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Abstract

The coenzyme NAD(+) is critical in cellular bioenergetics and adaptive stress responses. Its depletion has emerged as a fundamental feature of aging that may predispose to a wide range of chronic diseases. Maintenance of NAD(+) levels is important for cells with high energy demands and for proficient neuronal function. NAD(+) depletion is detected in major neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, cardiovascular disease and muscle atrophy. Emerging evidence suggests that NAD(+) decrements occur in various tissues during aging, and that physiological and pharmacological interventions bolstering cellular NAD(+) levels might retard aspects of aging and forestall some age-related diseases. Here, we discuss aspects of NAD(+) biosynthesis, together with putative mechanisms of NAD(+) action against aging, including recent preclinical and clinical trials.
Feature Review
NAD
+
in Aging: Molecular
Mechanisms and Translational
Implications
Evandro F. Fang,
1,2,7
Soe Lautrup,
1,3,7
Yujun Hou,
1
Tyler G. Demarest,
1,4
Deborah L. Croteau,
1
Mark P. Mattson,
4,5
and Vilhelm A. Bohr
1,6,
*
The coenzyme NAD
+
is critical in cellular bioenergetics and adaptive stress
responses. Its depletion has emerged as a fundamental feature of aging that
may predispose to a wide range of chronic diseases. Maintenance of NAD
+
levels is important for cells with high energy demands and for procient
neuronal function. NAD
+
depletion is detected in major neurodegenerative
diseases, such as Alzheimers and Parkinsons diseases, cardiovascular dis-
ease and muscle atrophy. Emerging evidence suggests that NAD
+
decrements
occur in various tissues during aging, and that physiological and pharmaco-
logical interventions bolstering cellular NAD
+
levels might retard aspects of
aging and forestall some age-related diseases. Here, we discuss aspects of
NAD
+
biosynthesis, together with putative mechanisms of NAD
+
action against
aging, including recent preclinical and clinical trials.
NAD
+
Has a Key Role in Human Health
Nicotinamide adenine dinucleotide (NAD
+
) is a cofactor for numerous enzymes involved in
cellular energy metabolism, and for adaptive responses of cells to bioenergetic and oxidative
stress. Over 110 years ago, NAD
+
was discovered as a cozymasenecessary for fermentation
(Figure S1 in the supplemental information online) [15]. In his 1930 Nobel lecture, Dr Hans von
Euler-Chelpin stated that cozymase (NAD
+
) is one of the most widespread and biologically
important activators within the plant and animal world[6]. NAD
+
is a necessary cofactor for
many metabolic pathways, such as glycolysis, fatty acid b-oxidation, and the tricarboxylic acid
cycle, while the reduced form of NAD
+
(NADH) is a primary hydride donor in the production of
ATP via anaerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) [7].
Recently, the importance of NAD
+
has expanded from a key element in intermediate metabo-
lism to a critical regulator of multiple cell signaling pathways, and is now a major player in aging
and age-related diseases [811].
Mounting evidence links compromised NAD
+
status to the hallmarks of aging (see Glossary).
Among the age-related cellular processes that may predispose to disease, impaired autoph-
agy has emerged as an important component [12,13]. Indeed, recent studies in human cells
and animal models have unveiled a novel role for NAD
+
in autophagy and mitophagy, in which
impairments in lysosome-targeting and recycling mechanisms result in the accumulation of
damaged molecules and mitochondria, leading to cell dysfunction and/or death [14]. Here, we
discuss the interconnected roles of autophagy (especially mitochondrial autophagy or mitoph-
agy), mitochondrial maintenance, DNA repair, and cell death relative to NAD
+
metabolism. We
summarize new insights into pathways of NAD
+
biosynthesis and consumption, and highlight
Trends
Recent discoveries have demon-
strated an age-dependent decrease
in cellular and/or tissue NAD
+
levels
in laboratory animal models. Moreover,
NAD
+
depletion has been linked to
multiple hallmarks of aging.
In premature aging animal models,
NAD
+
levels are decreased, while
NAD
+
replenishment can improve life-
span and healthspan through DNA
repair and mitochondrial maintenance.
Mitochondrial autophagy (mitophagy)
has a major role in clearance of
damaged and/or dysfunctional mito-
chondria, and compromised mito-
phagy has been linked to metabolic
disorders, neurodegeneration [includ-
ing Alzheimers disease (AD) and Par-
kinsons disease (PD)] in addition to
aging, and other age-related diseases.
New evidence suggests that NAD
+
precursors, such as nicotinamide
and nicotinamide riboside, forestall
pathology and cognitive decline in
mouse models of AD.
NAD
+
supplementation can inhibit
multiple aging features in animal mod-
els. This highlights essential roles for
NAD
+
in maintaining healthy aging, and
suggests that NAD
+
repletion may
have broad benets in humans.
1
Laboratory of Molecular Gerontology,
National Institute on Aging, National
Institutes of Health, Baltimore, MD
21224, USA
2
Department of Clinical Molecular
TRMOME 1265 No. of Pages 18
Trends in Molecular Medicine, Month Year, Vol. xx, No. yy http://dx.doi.org/10.1016/j.molmed.2017.08.001 1
Published by Elsevier Ltd.
TRMOME 1265 No. of Pages 18
how NAD
+
depletion might contribute to mammalian aging. We also examine how supple-
mentation of NAD
+
precursors might constitute a promising therapeutic strategy to counter
aging-associated pathologies and/or accelerated aging.
NAD
+
Biosynthesis and Consumption
Cellular NAD
+
Levels
NAD
+
is produced in all eukaryotic cells. The basal intracellular NAD
+
concentration can be up
to 800 mM in yeast [15], 100-400 mM in human HEK293 cells [16,17], and approximately
0.2 mmol/kg in mouse tibialis anterior muscle [18]. New methods have been developed to
enable the detection of subcellular NAD
+
levels (Box 1). NAD
+
is consumed in many catabolic
pathways: in the cytosol, NAD
+
is reduced to NADH by lactate dehydrogenase (LDH) during
anaerobic glycolysis [7]. In mitochondria, the three tricarboxylic acid cycle enzymes, isocitrate
dehydrogenase (IDH), a-ketoglutarate dehydrogenase (a-KGDH), and malate dehydrogenase
(MDH), reduce NAD
+
to NADH. NADH serves as the primary source of reducing equivalents for
complex I (NADH dehydrogenase) of the electron transport chain (ETC) to fuel OXPHOS,
generating NAD
+
, ultimately reducing oxygen to H
2
O and producing ATP [15]. Exercise and diet
can affect NAD
+
concentration in various tissues. For example, 6 weeks of exercise was shown
to improve glucose tolerance and increase muscle NAD
+
in a mouse model of high-fat diet
(HFD)-induced obesity [19,20]. Studies in both mice and humans have shown that exercise can
change circulating NAD
+
in a biphasic manner with moderate intensity exercise increasing, and
strenuous exercise reducing, NAD
+
[21]. In addition, while a high-fat diet decreases muscle
NAD
+
in HFD-induced obese mice, exercise and caloric restriction can increase NAD
+
in the
muscle and liver of obese or aged mice, respectively [19,20]. Thus, intracellular NAD
+
is not only
regulated by many cellular activities, including OXPHOS, mitochondrial metabolism, transcrip-
tion, and signaling, but can also be signicantly inuenced by diet, exercise, and other health
conditions.
NAD
+
Biosynthesis
In mammals, cellular NAD
+
is synthesized from a variety of dietary sources, including NAD
+
itself
(it is metabolized in the gut, then synthesized again in cells), and from one or more of its major
precursors: tryptophan (Trp), nicotinic acid (NA), nicotinamide riboside (NR), nicotinamide
mononucleotide (NMN), and nicotinamide (NAM) [22]. Food sources of NAD
+
and its precursors
are summarized in Box 1. Depending on the bioavailability of the precursors, there are three
Biology, University of Oslo and
Akershus University Hospital, 1478
Lørenskog, Norway
3
Danish Aging Research Center,
Department of Molecular Biology and
Genetics, University of Aarhus, 8000
Aarhus C, Denmark
4
Laboratory of Neurosciences,
National Institute on Aging, National
Institutes of Health, Baltimore, MD
21224, USA
5
Department of Neuroscience, Johns
Hopkins University School of
Medicine, Baltimore, MD 21205, USA
6
Danish Center for Healthy Aging,
University of Copenhagen,
Blegdamsvej 3B, 2200 Copenhagen,
Denmark
7
Co-rst authors
*Correspondence:
vbohr@nih.gov (V.A. Bohr).
