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Tag: aging

Therapeutic potential of boosting NAD+ in aging and age-related diseases


Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor in all living cells that is involved in fundamental biological processes. NAD+ depletion has been associated with hallmarks of aging and may underlie a wide-range of age-related diseases, such as metabolic disorders, cancer and neurodegenerative diseases. Emerging evidence implicates that elevation of NAD+ levels may slow or even reverse the aspects of aging and also delay the progression of age-related diseases. Here we discuss the roles of NAD+-synthesizing and -consuming enzymes in relationships to aging and major age-related diseases. Specifically, we highlight the contribution of NAD+ depletion to aging and evaluate how boosting NAD+ levels may emerge as a promising therapeutic strategy to counter aging-associated pathologies and/or accelerated aging.

1. Introduction

Nicotinamide adenine dinucleotide (NAD+) is an important cofactor in all living cells that is involved in fundamental biological processes, namely metabolism, cell signalling, gene expression, DNA repair, among others [1][2][3][4]. Originally, Harden and Young described NAD+ in 1906 as a molecular fraction (“cozymase”) that accelerated fermentation in yeast extracts. Over subsequent years, NAD+ was identified as a nucleoside sugar phosphate, which plays a role in redox reactions [5]. However, evidence stemming from recent studies have unveiled numerous roles of NAD+ metabolism on aging and longevity. In particular, an age-dependent decline in NAD+ levels have consistently been reported, possibly due to an imbalance in the synthesis and consumption of NAD+. Decreased levels of NAD+ are associated with the hallmarks of aging as well as several age-related diseases, including metabolic disorders, cancer and neurodegenerative diseases. Replenishment of NAD+ levels via administration of its precursors have been demonstrated to display beneficial effects against aging and age-related diseases. Importantly, boosting NAD+ levels have been shown to extend lifespan of various laboratory animal models including worms, flies, and rodents [3][5][6][7][8].

As a cofactor, NAD+ is found in abundance in the mitochondria, cytoplasm, and nucleus. It is essential for many cellular metabolism pathways that include: glycolysisfatty acid β-oxidation, and the tricarboxylic acid cycle. Whilst the reduced form of NAD+ (NADH) is a primary hydride donor in the production of ATP via anaerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) [6][9]. On the other hand, NAD+ is consumed by the NAD+-dependent sirtuins and the DNA damage sensors poly (ADP-ribose) polymerases (PARPs) in the processes of protein deacetylation and poly-ADP-ribosylation (PARylation), respectively. In addition, NAD+ glycohydrolases (i.e. CD38 and CD157) also consume NAD+ via conversion of NAD+ into ADP-ribose (ADPR) or cyclic-ADPR [1][2][3]. Thus, the importance of NAD+ has expanded from a key element in intermediate metabolism to a critical regulator of multiple cell signalling pathways; and is now a major player contributing to aging and age-related diseases [6].

In mammals, NAD+ is synthetized from a variety of dietary sources, including NAD+ itself (it is metabolized in the gut, then synthesized again in cells) as well as from one or more of its major precursors that include: tryptophan (Trp), nicotinic acid (NA), nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), and nicotinamide (NAM). Based upon the bioavailability of its precursors, there are three pathways for the synthesis of NAD+ in cells: (i) from Trp by the de novo biosynthesis pathway or kynurenine pathway; (ii) from NA in the Preiss–Handler pathway; and (iii) from NAM, NR, and NMN in the salvage pathway [5][6].

Accumulating evidence demonstrates an age-dependent decline in NAD+ levels and associate its depletion to several hallmarks of aging and age-related diseases (Fig. 1[6]. Here, we summarize the roles of NAD+-synthesizing and -consuming enzymes in aging and age-related diseases. Specifically, we highlight the contribution of NAD+ depletion to mammalian aging and evaluate how boosting endogenous NAD+ levels might emerge as a promising therapeutic strategy to counter aging-associated pathologies and/or accelerated aging.

Fig. 1. NAD+ decline at the core of hallmarks of aging. A schematic representation of age-dependent decline in NAD+ levels which contribute to ten hallmarks of aging, namely DNA damage, epigenetic alteration, deregulated nutrient-sensing, loss of proteostasis, altered cellular communication, cellular senescence, stem cell exhaustion, mitochondrial dysfunction, compromised autophagy, and possibly telomere attrition. Figure modified from Fang et al. 2017 [10].

2. Recent progress on the roles of NAD+ in aging

Mounting evidence has indicated that NAD+ levels decline with age in multiple types of tissues, which include the liver, skeletal muscle, adipose tissue, heart, brain, kidney, pancreas, lungs, spleen, skin, as well as extracellular fluids [3][10]. In addition, an age-dependent decline in NAD+ levels in Caenorhabditis elegans (C. elegans), mice, and human post-mortem tissues are reported. Thus, highlighting a universal age-dependent decrease of NAD+ across species. However, it remains elusive whether this is due to increased NAD+ consumption and/or decreased synthesis [6]. In this section, we provide an overview of methods that could potentially boost endogenous levels of NAD+.

