A NAD+/PARP1/SIRT1 axis in Aging

 

NAD+ levels decline with age in diverse animals from C. elegans to mice [1] [2] [3] [4] [5] and raising NAD+ levels has apparent anti‐aging effects [3] [4] [2] [6] [5]. Increasing NAD+ levels by dietary supplementation with NAD+ precursors Nicotinamide Riboside (NR) or Nicotinamide Mononucleotide (NMN) improved mitochondrial function [4], and muscle, neural and melanocyte stem cell function in mice. NR supplementation also increased lifespan modestly in mice [5].

Although the mechanisms of gradual NAD+ decline with age remain unclear, beneficial effects have been linked to mitochondrial rejuvenation via restoration of OXPHOS subunits via sirtuin SIRT1/HIF1a/c‐myc pathway [4], and stem cell function via stimulation of the mitochondrial unfolded protein response (UPSmt) [5] and synthesis of prohibitins, a family of mitochondrial stress response proteins linked to senescence of fibroblasts in mammals. Improvement of stem cell function by NAD+ is SIRT1 dependent[5].

NAD+ levels are significant for metabolic homeostasis in that they are important for redox reactions, and as substrates of sirtuins (SIRT1 to SIRT7) and other enzymes including PARPs ([poly(adenosine diphosphate– ribose) polymerases) as well as yet uncharacterized roles through other NAD+ interacting proteins.

Sirtuins have been implicated in aging, as well as in a large number of key cellular processes including mitochondrial biogenesis, stress resistance, inflammation, transcription via epigenomic modulation of histones, apoptosis [7] [8] and SIRT1 can even affect circadian clocks [9].

PARPs are NAD‐dependent pleiotropic enzymes that play a key role in detecting and responding to single‐stranded DNA break repair as well as in inflammation, apoptosis induction and DNA methylation. Increasing PARP activity has been correlated with differential longevity of 13 mammalian species with humans having about 5 fold more PARP activity than rats [10].

Intuitively, increasing PARP activity would seem to be beneficial to organisms and potentially be of benefit to delay aging by maintaining DNA integrity, but PARPs are major consumers of NAD+ and PARP activity can deplete sufficient NAD+ to detrimentally effect mitochondria function. In fact, in apparent contradiction to the species longevity results, PARP1 knockout in mice increases tissue

NAD+ levels, enhances mitochondrial function and blocks inflammation [11]. Moreover, ectopic expression of human PARP1 in mice, an enzyme which has about 2 fold more intrinsic PARP activity than mouse PARP1, induces a set of aging related phenotypes including cardiomyopathy, hepatitis, pneumonitis, kyphosis, nephropathy, adiposity, dermatitis, anemia, increased glucose resistance, and increased incidence of carcinomas.

Paradoxically human PARP1 transgenic mice even have delayed DNA repair [12]. One possible explanation is that human PARP is consuming more NAD+ in mice than can be compensated for metabolically, causing dysfunction.

Clearly decreased NAD+ levels that have been observed in aging animals could be detrimental by multiple mechanisms, which require careful elucidation.

Decreasing NAD+ levels with aging cause DBC1 to bind and inhibit PARP1 allowing accumulation of DNA damage.

Li et al. [13] have recently established that NAD+ levels control protein‐protein interactions of two NAD+ binding proteins: DBC1/CCAR2 and PARP1. At low NAD+ levels DBC1 binds to PARP1 and inhibits PARP activity. In aging mice, DBC1 increasingly binds to PARP1 causing the accumulation of DNA damage, linking reduced NAD+ levels directly to DNA damage.

DBC1 was already known to bind to and inhibit SIRT1, as well as histone deacetylase HDAC3 and methyltransferase SUV39H1. Since SIRT1 is regulated by NAD+, Li et al explored the hypothesis that DBC1 could also bind and inhibit PARP1, another NAD‐dependent enzyme. In HEK293T cells co‐ immunoprecipitation/Western studies showed that PARP1 and DBC1 physically interact. A catalytically inactive PARP1 also interacted with DBC1 in these studies demonstrating that PARP catalytic activity was not necessary for the interaction with DBC1.

However, unlike the DBC1/SIRT1 interaction, the DBC1/PARP1 interaction was sensitive to and broken by NAD+ in the physiological range (100um‐500um), while similar doses of nicotinamide or its structural analog 3‐AB had no effect. NADH and adenine had only weak effects, suggesting that DBC1/PARP1 interaction requires

low levels or the absence of NAD+. A catalytically inactive PARP1 behaved similarly to wild type suggesting that NAD+ cleavage or covalent attachment was not necessary for NAD+ to inhibit the DBC1/PARP1 interaction[13] .

In HEK293T, an inhibitor of NAD+ biosynthesis or depleting NAD+ by genotoxic stress increased the DBC1/PARP1 interaction. Various treatments that increased NAD+, decreased DBC1/PARP1 interaction. These data are strong evidence that NAD+ inhibits the formation of the DBC1/PARP1 complex [13].

In order to determine which domains of DBC1 and PARP1 were interacting, Li et al. expressed a variety of truncated mutants of DBC1 and PARP1. DBC1 lacking a nudix homology domain (NDH) did not bind to PARP1, but a DBC1 carrying only a NDH domain did bind to PARP1. Moreover, DBC1 interacted with a PARP1 mutant consisting only of a BRCT domain, but not with a PARP1 having only a catalytic domain, suggesting that the NDH domain of DBC1 binds to the BRCT domain of PARP1.

Homology modeling based on the known crystal structures of 5 NDH proteins suggested that NAD+ directly binds the DBC1 NHD, and this was confirmed by competition experiments in which radiolabeled or biotin labeled NAD+ bound to DBC1‐NHD and was then competed off with unlabeled NAD+ [13].