Box 1. NAD
+
Measurement, Subcellular Concentrations, and Molecular Precursors
Different assays are used to detect NAD
+
, including high-performance liquid chromatography (HPLC)-based methods
(e.g., HPLC/MALDI/MS) [16] and NAD
+
/NADH enzymatic cycling assays [28] (including commercial kits). The recent
development of genetically encoded uorescent biosensors, such as SoNar and a biosensor with a bipartite NAD
+
-
binding domain, allows the imaging of relative levels of free NAD
+
in subcellular compartments [17,120]. For example, in
HEK293T cells, the concentration of NAD
+
is similar in the cytoplasm and nucleus (approximately 100 mM), and higher in
the mitochondria (230 mM) [17]. These levels are consistent with other reports demonstrating that, in highly metabo-
lically active, postmitotic cells, such as neurons, mitochondria have higher NAD
+
levels compared with other subcellular
compartments [9,121]. Moreover, the nuclear and cytoplasmic NAD
+
pools are interchangeable, while the mitochon-
drial pool is relatively isolated, although a substantial decrease in the cytosolic pool may inuence the mitochondrial pool
[17].
NAD
+
precursors include nicotinamide (NAM), nicotinic acid (NA), tryptophan (Trp), nicotinamide riboside (NR), and
nicotinamide mononucleotide (NMN). Niacin (vitamin B3; NA and NAM) is abundant in eggs, sh, meat, diary, some
vegetables, and whole grains. Milk is a source of NR [122], and NMN is present in various types of food, including
broccoli (0.251.12 mg/100 g), avocado (0.361.60 mg/100 g), and beef (0.060.42 mg/100 g) [44]. NAD
+
, NADH,
NADPH, and NADP are also present in many foods, and ingested NAD
+
can be metabolized to precursors, including NR
(mild digestion), NMN (by intestinal brush border cells [22]), NAM, and NA (deaminated from NAM by gut bacterial
nicotinamidase), followed by absorption in the intestinal epithelial cells and transfer to the blood [123] (see Figure 1 in the
main text). The gut can also directly absorb these NAD
+
precursors from foods and dietary supplements.
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TRMOME 1265 No. of Pages 18
Glossary
3xTg AD mice: a model of AD. The
mice express the human genes
encoding Amyloid precursor protein
(APP) and Presenilin 1 (PS1), similar
to the APP/PS1 mouse, but also
express human Tau. The mice show
AD-like phenotypes, similar to APP/
PS1 mice, including tau tangles and
brils in the brain.
Amyloid beta (Ab) plaques: formed
from cleaved APP; Abaggregates to
form large rafts of proteins that are
toxic to neurons.
APP/PS1 mice: a model of AD.
Mice express the human genes
encoding APP and PS1. The mice
show severe Abplaque formation
and neurodegeneration and/or
cognitive impairments.
Ataxia telangiectasia (A-T): a rare,
untreatable, recessive inherited
human disease characterized by
severe neuromotor dysfunction,
telangiectasia, sterility, cancer, and
hypersensitivity to ionizing radiation.
It is caused by mutations in the ATM
gene, encoding ATM kinase, a
regulator of the DNA damage
response that is critical for genomic
stability, telomere maintenance, and
DNA double-strand break repair.
Autophagy: evolutionarily conserved
process where cytoplasmic
substrates are engulfed in an
autophagic vesicle, fused to
lysosomes, followed by degradation
and recycling. Autophagy is
necessary for cellular homeostasis
through a balance with apoptosis
and inammation. Compromised
autophagy occurs in many age-
related diseases.
Caloric restriction: diet where the
organism (e.g., mice) are not fed ad
libitum. This restriction of food intake
has been shown to increase lifespan.
Cockayne syndrome (CS): a rare
premature aging disease with
progressive neurodegeneration
caused predominantly by mutations
in genes encoding two DNA repair
proteins: Cockayne syndrome group
A (CSA) or Cockayne syndrome
group B (CSB).
De novo biosynthesis pathway/
kynurenine pathway: pathway from
which NAD
+
is produced from
tryptophan.
DNA damage response: the
response of a cell to DNA damage.
DNA damage activates a range of
cellular processes, enabling the cell
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 PreissHandler pathway; and (iii) from NAM, NR,
and NMN in the salvage pathway (Figure 1).
The de novo biosynthesis pathway and the PreissHandler pathway are well characterized
(recently reviewed elsewhere [8,9]). Here, we focus on the salvage pathway, which is important
from a translational research perspective because it produces NAD
+
from the precursors NAM,
NMN, and NR; these have shown potential as dietary supplements to increase intracellular
NAD
+
levels (see below). The major steps and key enzymes in this pathway are discussed in
Box 2.
While there is inherent redundancy among the three NAD
+
synthetic pathways, distinct
functions can be ascribed to the importance of specic metabolites and/or the tissue-specic
expression of some of the enzymes in each pathway. The NA mononucleotide transferases
(NMNATs), which are necessary for both the PreissHandler pathway and the salvage pathway
(Figure 1), have pivotal roles in embryonic development in mice [23] and in neuroprotection
across species [2325].Nmnat1
/
mice exhibit embryonic lethality [23] and studies in
Drosophila suggest a general neuroprotective function for NMNAT. Specically, overexpression
of NMNAT in a Drosophila model of spinocerebellar ataxia 1 (SCA1)-induced neurodegenera-
tion indicated that NMNAT could inhibit activity-induced neurodegeneration, injury-induced
axonal degeneration, as well as SCA1-induced neurodegeneration [24]. In addition, mice
lacking nicotinamide phosphoribosyltransferase (Nampt), a key enzyme in the salvage path-
way, also exhibit embryonic lethality [26]. Furthermore, murine neurons and pancreatic bcells
express low levels of Nampt protein compared with other cells and pancreatic bcells have been
reported to be particularly vulnerable to NAMPT inhibition [26,27]. The importance of NAMPT
has also been noted in mice with muscle-specic Nampt depletion; these mice experience ber
degeneration and loss of strength and endurance, whereas lifelong Nampt overexpression
increases NAD
+
and improves the physical function of aged mice [28]. Of note, the three major
NAD
+
-synthesizing pathways use different substrates for the generation of NAD
+
; thus, the
production of NAD
+
may be dependent on both substrate availability and the local levels of
synthesizing proteins (e.g., NAMPT, NMNATs, etc.).
NAD
+
-Consuming Enzymes
NAD
+
is a coenzyme for three groups of enzymes: (i) deacetylases in the sirtuin family (SIRTs); (ii)
ADP-ribosyltransferases, including poly(ADP-ribose) polymerases (PARPs); and (iii) cyclic ADP-
ribose synthases (cADPRSs) (Figure 1). SIRTs are a group of NAD
+
-dependent deacetylases
and ADP-ribosyltransferases that promote mitochondrial homeostasis, neuronal survival, stem
cell rejuvenation, and prevent certain aspects of the aging process, such as neurodegenera-
tion, loss of stem cells, and mitochondrial dysfunction [2931]. ADP ribosylation is an important
protein post-translational modication that affects DNA repair, epigenetic regulation, neuro-
degeneration, and aging [14]. ADP ribosylation of proteins is executed by ADP-ribosyltransfer-
ases through transfer of the ADP-ribose moiety from NAD
+
to target substrates. PARPs are
prominent members of the ADP-ribosyltransferase family, comprising 17 different enzymes in
mammals [32]. PARPs transfer the rst ADP-ribose unit from NAD
+
to target proteins, followed
by the sequential addition of ADP-ribose units to the preceding ones to form poly(ADP-ribose)
polymers (PARs) [32]. The cADPRSs include CD38 and its homolog CD157, in mammals and
birds: CD38 and CD157 are transmembrane proteins, localized to the plasma membrane and
to membranes of intracellular organelles, including the mitochondria, nucleus, and endoplas-
mic reticulum [33]. CD38 is expressed in immune cells, liver, testis, kidney, and brain [33]. It has
important roles in several physiological processes, such as nuclear Ca
2+
homeostasis, immu-
nity, inammation, as well as glucose and lipid homeostasis [3436]. cADPRSs can function as
glycohydrolases or NADases, hydrolyzing NAD
+
to NAM and ADP-ribose [9]. These NAD
+
-
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TRMOME 1265 No. of Pages 18
to survive and maintain genome
integrity.
Duchennes muscular dystrophy:
a genetic disorder caused by the
lack of dystrophin, which results in
muscle weakness and degeneration.
Hallmarks of aging: concepts or
cellular and/or organismal processes
all inuencing and contributing to the
process of aging.
Homologous recombination (HR)-
based double-strand break repair
(DSBR): repair process of DSBs by
the repair pathway HR. HR is
dependent on the cell cycle.
KK/H1J mouse model: a model
often used to study metabolic
syndromes because it presents
inherited glucose intolerance and
insulin resistance, which result in
hyperglycemia. KK/HlJ mice have a
strong tendency to develop type 2
diabetes mellitus in response to
certain dietary regimens (e.g., high-
fat diet) and aging.
Mdx mouse model: a model of
Duchennes muscle dystrophy with a
phenotype resembling that of human
patients, including muscle weakness
and degeneration.
Metabolic syndrome: a group of
risk factors and pathologies,
including heart disease, diabetes,
obesity, stroke, among others.