2.1. NMN and aging

NMN is a physically stable natural compound that serves as an efficient NAD+ precursor. In mammals, NMN is synthesized from nicotinamide, a form of water-soluble vitamin B3 and 5′-phosphoribosyl-1-pyrophosphate (PRPP), by the rate-limiting enzyme, nicotinamide phosphoribosyl transferase (NAMPT). In addition, it can also be synthesized from NR via NR kinases (NRKs)-mediated phosphorylation reactions. NMN is subsequently converted into NAD+ by NMN adenylyl transferases (NMNATs) [5]. Over the years, it has become increasingly evident that systemic administration of NMN in rodents enhances the biosynthesis of NAD+ in various peripheral tissues including liver, pancreas, adipose tissue, heart, skeletal muscle, kidney, eyes, and blood vessels [5][6][11][12][13][14][15][16][17][18][19]. Furthermore, NMN has also been shown to elevate levels of NAD+ in hypothalamus and hippocampus following an intraperitoneal injection, thereby indicating its ability to penetrate the blood-brain barrier (BBB) [20][21]. More importantly, long-term (1-year) oral administration of NMN (up to 300 mg/kg) has recently been shown to be well tolerated without any obvious deleterious or toxic effects in normal wild type C57BL/6 mice [22].

NMN has been shown to have remarkable beneficial effects that counter normal aging. In models of aging, long-term administration of NMN protects against age-associated functional decline as demonstrated by increases in energy metabolism, insulin sensitivitylipid metabolism, mitochondrial oxidative metabolism, eye function, bone density and immune function [10][22]. On the other hand, it suppressed age-related changes in gene expression and adipose tissue inflammation [22]. Moreover, it has been shown to maintain neural stem/progenitor cell population and restore skeletal muscle mitochondrial function as well as arterial function in aged mice [15][19][20]. In addition, loss of enzymes involved in NAD+ synthesis, namely NAMPT during the process of aging, led to decrease in NAD+ content and reduced SIRT1 activity; consequently, promoted cellular senescence in retinal pigment epithelium, which performs numerous functions critical to retinal health and visual function [23].

NMN administration may counteract age-predisposed metabolic diseases and neurodegeneration. In age-related pathophysiological conditions, NMN ameliorated impairments in glucose tolerance and promoted insulin secretion/sensitivity in age- or diet-induced diabetic mice, Nampt+/− mice, as well in aged wild-type and β cell-specific Sirt1-overexpressing (BESTO) mice [5][6][11][24][25][26]. Likewise, promotion and overexpression of the mitochondrial Nmnat3 in mice, also involved in NAD+ biosynthesis, resulted in improved glucose tolerance during the process of aging as well as in models of high-fat induced obesity [27]. The beneficial effect was suggested to be a result of improved mitochondrial function and by an independent mechanism of NAD+–SIRT1–PGC1α axis, which despite previously being reported to contribute to improved mitochondrial function, was not activated in these transgenic mice despite elevated levels of NAD+ [27]. Furthermore, NMN protects the heart and brain from ischaemia-induced damage [28][29]. In rodent models of Alzheimer’s disease (AD), administration of NMN decreased AD-associated β-amyloid (Aβ) pathology and improved cognitive function. In addition, it restored mitochondrial function and ameliorated inflammation, synaptic loss as well as protected against neuronal cell death [30][31][32]. The beneficial effect of NMN was also evident in premature aging conditions as demonstrated by extended lifespan and improved healthspan in the C. elegans model of xeroderma pigmentosum group A (XPA, a nucleotide excision DNA repair (NER) disorder with severe neurodegeneration) [33], and Ataxia telangiectasia (A-T, due to mutation of ATM which encodes a master regulator of DNA damage response) [34][35]. Moreover, in mice with hypomorphic BubR1 (a mitotic check-point kinase) exhibited characteristics of premature aging as evidenced by shorter lifespan, which was restored by NMN supplementation [17]. Altogether, these findings highlight NMN induced restoration of NAD+ levels serve as beneficial therapeutic strategy in countering aging as well as age-related pathological conditions in animal models.

2.2. NR and aging

NR is a natural NAD+ precursor. It can directly be converted into NMN via the activity of NRKs, thereby bypassing the requirement of the NAMPT in the salvage pathway, and therefore offers to provide an additional pathway for elevation of NAD+ levels. Similar to NMN, NR also exhibited beneficial effects in protection against aging and age-related diseases. It has been shown to promote longevity as well improve healthspan in multiple laboratory animal models [36][37][38][39][40][41]. In age-related disease, in particular obesity, diabetes and cardiovascular conditions NR was able to decrease weight gain, improve glucose tolerance and increase survival rates, respectively in rodents [42][43][44]. In addition, in models of diabetes and high-fat diet, NR was able to improve metabolic function and reduce lipid accumulation as well as increasing lifespan [40][44][45][46]. Furthermore, supplementation of NR reversed the progressive wasting syndrome and restored endurance in Nampt skeletal muscle knockout micemdx model of Duchenne’s muscular dystrophy [47][48].

