https://doi.org/10.

1038/s41580-020-00313-x

Supplementary information

NAD+ metabolism and its roles in cellular

processes during ageing
In the format provided by the
authors and unedited
Supplementary Box 1. NAD+ subcellular localization

Nicotinamide adenine dinucleotide (NAD+) functions both as a redox cofactor and as a substrate
for several families of enzymes and its intracellular localization is highly compartmentalized in the
cytoplasm, mitochondria and nucleus, which represent the main subcellular pools. Alternative
NAD+ compartments such as endoplasmic reticulum and peroxisome have been described but
remain largely uncharacterized 1. Additionally, there is evidence that intracellular NAD+
localization and concentration is not uniform, with the highest concentration of NAD+ being in the
mitochondria, and a lesser amount in the nuclear-cytosol space 2. Thus, a major question in the
field has been how these subcellular NAD+ pools are integrated and how they are differentially
regulated and involved in distinct NAD+-dependent processes. Consistent with each NAD+ pool
being regulated independently, the enzymes involved in the biosynthesis or degradation of NAD+
are highly compartmentalized as well.
Since most studies utilize whole-cell and tissue lysates to measure NAD+ via liquid
chromatography-Mass Spectrometry (LC-MS) and enzymatic assays, it has been extremely
challenging to study NAD+ levels in subcellular compartments and organelles. However, a novel
genetically encoded fluorescent biosensor, which was recently developed, has allowed for the
detection of NAD+ levels in multiple cellular compartments, and a greater understanding how the
subcellular NAD+ pools are maintained and regulated 2 . Using this novel tool, it was validated
that the cytosolic and nuclear NAD+ pools are considered to be exchangeable via diffusion through
the nuclear pore and are characterized by a free NAD+ concentration of around 100uM 2. The
largest NAD+ pool is in the mitochondria with concentrations around 250uM 2. In the mitochondria,
NAD+ was thought to be sequestered due to the impermeability of the inner mitochondrial
membrane and the lack of mitochondrial transporters. After the discovery of mitochondrial NAD+
transporters in plants, yeast, and bacteria, the mammalian NAD+ transporter SLC25A51 has been
recently identified 3,4. The identification of the mitochondrial NAD+ mammalian transporter
confirms previous studies suggesting that intact NAD+ can be transported into mitochondria from
the cytosolic pool 2,5. The development of second-generation NAD+ biosensors in tandem with
isotopic labeling and other novel technologies to study NAD+ levels rapidly and accurately 6,7 will
provide the NAD+ field with valuable tools to investigate the complex cellular NAD+ dynamics in a
more precise and refined manner. These will lead to even more breakthroughs and exciting
discoveries to reveal the major biosynthetic pathways, transporters, and enzymes that control
NAD+ production vs consumption rates in different cells, tissues and disease states.
Intracellular NAD+ can be released into the extracellular space upon cell lysis 8,9 or can
be actively released from various cell types, such as neuronal cells upon stimulation 10,11 or
prostate cancer cells 12. Thus, NAD+ can be found at submicromolar concentrations in an
extracellular pool which is largely uncharacterized 13,14. This extracellular NAD+ is mostly thought
to be degraded, and these by-products are imported inside the cells and converted back to NAD+
(see section “Cellular NAD+ metabolism” and Figure 1). However, the dynamic exchange of
intracellular pools of NAD+ to the extracellular space suggests a role for NAD+ as a potential
endocrine or paracrine signaling molecule. In support of NAD+ acting as a paracrine signal, a
direct exchange through the connexin 43 hemichannels on the plasma membrane has been
shown15. However, the biological function of extracellular NAD+ or cell to cell NAD+ exchange
between the intracellular and extracellular pools remains unclear. Although, it has been proposed
that extracellular NAD+ may also act as a danger signal, activating purinergic receptors similar to
ATP, to activate both innate and adaptive immune cells to initiate inflammatory responses 16,17.
Thus, more research is needed to understand what role extra cellular and subcellular tracking of
NAD+ plays in cell biology.
Supplementary Box 2. Cellular functions of NAD+ and their links to ageing