Mitophagy: a specialized form of
autophagy that regulates the
turnover of damaged and
dysfunctional mitochondria.
Nampt
/
mice: mice that do not
express NAMPT, a key protein in the
NAD
+
salvage pathway. They show
embryonic lethality.
Nicotinamide adenine
dinucleotide (NAD): a major
coenzyme and/or compound in all
human cells, exists in oxidized
(NAD
+
) and reduced (NADH) forms.
NAD
+
has major roles in cellular
energy metabolism, adaptive
responses of cells to bioenergetic
and oxidative stress, and aging.
Nonhomologous end joining
(NHEJ)-based DSBR: the only
DSBR pathway in postreplicative
cells, including neurons.
Nucleotide excision repair (NER):
a DNA repair pathway repairing bulky
DNA adducts introduced in DNA by
UV irradiation, environmental toxins,
and certain antitumor agents.
Parkin: a E3 ubiquitin ligase;
mutations in PARKIN, together with
PINK1 (a serine/threonine kinase),
are a leading cause of PD. PARKIN
Figure 1. Nicotinamide Adenine Dinucleotide (NAD
+
) Biosynthetic Pathways. NAD
+
is synthesized via three
major pathways in mammals. The rst is the de novo biosynthesis from tryptophan (Trp) (to the left) in a total of eight steps.
Four steps are shown in this gure, including the conversion of Trp to formylkinurenine (FK), and a spontaneous reaction
conversion of 2-amino-3-carboxymuconate semialdehyde (ACMS) to quinolinic acid (Qa). Qa is then converted to nicotinic
acid mononucleotide (NAMN) by quinolinate phosphoribosyltransferase (QPRT). The second pathway is the Preiss
Handler pathway, initiated by the conversion of NA to NAMN by NA phosphoribosyl-transferase (NAPRT). NAMN, an
intermediate in both de novo biosynthesis and the PreissHandler pathway, is then converted to form NA adenine
dinucleotide (NAAD) by NA mononucleotide transferases (NMNATs). The link between Trp and NA shown here is the
human pathway. The last step of these pathways is the conversion of NAAD to NAD
+
by NAD
+
synthase (NADS). The third
pathway is the Salvage pathway, generating NAD
+
from nicotinamide riboside (NR), which also includes the recycling of
nicotinamide (NAM) back to NAD
+
via NAM mononucleotide (NMN). Extracellularly, NAD
+
or NAM can be converted to
NMN, which is in turn dephosphorylated to NR, possibly by CD73. NR is transported into the cell via an as yet unknown
mechanism (possibly nucleoside transporters), where it is phosphorylated by nicotinamide riboside kinase (NRK)1 or NRK2
forming NMN. NMN is then converted to NAD
+
by NMNATs. The broken lines indicate that the mechanism and involved
proteins are not yet known. The lower part of the gure shows the major NAD
+
-consuming enzymes. From left: the cyclic
ADP-ribose synthases (cADPRSs) CD38 and CD157 hydrolyze NAD
+
to NAM; in addition, CD38 can degrade NMN to
NAM, removing NMN from NAD
+
synthesis. Poly(ADP-ribose) polymerases (PARPs), especially PARP1 and PARP2, use
NAD
+
as a co-substrate to PARylate target proteins, generating NAM as a by-product. The deacetylation activity of Sirtuin
(SIRT)1, SIRT3, and SIRT6 depend on NAD
+
, generating NAM as a by-product, which can inhibit the activity of SIRTs. The
enzyme NAM N-methyltransferase (NNMT) methylates NAM, using S-adenosyl methionine (SAM) as a methyl donor. This
removes NAM from recycling, and indirectly affects NAD
+
levels. Abbreviations: IDO, indoleamine 2,3-dioxygenase; Me-
NAM, methylated nicotinic acid mononucleotide; NAMPT, nicotinamide phosphoribosyl transferase (including extracellular
and intracellular ones); TDO, tryptophan 2,3-dioxygenase.
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TRMOME 1265 No. of Pages 18
and PINK1 are also involved in
mitophagy.
PARylation: post-translational
modication performed by PARPs,
mainly PARP1 in mammalian cells,
via the use of NAD
+
. PARylation is
also known as poly(ADP-ribosylation),
PreissHandler pathway: pathway
from which NAD
+
is produced from
nicotinic acid.
Premature aging syndromes
(accelerated aging/progeria): rare
diseases in which patients show
aspects of aging at a very early age.
Salvage pathway: primary pathway
from which NAD
+
is produced from
NR, NMN, or NAM.
Tau tangles: rafts or aggregates of
Tau proteins, often
hyperphosphorylated, causing
dysfunction and cell death of
affected neurons.
Xeroderma pigmentosum (XP): a
rare autosomal-recessive disorder
characterized by severe sun
sensitivity and skin cancer. The
etiology of XP is caused by mutation
of genes encoding a group of DNA
repair proteins, XP genes.
g-H2AX foci: foci that occur when
the histone H2A variant H2AX is
phosphorylated rapidly after a DSB
induction in DNA. Given that the
phosphorylation of H2AX occurs
rapidly after the induction of DSBs
and correlates well with DSBs, it is
often used as a DSB marker.
consuming enzymes regulate a spectrum of cellular activities, including mitochondrial mainte-
nance, DNA repair, and stem cell rejuvenation, processes that are critical for cellular health (see
also recent reviews [9,11,35]).
Crosstalk among NAD
+
-Consuming Enzymes: SIRTs, PARPs, and cADPRSs
The different classes of NAD
+
-consuming enzymes compete for bioavailable NAD
+
, which
affects their cellular functions in human health. Thus, hyperactivity of one protein might limit the
activities of the others and, conversely, inhibition of one protein may increase the NAD
+
pool for
the others [35]. For example, SIRT1 activity can be substantially decreased when there is
excessive PARP1 activation, and PARP1 or PARP2 deletion in human kidney cells and in in vivo
models (Parp2
/
mice) have been reported to increase SIRT1 activity [37,38]. Persistent
activation of PARP1, caused by DNA damage, has resulted in a greater than 50% decrease in
cellular NAD
+
in DNA repair-decient primary rat neurons and human neuroblastoma cells
[14,39]. Moreover, in DNA repair-decient human neuroblastoma cells, as well as in mouse and
Caenorhabditis elegans models, cellular NAD
+
and Sirt1 activity have been reported to increase
after treatment with Parp1 inhibitors {3-aminobenzamide, 3,4-dihydro-5-[4-(1-piperidinyl)
butoxyl]-1(2H)-isoquinolinone} or after supplementation with NAD
+
precursors (NR or NMN)
[14,39]. These studies suggest that PARP1 inhibition constitutes a potential therapeutic target
to sustain cellular NAD
+
and to maintain SIRT activity.
CD38 is another major NADase in tissues. In APP/PS1 mice, CD38 depletion and increased
NAD
+
led to neuroprotection, as evidenced from a reduction in amyloid beta (Ab) aggregates in
the brain, which was associated with improved learning [40]. In addition, CD38-decient mice
showed protection against HFD-induced obesity and metabolic syndrome, exhibiting higher
metabolic rates compared with wild-type mice [41]. This resistance to diet-induced obesity has
been attributed, at least in part, to NAD
+
-dependent activation of Sirt1 and the mitochondrial
regulator peroxisome proliferator-activated receptor gcoactivator (PGC-1a)[41]. Of note,
CD38-decient mice show signicant neuroprotection in the brain despite high levels of
PARylation [42], indicating that the available NAD
+
appears to be sufcient in enabling the
activity of Parp1, Sirt1, and other NAD
+
-dependent enzymes. A recent study showed an age-
dependent increase of CD38 in murine tissues, and documented (using CD38-knockout mice)
Box 2. The NAD
+
Salvage Pathway and Its Major Enzymes
The salvage pathway describes the formation of NAD+ from precursors including NR, NMN, and NAM. NR can be
transported into cells via nucleoside transporters, although a NR-specic transporter has not yet been identied in
mammals. In the cytoplasm, NR is phosphorylated by nicotinamide riboside kinases (NRK1-2) to form NMN [122]. NRKs
are not expressed in mitochondria, suggesting that the phosphorylation step occurs in the cytosolic compartment [124].
In addition, extracellular interconversion of NAD
+
and its precursors may occur (see Figure 1 in the main text).
Extracellular NMN can be converted from NAD
+
and NAM, with the latter in a nicotinamide phosphoribosyl-transferase
(NAMPT) (extracellular)-dependent manner. NMN can either enter the cells directly, although a mechanism that is still
unclear, or be converted to NR before transport into cells [124].
