NAD REQUIRED FOR DISEASE PREVENTION AND HEALTHY AGING

The intracellular concentration of NAD⁺ is a balance between NAD⁺ consumption and synthesis. The biosynthetic pathways of NAD⁺ play an important role in maintaining NAD⁺ pools, which are not only required for fueling redox metabolism but also to support NAD⁺-dependent signaling pathways. As NAD⁺-dependent signaling pathways involve the degradation of NAD⁺, cells require continuous replenishment of NAD⁺ that can be accomplished by an efficient NAD⁺ biosynthesis [10]. The three canonical pathways for the synthesis of NAD⁺ in mammalian cells are the Preiss-Handler pathway, de novo biosynthesis pathway, and NAD⁺ salvage pathway (Figure 1) [11,12]. The formation of nicotinamide (NAM) or nicotinic acid (NA) adenine dinucleotides from the corresponding mononucleotides and ATP constitutes a critical step for NAD generation [13]. The three isoforms of nicotinamide mononucleotide adenylyl transferase (NMNAT1, 2, and 3) that catalyze this reaction have different tissue and subcellular distributions [13]. NMNAT-1 is highly expressed in the skeletal muscle, heart, kidney, liver, kidney, and pancreas [14,15]. In contrast, NMNAT-2 is highly expressed in the brain and NMNAT-3 is highly expressed in the erythrocytes, lungs, and spleen [[16], [17], [18]]. NMNAT-3 and in some reports a minority of nicotinamide phosphoribosyltransferase (NAMPT) [19], which lies upstream of NMNAT-3, were found to be mitochondrial, yet their localization status remains controversial. In contrast, all of the other enzymatic activities associated with NAD⁺ biosynthesis occur in the cytosol and/or nucleus [20]. To identify the factors that contribute to the reduction of NAD⁺ levels associated with aging and its related pathologies, including cancer, it is important to review the NAD⁺ biosynthesis pathways.

Reduced levels of NAD⁺ coupled with a shift toward NADH are considered a hallmark of aging, although the underlying causes remain unclear [39]. Preclinical studies in aging models show that increasing NAD⁺ levels reduces age-related immune and metabolic changes and could potentially be used as a therapeutic strategy for treating aging-associated pathologies [12,[40], [41], [42]]. The role of NAD⁺ in various diseases has been evaluated using genetically modified mice or by boosting or replenishing NAD⁺ levels by administering precursors of NAD⁺ biosynthesis [43,44]. One crucial role of NAD⁺ in cellular maintenance is related to sirtuins. Sirtuins are NAD⁺-dependent deacylases and ADP ribosyltransferases that consume NAD⁺ and are crucial to maintaining and regulating cellular homeostasis. They may be affected by age-dependent declines in NAD⁺ levels [41,45]. The loss of sirtuins can lead to mitochondrial dysfunction, which can cause redox imbalance, leading to damaged cell proteins, lipids, and DNA. Damage to DNA can cause chromosomal aberrations and gene mutations, leading to the development of several chronic diseases, including cancer [46]. Reduced levels of sirtuins also lead to age-related diseases, as members of the sirtuin family such as sirtuin 1 (SIRT1) delay aging processes by catalyzing histone deacetylation and regulating transcription factors [47]. Nutritional and environmental factors significantly affect intracellular NAD⁺ levels. Declining levels of cellular NAD⁺ can impair sirtuin activities and alter the epigenetic chromatin structure [48] and mitochondrial metabolism, leading to increased oxidative stress and decreased ATP production. This increase in oxidative stress promotes inflammation, aggravating cellular injury [49] (Figure 2). Aging is characterized by a continued shift in metabolic activity, which increases from childhood to adulthood and declines afterward [50]. Although age-related metabolic activity changes correlate with cellular NAD⁺ levels [51], the exact mechanisms underlying decreased NAD⁺ levels remain unclear [39]. The expression and activity of NADase CD38 have also been associated with an age-related decline in NAD⁺ and may serve as potential therapeutic targets for age-related diseases [51]. Researchers are focusing on unraveling the molecular mechanisms involved in the physiological decline of NAD⁺ levels. Measuring changes in NAD⁺ levels in vivo may provide information about the altered redox potential associated with age-related diseases and can subsequently be linked with altered cellular metabolism (Figure 2). The decline in NAD⁺ with aging and in progeroid states (as reported in Werner and Cockayne syndromes) [52,53] can result in the development of various metabolic abnormalities [51]. As nuclear NAD + -dependent enzymes regulate mitochondrial function, it is suggested that an impairment in the oxidative phosphorylation (OXPHOS) mechanism during aging might be accelerated by the depletion of nuclear NAD⁺ [54]. Thus, to understand the molecular mechanisms underlying the aging process, it is crucial to study the effects of decreased cellular NAD⁺ levels and understand how different factors such as oxidative stress, inflammation, and DNA damage impact the cellular metabolism of NAD⁺ during aging. Many studies show a relationship between age-related illness, its comorbidities, and metabolic changes.