Metabolic reactions
NAD+ exists in two forms including in an oxidized state known as NAD+, and its reduced form
NADH. NAD+, as well as its phosphorylated version NADP+, is able to accept two electrons in the
form of a hydride ion, making this molecule a critical redox coenzyme essential for redox
homeostasis and multiple cellular functions. One of the most important roles of NAD+ in our cells,
if not the most important, is the role of NAD+ in the catabolism of nutrients in metabolic reactions
such as glycolysis, TCA cycle, glutaminolysis, and β-oxidation. Conversely, NADP+, and more
specifically its reduced version NADPH is primarily used for anabolic processes such as lipid and
cholesterol synthesis, as well as in ROS production and maintenance of antioxidant defense
mechanisms such as reducing glutathione.
During aerobic glycolysis high energy hydride ions are harvested from glucose. NAD+ is
critical for the sixth step of glycolysis, conversion of glyceraldehyde 3-phosphate to D-glycerate
1,3-bisphosphate by the enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which
utilizes NAD+ as an electron acceptor forming NADH. However, during periods of high rates of
glucose metabolism, such that occur during the Warburg effect, NAD+/NADH ratios can become
too low and NADH must be reoxidized to sustain glycolysis. Thus, our cells have adapted multiple
mechanisms to restore NAD+/NADH ratios to regulate metabolic flux of carbons. For example,
electrons from cytosolic NADH (which is impermeable to the mitochondrial membrane) can be
transferred to NAD+ molecules or the electron transport chain in the mitochondria via the malate-
aspartate shuttle or the glycerol-3-phosphate shuttle, respectively, where they will be used to
make ATP via oxidative phosphorylation. Additionally, NAD+ can be restored in the cytosol by the
diversion of pyruvate to lactate by lactate dehydrogenase (LDH) which converts NADH to NAD+.
Another mechanism recently shown to regulate NAD+ redox balance is via the ability of nuclear
NAD+ levels to regulate the expression of mitochondrial ox-phos genes, via a SIRT1-HIF1a
dependent mechanism, affecting the NAD+/NADH redox state in the mitochondria 18.
This highly orchestrated maintenance of the NAD+/NADH ratio can effectively control the
rate of glycolysis, as well as other metabolic reactions that utilize dehydrogenase enzymes such
as glutamate dehydrogenase which is necessary for glutamine catabolism, the TCA cycle
dehydrogenase enzymes (isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, and malate
dehydrogenase) and in β-oxidation (3-hydroxy-acyl-CoA dehydrogenase). It is important to note
that when the NAD+/NADH ratio becomes too high these reactions can also occur in the opposite
reaction, for example LDH can catalyze the conversion of lactate to pyruvate, converting NAD+ to
NADH in the process. Interestingly, the redox state of NAD may also become dysregulated during
aging (see Redox Reactions supplementary section), however what impact this may have on
metabolic reactions is still unclear and remains an interesting area of future investigation.
Finally, the ability of NAD+ to act as a hydride acceptor and NADH to act as an electron
donor, makes NAD+ an energy reservoir and an efficient carrier of energy from one organelle to
another and one chemical reaction to another 19. Ultimately the potential energy stored in NADH
can be cashed in by transferring the electrons to the electron transport chain in the mitochondria
to make ATP using oxidative phosphorylation, with oxygen as the final electron acceptor.