(Intracellular) NAMPT rst converts NAM to NMN, which is then adenylated to NAD
+
by NMNATs. NMNATs are the nal
step in NAD
+
synthesis and are also involved in the PreissHandler pathway. In humans, there are three NMNAT
isoforms with different subcellular localizations. NMNAT1 is localized in the nucleus, and shows the highest expression
in skeletal muscle, heart, kidney, liver, and pancreas [125]. NMNAT2 is primarily found in the cytosol, tethered to the
outer membrane of the Golgi apparatus by palmitoylation. NMNAT3 is localized to mitochondria and expressed at high
levels in skeletal muscle and heart tissue [125]. Thus, intracellular NMN can be converted to NAD
+
in the nucleus,
cytoplasm, or the mitochondria by one of the three NMNATs. It is still unclear how cytoplasmic NMN is imported into
mitochondria.
NAM is not only an ingested NAD
+
precursor, but is also a byproduct of the degradation of cellular NAD
+
by NAD
+
-
consuming enzymes (detailed below). In mammalian cells, nicotinamide N-methyltransferase (NNMT) removes NAM
from the NAD
+
biosynthesis pathway through methylation of NAM, which is further degraded into N-methyl-2-pyridone-
5-carboxamide (Me2PY) and N-methyl-4-pyridone-5-carboxamide (Me4PY) [77].
Trends in Molecular Medicine, Month Year, Vol. xx, No. yy 5
TRMOME 1265 No. of Pages 18
that CD38 was required for the observed age-related NAD
+
decline [43]. This is interesting
because CD38 not only consumes NAD
+
, but can also degrade the NAD
+
precursor NMN [43].
Thus, there appears to be a delicate balance between NAD
+
consumption and bioavailability,
which is important for cellular function and survival. However, further studies of the functional
signicance of the interconnected networks between NAD
+
-consuming enzymes is warranted.
NAD
+
in Aging and Age-Related Diseases
Age-Dependent Decrease of NAD
+
in Animals and Humans
Mice display an age-dependent decrease of NAD
+
in multiple organs, including brain, liver,
muscle, pancreas, adipose tissue, and skin [14,4446].InC. elegans, an age-dependent
reduction of NAD
+
has also been reported [14,39]. There is also evidence of decreased NAD
+
in
aged human tissues. Specically, in vivo NAD
+
assays have been used to demonstrate that
intracellular NAD
+
declines with age in the human brain [47]. Also, NAD
+
in post-pubescent
males and females negatively correlates with age [48]. Together, these data suggest that there
is a universal age-dependent decrease of cellular NAD
+
across species. However, it is not clear
whether this is due to increased NAD
+
consumption and/or decreased production.
NAD
+
Replenishment Can Improve Lifespan and Healthspan
The effects of different NAD
+
precursors, including NR and NMN, on the lifespan and health-
span of yeast, C. elegans,Drosophila, and mice have been investigated. One study showed
that 10 mM NR could extend the replicative lifespan of wild-type yeast by more than ten
generations [15]. In this yeast model, two NAD
+
synthetic pathways appeared to be necessary
for the NR-induced lifespan extension, the tNrk1 and the Urh1/Pnp1/Meu1 pathways [15].InC.
elegans, 500 mM NR extended the average lifespan of wild-type worms (N2) via the SIR-2.1
(ortholog to mammalian SIRT1) pathway [39]. For Drosophila, no information is available
regarding a direct effect of NAD
+
precursors on lifespan, but genetic overexpression of an
NAD
+
synthetic enzyme nicotinamidase (D-NAAM) has been reported to extend lifespan [49].
D-NAAM, an ortholog of yeast PNC1, functions in the NAD
+
salvage pathway and converts
NAM to NA [49].InDrosophila, overexpression of D-NAAM can increase the NAD
+
:NADH ratio,
as well as the mean and maximal lifespan by up to 30% in a Sir2-dependent manner [49].
Notably, NR has been shown to improve mouse lifespan, even when administered late in life. At
approximately 2 years of age, C57BL/6J mice were given NR, resulting in a signicant increase
in lifespan (5%) [46]. Supplementation with NAD
+
precursors not only extends lifespan, but also
improves healthspan in yeast, ies, worms, and mice, as shown by various features, including
improved mitochondrial health, muscle strength, and motor function [15,39,46,4951].In
summary, these data suggest that NAD
+
replenishment delays normal aging in laboratory
animal models.
NAD
+
may also delay the onset of aging in some premature aging diseases. These are a group
of rare diseases in which patients exhibit aging features at a younger age. DNA repair
impairment is a cause of many of these diseases, and some patients exhibit severe neuro-
degeneration, as in the case of Xeroderma pigmentosum group A (XPA), Cockayne
syndrome (CS), and Ataxia-telangiectasia (A-T)[14]. XPA is caused by mutations in the
XPA gene, which participates in nucleotide excision DNA repair (NER) [52,53]. The etiology
of CS has been related to mutations in two proteins, CS group A (CSA) and CS group B (CSB)
[54,55]. A-T is a multifaceted disease caused by mutations in A-T mutated protein (ATM), a
master regulator of the DNA damage response. ATM has a key role in DNA double-strand
break repair (DSBR). Interestingly, all three premature aging diseases show mitochondrial
dysfunction and NAD
+
depletion (demonstrated in C. elegans, mice, and human cells) [14].
Furthermore, NAD
+
replenishment, using NR and/or NMN, improved the lifespan and health-
span of C. elegans in relevant models of XPA, A-T, and CS, relative to controls [14,39,56].
Remarkably, NR extended the lifespan in Atm
/
mice (B6;129S4-Atm
tm1Bal
/J), which typically
6Trends in Molecular Medicine, Month Year, Vol. xx, No. yy
TRMOME 1265 No. of Pages 18
perish at 35 months of age. Specically, NR supplementation was given at 12 mM in drinking
water after weaning, resulting in 80% survival at 10 months of age [39].InC. elegans, NAD
+
supplementation has been shown to improve neuronal DNA repair through deacetylation of the
DNA repair protein Ku70, and to restore mitochondrial homeostasis via the mitophagy regulator
NIX (DCT-1 in C. elegans)[39]. The DCT-1 mechanism of mitochondrial homeostasis was
implicated based on evidence that DCT-1 co-localized with mitochondria in Atm-1 worms [39].
Of note, NR and NMN exhibited similar benecial effects in A-T models of C. elegans and mice
[39]. In another example, mice hypomorphic for BubR1 (a mitotic checkpoint kinase) also
presented with signs of premature aging, and mice overexpressing BubR1 exhibit an extended
lifespan [57]. This is pertinent because loss of BubR1 during aging can result from NAD
+
depletion and decreased Sirt2 activity, rendering Sirt2 unable to deacetylate BubR1 (normally
Sirt2 deacetylates BubR1, targeting it for ubiquitination and degradation) [57]. Both over-
expression of Sirt2 or NMN treatment increased the lifespan of BubR1 mice, suggesting that
BubR1 stabilization is important for achieving an increased lifespan [57]. In summary, restora-
tion of intracellular NAD
+
has been shown to improve lifespan and healthspan in normal and
prematurely aged laboratory organisms, but whether these ndings can be translated to
humans remains unproven.
NAD
+
and Neurodegeneration
Age is the greatest risk factor for neurodegenerative disorders, including Alzheimers disease
(AD), Parkinsons disease (PD), and hearing loss [58]. Recent studies in AD animal models
suggest that Abplaques,tau tangles, and mitochondrial dysfunction (due to compromised
mitophagy) are among the key features of AD [59]. Strategies to increase intracellular NAD
+
are
considered as novel potential therapeutic interventions in AD. For example, 3 months of NR
treatment has been documented to attenuate cognitive deterioration through Abreduction in
the cortex and hippocampus in a mouse model (AD mouse crossed with a PCG-1aKO mouse)
[60]. The study suggests that NR treatment promotes PGC-1aactivity and induces the
ubiquitin proteasome system, leading to degradation of Abaggregates [60]. Similarly to
NR, NMN has also been found to ameliorate mitochondrial dysfunction and neuronal death
in APP/PS1 mice, as evidenced from restored oxygen consumption rates, increased levels of
Sirt1 and PGC-1a, and normalization of morphology of brain mitochondria from NMN-treated
APP/PS1 mice [61]. NMN was also found to protect against Aboligomer-induced cognitive
impairment, neuronal death, and cognitive dysfunction in a rat model of AD [62]. In another
study, treatment with a different NAD
+
precursor, NAM, delayed pathology and cognitive
decline in 3xTg AD mice, through upregulation of neuronal bioenergetics, including neuro-
plasticity-involved kinases and transcription factors, as well as by improving autophagy
processing (reduced autophagosome accumulation mediated by enhanced lysosome and/
or autolysosome acidication) [63]. At the molecular level, NAD
+
-dependent reduction of AD
phenotypes may be attributed to the upregulation of autophagy and/or mitophagy because
NAD
+
/SIRT1 are able to upregulate autophagy through deacetylation of the major autophagy
proteins Atg5, Atg7, and Atg8 in human cells, murine neurons, and C. elegans [39]. Further-
more, NAD
+
/SIRT1 has been shown to upregulate mitophagy through the forkhead box-O3
(FOXO3)-NIX (BNIP3L/DCT-1) axis in C. elegans and in calorically restricted mice, or indirectly
through an interaction between PGC-1aand Parkin, as evidenced from co-expression of
PGC-1aand Parkin in murine cortical neurons that led to improved mitochondrial biogenesis
and mitophagic activity [39,6466]. Accordingly, upregulation of autophagy and/or mitophagy
has been reported to clear Abplaques, tau tangles, and damaged mitochondria in mice,
leading to improved mitochondrial function and neuronal survival [59,63,67].