NR ameliorates neurodegeneration in animal models. In animal models of age-related neurodegenerative diseases such as AD and Parkinson’s disease (PD), NR has been shown to improve memory, learning, motor function and mitochondrial function as well as protected against neuronal cell death [49][50][51][52]. In particular, chronic NR administration in the amyloidogenic models of AD delayed the development and progression of Aβ pathology in AD mice, SH-SY5Y cells, and AD C. elegans [50][52]. In addition, it was able to promote longevity and inhibit/delay cognitive decline in AD C. elegans and mice, with the enhanced process of mitochondrial proteostasis and modulation of β-secretase 1 (BACE-1) activity via peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC)-1alpha highlighted as possible underlying mechanisms [50][52]. Further evidence reinforcing the beneficial effects of NR as a therapeutic strategy in AD was provided by a recent study using triple transgenic model of AD (3 × Tg), which exhibited not only reduced phosphorylated tau pathology and inhibited cognitive decline; but also, normalised AD-associated neuroinflammation and synaptic dysfunction. Moreover, DNA damage was reduced, in addition, following chronic administration of NR in a DNA repair-deficient 3 × Tg/Polβ +/− mouse model [6][51]. The underlying mechanism proposed was the reduction in DNA damage results in reduced activity of NAD+ consuming enzyme PARPs that is involved in DNA repair; thereby increase in the levels of NAD+, which in turn contributes to neurogenesis and inhibits AD-associated pathology, neuroinflammation and mitochondrial dysfunction [51]. Likewise, NR treated induced pluripotent stem cells (iPSCs) derived from PD patients harbouring mutations in the lysosomal enzyme β-Glucocerebrosidase (GBA) gene (GBA-PD), the most common genetic risk for PD, resulted in elevated levels of NAD+ and NAM which coincided with improved mitochondrial morphology and function [49][53]Mitophagy was suggested to be a possible mechanism promoted by NR, which may underlie improved mitochondrial quality control [49]. In addition, flies model of GBA-PD expressing human N370S GBA raised on food containing NR displayed improved motor function and significantly decline in loss of dopamine-containing neuronal population [49]. Additionally, NR has shown significant neuroprotection in a series of DNA repair-deficient premature aging diseases, including XPA, A-T, and Cockayne syndrome (CS, due to impairment of NER) [33][34][54].

2.3. NAM and aging

NAM is also a precursor for NAD+ and a key molecule involved in energy metabolism. Low doses of NAM have been shown to increase lifespan in yeast and C. elegans, however, higher doses have been associated with reduced lifespan via inhibition of Sir2 activity [39][55][56][57][58]. In models of aging and high fat diet-induced obesity, NAM improved healthspan although it failed to extend lifespan as illustrated by comparable mean and maximum lifespan [59]. In the model of obesity, it has able to restore glucagon storage to similar levels as age-matched standard-diet mice as well as ameliorate diet-induced hepatosteatosisoxidative stress and inflammation [59]. It was suggested that the beneficial impact of NAM may be attributes to improved mitochondrial function and countering age and high fat diet induced DNA damage [59]. Further evidence reinforcing the beneficial impact of NAM stemmed from a mouse model of glaucoma, which inhibited the development glaucoma in the eyes [60]. Moreover, these findings were replicated by Nmnat1 gene therapy whereby an intravitreal administration of adeno-associated virus AAV2.2 carrying a plasmid to overexpress murine Nmnat1 under a CMV promoter was performed in D2 eyes [60]. The improvement of mitochondrial health and metabolism was suggested to be the underlying mechanism for countering glaucoma mediated by NAM supplementation and Nmnat1 gene therapy [60].

3. Other approaches to regulate NAD+

3.1. PARP inhibition

The NAD+ consuming enzymes, PARPs, cleave NAD+ into NAM and ADP-ribose (ADPR), as a result generating a chain of ADPR. PARP1 is the most abundant PARPs, which is ubiquitously expressed and is a major consumer of NAD+ in response to DNA damage whereby it contributes to facilitation of the DNA repair process [61]. Both PARPs and sirtuins share NAD+ as a common substrate thus compete for its consumption. It has been reported that PARP1 activity increased with the inevitable process of aging possibly due accumulation of DNA damage [33]. Consequently, the NAD+ pool is depleted which results in reduced activity of sirtuins [62]. Evidence stemming from genetic deletion of PARP1 in mice as well as pharmacological inhibition of PARPs revealed increase in NAD+ content and enhanced activities of sirtuins, in particular SIRT1 and SIRT6 [33][34][39]. Elevated SIRT1 activity was associated with increased mitochondrial content and oxidative metabolism as well as protection against metabolic dysfunction, DNA damage, and neurodegeneration [4][10][62].

In addition, increased PARP1 activity has been reported in animal models of age-related neurodegenerative diseases, namely AD and PD [63][64][65]. Deletion of PARP1 in AD mice protected against cognitive decline as well as attenuated neuroinflammation and Aβ-induced neurotoxicity [66]. In PD rodents, PARP1 pharmacological inhibitors or deletion resulted in resistance to the toxic effects and loss of dopamine-containing neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA), respectively [67][68][69]. Thus, implicating the contribution of PARP1 hyperactivity in processes leading to neurodegeneration. Moreover, hyperactivity of PARP1 is reported in models of premature aging that resulted decrease in SIRT1 activity, a feature that was reversed by replenishment of NAD+ using its precursors [33][34]. Therefore, a potential therapeutic counter for depleted NAD+ pool in aging and age-related diseases could be inhibition of its consumer, PARPs, which will enable activity of sirtuins that plays a pivotal role in regulating cellular processes.

3.2. CD38 inhibition

CD38 is one of the primary NADases in mammals. It can modulate the levels of NAD+ via hydrolysis of NAD+ itself as well degradation of its precursors, NMN and NR. Primarily, CD38 hydrolyses NAD+ to produce of ADPR and NAM [70][71][72]. It is a key player in several physiological processes, including nuclear Ca2+ homoeostasis, immunity, inflammation, glucose and lipid homoeostasis, transferring of mitochondria from astrocytes to neurons, as well as social behaviour [6][73][74][75][76][77]. An age-dependent increase in levels of CD38 protein has been reported in multiple tissues and organs, which as a result contributes to NAD+ decline [78]. Therefore, CD38-dependent modulation of NAD+ can alter the activity of NAD+-consuming enzymes and affect cellular signalling and metabolism [71][72]. Inhibition of CD38 can also promote NAD+ levels and improve glucose and lipid metabolism, which protects against age- and diet-induced diabetes and obesity [72][75]. In APP/PS1 model of AD, CD38 depletion resulted in elevation of NAD+ levels that were associated with decrease in Aβ pathology and associated neuroinflammation accompanied by improvement spatial learning behaviour [79]. However, due to the reported important neuroprotective activities of CD38, further stringent and comprehensive evaluation of the procedures of CD38 inhibition as a safe anti-aging strategy [76][77].