Redox reactions
During ageing, absolute NAD pools and the NAD+/NADH ratio have been shown to decline in
multiple tissues and across species during the natural ageing process and in progeria mouse
models 20–23. Although increased consumption of NAD+ by NADase enzymes such as CD38 and
PARP1, two of the major NADase responsible for degrading NAD+ during ageing, may explain
why total NAD pools decline, it is not clear why NAD+/NADH ratios are affected during ageing.
Enhanced activity of PARP and CD38 have been shown to significantly lower the NAD+/NADH
ratio compared to WT mice, suggesting enhanced NADase activity is sufficient to not only
decrease the absolute NAD+ levels but also affect the NAD redox state 21,24. In addition to
increased NADase activity, another potential mechanisms is that in ageing mice the decline in
nuclear NAD+ levels inhibits SIRT1 activity and this leads to reduced gene expression of
mitochondrial genes, resulting in lower rates of ox-phos and decreased NAD+/NADH in the
mitochondria of muscle tissue of old mice 18. Taken together, multiple mechanisms may be
involved in the regulation of the NAD redox states during ageing which may be tissue and context
dependent.
In addition to ageing, altered NAD pools and NAD+/NADH ratios have been observed in
metabolic disease, cellular senescence and in cancer 25–27. In obese conditions when the
NAD+/NADH ratio is low, it inhibits the activity of dehydrogenase enzymes and can lead to
reductive stress, which has been shown to further promote metabolic disease and insulin
resistance 19. In ageing, the physiological consequences of lower NAD+/NADH ratios, and the role
of reductive stress in ageing-related disease is unclear 28. However, interventions that promote
healthy ageing such as caloric restriction and supplementation with NAD+ precursors (NR/NMN)
have been shown to increase the NAD+/NADH ratio 18,29–31. Thus, it is tempting to speculate that
restoring NAD+/NADH ratios to a healthy level may be beneficial to promoting healthy ageing.
However, what this proper ratio is, is still unclear, and it is likely to be cell specific and dependent
on the metabolic demands of each cell type.
One limitation in studying the role of NAD+/NADH redox balance in vitro and in vivo is the
lack of tools to reliably measure and quantify NAD+/NADH ratio in subcellular compartments over
time and in a high throughput manner. However, as discussed in “techniques to study NAD+
metabolism” these barriers have been broken the last few years with new technology such as
NAD(P)-Snifits 7, SoNar 25 and biosensor with a bipartite NAD+- binding domain 2, that allows the
relative quantification of NAD+/NADH and NADP+/NADPH in live cells and in different subcellular
fractions under varying conditions. These tools will be of critical importance to further our
knowledge on the cellular mechanisms that regulate the NAD+/NADH balance and how this
balance in turn influences other cellular functions that impact ageing.
Lastly, in addition to tools to measure the NAD+/NADH ratio, another recent genetic tool
includes the use of the Lactobacillus brevis (Lb)NOX, a bacterial water forming NADH oxidase,
that when expressed in mammalian cells can increase the NAD+/NADH ratio 28, increasing the
flux and directionality of multiple metabolic pathways. Recent work using this tool to tissue
specifically increase the NAD+/NADH ratio in hepatocytes was able to lower reductive stress in
the livers of mice fed a high fat diet and improve glucose metabolism and insulin resistance 27,28.
This data suggests reductive stress not only occurs during metabolically stressful conditions such
as obesity and ageing, but is also causative in promoting metabolic and mitochondrial dysfunction,
and disease. Thus, targeting the NAD+/NADH redox balance may be a potential therapeutic target
to treat metabolic diseases including ageing. Thus, future work using this tool, along with the
others mentioned above, to tissue specifically measure and impact NAD(P) redox states and how
they influence metabolic health and lifespan in ageing mice will be exciting to follow.