PD appears to involve impairment of mitochondrial complex I, as well as compromised
mitophagy in vulnerable neurons of the brain, including midbrain dopaminergic neurons
[68]. There are several promising studies that suggest that NAD
+
precursors have therapeutic
Trends in Molecular Medicine, Month Year, Vol. xx, No. yy 7
TRMOME 1265 No. of Pages 18
potential in PD. Early clinical studies showed that supplementation (intravenous infusion or oral
capsules) with the reduced form of NAD
+
(NADH) improved motor disability in patients with PD
[69]. Recently, in models of PD in vitro (rotenone-treated PC12 cells) and in vivo,inDrosophila
bearing pink1 mutations, studies have shown that NR and NAM administration could amelio-
rate PD phenotypes [70,71]. For example, dietary NAM (5 mM) rescued thoracic defects and
inhibited the loss of dopaminergic neurons in pink1 mutant Drosophila, possibly through
maintenance of a healthy mitochondrial pool, as evidenced from improved mitochondrial
morphology and respiration [71]. However, further studies of NAD
+
precursor treatments in
PD mice are necessary to elucidate the underlying molecular mechanisms and functional roles
of NAD
+
in PD pathology.
Hearing loss is a common feature associated with advanced age, and noise exposure is a major
cause of such loss [72]. Intense noise exposure can result in direct mechanical damage
(acoustic trauma) to cochlear hair cells, and can trigger the delayed death of these auditory
sensory neurons. NR administration (intraperitoneal injection) has been found to protect mice
from transient and permanent noise-induced hearing loss and spiral ganglia neurite degener-
ation, as shown by the retraction of neurite ends from inner hair cells in the cochlea [73]. These
effects appear to be mediated by NAD
+
-dependent mitochondrial Sirt3, since the effect of NR
was reduced in Sirt3-decient mice [73]. This role of Sirt3 is supported by studies showing that
caloric restriction prevents hearing loss in wild-type mice, but not in Sirt3-decient mice [74].
Moreover, ototoxicity, caused by chemical damage to the inner ear, has been reported to be
prevalent in patients with cancer receiving cisplatin chemotherapy [75,76]. Cisplatin causes
DNA damage by crosslinking the two DNA strands. Increased NAD
+
has been reported to
prevent cisplatin-induced cochlear damage through suppression of oxidative stress, DNA
damage, and inammatory responses in mice, as shown by a decrease in g-H2AX signals,
increased Sirt1 activity, and activation of p53 and NF-kB, proteins involved in inammation in
cochlear tissue [75,76]. These studies support the hypothesis that NAD
+
supplementation
could provide a putative treatment option to preserve hearing during normal aging, or in
individuals undergoing chemotherapeutic treatment. However, rigorous testing will be required
to determine whether NAD
+
supplementation might modify the effectiveness of the chemo-
therapeutic treatment.
NAD
+
Can Mitigate Age-Associated Muscle Atrophy and Metabolic Disorders in Laboratory
Animal Models
Studies in C. elegans and mice suggest that NAD
+
supplementation delays the onset of muscle
atrophy, vision loss, as well as certain age-related diseases that might include metabolic
disease, heart dysfunction, and cancer [39,44,50,73,7782].InTable 1 (Key Table), we
summarize the known organismal benets of NAD
+
supplementation for specic animal
models, humans, NAD
+
precursor doses, and putative molecular mechanisms underlying
these improvements.
Skeletal muscle mass and strength is reduced with aging as a result of muscle atrophy, leading
to signicant susceptibility to injury and reduced quality of life [83]. In the mdx mouse model of
Duchennes muscular dystrophy, which exhibits low muscle NAD
+
, NR protects against
disease progression through maintenance of muscle stem cell function and regeneration,
where Sirt1-dependent enhancement of mitochondrial function and energetics as well as a
reduction of PARylation have been implicated [46,77]. In another study, muscle-specic
depletion of Nampt
/
in mice (mNampt
/
mice) caused myocyte necrosis and progressive
loss of muscle function, as well as inducing proinammatory and regenerative transcriptional
programs coincident with alterations in glucose metabolism [28]. In this model, oral NR
supplementation reversed decits in muscle mass, strength, and exercise capacity of
mNampt
/
mice to wild-type levels [28]. In combination, these studies suggest that NAD
+
8Trends in Molecular Medicine, Month Year, Vol. xx, No. yy
TRMOME 1265 No. of Pages 18
Key Table
Table 1. Known Benets and Mechanisms of Action of NAD
+
Precursors in Humans and
Animal Models
Tissue Supplement dose Benets Pathways affected Refs
Human
Neurons NR 500 mM Decreased AT pathology and restored
mitochondrial function in ATM KD cells
"NAD
+
, SIRT1 activation, BDNF levels
and CREB activation, #DNA damage
[38]
Blood Acipimox 250 mg/3/d #triglycerides and glucose in plasma
of patients with T2DM; restored levels
of NEFA
2-fold "NAD
+
[11]
NR 1000 mg/d/7 d 45-fold "NAAD [7]
Muscle Acipimox 250 mg/3/d Improved mt function in patients with
T2DM; "lipid content in patient skeletal
muscle, due to "NEFA
Gene sets affected were similar to
those affected by NR and NMN in
animal models
[6,11]
Worm
Neurons NR 500 mM Improved long- and short-term
memory in Atm worms
"NAD
+
, activation of sir-2.1,
"CREB, HSP-6
[38]
Muscle NR 500 mM Improved mt network in Atm worms. "NAD
+
, sir-2.1 activation,
CREB, HSP-6
[38,45,76]
Lifespan NR 500 mM; NAM 200 mM;
NR 500 mM
"Lifespan and improved tness
with age in wild-type N2 worms;
"lifespan of Atm worms
"NAD
+
, sir-2.1, CREB.
HSP-6, mt content and ATP,
improved metabolism
[38,44]
Mouse
Brain NR 400 mg/kg/d "Neurogenesis; #cognitive deterioration
and Abproduction; "synaptic plasticity
"NAD
+
, activation of Sirt1 and
PGC-1aand degradation of Bace1;
"LC3-II and altered ssion/fusion
balance
[45,59]
NR 250 mg/kg/d
NR or NMN 12 mM NR abolished metabolic prole of cerebellum
from Atm KO mice; NR and NMN improved
mt morphology and health, leading to
neuron protection
Ear/cochlear NR 1000 mg/kg/2/d Prevented transient and permanent hearing
loss (noise-induced) by preventing degeneration
of spiral ganglia neurites
"NAD
+
and Sirt3 activation [38,72]
Eye NMN, 100 or 300 mg/kg/d Prevented age-associated decline of rod and
cone cell function; increased tear production.
Likely due to "NAD
+
level and sirtuin
activity
[43]
Muscle NR 400 mg/kg/d "NAD
+
and mt content, prevented mt
myopathy
Sirt1 and Sirt3 activation, and their
targets Foxo1, Sod2, PGC1a, UPR
mt
,
Fgf21; improved mt function and
mitophagy mediated via Sirt1 and EglN;
"mtDNA encoded proteins and/or
nuclear encoded proteins
[43,45,49,7
6,80,84]
NR 750 mg/kg/d "Lifespan of Trf1
hrt/hrt
mutants and improved
cardiac function
NMN 500 mg/kg/d Reversed age-associated muscle atrophy and
inammation, impaired insulin signaling, and
insulin-stimulated glucose uptake
NMN 300 mg/kg/d Improved mitonuclear signaling and mt function
Stem cells NR 400 mg/kg/d Rejuvenated muscle stem cells; attenuated
senescence in neuronal and melanocyte
stem cells
Activation of UPR
mt
, prohibitin pathways [45,76]
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TRMOME 1265 No. of Pages 18
supplementation inhibits muscular dystrophy in mice, which may have interesting implications
for aged individuals.
NAD
+
replenishment has also been shown to have benecial affects against obesity and/or
other metabolic diseases. NR can enhance oxidative metabolism and protect mice against
HFD-induced obesity and nonalcoholic fatty liver disease (NAFLD) by activating Sirt1 and Sirt3,
and enhancing the energy expenditure and oxygen consumption rate of mitochondria [84,85].