Of significance is that PARP1 activity was inhibited by DBC1 interaction in vitro and stimulated in cell culture by siRNA knockdown of DBC1. DBC1 but not DBC1Q391A, a mutant of DBC1 that binds NAD+ poorly and does not interact well with PARP1, reduced PARP1 activity. Reducing DBC1 increased DNA repair after paraquat treatment, as assessed by lower gammaH2AX, reduced DNA fragmentation, increased NHEJ and HR recombination pathway activity and cell viability. NMN. which raises NAD+ levels. had similar effects on these cells. These data support the hypothesis that NAD+ binding to DBC1‐NHD regulates two key pathways of DNA repair through controlling PARP1 activity [13].

DNA repair capability decreases with increasing age [14]. Similarly PARP1 activity has been reported to decrease with age [10], suggesting a possible connection between PARP1 activity and overall DNA repair. Li et al hypothesize that increased DBC1/PARP1 binding occurs with reduced NAD+ levels during aging to reduce DNA repair and increase DNA damage.

In 22‐month old mice, levels of NAD+ were reduced in the liver compared to young mice. Old mice had increased amounts of DBC1‐PARP and increased staining for DNA‐damage associated gammaH2AX. A week of daily NMN treatment (500 mg/kg) intraperitoneally increased NAD+ levels, reduced the number of DBC1‐PARP1 complexes and decreased DNA damage as reported by decreased levels gammaH2AX in old mice. Reduced PARP1 activity in old mice was restored by NMN treatment.

To show that repair was affected, old mice were subjected to gamma irradiation. Irradiated mice were observed to have reduced DNA damage when treated with NMN either before irradiation or even one hour after irradiation[13]. These data support the hypothesis that reduced NAD+ with aging causes increased DBC1/PARP1 inhibiting PARP1‐mediated repair of DNA damage.

The authors reasonably speculate that DBC1 evolved as a buffer to prevent PARP1 from depleting NAD+ levels to cytotoxic levels [15]. They also point out that DBC1 is often mutated or downregulated in cancer and that increased repair capability and protection from radiation are potential mechanisms by which elimination of DBC1 could help cancer cells evade treatment[13]. Another possible mechanism for the tumor suppressor DBC1 is that the known stabilizing interaction of DBC1 with p53 is important[16].

Medical Implications:

Evidence is accumulating that diminished NAD+ during aging results in mitochondrial and stem cell dysfunction, as well as reduced DNA repair activity. Supplementation with NMN or especially NR at doses of 100, 300 or 1000 mg can increase NAD+ levels systemically in humans [17] and may counteract NAD+ aging‐associated phenotypes, although more preclinical and human studies are needed to establish efficacy of NR or NMN as anti‐aging interventions.

 A key question arises, if NAD+ levels and in particular PARP activity levels are so important to maintaining homeostasis and contribute to the biological processes called aging, why did treatment with NR, although beneficial to stem cell function, only increase mean and maximum longevity modestly in one study: about a 5% increase when treatment was initiated in very old mice (24 months) [5].

One point is that the intervention was started very late, which may not allow reversal of aging associated damage or loss of epigenomic information. On the other hand, the effect of increasing NAD+ is significantly less than that achieved by rapamycin, for example[18]. The simplest explanation is that decreased NAD+ represents only one of a set of key regulatory changes that result in the effects of aging.

However, it is possible that the role of PARP is more profound. Given the apparent correlation of PARP activity with mammalian longevity for diverse species, it is quite possible that increasing PARP1 expression by genetic or pharmaceutical means in the context of increasing NAD+ through supplementation may have profound effects on aging.

One interesting experiment would be to supplement the human PARP1 transgenic mice [12], which express a PARP1 2‐fold more active than mouse PARP1, with NR or NMN from birth to overcome the potential detrimental effects of decreased NAD+ levels from the overactive human PARP1. We hypothesize that these mice would not only not be sick, but would thrive and potentially live longer than wild‐type animals.

The significance of PARP1 activity to aging is at least somewhat tied to its DNA repair activity. It is controversial whether that the accumulated effects of DNA mutations alone are sufficient to drive aging [19], normal cells apparently accumulate hundreds to a few thousand mutations over a lifetime of cell divisions[20].

Non‐dividing cells should possess significantly fewer mutations. While some premature aging syndromes and cancer are driven by increased genome instability, it is unclear that accumulation of ~1000 genetic substitutions would sufficiently degrade cell function. On the other hand, because decreased PARP1 activity is expected to also cause increased chromosomal instability through reduced NHEJ and HR DNA repair, PARP1 activity probably plays a significant role in preventing age‐associated cancer.

If PARP1 plays a central role in aging via its role in DNA repair, it could do so indirectly, through effects on the epigenome. PARP1 and other members of the PARP family have been shown to have profound effects on the epigenome through Poly(ADP‐ribosyl)ation (PARylation), affecting DNA methylation/demethylation via effects on DNMT1 and CTCF, histone acetylation, histone methylation, and organizing chromatin domains [21].

DNA repair events initiated by single‐ stranded DNA breaks could change the balance of PARP1 activities changing the epigenome in defined ways. Animals with higher intrinsic PARP1 activity may have less DNA damage and fewer epigenomic alterations over a given amount of chronological time, resulting in a younger biological “age”.

Superimposed upon the effects of decreased NAD+ levels, it does not escape attention that the NAD+/PARP1/SIRT1 axis could contribute to creating the apparent epigenetic aging “clocks” that are reflected in the tissue‐invariant and tissue‐specific DNA methylation patterns observed to strongly correlate biological age in humans, chimpanzees and mice [22][23] [24] through modulation of DNA methylation through DNMT1 and CTCF.

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