3.3. NNMT knockdown

Nicotinamide N-methyltransferase (NNMT) catalyses the methylation of NAM N1-methyl-2- pyridone-5-carboxamide (2py) and N1-methyl-4-pyridone-3-carboxamide (4py) using the universal methyl donor S-adenosyl methionine (Met) (SAM). Both products of NAM methylation are eventually excreted in the urine; thus, NNMT removes NAM from the NAD+ biosynthesis pathway and thereby contributes to decrease in levels of NAD+ [80]. It is predominantly expressed in the liver and adipose tissue but is also found in other tissues including kidney, lung, muscle, heart, brain, and tumour cells. NNMT has been shown to be involved in various disease conditions such as metabolic disorders, neurodegenerative diseases and cancer [80]. In conditions such as obesity and diabetes, NNMT levels have been reported to be upregulated significantly, which in turn are associated with the disease phenotype [81][82]. In fact, genetic knockdown as well as pharmacological inhibition of NNMT was shown to be beneficial in protection against obesity in rodent models of obesity [83][84].

3.4. Genetic promotion of NAD+ biosynthesis

In addition to abovementioned techniques that have shown to enhance NAD+ biosynthesis, genetic tools in the form of Lactobacillus brevis (LbNOX), a water-forming NADH oxidase, has been demonstrated to also induce an increase in compartment-specific levels of NAD+/NADH ratio in HeLa cells [85]. It catalyses a four-electron reduction of oxygen to water (2 NADH + 2H+ + O2 → 2 NAD+ + 2 H2O) [85][86]. The NAD+/NADH ratio plays a role in cellular metabolism by affecting the activity of NAD+-dependent enzymes such as sirtuins. LbNOX was shown to ameliorate proliferative and metabolic defects induced by a dysfunctional electron transport chain (ETC) by recycling the pool of NAD+ [85]. This, therefore offers to be a novel approach whereby NAD+ levels could potentially be boosted via genetic manipulation in order to understand the fundamental molecular mechanisms in models of aging and age-related diseases.

4. Methods to detect subcellular NAD+

In view of the importance of NAD+ in life, aging, and diseases, it is necessary to accurately detect subcellular NAD+ levels to further unveil its intracellular functions as well as to develop sub-cellular organelle-targeted therapeutic approaches. Traditionally, several assays, such as high-performance liquid chromatography (HPLC)-based methods (e.g., HPLC/MALDI/MS) and fluorometric-based commercial kits have been utilised to detect whole NAD+ [26][48] at both cellular levels and/or sub-cellular levels (by isolating sub-cellular fractions ahead). There have some challenges of using these methods to accurately detect sub-cellular NAD+ levels because of the highly instability of NAD+ as well as impossibility of NAD+ detection in live cells/tissues. The recent development of genetically encoded fluorescent biosensors such as SoNar and a biosensor with a bipartite NAD+-binding domain have enabled imaging of relative levels of free NAD+ in the subcellular compartments [87][88]. Quantification of NAD+ using fluorescent biosensor targeted to different compartments of mammalian cells showed that mitochondria contain more than twice as much free NAD+ as other compartments [87]. Specifically, the concentration of NAD+ was reported to be approximately 110 μM in the cytoplasm and the nucleus relative to 230 μM in the mitochondrion [87]. These levels are consistent with other reports demonstrating that, in highly metabolically active, post mitotic cells, such as neurons, mitochondria have higher NAD+ levels compared with other sub-cellular compartments [1]. It was further demonstrated genetic and pharmacologic inhibition of NAMPT result in a reduction in NAD+ concentration in all compartments, but depletion of mitochondrial NAD+ occurred at a slower rate. Furthermore, the nuclear and cytoplasmic pools were shown to be readily exchangeable, whilst the mitochondrial pool may maintain mitochondrial NAD+ levels via NAD+ biogenesis by mitochondrial isoform NMNAT3 and import from the cytoplasm [87]. Another recent development has been offered in the form of NAD+ flux quantification that is isotope-tagged and used for analysis of NAD+ metabolism. It demonstrated that approximately 50% decrease in NAD+ consumption following treatment with SIRT1/2 and PARP1/2 inhibitors; thereby implicating both are major consumers of NAD+ [89]. The use of such approaches in models of aging and age-related diseases in combination with NAD+ promoting methods would allow the identification of subcellular localisation as well as consumption, which in turn would allow to identify the underlying molecular mechanisms and pathways attributed to NAD+ benefits.