NR induced a Sirt1- and Sirt3-dependent mitochondrial unfolded protein response, thereby
improving mitochondrial metabolism (increasing mitochondrial complex formation and activity)
[85]. NR administration (via an osmotic pump) lowered serum insulin levels and serum choles-
terol concentrations in the KK/H1J mouse model of type 2 diabetes mellitus [86]. Other NAD
+
precursors, such as NMN (delivered intraperitoneally), have been reported to improve diet- and
age-induced diabetes in 2-year-old mice by enhancing hepatic insulin sensitivity and antiox-
idative responses, including the production of glutathione S-transferase, among others [51].
NAD
+
precursors can also act partly through Sirt1 activation, as demonstrated by the abro-
gated effect of NMN via Sirt1 inhibition with EX527 (a Sirt1-specic inhibitor) [51]. NMN
administration also ameliorated the impairment in glucose tolerance and glucose-stimulated
insulin secretion in Nampt
+/
mice [26].
The aforementioned data stronglysuggest that NAD
+
replenishmenthas systemic benetsin aged
laboratory animals and disease models in mice, although further testing is required. Importantly,
translational human intervention studies with NAD
+
supplementation are already in progress
i
.
Potential NAD
+
-Mediated Mechanisms to Counter Aging
While phenotypic studies of NAD
+
replenishment on aging have been extensively explored in
laboratory animals, the exact molecular mechanisms responsible for their benecial effects are
not yet understood. To dissect the interconnected mechanisms of the multifaceted functions of
NAD
+
to aging, we can associate NAD
+
with most of the nine hallmarks of aging[58] (Table 2).
We also consider autophagy impairment as a NAD
+
-related hallmark of aging.
Table 1. (continued)
Tissue Supplement dose Benets Pathways affected Refs
Fat BAT: NR 400 mg/kg/d "Mt content and mt respiratory capacity,
reduced fat mass
"NAD
+
levels, Sirt1 and Sirt3 activation [36,37,
50,84]
WAT: IP NMN 500 mg/kg/d Restored NAD
+
levels in diabetic mice and
normalized glucose tolerance
Liver NR 400 mg/kg/d, 500 mg/kg/d "Oxidative prole, biogenesis, content and
activity of mt; decreased tumorigenesis and
DNA damage; prevented fatty liver and
inammation induced by high-fat high-sucrose diet
Sirt1 and Sirt3 activation, and Sirt1
target genes including Akt, prevention
of NAFLD, decreased DNA damage
[43,50,
84,85]
NMN 500 mg/kg/d,
300 mg/kg/d
Improved hepatic insulin sensitivity, decreased
oxidative stress, and improved inammatory
response, immune response, and lipid metabolism
Pancreas NMN 500 mg/kg/d Improved glucose-stimulated insulin secretion "NAD
+
and Sirt1 activation [50]
Lifespan NR 400 mg/kg/d "Lifespan of mice "NAD
+
, Sirt1 and prohibition
activity; improved mt function;
mitophagy, DNA repair, and
anticancer potential
[38,45]
12 mM NR throughout life "Lifespan of Atm KO mice
Abbreviations: BAT, brown adipose tissue; KO knockout; mt, mitochondrial; T2DM, type 2 diabetes mellitus; UPR, unfolded protein response; WAT, white adipose
tissue; "increase; #decrease.
10 Trends in Molecular Medicine, Month Year, Vol. xx, No. yy
TRMOME 1265 No. of Pages 18
Effects of NAD
+
Supplementation on DNA Repair
Cumulative evidence indicates that impaired genomic maintenance may causally contribute to
aging. An age-dependent accumulation of DNA damage occurs in humans, possibly due to
impaired DNA repair [87]. This suggests that maintenance of efcient DNA repair may delay the
onset of aging and age-related diseases [88,89]. NAD
+
replenishment can improve DNA repair
in cells, C. elegans, and mice. An early study reported that suitable doses of NAM (3 mM)
signicantly enhanced DNA repair in gamma-irradiated XP cells in vitro [90]. In line with this
nding, studies in human aortas suggest that the NAM-consuming enzyme NAMPT has a
signicant role in DNA repair to maintain genome integrity, as demonstrated from an increase in
DNA oxidative DNA lesions and DNA DSBs in murine Nampt-decient murine smooth muscle
cells [91]. In addition to NAM, NA and NR also improve DNA repair in vitro. Indeed, studies
revealed increased DNA repair in vitro in peripheral blood mononuclear cells, where NA
treatment increased DNA repair efciency and decreased micronuclei numbers following X-
ray irradiation, and in murine Atm-decient neurons, following NR administration [39,92]. NAD
+
supplementation can also increase DNA repair in vivo, because wild-type mice treated with NR
showed decreased DNA damage, as evidenced from lower global PARylation and g-H2AX
foci relative to controls [46,77]. As previously mentioned, NAD
+
replenishment can also
improve aging features of some DNA repair-decient premature aging disorders (XPA, CS,
and A-T), such as neurodegeneration, possibly through the upregulation and/or activation of
DNA repair and mitophagy [14,39,93]. For example, due to the dysfunction of the DSBR protein
ATM, accumulation of DNA damage occurs in both nuclear and mitochondrial genomes, as
revealed from human patient broblasts and brain-tissue from Atm-decient mice [94,95].NR
supplementation improved genomic stability in murine Atm-decient neurons and C. elegans
models of A-T, at least partially through Sirt1/Sirt6-dependent DSBR, upon protection against
ionizing irradiation [39]. In neurons, there is no homologous recombination (HR)-based
DSBR, only nonhomologous end joining (NHEJ)-based DSBR [96]. In murine Atm-de-
cient neurons, NR increases DNA-PKC-associated NHEJ through deacetylation of Ku70 [39].
These ndings suggest that enhancing NAD
+
bioavailability can also target and increase DNA
repair.
NAD
+
Maintains Mitochondrial Health
Mitochondrial abundance and quality are pivotal for health, and mitochondrial dysfunction is a
hallmark of aging, detected in a broad spectrum of age-associated diseases [97]. NAD
+
replenishment can inhibit age-dependent mitochondrial decline or production in both C.
elegans and mice [45,46]. For example, dietary NR treatment can compensate for a respiratory
chain defect and reverse exercise intolerance in the mitochondrial disease mouse model
Sco2
KOKI
[98]. NAD
+
can also bolster mitochondrial function by enabling mitochondrial bio-
genesis and mitophagy; mitochondrial biogenesis and respiration are induced by PGC-1a
through the transcriptional upregulation of Nrf1 and Nrf2 in mouse myoblasts [99]. Thus, NAD
+
replenishment has been documented to induce mitochondrial biogenesis through the NAD
+
/
Sirt1-PGC1apathway in aged mice, murine muscle stem cells, and C. elegans [45,46]. Indeed,
NAD
+
can promote the removal of damaged and/or dysfunctional mitochondria via mitophagy,
as detailed below.
NAD
+
in Autophagy Induction
Autophagy has multifaceted roles in health and aging, including the maintenance of cellular
homeostasis, cellular energy (especially during nutrient starvation), neuroprotection, and anti-
inammation [13,67,100]. Compromised autophagy is a common signature of aging and
contributes to age-related diseases, such as AD and PD [13,35,67]. Upregulation of autophagy
can inhibit disease progression in animal models of AD and PD (3xTgAD models) [59,60,63,67].