The systemic decline in NAD⁺ has been associated with many hallmarks of aging. In addition to age-related mitochondrial content changes such as a change in the volume, integrity, and functionality of mitochondrial DNA due to increased ROS accumulation, other mechanisms also contribute to age-related declines in NAD⁺ [39,55]. The most recognized mechanisms include an increase in NAD⁺-consuming enzymes such as poly-ADP-ribose-polymerase 1 (PARP1) and SIRT1 [56,57] due to inflammation or DNA damage. PARP1 activation allows the recruitment of DNA repair proteins to repair damaged DNA. Although PARP1 activation is crucial for genomic maintenance, hyperactivation of PARP1 can cause a reduction in NAD⁺ levels [58]. NAD⁺ reduction leads to reduced SIRT1 activity, increasing proliferator-activated receptor gamma coactivator 1-alpha (PGC)-1α acetylation and decreasing transcriptional factor A mitochondria (TFAM) levels [59]. This decline in NAD⁺ levels can lead to cellular dysfunction and DNA damage and aggravate age-related pathologies. Changes in the mitochondrial content of cells with age can also compromise mitochondrial function and contribute to age-related NAD⁺ decline (Figure 3). Possible explanations for the age-related decline in NAD⁺ levels include increased expression of CD38 protein, dysregulation of circadian rhythms that reduce the expression of NAMPT, and high levels of PARP activity due to DNA damage, inflammation, or metabolic stress [[60], [61], [62]]. Studies show that blocking PARP activity can help recover NAD⁺ levels and mitochondrial function [54,62]. Pharmacological inhibition or deletion of the PARP1 gene enhances mitochondrial content and oxidative metabolism in mice [33,63].

Mitochondrial dysfunction in neurons is a significant contributor to age-related neurodegenerative disorders such as AD and Parkinson’s disease (PD) [73]. Aging contributes to the progression of neurodegenerative disorders, as it accelerates the production and accumulation of reactive oxygen species (ROS), triggering various cellular processes that contribute to DNA damage and impairment in mitochondrial functioning [74]. Several studies have documented NAD + – and NAD-dependent sirtuins’ neuroprotective role in many neurodegenerative disorders [75,76]. Being the only de novo NAD⁺ synthesis pathway in mammals, the KYN pathway modulates neuronal functions and acts as a double-edged sword as it generates both neuroprotective metabolites and neurotoxic intermediates [77] (Figure 4). The maintenance of KYN pathway metabolites is determined by enzymes localized in the astrocytes and microglia in the brain, and any fluctuations in levels of these metabolites can impair neurotransmitter systems, which can lead to various neurological disorders [78]. For NAD⁺ depletion associated with aging-related neurodegenerative disorders, various NAD⁺ augmentation strategies such as supplementation/treatment with NAD⁺ precursors, PARP inhibitors (PARPi), or sirtuin activators can help restore mitochondrial function and enhance neuronal function, which can improve cognitive function [77]. Preclinical studies suggest that PARP1 inhibition helps treat AD, PD, and Huntington’s disease (HD) [79,80]. Axonal degeneration is a hallmark of many neurological diseases, including neurodegenerative disorders, and delaying axonal degeneration can increase survival and decrease disease progression [81]. Activation of SARM1 is found to promote axonal degeneration by initiating a local destruction program that depletes NAD⁺ [82]. In contrast, the overexpression of NMNAT1 is found to inhibit axonal degradation by blocking SARM1-mediated NAD⁺ depletion [83]. Interestingly, elevated levels of NAD⁺ in CD38- and PARP1-deficient mice are found to have no effect on axonal degeneration and do not provide axonal protection, suggesting that the loss or deletion of these NAD⁺-consuming enzymes has no effect on NMNAT1-mediated axonal protection [81].

Cancer cells prefer aerobic glycolysis over OXPHOS, even in the presence of sufficient oxygen for energy metabolism [167] due to increased glucose uptake and glycolytic flux but reduced activity of the pyruvate dehydrogenase enzyme, which results in reduced pyruvate to acetyl CoA [168]. However, recent studies demonstrated that OXPHOS is upregulated in certain cancers [169]. Enhanced glycolysis is characterized by increased nutrient uptake and increased protein, lipid, and nucleic acid synthesis rates to support high rates of cancer cell proliferation (Figure 6). Elevated levels of NAD⁺ augment the process of anaerobic glycolysis through glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) and contribute to cancer cell proliferation [170].