Regulation of DNA repair response and genomic stability

A key hallmark of ageing includes a reduced capacity to repair DNA damage and an
accumulation of DNA mutations and genomic instability 32. Excess DNA damage or unresolved
DNA damage, such as that occur during ageing or progeria, results in prolonged PARP1 activation
and depletion of NAD pools 33. Decreased NAD pools from PARP1 activation have downstream
consequences and affect the activity of other NAD+-dependent cellular processes and NAD+
enzymes, including SIRT1, resulting in reduced DNA repair capacity, reduced mitophagy and
altered mitochondrial metabolism and homeostasis 33–35. Therefore, restoring NAD+ levels has
emerged as a potential therapeutic approach to restore or mitigate the negative consequences of
hyper PARP1 activation in response to DNA damage during ageing or other DNA-damaging
insults. In this section, we will explain the emerging evidence for the role of NAD+ in the DNA
damage response and its consequences.
In support of a major role for NAD+ in DNA repair during ageing, repletion with NAD+
precursors, such as NMN and NR, or inhibition of the major NAD+ consumers, such as CD38, are
effective in extending the healthspan and lifespan of many progeroid models, including ataxia-
telangiectasia, Cockayne syndrome and p44+/+ mice 21,36,37. Additionally, NR and NMN treatment
help maintain telomere length in mice lacking telomerase 38 and promote DNA repair in mice with
atherosclerotic lesions, radiation exposure, and even in naturally old wild-type mice, leading to
reduced DNA damage and decreased numbers of senescent cells 39–41. Thus, increasing NAD+
levels in cells experiencing high levels of DNA damage (such as ageing cells) may support DNA
repair and overall genomic stability. However, NADs role in the DNA damage response seems to
be much more complicated than simply acting as a co-substrate for PARP1. For example, when
NAD+ levels are low, PARP1 is bound and sequestered by deleted in breast cancer 1 (DBC1)
protein, which restricts PARP1 activity 39. Interestingly, DBC1 also inhibits SIRT1 activity 42,43, but
this interaction seems to be independent of NAD+ 39. Thus, these results show that, in addition to
NAD’s role as a coenzyme and cosubstrate, NAD+ facilitates protein-protein interactions that
influence DNA repair. In support of this, NAD+ facilitates the interaction of the DNA repair protein
HPF1 and PARP1/2, which completes the PARP activation site and is essential for substrate
PARylation and the DNA repair response 44. These results suggest how decreased NAD+ levels
in ageing leads to diminished PARP1 activity and DNA repair, and how targeting NAD+
consumption routes or administration of NAD+ precursors may promote more efficient DNA repair
mechanisms. While the clinical benefits of targeting NAD+ metabolism remain unclear in humans,
it may be an effective therapeutic route for treating patients with DNA repair defects and progeroid
diseases.
Lastly, sirtuins play indirect and direct roles in genome stability and DNA repair. For
example, the mitochondrial localized SIRT3 suppresses production of mitochondrial derived ROS,
an endogenous source of DNA damage 45–47. Whereas the nuclear localized sirtuins 1, 6, and 7
are integral parts of the DNA repair response to double-stranded breaks. For example, mice
lacking SIRT1, SIRT6, and SIRT7 exhibit genome instability and die within a few weeks after birth
48–50
. Conversely, mice overexpressing these sirtuins have increased healthspan and lifespan 51,52.
SIRT1 is activated by DNA damage and promotes DNA repair via nuclear excision repair 53–55. In
agreement with the role of SIRT1 in regulating genome stability, mice overexpressing SIRT1 are
protected from cancer 56. Similarly, SIRT6 is activated by DNA damage and helps in the repair of
double-stranded breaks by deacetylating histones, recruiting chromatin remodelers, and
promoting PARP1 activation 48,57–59. Interestingly, recent work found that rodent lifespan is
positively correlated with the increased efficiency of SIRT6-dependent repair of double-stranded
breaks 60. Additionally, L1 retrotransposable elements are upregulated during cellular senescence
and are emerging as a key source of DNA damage and genomic instability during ageing 61.
Interestingly, SIRT6 represses L1 retrotransposons by ribosylating the KRAB-associated protein
1 (KAP1), which promotes viral and retrotransposon latency 62. In agreement with this, mice
lacking SIRT6 have shortened lifespan and healthspan that can be extended twofold by treating
SIRT6 KO mice with nucleoside RT inhibitors, antiviral drugs that reduce L1-dependent DNA
damage and genomic instability 63. These studies show that a major function of sirtuins is to
maintain genomic stability and promote DNA repair.
Sirtuin function is tightly linked to NAD+ levels, however in humans it is still unknown
whether augmenting NAD+ levels during ageing can enhance sirtuin dependent DNA repair
mechanisms in addition to those regulated by PARPs. However, it is tempting to speculate that
increasing NAD+ levels will provide therapeutic benefits to human healthspan by activating
multiple downstream NAD+-dependent DNA protective processes discussed above and/or DNA
repair pathways. Thus, an open question in the ageing field is whether NAD+ boosting
supplements can be used as a way to prevent the accumulation of genotoxic stress in the clinic.
Future work untangling how NAD+ influences DNA repair mechanisms will be of utmost
importance to better understand how we can more efficiently target NAD+ metabolism and NAD+-
dependent proteins, such as sirtuins and PARPs, to treat ageing and other diseases associated
with DNA damage.