Moreover, mutations of proteins in the autophagy pathway, including Atg1 (unc-1 in C. elegans)
and other proteins encoded by the Atg genes, can lead to premature aging in yeast,
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TRMOME 1265 No. of Pages 18
Table 2. NAD
+
and the Hallmarks of Aging
Hallmarks of aging Pathways and conditions affected by changes in NAD
+
levels Refs
Genomic instability "NAD
+
levels lead to "DNA repair capacity; #NAD
+
leads to "ROS accumulation [11,14,38,55]
Mutations in DNA repair genes associated with premature aging disorders (e.g., XPA and CS), lead
to #NAD
+
, PARP1 hyperactivation, neurodegeneration, and mitochondrial dysfunction
SIRT1 interacts with DNA repair enzymes Ku70, PARP1, and WRN. PARP1 is involved in both BER and
NER; SIRT3 enhances NHEJ and HR via interaction with PARP1
Telomere attrition Data not available
Epigenetic alterations Treatment with NAD
+
precursors NR and NMN activates SIRTs, which deacetylate and activate
transcription factors, including PGC-1a, FOXOs, and others, all related to aging
[8,9,44,49,59]
PARP1 may be involved in chromatin structure modulation and insulation promotion, associated
with changes in gene expression. Also, PARP-1 may function as a transcriptional co-factor. PARP-2
transcriptionally regulates SIRT1, indirectly connecting NAD
+
levels to epigenetic alterations
Stem cell exhaustion NR treatment rejuvenated muscle, neuronal, and melanocyte SC pools through induction of UPR
mt
and
synthesis of prohibitin proteins; leads to "oxidative respiration and/or ATP levels and higher
mitochondrial membrane potential
[45,76]
NR treatment prevented senescence of muscle SC in mouse model of muscular dystrophy
SIRT1 maintains naïve state of pluripotent SC by deacetylating Oct4. Restoration of NAD
+
in
aged somatic cells (overexpression of NNT or NMNAT3) enhanced reprogramming efciency
and prolonged lifespan of mesenchymal SC by delaying senescence
Loss of proteostasis NAD
+
precursor NR, or PARP1 inhibitors, activate UPR
mt
, causing translocation of FOXO transcription
factors, triggering "antioxidant defenses in mice and worms, prolonging lifespan and health. UPR
mt
activation also observed in yeast
[26,38,39,44,59]
NR treatment of an AD mouse model (Tg2576) "NAD
+
levels and PGC-1a-mediated degradation of
Bace1 leading to #Abproduction. AD mice crossed with CD38 KO mice showed attenuated AD
pathology, suggesting that "NAD
+
leads to #aggregated dysfunctional proteins, such as Ab
Mitochondrial dysfunction Mouse and worm models of XPA, CS, and A-T show impaired mitophagy and mitochondrial biogenesis,
likely due to #activity of NAD
+
-SIRT1-PGC-1aaxis. These defects can be restored by "NAD
+
with
NAD
+
precursors
[14,40,49,50,84]
"NAD
+
in aged mice restores mitochondrial function to that of young mice in a Sirt1-dependent
manner either via PGC-1a/bor AMPK
#NAD
+
leads to #TFAM signaling, likely via HIF-1astabilization, resulting in #mitochondrial biogenesis
and loss of mitochondrial homeostasis. Short-term treatment with NMN "NAD
+
and restored
mitochondrial homeostasis in mice via Sirt1-PGC-1aactivation
Deregulated nutrient sensing NAD
+
levels affected in DIO. NR treatment can help to prevent high-fat DIO by "NAD
+
and
stimulating SIRT1 activity
[9,40,49,50,84]
NNMT deciency protects against DIO by "SAM and NAD
+
in adipose and liver. Benecial effects of
calorie restriction are lost when SIRT1 and SIRT3 are inhibited
NMN treatment of a diabetes mouse model ameliorated glucose intolerance and "hepatic insulin
sensitivity or secretion by restoring NAD
+
levels (likely due to "SIRT1 activity)
NR/NMN treatment of mice fed a HFD "use of lipids as substrates, "energy expenditure,
and improved insulin sensitivity
Altered cellular communication NR treatment prevented noise-induced hearing loss and led to regeneration of neurite ganglia
mediated by NAD
+
-dependent SIRT3 activity. "NAD
+
directed SIRT1 activity delays axon degeneration
[25,40,50,72]
NMN treatment reversed age-related changes in expression of genes related to inammation, partly by
increasing SIRT1 activity. Parp1 KO mice, CD38 KO mice, and NNMT KO mice exhibit "NAD
+
levels and SIRT1 activation, correlating with #risk of high-fat DIO
Cellular senescence NAD
+
concentrations #during senescence [8,45,47]
NAD
+
levels #in aged human tissues, resulting in changes in oxidative stress and cellular metabolism,
suggesting link between NAD
+
metabolism and senescence
12 Trends in Molecular Medicine, Month Year, Vol. xx, No. yy
TRMOME 1265 No. of Pages 18
nematodes, and Drosophila, and treatment with autophagy inhibitors can reduce their lifespan
[101103].
Autophagy induction may delay the onset of aging and potentially slow the initiation or
progression of age-related diseases [13]. For example, upregulation of autophagy inhibits
major markers of aging, including inammation and senescence. Indeed, autophagy can inhibit
inammation through the regulation of immune mediators and their interaction with innate
immune signaling pathways by removing endogenous inammasome agonists [104]. In senes-
cent cells, AMP-activated protein kinase (AMPK) can be inactivated; however, pharmacological
activation of AMPK has been found to inhibit cellular senescence through NAD
+
/SIRT1
induction and autophagy upregulation [105]. At the organismal level, pharmacological or
genetic upregulation of autophagy has been reported to extend healthspan and lifespan in
laboratory animal models [13]. Impaired autophagy might occur both upstream and down-
stream of other aging hallmarks, including loss of proteostasis and stem cell exhaustion.
Moreover, mitophagy, which has a major role in mitochondrial maintenance, increases in
an age-dependent manner [66].
NAD
+
has a fundamental role in the initiation of autophagy and is likely a key regulator in the
molecular mechanism of autophagy induction. For instance, the NAD
+
/SIRT1 signaling path-
way stimulates autophagy, and several ndings support a crucial role for SIRT1 in autophagy: (i)
SIRT1 is required for autophagy induction and SIRT1 overexpression increases autophagic
ux; (ii) the SIRT1 activator resveratrol induces autophagy; and (iii) autophagy is required for the
lifespan prolonging effect of SIRT1 [13,106,107]. Several mechanisms have been implicated in
NAD
+
/SIRT1-mediated autophagy. For instance, the NAD
+
/SIRT1 pathway may upregulate
macroautophagy through the deacetylation of autophagy proteins, including Atg5, Atg7, and
Atg8/LC3 [108]. NAD
+
/SIRT1 may also stimulate autophagy and/or mitophagy through acti-
vation of the AMPK pathway: activated AMPK can upregulate autophagy and/or mitophagy
through the phosphorylation of ULK1, the human autophagy protein 1 (hATG1) [109]. Given the
strong association between NAD
+
and AMPK [18,105,110], it is likely that NAD
+
may also
induce autophagy and/or mitophagy through activation of AMPK. In agreement, the upregu-
lation of the rate-limiting enzyme in NAD
+
synthesis, Nampt, induces autophagy through the
NAD
+
/Sirt1 pathway in primary rat cortical neurons [111]. Specically, NAD
+
/Sirt1 can activate
AMPK in primary rat cortical neurons, which then phosphorylates Tuberous Sclerosis Complex
2 (Tsc2) at Ser1387, leading to inhibition of the autophagy inhibitor mTOR [111]. In addition,
Sirt1 can deacetylate Foxo1, leading to upregulation of the autophagic protein Rab7, which
mediates late autophagosomelysosome fusion in murine cardiomyocytes [112]. In addition to
NR, another NAD
+
precursor, NAM, can also induce mitophagy and/or autophagy in human
broblasts, murine cortical neurons, and 3xTg AD mice [63,113].
Table 2. (continued)
Hallmarks of aging Pathways and conditions affected by changes in NAD
+
levels Refs
NR #senescence in both neuronal and melanocyte SC by improving mitochondrial function,
dependent on SIRT1 function
"NAD
+
(overexpression of NNT and NMNAT3) delays senescence in mesenchymal SC
Comprised autophagy Exogenous NAD
+
administration "autophagy in retinal pigment epithelium [105]
"NAD
+
synthesis, caused by AMPK activation through SIRT1/mTOR activation,
leads to induction of autophagy in senescent cells
Abbreviations: BER, base excision repair; DIO, diet-induced obesity; KO, knockout; mt, mitochondrial; ROS, reactive oxygen species; SAM, S-adenosyl methionine;
SC, stem cell; UPR, unfolded protein response; "increase; #decrease.
Trends in Molecular Medicine, Month Year, Vol. xx, No. yy 13
TRMOME 1265 No. of Pages 18
Recent studies suggest that NR induces mitophagy in both wild-type mice and C. elegans,
and in some DNA repair-decient premature aging diseases, including XPA, A-T, and CS.
Specically, nuclear DNA damage can induce mitochondrial dysfunction, which could be a
common cause of the neurodegenerative phenotypes seen in XPA, A-T, and CS [14,39,93].
Cross-species studies using C. elegans, mice, and human patient cells reported increased
mitochondrial reactive oxygen species (ROS) and accumulation of damaged mitochondria in
models of XPA, A-T, and CS [14,39,93].Morespecically, in atm-1 C. elegans, NR treatment
was shown to ameliorate mitochondrial dysfunction through mitophagy in a SIR2.1-DAF16-
DCT-1-dependent manner [39]. The mammalian orthologs of DAF16 and DCT-1 are FOXO3
and NIX (BNIP3L), and this pathway is conserved in mammalian cells, because SIRT1-
FOXO3-BNIP3L-dependent mitophagy is necessary to protect against hypoxia-induced
mitochondrial damage, (revealed in mice) [64]. As mentioned above, NAD
+
replenishment
can also improve DNA repair and, thus, we suggest that NAD
+
serves to link nuclear DNA
repair and mitochondrial maintenance [35,108]. Also, NAD
+
can stimulate autophagy and/or
mitophagy, which can help delay aging and extend longevity in certain models and across
species.