NAMPT is overexpressed in many types of cancers [171]. Overexpression of NAMPT accompanied by increased levels of NAD⁺ promotes cancer cell survival by making cells resistant to anti-cancer reagents. Expression profiles of NAMPT in nine different cancer types are shown in Figure 7. The well-known oncogene c-MYC regulates the expression of NAMPT and enhances the process of glycolysis and lactate production, leading to Warburg effects [172,173]. Furthermore, NAMPT-specific inhibitors reduce NAD⁺ levels by inhibiting energy metabolism pathways such as glycolysis, citric acid cycle, and OXPHOS, contributing to the suppression of cancer cell proliferation. Studies also reported the association of eNAMPT with the progression of cancers such as leukemia, hepatocellular carcinoma, and melanoma [[174], [175], [176], [177]] and in the promotion of cancer-related inflammation and epithelial to mesenchymal transition [178]. NAD⁺ also regulates DNA repair mechanisms, stress responses, and gene expression by serving as a substrate for enzymes such as sirtuin, PARP, and NAD glycohydrolase [170]. An interesting study showed that restoring NAD⁺ pools with NR can prevent DNA damage and tumor formation induced by unconventional prefoldin RPB5 interactor (URI) in hepatocellular carcinoma [179]. Overall survival rates in high- and low-NAMPT expression groups in nine cancer types are shown in Figure 8. NAPRT, a key enzyme mediating NAD biosynthesis from nicotinic acid, plays an important role in cancer cell metabolism. Studies reported downregulation of the NAPRT gene in cancers such as glioblastoma and neuroblastoma [180,181]. A recent study reported overexpression of the NAPRT gene in a subset of common types of cancer such as breast, prostate, liver, pancreatic, ovarian, and head and neck cancers, where it promotes cancer cell metabolism and reduces the susceptibility to NAMPT inhibitors and DNA-damaging drugs [182]. A recent study showed that NAPRT silencing reduces the NAD⁺ pool in human ovarian and pancreatic cancer cells and sensitizes cells to NAMPT inhibitors [182]. Silencing of NAPRT in cancer cells reduces OXPHOS, protein synthesis, and ATP levels due to a reduction in mitochondrial NADH [182]. Thus, NAPRT could prove beneficial to understanding the function of NAD⁺ biosynthetic mechanisms in cancer and likely serve as a potential therapeutic target. PARPs are major mediators of cellular responses associated with DNA repair and genomic stability. Activation of PARP in cells with DNA damage or external stressors leads to NAD depletion, as PARP consumes large amounts of NAD⁺ during DNA repair processes [183]. PARPi is an effective therapeutic agent against BRCA1- and BRCA2-associated cancers, as BRCA mutations lead to DNA double-strand breaks that cannot be efficiently repaired, thus leading to the death of cancer cells. The regulation of PARP1 activity has a significant impact on the metabolism of NAD⁺ [184]. The endogenous PARP1 inhibitor macroH2A1.1 decreases the consumption of NAD⁺ in differentiating cells and increases the availability of mitochondrial NAD⁺, thereby enabling increased OXPHOS [184]. However, PARPi is only effective against certain cancers. The combination of PARPi and β-lapachone, an NAD(P)H:quinone oxidoreductase 1 (NQO1) bioactivatable drug, has synergistic anti-tumor activity and blocks PARP-dependent DNA repair in NQO1-overexpressing cancers such as non-small cell lung cancer, breast cancer, and pancreatic cancer, causing tumor-selective apoptosis [185]. However, any disturbances in levels of NAD⁺ influence the suppression of tumor formation mediated by sirtuins. NAD⁺ and NAMPT are involved in the activation of SIRT1 [186]. NAMPT controls the activity of SIRT1 by regulating NAD⁺ levels. The overexpression of NAMPT increases survival in a SIRT1-dependent manner, whereas inhibition of NAMPT contributes to premature senescence [187]. SIRT1 with NAMPT is overexpressed in many cancers such as colorectal cancer, prostate cancer, and gliomas [[188], [189], [190]]. Moreover, mitochondrial NAD⁺ is a cosubstrate for mitochondrial-localized SIRT3, SIRT4, and SIRT5. The overexpression of nicotinamide nucleotide transhydrogenase (NNT), a mitochondrial enzyme involved in the generation of NADPH from NADH and found to enhance anti-oxidant capacity, induces differentiation and reduces clonogenicity of glioblastoma tumor-initiating cells [191], whereas a reduction in NAD⁺ facilitates metastasis of hepatocellular carcinoma cells [192]. SIRT2 is overexpressed in several cancers such as gastric cancer [193] and hepatocellular carcinoma [194]. SIRT2 enhances NADPH production and promotes leukemia cell proliferation by deacetylation and activation of glucose-6-phosphate dehydrogenase [195]. Consumption of large amounts of intracellular NAD⁺ by PARPs reduces the availability of NAD⁺ for sirtuins, which can lead to aberrant deacetylation of tumor suppressor proteins such as p53 [196]. As p53 is essential for the effective regulation of different cellular processes such as apoptosis, autophagy, and senescence, p53 mutations can result in cancer cell growth by enabling cancer cell survival under nutrient-limiting conditions [197]. Additionally, the expression of p53 depends on the concentration of NAD⁺.