5. Clinical translation

Encouraged by significant and replicable benefits of NAD+ precursors, NR and NMN, in aging and disease animal models, a series of clinical trials of NR and NMN in normal aged population and individuals with diseases have been performed [10]. NAD+ precursors, in particular NR has been demonstrated to elevate blood concentration of NAD+ in healthy individuals in a dose-dependent manner and without any toxic effects [90]. In particular, single oral self-administration of NR (1000 mg) over a period of seven days in a 52 years old male increased blood concentration of NAD+ by 2.7-folds and 45.5-fold increase in nicotinic acid adenine dinucleotide (NAAD), an NAD+ biosynthesis intermediate [90]. In addition, a randomized double-blind pharmacokinetic study of single oral administration NR (doses: 100 mg, 300 mg, and 1000 mg) with seven-days gap conducted in 12 healthy patients (aged 30–55 years old) revealed a dose-dependent increase in NAD+ and NAAD levels, with no reported adverse effects [90]. The encouraging results in animal models of aging and age-related diseases of chronic administration of NAD+ precursors have led to studies in humans [91][92]. An eight-week randomized, double-blinded, placebo-controlled study in 120 healthy adults (60–80 years old) demonstrated NR (250 mg and 500 mg) induced dose-dependent increase of blood NAD+ level that becomes apparent after 4-weeks and is sustained till the end of the study [91]. Importantly, no serious adverse effects were reported, thereby implicating the chronic administration of NR is a safe and effective way to increase NAD+ levels [91]. These findings are reinforced by a 2 × 6-week randomized, double-blind, placebo-controlled crossover clinical trial conducted in 55–79 years old individuals that showed NR (oral 500 mg, twice a day) to be well tolerated and able to effectively elevates NAD+ levels in healthy adults [92]. Moreover, it was able to reduce systolic blood pressure and aortic stiffness, which are considered measures of cardiovascular disease [92]. Thus, not only the NAD+ precursors are safely administrated but they may also recapitulate the beneficial effects that were evident in animal models, which is an exciting prospect for future clinical trials. However, in conditions such as pancreatic cancer, cell growth has been shown to be dependent of the NAD+ salvage pathway [93]. Hence, inhibition of NAD+ synthesis (via Nampt inhibition) and/or promotion of its consumption (via CD38 NADase) prevented cancer cell growth [93][94]. Therefore, implicating that there should be a thorough evaluation for the use of approaches that promote NAD+ biosynthesis as it may be contributor rather than a counter-mechanism in certain conditions. Clinical trials of NAD+ precursors on age-related diseases, such as diabetes, premature aging diseases, and neurodegenerative diseases are in progress [10].

6. Outstanding questions and future perspectives

Age is the primary cause of the majorly of human diseases and interventional strategies/therapeutics targeting on human aging is arguably the most efficient approach to achieve healthy aging and the improvement of the quality of life worldwide. Although, maintaining a healthy diet, fasting, and exercise may improve the quality of life, it may not be feasible option for all individuals. Therefore, the beneficial effects of NAD+ discussed in the present review, highlight possible ways for improving the quality of life via hindering numerous pathological hallmarks of aging and thereby improving the quality of life and delay age-related diseases (summarized in Fig. 2). Preclinical evidence of NAD+ replenishment that could potentially delay and/or prevent metabolic conditions, hearing loss, muscle atrophy, and cognitive decline are really encouraging for future perspectives. Moreover, NAD+ precursors, in particular NR has been shown to be safely administrated and also able to demonstrate improvement of cardiovascular functions in human. Thus, implicating a possible translational aspect of preclinical benefits of NAD+ supplementation, which is an exciting prospect and opens avenues for future studies to test the impact of elevated NAD+ biosynthesis in aging and age-associated diseases in human.

Fig. 2. Physiological and pharmacological strategies for boosting NAD+ levels. Inhibiting the age-related decline in NAD+ levels is critical for preventing age- or disease-related frailties. Physiological strategies that could potentially boost NAD+ levels include exercise, fasting, and maintaining a healthy diet. Pharmacologically, boosting NAD+ can be achieved via either supplementation of its precursors, NR and NMN, or inhibition of its consumers by use of CD38 and PARPs inhibitors. Figure modified from Fang et al. 2017 and Fivenson et al. 2017 [10][95].

Despite extensive research on NAD+ biosynthesis and its implications in health and disease, there are major questions that are yet to be explored. Firstly, what levels of NAD+ are to be associated with healthy aging and age-related diseases? In particular, it is of great relevance to elucidate organ and sub-cellular localisation as well levels of NAD+ in health and disease. Such observations would allow to map health- and disease-specific alterations of NAD+, which could be utilised to develop therapeutic interventions that promote NAD+ in a region-specific manner as a counter-mechanism. Decline in NAD+ has been implicated during the process of aging and age-associated diseases, thereby may be affect various processes that are likely to key contributors and drivers of associated dysfunction. Thus, preclinical studies driven towards unveiling the pathways and mechanisms underlying the beneficial effects of NAD+ replenishment in healthy aging and models of disease are required in order to understand how NAD+ contributes to delay and/or prevent hallmarks associated with aging. In particular, it is important to elucidate whether individual or multiple hallmark(s) mechanisms associated with aging are countered by NAD+ replenishment. This would allow understanding of the mode of NAD+ action and the interconnection and contribution of the various hallmarks of aging. NAD+ precursors have thus far shown to be safely and effectively administrated in healthy old humans, elevating NAD+ levels in blood, but its safety and tolerance is yet to be determined in individuals with age-related diseases. Therefore, future clinical trials are required to assess the safety of NAD+ precursors in patients with age-associated diseases such as diabetes and AD. Though, as abovementioned, careful evaluation of the role of NAD+, whether friend or foe in disease, must be taken into account for each disease-condition. Altogether, NAD+ replenishment may serve as a potential therapeutic strategy for aging and multiple conditions to improve the quality of life of the increasing aged population.

Conflicts of interest

The authors declare there is no conflict of interest.


