Augmentation of cellular NAD+ by NQO1 enzymatic action improves age-related hearing impairment.
ABSTRACT: Age-related hearing loss (ARHL) is a major neurodegenerative disorder and the leading cause of communication deficit in the elderly population, which remains largely untreated. The development of ARHL is a multifactorial event that includes both intrinsic and extrinsic factors. Recent studies suggest that NAD+ /NADH ratio may play a critical role in cellular senescence by regulating sirtuins, PARP-1, and PGC-1?. Nonetheless, the beneficial effect of direct modulation of cellular NAD+ levels on aging and age-related diseases has not been studied, and the underlying mechanisms remain obscure. Herein, we investigated the effect of ?-lapachone (?-lap), a known plant-derived metabolite that modulates cellular NAD+ by conversion of NADH to NAD+ via the enzymatic action of NADH: quinone oxidoreductase 1 (NQO1) on ARHL in C57BL/6 mice. We elucidated that the reduction of cellular NAD+ during the aging process was an important contributor for ARHL; it facilitated oxidative stress and pro-inflammatory responses in the cochlear tissue through regulating sirtuins that alter various signaling pathways, such as NF-?B, p53, and IDH2. However, augmentation of NAD+ by ?-lap effectively prevented ARHL and accompanying deleterious effects through reducing inflammation and oxidative stress, sustaining mitochondrial function, and promoting mitochondrial biogenesis in rodents. These results suggest that direct regulation of cellular NAD+ levels by pharmacological agents may be a tangible therapeutic option for treating various age-related diseases, including ARHL.
Project description:NAD<sup>+</sup> was discovered during yeast fermentation, and since its discovery, its important roles in redox metabolism, aging, and longevity, the immune system and DNA repair have been highlighted. A deregulation of the NAD<sup>+</sup> levels has been associated with metabolic diseases and aging-related diseases, including neurodegeneration, defective immune responses, and cancer. NAD<sup>+</sup> acts as a cofactor through its interplay with NADH, playing an essential role in many enzymatic reactions of energy metabolism, such as glycolysis, oxidative phosphorylation, fatty acid oxidation, and the TCA cycle. NAD<sup>+</sup> also plays a role in deacetylation by sirtuins and ADP ribosylation during DNA damage/repair by PARP proteins. Finally, different NAD hydrolase proteins also consume NAD<sup>+</sup> while converting it into ADP-ribose or its cyclic counterpart. Some of these proteins, such as CD38, seem to be extensively involved in the immune response. Since NAD cannot be taken directly from food, NAD metabolism is essential, and NAMPT is the key enzyme recovering NAD from nicotinamide and generating most of the NAD cellular pools. Because of the complex network of pathways in which NAD<sup>+</sup> is essential, the important role of NAD<sup>+</sup> and its key generating enzyme, NAMPT, in cancer is understandable. In the present work, we review the role of NAD<sup>+</sup> and NAMPT in the ways that they may influence cancer metabolism, the immune system, stemness, aging, and cancer. Finally, we review some ongoing research on therapeutic approaches.
Project description:Age-dependent decrease of mitochondrial energy production and cellular redox imbalance play significant roles in age-related hearing loss (ARHL). Lactate dehydrogenase B (LDHB) is a key glycolytic enzyme that catalyzes the interconversion of pyruvate and lactate. LDH activity and isoenzyme patterns are known to be changed with aging, but the role of LDHB in ARHL has not been studied yet. Here, we found that LDHB knockout mice showed hearing loss at high frequencies, which is the typical feature of ARHL. LDHB knockdown caused downregulation of mitochondrial functions in auditory cell line, University of Bristol/organ of Corti 1 (UB/OC1) with decreased NAD<sup>+</sup> and increased hypoxia inducing factor-1?. LDHB knockdown also enhanced the death of UB/OC1 cells with ototoxic gentamicin treatment. On the contrary, the induction of LDHB expression caused enhanced mitochondrial functions, including changes in mitochondrial respiratory subunits, mitochondrial membrane potentials, ATP, and the NAD<sup>+</sup>/NADH ratio. Thus, we concluded that suppression of LDHB activity may be closely related with the early onset or progression of ARHL.
Project description:<h4>Significance</h4>Sirtuins are an evolutionarily conserved family of NAD<sup>+</sup>-dependent lysine deacylases and ADP ribosylases. Their requirement for NAD<sup>+</sup> as a cosubstrate allows them to act as metabolic sensors that couple changes in the energy status of the cell to changes in cellular physiological processes. NAD<sup>+</sup> levels are affected by several NAD<sup>+</sup>-producing and NAD<sup>+</sup>-consuming pathways as well as by cellular respiration. Thus their intracellular levels are highly dynamic and are misregulated in a spectrum of metabolic disorders including cerebral ischemia. This, in turn, compromises several NAD<sup>+</sup>-dependent processes that may ultimately lead to cell death. Recent Advances: A number of efforts have been made to replenish NAD<sup>+</sup> in cerebral ischemic injuries as well as to understand the functions of one its important mediators, the sirtuin family of proteins through the use of pharmacological modulators or genetic manipulation approaches either before or after the insult. Critical Issues and Future Directions: The results of these studies have regarded the sirtuins as promising therapeutic targets for cerebral ischemia. Yet, additional efforts are needed to understand the role of some of the less characterized members and to address the sex-specific effects observed with some members. Sirtuins also exhibit cell-type-specific expression in the brain as well as distinct subcellular and regional localizations. As such, they are involved in diverse and sometimes opposing cellular processes that can either promote neuroprotection or further contribute to the injury; which also stresses the need for the development and use of sirtuin-specific pharmacological modulators. Antioxid. Redox Signal. 28, 691-710.