Epigenetic regulation
The ageing process is characterized by major epigenetic changes, also known as “epigenetic
drift” 64,65 , including reduced global heterochromatin 66,67, changes in histone modification pattern
68
and global DNA hypomethylation 69,70,71, which together affect the genomic stability, chromatin
state and gene expression.
The discovery that enzymes involved in epigenetic regulation, such as the histone
deacetylases sirtuins, use NAD+ as a cofactor highlighted the pivotal role of this metabolite in the
crosstalk between metabolic state of the cell and epigenetic regulation of gene expression 72,73.
Increased NAD+ levels in response to caloric restriction (CR) 74,75 and physical exercise 76,
increase sirtuin activity which transduce the signal via deacetylation of specific histone residues
(e.g., H3K9, H3K14 and H4K16) 77 and transcription factors (e.g., p53, NF-kB, PGC1a and FOXO
) 78–80 resulting in beneficial anti-ageing effects. An extensive body of evidence shows in particular
the role of SIRT1 in modulating the age-related epigenetic landscape and the potential of targeting
this enzyme with activating compounds, such as resveratrol, which reduce genomic instability and
histone acetylation (e.g., H3K9 H4K16, and H3K56) and increased heterochromatin formation
55,81,82
. Moreover, the circadian regulation of NAD+ levels orchestrated by the clock machinery
CLOCK:BMAL1 and SIRT1 reveal a circadian regulation of the SIRT1 epigenetic targets such as
H3K9 and H3K14 at multiple loci 83 83,84. Beside the established role of SIRT1 as a NAD+-
dependent biosensor for the nutrient state of the cell and epigenetic regulator, a growing body of
evidence shows the involvement of other members of the sirtuin family including SIRT2 85, -6 86,87
and -7 88 (more extensively reviewed here 89). Moreover, accumulating evidence suggests the
involvement of PARPs and PARylation in epigenetic regulation, in particular in heterochromatin
integrity and architecture (reviewed in 90), however its role in ageing is still unclear. Decreased
PARylation has been associated with hypoacetylation 91 possibly due to the reduced NAD+
availability for SIRT1 mediated by PARP1 92.
One key concept related to age-related epigenetic changes is the relocalization of
chromatin modifiers (RCM) hypothesis, suggesting that in ageing cells the epigenetic modifiers
recruited in the repair process do not return to their original site once the repair process is
completed 93. This results in an altered landscape of these chromatin modifiers which affects the
gene expression profile and contributes to loss of cell identity and senescence 94. The involvement
of sirtuins in the RCM process has been demonstrated in yeast 55,58, however, the contribution of
the RCM to mammalian ageing and the role of NAD+ metabolism is still largely unexplored.
One exciting area in the ageing/epigenetics field is the discovery that changes in DNA
methylation occurs at a predictable rate, which can be used as a biological clock (known as the
Horvath Clock) to predict and determine the rate of ageing in humans 95. Interestingly, a recent
small clinical study, consisting of 9 middle aged men were given a cocktail of three drugs
(recombinant human growth hormone, dehydroepiandrosterone (DHEA) and metformin) for one
year to try to reverse immunosenescence and ageing as predicted by the Horvath clock 96.
Interestingly, analysis of peripheral blood immune cells in these patients showed that this cocktail
was able to reduce the DNA methylation clock on average by 2.5 years, and also able to reduce
the expression of CD38 in peripheral blood monocytes in these patients suggesting this reversal
may be dependent on NAD+ levels. Thus, this exciting study hints, but does not prove, that
targeting NAD+ metabolism may be used to reverse specific age-related epigenetic features.
Therefore, future studies are required to advance our understanding of its real therapeutic
potential.
 Taken together, accumulating evidence indicates the beneficial effect of increased NAD+
levels via upregulation of epigenetic regulators (e.g., SIRT1) in reversing age-related epigenetic
changes and increasing healthspan 97–100 . Thus, it remains to be determined whether other NAD+
boosting strategies such as supplementation with NAD+ precursors could have the same
beneficial effects.