Clinical Translation
NAD
+
precursors can delay aging and counteract a broad spectrum of age-related disease;
however, the most important question is whether their benecial effects will translate to
humans. Preclinical and clinical safety assessments of some NAD
+
precursors in mice, rats,
and humans have or are being conducted. In mice, there is no detectable toxicity of short-
term (500 mg/kg body weight/day for 14 days) [14] or long-term NR treatment (400 mg/
kg/day NR treatment in drinking water for 6 weeks, or 570590 mg/kg/day for over 10
months) [28,39]. The dose of 570590 mg/kg/day in mice is equivalent to 3.193.30 g in
humans based on weight [114]. Studies in rats reported no observed adverse effects of
300 mg/kg/day, and the lowest dose of NR that induced observed adverse effects was
1000 mg/kg/day, with target organs for toxicity assessment being the liver, kidney, ovaries,
and testes [115]. Similarly, there is no detectable toxicity of short-term (500 mg/kg/day,
intraperitoneal injection for 7 consecutive days) [50] or 12-month-long NMN treatment in
wild-type C57BL/6N mice (100 mg/kg/day or 300 mg/kg/day) [44]. Consequently, the low
toxicity of NAD
+
precursors in mammals may render these good candidates for clinical
intervention.
Acipimox is a NA analog, and a 2015 report showed that it improved skeletal muscle
mitochondrial function in patients with type 2 diabetes mellitus [116].Intherst controlled
clinical trial of NR, researchers demonstrated that the compound was safe for humans, and
increased blood levels of NAD
+
were detected relative to control subjects [117]. This trial
involved six healthy men and six healthy women. Each participant received a single oral
dose of 100 mg, 300 mg, or 1000 mg of NR with a 7-day gap between doses. The data
indicated that NR administration increased NAD
+
in a dose-dependent manner with no
serious side effects at any tested dose [117,118]. In addition, 1000 mg/day of NR taken
orally resulted in a 2.7-fold increase of NAD
+
and 45-fold increase of the NAD
+
intermediate,
NAAD [118]. Studies in mice also suggest that NR is more effective at increasing intracel-
lular NAD
+
than other NAD
+
precursors, such as NAM and NA [118]. In 2016, a follow-up
clinical trial was initiated with 140 healthy adults (aged 4060 years), examining the benets
of 8 weeks of NR treatment [118]. However, the results are not yet available. Some recent
clinical trials
i
have been designed to evaluate the efcacy of NAD
+
supplementation for the
treatment of metabolic and age-related diseases. Over ten clinical studies have assessed or
are currently assessing the safety and efcacy of NR (Box 3). It will be exciting to see
whether NAD
+
supplementation has any effects on some of these human conditions going
forward.
14 Trends in Molecular Medicine, Month Year, Vol. xx, No. yy
TRMOME 1265 No. of Pages 18
Concluding Remarks
The increasing population of older individuals presents a serious socioeconomic burden for
families, societies, and the healthcare system. Lifestyle interventions, such as a healthy diet,
fasting, and exercise, are ways to improve health and the quality of life [35,59,119]. However,
not every individual at risk of developing an age-associated ailment may be willing or able to
follow these lifestyle interventions. Therefore, the benecial effects of NAD
+
precursors dis-
cussed in this review may promote healthy aging and delay various age-related diseases.
Especially encouraging is the evidence demonstrating that NAD
+
replenishment is benecial in
multiple organs with varying disease conditions. The observations that NAD
+
replenishment
delays or prevents muscle atrophy, hearing loss, and cognitive decline are remarkable.
Moreover, preclinical data suggest that NAD
+
precursor treatment is a promising therapeutic
strategy to improve clinical characteristics of the AD phenotype. Moreover, NAD
+
precursors,
such as NR and NMN, are relatively safe and orally bioavailable. Thus, NAD
+
precursors may
serve as promising candidates to combat normal aging and age-related disease. However,
extensive research is warranted to validate their potential, particularly in humans.
While many studies on NAD
+
precursors are ongoing, major questions remain (see Outstanding
Questions and Box 3). What predictive value does the alteration in NAD
+
levels have in normal
aging and age-related disorders? Further studies evaluating alterations in cellular NAD
+
as a
hallmark of aging are necessary. This task requires the development of new technologies to
simultaneously detect NAD
+
and its metabolites in humans. Moreover, the pleotropic role of
NAD
+
in human physiology is complex and requires further mechanistic insight. For example,
the tissue and subcellular specicity of NAD
+
precursors need to be carefully evaluated. It is also
possible that some unforeseen side effects may present in certain human populations. Thus,
highly stringent and carefully designed clinical trials are necessary to ensure safety. Indeed, a
decline in NAD
+
concentrations may have numerous roles in human physiology, some of which
we are only beginning to understand. Some of these roles appear to be important in the aging
process and are likely to be important drivers of aging and age-related dysfunction. With careful
scientic evaluation, NAD
+
replenishment strategies might serve as a promising multifunctional
approach to improve the quality of life for an increasingly aged population.
Disclaimer Statement
The Bohr laboratory has CRADA arrangements with ChromaDex and GlaxoSmithKline.
Box 3. Clinicians Corner
NAD
+
depletion may contribute to a wide spectrum of age-predisposed diseases, including neurodegenerative
diseases, muscle atrophy, and progeria. Recent progress in animal studies support the hypothesis that NAD
+
replenishment may inhibit metabolic diseases, AD, hearing loss, and muscle atrophy, among others. However, further
research elucidating the molecular mechanisms of the functions of NAD
+
precursor in delaying aging is warranted.
Over ten clinical studies have or are currently assessing the safety and efcacy of NR in humans. In addition to the focus
on safety (ClinicalTrials.gov identier number: NCT02678611), pharmacokinetics (NCT02300740 and NCT02191462),
and bioavailability (NCT02712593), there are trials focusing on metabolic disturbance (NCT02689882), aging
(NCT02921659 and NCT02950441), obesity, diabetes, or coronary artery disease (NCT02835664, NCT02812238,
and NCT02303483), concussion (NCT02721537), and mild cognitive impairment (NCT02942888). Some of these
clinical trials have been completed but have not yet been published. The rst NMN clinical trial was launched in Japan in
2016, with a focus on safely and bioavailability of NMN in ten healthy humans [126]. In addition to NR and NMN, there are
some clinical studies on NAM. Based on the benets of NAM on AD mice [63], a safety study of the use of NAM to treat
human AD, which involved 50 participants aged from 50 to 95 years, has just ended (NCT00580931). Even though
some NAD
+
precursors (NR, NMN, and niacin/vitamin B3) are available in the market as dietary supplements, results
from these clinical studies will determine the broad applications of NAD
+
in the aging population.
However, many questions remain. What are the therapeutic doses of NR/NMN needed for different diseases in clinical
trials? Does long-term supplementation with NR/NMN have any side effects in humans? If NR and/or NMN show clinical
benet, what other clinical studies or combinational drugs should be pursued?
Outstanding Questions
What are the precise molecular mech-
anisms by which NAD
+
acts on mito-
chondrial homeostasis?
What molecular mechanisms underlie
the autophagy and/or mitophagy-
inducing activity of NAD
+
?
In addition to deacetylation of major
autophagic proteins (such as Atg-5,
Atg-7, and Atg-8) and upregulation
of certain autophagic proteins (e.g.,
DCT-1), are there other molecular
mechanisms whereby NAD
+
modu-
lates autophagy and/or mitophagy?
How are NA and NR transported into
the cytosol from the extracellular
milieu?
What cell membrane transport does
NR use to enter the cells?
Which are the intracellular transporters
for different NAD
+
precursors, espe-
cially for NR and NMN?
Trends in Molecular Medicine, Month Year, Vol. xx, No. yy 15
TRMOME 1265 No. of Pages 18
Acknowledgments
We acknowledge the valuable work of the many investigators whose published articles we were unable to cite owing to
space limitations. We appreciate personal communications of NAD
+
consumption with Charles M. Brenner. We thank
Rachel Abbotts and Beverly Baptiste for critical reading of the manuscript, and Marc Raley for generation of the gures.
This research was supported by the Intramural Research Program of the National Institute on Aging, including a 2015-
2016, 2016-2017 NIA intra-laboratory grant (E.F.F./V.A.B.). E.F.F. was supported by HELSE SOR-OST RHF (Project No:
2017056) and the Research Council of Norway (Project No: 262175). S.L. was sponsored by The Oticon Foundation, Ib
Henriksens Foundation, Her Majesty Queen Margrethe IIs Travel Grant, and The Velux Foundation.
Resources
i
https://clinicaltrials.gov/ct2/results?term=nad%2B&Search=Search
Supplemental Information
Supplemental information associated with this article can be found online at http://dx.doi.org/10.1016/j.molmed.2017.08.
001.
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18 Trends in Molecular Medicine, Month Year, Vol. xx, No. yy
... Age-related decline in DNA repair capacity correlates with metabolic dysfunction and increased oxidative stress [97]. The NAD+ depletion that occurs with aging has been shown to impair both DNA repair capacity and metabolic homeostasis, creating a vicious cycle that accelerates aging-related pathologies [98]. ...
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