Project description:Acute pancreatitis (AP) is a complicated disease without specific drug therapy. The cofactor nicotinamide adenine dinucleotide (NAD<sup>+</sup>) is an important regulator of cellular metabolism and homeostasis. However, it remains unclear whether modulation of NAD<sup>+</sup> levels has an impact on caerulein-induced AP. Therefore, in this study, we investigated the effect of increased cellular NAD<sup>+</sup> levels on caerulein-induced AP. We demonstrated for the first time that the activities and expression of SIRT1 were suppressed by reduction of intracellular NAD<sup>+</sup> levels and the p53-microRNA-34a pathway in caerulein-induced AP. Moreover, we confirmed that the increase of cellular NAD<sup>+</sup> by NQO1 enzymatic action using the substrate ?-Lapachone suppressed caerulein-induced AP with down-regulating TLR4-mediated inflammasome signalling, and thereby reducing the inflammatory responses and pancreatic cell death. These results suggest that pharmacological stimulation of NQO1 could be a promising therapeutic strategy to protect against pathological tissue damage in AP.
Project description:β-lapachone (b-lap) is an anticancer agent that selectively induces cell death in human cancer cells. A mechanism for b-lap cytotoxicity was shown to be mediated by the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation in several tumour cells. To further characterise the molecular effects of b-lap action, we compared the gene expression profile of yeast cells treated or not with b-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were enriched in the set of differentially expressed genes. Accordingly, b-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, and inhibitor of NADH dehydrogenases (NADH-DH). In addition, a mutant defective in the mitocondrial NADH dehydrogenase Nde2p was found to be resistant to b-lap treatment, thus resembling tumour cell responses to b-lap exposure. However, b-lap treatment elicited similar ROS production in WT and nde2 cells, and dicoumarol did not affect cell viability in b-lap treated yeasts. Most interestingly, DNA damage responses triggered by b-lap were abolished in the nde2 mutant. Furthermore, the nde2 mutant was sensitive to DNA damaging agents other than b-lap. These data indicated that ROS production did not mediate b-lap cytotoxicity and highlighted a role of Nde2p in DNA damage checkpoints. Amino acid biosynthesis genes were also differentially expressed in b-lap treated cells, suggesting that b-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Supporting this, b-lap treatment increased phosphorylation of eIF2 alpha in a Gcn2p-dependent manner. eIF2alpha phosphorylation required Gcn1p and Gcn20p and surprisingly Nde2p. Gcn2p was also shown to be required for the G1 checkpoint triggered by b-lap and for cell survival. Remarkably, b-lap treatment also increased phosphorylation of eIF2 alpha in breast tumour cells, which was partially dependent on the Nde2p orthologue AMID. These findings i) suggest that Gcn2p and Nde2p are key determinants of b-lap toxicity in yeast and human cells, ii) uncover a new functional connection between Nde2p, Gcn2p and DNA damage checkpoints conserved throughout evolution and iii) suggest that pharmacological modulation of Gcn2p could provide an effective strategy for antitumour drug discovery. Direct comparison between wt samples and b-lapachone treated-wt samples. Four biological replicates. Dye was swapped in two of them
Project description:Mono(ADP-ribose) transferases and mono(ADP-ribosyl)ating sirtuins use NAD<sup>+</sup> to perform the mono(ADP-ribosyl)ation, a simple form of post-translational modification of proteins and, in some cases, of nucleic acids. The availability of NAD<sup>+</sup> is a limiting step and an essential requisite for NAD<sup>+</sup> consuming enzymes. The synthesis and degradation of NAD<sup>+</sup>, as well as the transport of its key intermediates among cell compartments, play a vital role in the maintenance of optimal NAD<sup>+</sup> levels, which are essential for the regulation of NAD<sup>+</sup>-utilizing enzymes. In this review, we provide an overview of the current knowledge of NAD<sup>+</sup> metabolism, highlighting the functional liaison with mono(ADP-ribosyl)ating enzymes, such as the well-known ARTD10 (also named PARP10), SIRT6, and SIRT7. To this aim, we discuss the link of these enzymes with NAD<sup>+</sup> metabolism and chronic diseases, such as cancer, degenerative disorders and aging.