Autophagy
Autophagy is a highly conserved catabolic process that mediates the degradation of defective
organelles and protein aggregates, particularly during conditions when nutrients are low, for
recycling in the lysosomes, and is thought to promote beneficial effects for cellular homeostasis
and more largely to organismal fitness. However, defective autophagy is a common signature of
ageing and contributes to age-related diseases, including metabolic and cardiac disorders and
neurodegeneration 101,102,103. Autophagy is controlled by multiple nutritional and stress-related
cues and by a set of highly conserved autophagy (Atg) proteins 104. The reasons why autophagy
decreases with age are unclear but down-regulation of these Atg genes such as Atg5 and Atg7
have been detected in human tissues during normal ageing 105.
Increasing data indicate that NAD+/SIRT1 signaling pathway increases autophagic flux by
regulating the molecular mechanisms of autophagy induction and initiation 106 through the
deacetylation of several ATG proteins, including ATG7, ATG6 107 and LC3 108 . In addition, the
NAD+/SIRT1 pathway also induces autophagy through the deacetylation of FOXO1 and
consequently the upregulation of Rab7 in cardiomyocytes 109 and the activation of AMPK in
primary neurons, leading to inhibition of mTOR 110 . Thus, these data link multiple nutrient sensing
pathways, including those of NAD+ via SIRT1, to control of autophagy. In support of NAD levels
influencing autophagy, it has been demonstrated that cardiac CD38 expression is elevated under
hypoxia/Ischemia injury, leading to reduced NAD+ levels, and cardiac dysfunction by inhibiting
autophagic flux via NAD+-dependent Rab7 and non-NAD+-dependent PLEKHM1 downregulation
111
. Thus, autophagy is a major cellular mechanism that is directly regulated by NAD+ levels and
can be potentially improved or made more efficient in ageing patients by targeting NAD+
metabolism pathways.
Mitophagy is a selective autophagy mechanism that plays a major role in specific
recognition and degradation of damaged or ROS-producing mitochondria. NAD+ depletion has
been associated to loss of mitophagy and mitochondrial dysfunction in several studies of
premature ageing syndromes including Werner syndrome 112 Xeroderma Pigmentosum 33,
Cockayne syndrome 21 and Ataxia-telangiectasia 113 due to PARP1 hyperactivation induced by
DNA damage and the impairment of sirtuin activity. Moreover, it was demonstrated that
supplementation with NAD+ precursors such as NR and NMN promotes the sirtuin dependent
mitochondrial recycling and clearance of protein aggregates in relevant models of
neurodegenerative disorders including AD 114,115 and PD 116 via increased TFEB- and FOXO-
dependent expression of autophagy/mitophagy genes. Lastly, the NAD+-consuming enzyme
SARM1 promotes mitophagy by forming a complex with PTEN-induced putative kinase 1 (PINK1)
and TRAF6 (TNF receptor associated factor 6) that stabilizes PINK1 on depolarized mitochondria
supporting a link between the deregulation of complex formation and PD pathogenesis 117. Thus,
multiple NAD+ dependent pathways and enzymes converge to control mitophagy, suggesting
there are multiple therapeutic approaches targeting NAD+ metabolism that may promote
mitophagy and better ageing.
Supplementary Box 3. NAD+ in cancer