This research was supported by the HELSE SøR-ØST (E.F.F., #2017056), The Research Council of Norway (E.F.F., #262175 and #277813), and two NIA Intra-laboratory grants (2016, 2017 to E.F.F. and V.A.B.). The E.F.F. laboratory has CRADA arrangements with ChromaDex. We thank Øystein Horgmo (Senior Photographer, Medical Photography and Illustration Service, UiO) for making some elements in Fig. 1Fig. 2.


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Originally published here:

Rewinding the Clock

Treatment restores blood vessel growth, muscle vitality, boosts exercise endurance in aging animals  

By EKATERINA PESHEVA March 22, 2018 Research

We are as old as our arteries, the adage goes, so could reversing the aging of blood vessels hold the key to restoring youthful vitality?

The answer appears to be yes, at least in mice, according to a new study led by investigators at Harvard Medical School.

The research, published March 22 in Cell, identifies the key cellular mechanisms behind vascular aging and its effects on muscle health and has successfully reversed the process in animals.

The findings pinpoint a glitch in the normal crosstalk that occurs between muscles and blood vessels and keeps both tissues healthy.

Using the synthetic precursors of two molecules naturally present in the body, the scientists also managed to reverse blood vessel demise and muscle atrophy in aging mice, boosting their exercise endurance in the process.

The achievement, the team said, paves the way to identifying related therapies for humans.

“We’ve discovered a way to reverse vascular aging by boosting the presence of naturally occurring molecules in the body that augment the physiological response to exercise,” said study senior investigator David Sinclair, professor in the Department of Genetics at Harvard Medical School and co-director of the Paul F. Glenn Center for the Biology of Aging at Harvard Medical School.

“The approach stimulates blood vessel growth and boosts stamina and endurance in mice and sets the stage for therapies in humans to address the spectrum of diseases that arise from vascular aging,” added Sinclair, who is also a professor at the University of New South Wales School of Medical Sciences in Sydney, Australia.

The researchers caution that many promising treatments in mice don’t have the same effect in humans due to critical differences in biology. However, the results of the experiments were dramatic enough to prompt the research team to pursue experiments in humans. Clinical trials for safety are already under way, Sinclair said.

As old as our blood vessels

Sinclair and team set out to unravel the mechanisms behind one of biology’s inevitabilities: aging.

As we grow old, we become weak and frail. A constellation of physiological changes—some subtle, some dramatic—precipitate this inevitable decline. What exactly happens inside our cells to cause the biological shifts that lead to aging? It’s a question that has vexed Sinclair and team for years.

As we age, our tiniest blood vessels wither and die, causing reduced blood flow and compromised oxygenation of organs and tissues. Vascular aging is responsible for a constellation of disorders, such as cardiac and neurologic conditions, muscle loss, impaired wound healing and overall frailty, among others. Scientists have known that loss of blood flow to organs and tissues leads to the build-up of toxins and low oxygen levels. The so-called endothelial cells, which line blood vessels, are essential for the health and growth of blood vessels that supply oxygen-rich and nutrient-loaded blood to organs and tissues. But as these endothelial cells age, blood vessels atrophy, new blood vessels fail to form and blood flow to most parts of the body gradually diminishes. This dynamic is particularly striking in muscles, which are heavily vascularized and rely on robust blood supply to function.

Muscles begin to shrivel and grow weaker with age, a condition known as sarcopenia. The process can be slowed down with regular exercise, but gradually even exercise becomes less effective at holding off this weakening.

Sinclair and team wondered: What precisely curtails the blood flow and precipitates this unavoidable decline? Why does even exercise lose its protective power to sustain muscle vitality? Is this process reversible?

In a series of experiments, the team found that reduced blood flow develops as endothelial cells start to lose a critical protein known as sirtuin1, or SIRT1. Previous studies have shown that SIRT1 delays aging and extends life in yeast and mice.

SIRT1 loss is, in turn, precipitated by the loss of NAD+, a key regulator of protein interactions and DNA repair that was identified more than a century ago. Previous research by Sinclair and others has shown that NAD+, which also declines with age, boosts the activity of SIRT1.

A stimulating conversation

The study reveals that NAD+ and SIRT1 provide a critical interface that enables the conversation between endothelial cells in the walls of blood vessels and muscle cells.

Specifically, the experiments reveal that in young mouse muscle, SIRT1 signaling is activated and generates new capillaries, the tiniest blood vessels in the body that supply oxygen and nutrients to tissues and organs. However, as NAD+/SIRT1 activity diminishes over time, the study found, so does the blood flow, leaving muscle tissue nutrient-deprived and oxygen-starved.

Indeed, when researchers deleted SIRT1 in the endothelial cells of young mice, they observed markedly diminished capillary density and decreased number of capillaries, compared with mice that had intact SIRT1. Mice whose endothelial cells lacked SIRT1 had poor exercise tolerance, managing to run only half the distance covered by their SIRT1-intact peers. 

To determine SIRT1’s role in exercise-induced blood vessel growth, the researchers observed how SIRT1-deficient mice responded to exercise. After a month-long training regimen, the hind-leg muscles of SIRT1-deficient mice showed markedly diminished ability to form new blood vessels in response to exercise compared with same-age mice that had intact SIRT1 in their endothelial cells.

Exercise-induced blood vessel formation is known to occur in response to growth-stimulating proteins released by muscles under strain. SIRT1, however, appears to be the key messenger relaying growth-factor signaling from muscles to blood vessels, the study found. 

Experiments showed that endothelial cells lacking SIRT1 were desensitized to the growth-stimulating proteins released by exercised muscles.

“It’s as if these cells had grown deaf to the signals that muscles sent their way,” Sinclair said.

