles, and NAD+ was required for ATP production around lysosomal vesicles. Thus, we conclude that NAD+ content affected by mitochondrial dysfunction is essential for lysosomal maintenance.
... Due to its critical function within mitochondria, there has been a steady stream of studies leading to the discovery of the source of the mitochondrial NAD + pool [5] and, eventually, the mammalian mitochondrial NAD + transporter [1][2][3]. Specifically, previous studies reported that mammalian cells cannot synthesize NAD + within mitochondria [6], and that NAD + is transported into mitochondria [7]. Girardi et al. and Kory et al. recently used gene co-essentiality data to generate the hypothesis that SLC25A51 is the mitochondrial NAD + transporter [1,2]. ...
Recently, three groups, Girardi et al., Kory et al., and Luongo et al., independently identified solute carrier (SLC) 25A51 as the long-sought, major mitochondrial NAD⁺ transporter in mammalian cells. These studies not only deorphan an uncharacterized transporter of the SLC25A family, but also shed light on other aspects of NAD⁺ biology.
... NAD + is produced in each compartment by the resident NMNAT [24]. NMNAT3 is not essential to maintain NAD + levels in mitochondria, thus other NMNATs may have a major role [25]. In humans, the gene nmnat3 codes for two mRNAs, NMNAT3v1 and FKSG76, through alternative splicing: NMNAT3v1 protein is cytosolic and inactive, whereas FKSG76, localized to mitochondria, cleaves NAD + in the reverse reaction, more favorable than NAD + synthesis [25]. ...
... NMNAT3 is not essential to maintain NAD + levels in mitochondria, thus other NMNATs may have a major role [25]. In humans, the gene nmnat3 codes for two mRNAs, NMNAT3v1 and FKSG76, through alternative splicing: NMNAT3v1 protein is cytosolic and inactive, whereas FKSG76, localized to mitochondria, cleaves NAD + in the reverse reaction, more favorable than NAD + synthesis [25]. NMN production with NAD + degradation has also been reported for NMNAT2 [26]. ...
... De novo NAD + synthesis or increased availability of NAD precursors may support health quality during aging [147] and improve cognitive functions through a decrease of Aβ fibrils. Nicotinamide or NMN treatment preserved mitochondrial integrity in mouse models of AD [62,148], and may function in the therapy of pathological processes such as diabetes, ischemia-reperfusion injury, heart failure are age-related diseases [25,149]. The application of NMN and NR may sustain the need for higher levels of NAD + , either due to higher consumption, or to reduced synthesis. ...
Mitochondrial dysfunction and oxidative stress are prominent features of a plethora of human disorders. Dysregulation of mitochondrial functions represents a common pathogenic mechanism of diseases such as neurodegenerative disorders and cancer. The maintenance of the Nicotinamide adenine dinucleotide (NAD+ ) pool, and a positive NAD+ /NADH ratio, are essential for mitochondrial and cell functions. The synthesis and degradation of NAD+ and transport of its key intermediates among cell compartments play an important role to maintain optimal NAD levels, for regulation of NAD+ -utilizing enzymes, such as sirtuins (Sirt), poly-ADP-ribose polymerases, and CD38/157 enzymes, either intracellularly as well as extracellularly. In this review, we present and discuss the links between NAD+ , NAD+ -consuming enzymes, mitochondria functions, and diseases. Attempts to treat various diseases with supplementation of NAD+ cycling intermediates and inhibitors of sirtuins and ADP-ribosyl transferases may highlight a possible therapeutic approach for therapy of cancer and neurodegenerative diseases.
... Consequently, the enzyme may not be solely involved in mitochondrial NAD + biosynthesis. In fact, the NMNAT-catalyzed reaction is fully reversible 38,83 , the equilibrium favoring the production of NMN and ATP from NAD + and pyrophosphate (PPi) (Fig. 7E). Therefore, in cooperation with SLC25A51/MCART1, NMNAT3 could act as a rheostat buffering cellular NAD + levels according to demand (Fig. 7E). ...
The coenzyme NAD is consumed by signaling enzymes including poly-ADP-ribose-polymerases (PARPs) and sirtuins. Understanding the mechanisms of aging-associated NAD decline and how cells cope with decreased NAD concentrations requires model systems reflecting chronic NAD deficiency. To evoke compartment-specific over-consumption of NAD, we have engineered cell lines expressing PARP activity in mitochondria, the cytosol, endoplasmic reticulum, or peroxisomes. Irrespective of the compartment targeted, total cellular NAD concentrations declined by ~40%. Isotope-tracer flux measurements and mathematical modeling showed that the lowered NAD concentration limits total NAD consumption kinetically. Moreover, NAD biosynthesis rate and capacity remained unchanged, thereby also precluding an increase of total NAD turnover. The chronic NAD deficiency was surprisingly well tolerated unless the mitochondria were targeted. Oxidative phosphorylation and glycolysis were little affected by NAD over-consumption in the other compartments. Likewise, peroxisomal NAD over-consumption was balanced by mitochondrial NAD decrease to maintain beta-oxidation of very long chain fatty acids in peroxisomes. We propose that subcellular NAD pools are interconnected, with mitochondria acting as a rheostat to facilitate NAD-dependent processes in organelles with excessive consumption.