Ageing is one of the main risk factors for several types of cancer, which are characterized by age-
related pathophysiological processes regulated by NAD+ levels, including oxidative stress and
DNA damage 118 , metabolic dysfunction 119, cellular senescence 120 and inflammation 121,122. NAD+
metabolism is emerging as a potential target for cancer treatment since many NAD+-related
enzymes are dysregulated in carcinogenesis.
Both iNAMPT and eNAMPT are overexpressed in numerous malignancies 123 including
prostate 124, breast 125 and colorectal cancers 126 resulting in increased NAD+ levels which promote
cell proliferation and cancer initiation and progression. Several NAMPT inhibitors (i.e. CHS828 127
and FK866 128) have been developed and showed promising antineoplastic activity in vitro but
very low efficacy and high toxicity in clinical trials 129. The role of other NAD+ biosynthetic enzymes
is less clear but NMNAT2 overexpression in cancer has been reported 130,131. Overall, in contrast
with the pathological role of NAD+ decline in ageing, this evidence suggests the detrimental role
of an upregulated NAD+ biosynthesis in cancer.
The role of NAD+-consuming enzymes in cancer may vary from tumour promoters to
tumour suppressors. Evidence showing that sirtuins may both promote and suppress
tumorigenesis targeting multiple pathways has been extensively reported and recently reviewed
132
. For example, while SIRT1 overexpression is associated with poor disease progression and
poor prognosis in colorectal cancer 133, its expression in non-small cell lung cancer seems to
attenuate tumour progression via NF-kB signaling regulation 134. SIRT1 was also reported to
downregulate several tumour suppressors including p53 135,136 and PTEN 137, and stabilize
oncogenes such as MYCN 138.
Since genomic instability and DNA damage are key underlying factors in most types of
cancers 139 but also side effects of chemotherapeutic agents, the role of PARPs in DNA repair
and cancer has been extensively studied. Remarkably, targeting PARPs shows encouraging
results in breast and ovarian cancers with BRCA mutations characterized by a defective
homologous recombination repair system and several PARP inhibitors were developed and FDA-
approved 140–142. In BRCA deficient cancers, the inhibition of PARP activity promotes synthetic
lethality 143,144 via different mechanisms including “PARP trapping” 145,146, however the role of
NAD+ level in this process is unclear.
Lastly, CD38 is overexpressed in several hematologic malignancies, and its expression is
often associated with poor prognosis 147–150,151. One of the blood cancers with the highest and
most frequent expression of CD38 is in patients with multiple myeloma (MM), where CD38 is
highly expressed on malignant plasma cells 147–150. As a result, in the last decade, several anti-
CD38 antibodies were developed to efficiently and selectively target CD38 in MM patients. So far,
the clinical results have been extremely promising 152–154. Interestingly, CD38 KO in the ARH1
multi-tumour mouse model shows higher NAD+ levels and strong inhibition of tumour development
151
. Recent evidence shows the detrimental role of CD38 in suppressing anti-tumour effector
functions mediated by several immune populations. Th1/17 hybrid T cells with high levels of
NAD+-dependent SIRT1 and reduced CD38 expression exhibited enhanced anti-tumour activity,
therefore suggesting that blocking CD38 augments adoptive T-cell transfer in cancer patients 155.
Moreover, a new role of CD38 was proposed in the CD8-mediated immune control of tumours
treated with the checkpoint inhibitors PD-1/PD-L1 antagonist antibodies 156,157. After PD-1/PD-L1
blockade therapy, CD38 is overexpressed by CD8 T cells, leading to CD8 T-cell exhaustion, poor
antitumour immunity and ultimately resistance. Interestingly, targeting CD38 on this dysfunctional
immune population enhanced tumour control in in vivo models with poor therapeutic responses
to PD-1/PD-L1 blockade therapy 156, 157. The resistance mechanisms driven by CD38 are still
unclear; however, they seem to involve a perturbation of the adenosine receptor signaling 156,157.
Overall, CD38 may represent an innovative target in combination with checkpoint inhibitors to
improve the T-cell-mediated immune response to tumours. Unlike the case of CD38, data on the
role of CD157 in diseases are still limited. In hematologic malignancies, CD157 has a restricted
pattern of expression, and it’s mostly associated with ovarian cancer158 and acute myeloid
leukemia (AML) where an anti-CD157-based therapy shows encouraging results159.