The observation, he added, explains why age-related loss of SIRT1 leads to muscle atrophy and blood vessel demise.

Since the experiments revealed the critical role of SIRT1 in exercise-induced blood vessel formation, the researchers wondered whether boosting SIRT1 levels would stimulate blood vessel growth and stave off muscle wasting.

Exercise in a pill?

The scientists set their sights on NAD+, a molecule conserved across many life forms, known to decline with age and previously shown to stimulate SIRT1 activity.

“We reasoned that declining NAD+ levels reduce SIRT1 activity and thus interfere with aging mice’s ability to grow new blood vessels,” said study first author Abhirup Das, who conducted the work as a post-doctoral fellow in Sinclair’s lab, currently a visiting scholar in genetics at Harvard Medical School and a post-doctoral research fellow at the University of South New Wales School of Medical Sciences.

To test this premise, scientists used a chemical compound called NMN, a NAD+ precursor, previously shown to play a role in repairing cellular DNA and maintaining cell vitality.

In lab dish experiments, endothelial cells from humans and mice treated with NMN showed enhanced growth capacity and reduced cell death.

Next, the team gave NMN over two months to a group of mice that were 20 months old—the rough equivalent of 70 in human years. NMN treatment restored the number of blood capillaries and capillary density to those seen in younger mice. Blood flow to the muscles also increased and was significantly higher than blood supply to the muscles seen in same-age mice that didn’t receive NMN.

The most striking effect, however, emerged in the aging mice’s ability to exercise. These animals showed between 56 and 80 percent greater exercise capacity, compared with untreated mice the study showed. The NMN-treated animals managed to run 430 meters, or about 1,400 feet, on average, compared with 240 meters, or 780 feet, on average, for their untreated peers.

To see whether the effects of NMN could be further augmented, the researchers added a second compound to the treatment regimen. The compound, sodium hydrosulfide (NaHS), is a precursor to hydrogen sulfide, which also boosts the activity of SIRT1.

A group of 32-month-old mice—the rough equivalent to 90 in human years—receiving the combo treatment for four weeks were able to run, on average, twice as long as untreated mice. In comparison, mice treated with NMN alone ran 1.6 times farther, on average, than untreated animals.

“These are really old mice so our finding that the combo treatment doubles their running capacity is nothing short of intriguing,” said study co-author James Mitchell, associate professor of genetics and complex diseases at the Harvard T. H. Chan School of Public Health.  Research led by Mitchell and published in the same issue of Cell also found sodium hydrosulfide to augment blood vessel formation in the muscles of mice.

Interestingly, the NMN treatment did not improve blood vessel density and exercise capacity in young sedentary mice. However, it did boost blood vessel formation and exercise capacity in young mice that had been exercising regularly for a month.

“This observation underscores the notion that age plays a critical role in the crosstalk between blood vessels and muscles and points to a loss of NAD+ and SIRT1 as the reason behind loss of exercise effectiveness after middle age,” Das said.

The researchers say their findings may pave the way to therapeutic advances that hold promise for the millions of older people for whom regular physical activity is not an option.

“Even if you’re an athlete, you eventually decline,” Sinclair said. “But there is another category of people—what about those who are in a wheelchair or those with otherwise reduced mobility?”

The team’s ultimate goal is to replicate the findings and, eventually, move toward developing small-molecule, NMN-based drugs that mimic the effects of exercise—enhanced blood flow and oxygenation of muscles and other tissues. Such therapies may even help with new vessel growth of organs that suffer tissue-damaging loss of blood supply and oxygen, a common scenario in heart attacks and ischemic strokes, the team said.

Neo-vascularization—the formation of new blood vessels—should be treated with caution, the researchers say, because increased blood supply could inadvertently fuel tumor growth.

“The last thing you want to do is provide extra blood and nourishment to a tumor if you already have one,” said study co-author Lindsay Wu, at the University of New South Wales School of Medical Sciences.

Sinclair and Wu point out that experiments done as part of the current study provide no evidence that treatment with NMN stimulated tumor development in animals treated with the compound.

Co-investigators included George Huang, Michael Bonkowski, Alban Longchamp, Catherine Li, Michael Schultz, Lynn-Jee Kim, Brenna Osborne, Sanket Joshi, Yuancheng Lu, Jose Humberto, Trevine-Villareal, Myung-Jin Kang, Tzong-tyng Hung, Brendan Lee, Eric Williams, Masaki Igarashi, James Mitchell, Nigel Turner, Zolt Arany and Leonard Guarente.

The work was supported by the Glenn Foundation for Medical Research (grants RO1 AG028730 and RO1 DK100263), and by the National Institutes of Health/National Heart, Lung and Blood Institute (grant RO1 HL094499).


NAD+ and sirtuins in aging and disease


Nicotinamide adenine dinucleotide (NAD(+)) is a classical coenzyme mediating many redox reactions. NAD(+) also plays an important role in the regulation of NAD(+)-consuming enzymes, including sirtuins, poly-ADP-ribose polymerases (PARPs), and CD38/157 ectoenzymes. NAD(+) biosynthesis, particularly mediated by nicotinamide phosphoribosyltransferase (NAMPT), and SIRT1 function together to regulate metabolism and circadian rhythm. NAD(+) levels decline during the aging process and may be an Achilles’ heel, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies. Restoring NAD(+) by supplementing NAD(+) intermediates can dramatically ameliorate these age-associated functional defects, counteracting many diseases of aging, including neurodegenerative diseases. Thus, the combination of sirtuin activation and NAD(+) intermediate supplementation may be an effective antiaging intervention, providing hope to aging societies worldwide.