Project description:?-Lapachone (?-lap) is a novel anticancer agent that selectively induces cell death in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation. We characterized the gene expression profile of budding yeast cells treated with ?-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were differentially expressed in ?-lap treated cells. ?-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in the mitochondrial NADH dehydrogenase Nde2p was found to be resistant to ?-lap treatment, despite inducing ROS production in a WT manner. Most interestingly, DNA damage responses triggered by ?-lap were abolished in the nde2? mutant. Amino acid biosynthesis genes were also induced in ?-lap treated cells, suggesting that ?-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Accordingly, ?-lap treatment increased phosphorylation of eIF2? subunit in a manner dependent on the Gcn2p kinase. eIF2? phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required for cell survival upon exposure to ?-lap and to elicit checkpoint responses. Remarkably, ?-lap treatment increased phosphorylation of eIF2? in breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2 kinase PERK. These findings uncover a new target pathway of ?-lap in yeast and human cells and highlight a previously unknown functional connection between Nde2p, Gcn2p and DNA damage responses.
Project description:Nicotinamide adenine dinucleotide (NAD<sup>+</sup>) and its reduced form (NADH) are coenzymes employed in hundreds of metabolic reactions. NAD<sup>+</sup> also serves as a substrate for enzymes such as sirtuins, poly(ADP-ribose) polymerases (PARPs) and ADP-ribosyl cyclases. Given the pivotal role of NAD(H) in health and disease, studying NAD<sup>+</sup> metabolism has become essential to monitor genetic- and/or drug-induced perturbations related to metabolic status and diseases (such as ageing, cancer or obesity), and its possible therapies. Here, we present a strategy based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the analysis of the NAD<sup>+</sup> metabolome in biological samples. In this method, hydrophilic interaction chromatography (HILIC) was used to separate a total of 18 metabolites belonging to pathways leading to NAD<sup>+</sup> biosynthesis, including precursors, intermediates and catabolites. As redox cofactors are known for their instability, a sample preparation procedure was developed to handle a variety of biological matrices: cell models, rodent tissues and biofluids, as well as human biofluids (urine, plasma, serum, whole blood). For clinical applications, quantitative LC-MS/MS for a subset of metabolites was demonstrated for the analysis of the human whole blood of nine volunteers. Using this developed workflow, our methodology allows studying NAD<sup>+</sup> biology from mechanistic to clinical applications.
Project description:NAD+ is a dinucleotide cofactor with the potential to accept electrons in a variety of cellular reduction-oxidation (redox) reactions. In its reduced form, NADH is a ubiquitous cellular electron donor. NAD+, NADH, and the NAD+/NADH ratio have long been known to control the activity of several oxidoreductase enzymes. More recently, enzymes outside those participating directly in redox control have been identified that sense these dinucleotides, including the sirtuin family of NAD+-dependent protein deacylases. In this review, we highlight examples of non-redox enzymes that are controlled by NAD+, NADH, or NAD+/NADH. In particular, we focus on the sirtuin family and assess the current evidence that the sirtuin enzymes sense these dinucleotides and discuss the biological conditions under which this might occur; we conclude that sirtuins sense NAD+, but neither NADH nor the ratio. Finally, we identify future studies that might be informative to further interrogate physiological and pathophysiological changes in NAD+ and NADH, as well as enzymes like sirtuins that sense and respond to redox changes in the cell.
Project description:Aldehyde dehydrogenase 1A1 (ALDH1A1) is a member of the aldehyde dehydrogenase superfamily that oxidizes aldehydes to their corresponding acids, reactions that are coupled to the reduction of NAD<sup>+</sup> to NADH. We report here that ALDH1A1 can also use glutathione (GSH) and dihydrolipoic acid (DHLA) as electron donors to reduce NAD<sup>+</sup> to NADH. The GSH/DHLA-dependent NAD<sup>+</sup>-reduction activity of ALDH1A1 is not affected by the aldehyde dehydrogenase inhibitor or by mutation of the residues in its aldehyde-binding pocket. It is thus a distinct biochemical reaction from the classic aldehyde-dehydrogenase activity catalyzed by ALDH1A1. We also found that the ectopic expression of ALDH1A1 decreased the intracellular NAD<sup>+</sup>/NADH ratio, while knockout of ALDH1A1 increased the NAD<sup>+</sup>/NADH ratio. Simultaneous knockout of ALDH1A1 and its isozyme ALDH3A1 in lung cancer cell line NCI-H460 inhibited tumor growth in a xenograft model. Moreover, the ALDH1A1 mutants that retained their GSH/DHLA-dependent NAD<sup>+</sup> reduction activity but lost their aldehyde-dehydrogenase activity were able to decrease the NAD<sup>+</sup>/NADH ratio and to rescue the impaired growth of ALDH1A1/3A1 double knockout tumor cells. Collectively, these results suggest that this newly characterized GSH/DHLA-dependent NAD<sup>+</sup>-reduction activity of ALDH1A1 can decrease cellular NAD<sup>+</sup>/NADH ratio and promote tumor growth.