Supplementary Box 4. Techniques to study NAD+ metabolism

Implementing current analytical methods to detect NAD+-related metabolites is one of the main
topics in the NAD+ field. Over the past decades, different techniques have been employed
including NAD+/NADH enzymatic cycling assays 160, a multitude of high-performance liquid
chromatography (HPLC) based-methods, such as HPLC with UV 160,161 or fluorometric detection
162
, and more sophisticated LC/MS/MS 163 or HPLC/MALDI/MS 164 systems. Despite the
advantages achieved in terms of sensitivity and accuracy with the mass-spectrometry based
technologies, quantification of NAD(P)+ (as well as detection of its reduced form) in different
cellular compartments remains a challenge due to the complexity of subcellular fractionations,
instability of the redox state during metabolite extraction, and suboptimal metabolite isolation
procedures.
However, the recent development of semisynthetic fluorescent biosensors, such as BRET-
based biosensors 6, NAD(P)-Snifits 7, SoNar 25 and biosensor with a bipartite NAD+- binding
domain2, allows the relative quantification of free NAD+/NADH and NADP+/NADPH in live cells
and in different subcellular fractions. Nevertheless, this method has been established for in vitro
experiments and further studies are needed to translate it in animal models.
Estimating NAD+ synthesis and consumption rates based on unlabeled analyte
concentrations or biochemical assays is inadequate to narrow down the different pathways of
generating and salvaging NAD+. Although tracing experiments with decaying isotopically labeled
NAD+ have been reported since the 80s 165 , the advance in mass spectrometry nowadays allows
flux measurements using stable isotopes, with quantitative measurement of unlabeled and
labeled forms of different NAD+-related metabolites.
Single labeled isotopic forms (2H and 13C) of nicotinamide, nicotinic acid, NAD+ and
tryptophan are commercially available whereas a new era of dedicated enzymatic or chemical
syntheses of doubly labelled NAD+-related metabolites (2H, 18O, 13C and 15N isotopes) in which
one heavier isotope is incorporated on the nicotinamide ring and one heavier isotope on the ribose
moieties is taking place. Indeed, this type of doubly labeling provides valuable information
regarding flows of metabolites in the NAD+ biosynthetic and turn-over pathways. For instance,
doubly labeled NMN or NR were employed in vitro and in vivo experiments to elucidate the direct
uptake and incorporation into NAD+ 166,167,168 as well as isotopic labeling experiments using
nicotinic acid riboside were performed to discern the ability of mitochondria to directly import intact
NAD+ 5.
Supplementary Table 1. NAD+ and hallmarks of ageing
Hallmarks of ageing
Genomic instability
Telomere attrition
Epigenetic alterations
Loss of proteostasis
Deregulated nutrient sensing
Mitochondrial dysfunction
Pathways and conditions References
affected by NAD+ impairment
or NAD+-dependent enzymes
33,169,170,39
In case of premature ageing
diseases (XPA, CS, A-T), mutation in
DNA repair mechanisms leads to
PARP1 hyperactivation and
reduction in sirtuin activity.
Age-dependent NAD+ decline
decreases PARP1 activity by
promoting the interaction with
DBC1.
38
Telomere shortening in livers of
TERT KO mice leads to a p53 -
dependent repression of all seven
sirtuins.
171
SIRT1-dependent gene regulation
through histone modification, DNA
methylation, and the modulation of
chromatin structure is affected by
NAD+ decline during ageing
172, 113,169,173
In AD models, mitophagy is
enhanced by NAD+ boosting
strategies via a mechanism
mediated by pink1, pdr-1, and dct-1.
In mice and worm, Sirtuin-mediated
UPRmt response and FOXO
signaling is activated by NAD+
augmentation or PARP inhibition
NAD+ decrease affects 174, 175, 176
AMPK/sirtuin/PGC1α pathways.
24, 18
Age-dependent CD38 increase
affects the availability of NAD+ to
mitochondrial enzymes including
SIRT3, leading to impairment of
oxygen consumption
 In worm and mice, decline in NAD+
and accumulation of HIF-1α causes
loss of OXPHOS subunits via
impaired SIRT1-PGC-1a signaling

177,178
Cellular senescence Senescence associated secretory
proteins (SASP) secreted by
senescent cells induce CD38-
NADase activity in non-senescent
cells (macrophages or endothelial
cells) that ultimately lead to tissue
NAD+ decline

Stem cell exhaustion NAD+ supplementation rejuvenates 41,179–181

aged gut, muscle and hematopoietic

stem cell pools by enhancing sirtuin
1 activity or mitochondria clearance

Altered intercellular The effect of NAD+ augmentation on 114, 115, 182

communication inflammation has also been

demonstrated in age-associated
disease including diabetes and AD
by reducing pro-inflammatory
cytokines and major inflammasome
NLRP3
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