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Critical step found in DNA repair, cellular aging

DNA repair is essential for cell vitality, cell survival, and cancer prevention, yet cells’ ability to patch up damaged DNA declines with age for reasons not fully understood.

Now, research led by scientists at Harvard Medical School (HMS) reveals a critical step in a molecular chain of events that allows cells to mend their broken DNA.

The findings, to be published March 24 in Science, offer a critical insight into how and why the body’s ability to fix DNA dwindles over time and point to a previously unknown role for the signaling molecule NAD as a key regulator of protein-to-protein interactions in DNA repair. NAD, identified a century ago, is already known for its role as a controller of cell-damaging oxidation.

Additionally, experiments conducted in mice show that treatment with the NAD precursor NMN mitigates age-related DNA damage and wards off DNA damage from radiation exposure.

The scientists caution that the effects of many therapeutic substances are often profoundly different in mice and humans owing to critical differences in biology. However, if affirmed in further animal studies and in humans, the findings can help pave the way to therapies that prevent DNA damage associated with aging and with cancer treatments that involve radiation exposure and some types of chemotherapy, which, along with killing tumors, can cause considerable DNA damage in healthy cells. Human trials with NMN are expected to begin within six months, the researchers said.

“Our results unveil a key mechanism in cellular degeneration and aging, but beyond that they point to a therapeutic avenue to halt and reverse age-related and radiation-induced DNA damage,” said senior author David Sinclair, professor in the Department of Genetics at HMS, co-director of the Paul F. Glenn Center for the Biology of Aging, and professor at the University of New South Wales School of Medicine in Sydney.

A previous study led by Sinclair showed that NMN reversed muscle aging in mice.

A plot with many characters

The investigators started by looking at a cast of proteins and molecules suspected to play a part in the cellular aging process. Some of them were well-known characters, others more enigmatic figures.

The researchers already knew that NAD, which declines steadily with age, boosts the activity of the SIRT1 protein, which delays aging and extends life in yeast, flies, and mice. Both SIRT1 and PARP1, a protein known to control DNA repair, consume NAD in their work.

Another protein, DBC1, one of the most abundant proteins in humans and found across life forms from bacteria to plants and animals, was a far murkier presence. Because DBC1 previously had been shown to inhibit vitality-boosting SIRT1, the researchers suspected DBC1 may also somehow interact with PARP1, given the similar roles PARP1 and SIRT1 play.

“We thought if there is a connection between SIRT1 and DBC1, on one hand, and between SIRT1 and PARP1 on the other, then maybe PARP1 and DBC1 were also engaged in some sort of intracellular game,” said Jun Li, first author on the study and a research fellow in the Department of Genetics at HMS.

They were.

To get a better sense of the chemical relationship among the three proteins, the scientists measured the molecular markers of protein-to-protein interaction inside human kidney cells. DBC1 and PARP1 bound powerfully to each other. However, when NAD levels increased, that bond was disrupted. The more NAD was present inside cells, the fewer molecular bonds PARP1 and DBC1 could form. When researchers inhibited NAD, the number of PARP1-DBC1 bonds went up. In other words, when NAD is plentiful, it prevents DBC1 from binding to PARP1 and meddling with its ability to mend damaged DNA.

What this suggests, the researchers said, is that as NAD declines with age, fewer and fewer NAD molecules are around to stop the harmful interaction between DBC1 and PARP1. The result: DNA breaks go unrepaired and, as these breaks accumulate over time, precipitate cell damage, cell mutations, cell death, and loss of organ function.

Averting mischief

Next, to understand how exactly NAD prevents DBC1 from binding to PARP1, the team homed in on a region of DBC1 known as NHD, a pocket-like structure found in some 80,000 proteins across life forms and species whose function has eluded scientists. The team’s experiments showed that NHD is an NAD binding site and that in DBC1, NAD blocks this specific region to prevent DBC1 from locking in with PARP1 and interfering with DNA repair.

Sinclair said that since NHD is so common across species, the finding suggests that by binding to it, NAD may play a similar role averting harmful protein interactions across many species to control DNA repair and other cell survival processes.

To determine how the proteins interacted beyond the lab dish and in living organisms, the researchers treated young and old mice with the NAD precursor NMN, which makes up half of an NAD molecule. NAD is too large to cross the cell membrane, but NMN can slip across it easily. Once inside the cell, NMN binds to another NMN molecule to form NAD.

As expected, old mice had lower levels of NAD in their livers, lower levels of PARP1, and a greater number of PARP1 with DBC1 stuck to their backs.

After receiving NMN with their drinking water for a week, however, old mice showed marked differences both in NAD levels and PARP1 activity. NAD levels in the livers of old mice shot up to levels similar to those seen in younger mice. The cells of mice treated with NMN also showed increased PARP1 activity and fewer PARP1 and DBC1 molecules binding together. The animals also showed a decline in molecular markers that signal DNA damage.

In a final step, scientists exposed mice to DNA-damaging radiation. Cells of animals pre-treated with NMN showed lower levels of DNA damage. Such mice also didn’t exhibit the typical radiation-induced aberrations in blood counts, such as altered white cell counts and changes in lymphocyte and hemoglobin levels. The protective effect was seen even in mice treated with NMN after radiation exposure.

Taken together, the results shed light on the mechanism behind cellular demise induced by DNA damage. They also suggest that restoring NAD levels by NMN treatment should be explored further as a possible therapy to avert the unwanted side effects of environmental radiation, as well as radiation exposure from cancer treatments.