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.

References

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NAD+ metabolism: Bioenergetics, signaling and manipulation for therapy

1. Historical background of Vitamin B3 and NAD+
Over two centuries ago, a Spanish doctor Gasper Casal described a disease in poor farmers, whose diet was poor in meat and mainly dependent on Indian corn or maize [1]. This disease was characterized by dermatitis, diarrhea, dementia and death and was later termed pellagra [2]. Pellagra was a peril amongst the malnourished populations in southern Europe for over two hundred years and became an epidemic in the southern states of the U.S. in the early 1900s [3]. By 1912, South Carolina alone had over 30,000 cases of pellagra with 40% eventual mortality [4]. Between 1907 and 1940, 100,000 Americans died from pellagra [4]. In 1914, Joseph Goldberger related pellagra with a nutrient deficiency associated with a corn-based diet [5]. He later suggested that a deficiency in a specific amino acid caused pellagra [6], and identified a water soluble substance as the “pellagra preventive factor” in the 1920s [7]. He also recommended the use of dried yeast as a cheap dietary source to prevent the disease [8]. In 1937, Conrad Elvehjem discovered that nicotinic acid and nicotinamide alternatively, cured “black tongue”, a correlated disease to pellagra in dogs [9]. Nicotinic acid and nicotinamide are now collectively recognized as Vitamin B3. It is now clear that the chronic lack of dietary Vitamin B3 and the amino acid tryptophan, precursors to nicotinamide adenine dinucleotide (NAD+), are the cause of pellagra. Vitamin B3 obtained from NAD+ and NADP+ hydrolysis in meat and tryptophan are markedly deficient in a corn-based diet. Eventually, due to key breakthroughs in knowledge and diet, the epidemic of pellagra was relieved in the U.S. especially with the fortification of Vitamin B3 in bread starting in 1938 [10].

While the clinical investigation of pellagra was underway, the metabolic importance of nicotinamide adenine dinucleotide (NAD+) was also being recognized. In 1906, Harden and Young discovered that yeast extract that was boiled and filtered facilitated a rapid alcoholic fermentation in unboiled yeast extract [11]. They named the responsible component in the extract coferment or cozymase, which was a mixture of components that are essential to carbohydrate utilization.

In 1923, von Euler-Chelpin and Myrbäck purified cozymase, from which they identified a nucleoside sugar phosphate [12]. A decade later, Otto Warburg isolated NAD+ from cozymase and showed its role in hydrogen transfer during fermentation [13, 14]. The chemistry of NAD+ and discovery of its key role in human health converged with the discovery of the cure of pellagra. The biosynthetic pathways to produce NAD+ in cells was more fully elucidated by the work of Arthur Kornberg in the 1940s [15]

and by the work of Preiss and Handler in the late 1950s [16, 17]. In the meantime, the role of NAD+ in metabolism was being elucidated by scientists such as Krebs [18], and this included fuller descriptions of its integral roles in glycolysis, the TCA cycle and mitochondrial oxidative phosphorylation (See Figure 1). In more recent decades, non-redox roles for NAD+ have been elucidated. NAD+ possess multiple crucial functions in cell signaling pathways including ADP-ribosylation reactions and sirtuin activities. Remarkably, NAD+ homeostasis is not only important for the prevention of pellagra, but is also associated with extended lifespan, increased resistance against infectious and inflammatory diseases [19, 20] and is likely very important in resisting a number of other disease processes [21] such as cardiovascular disease, metabolic syndrome, neurodegenerative diseases and even cancer.

2. Roles of NAD+ in energy producing metabolic pathways
NAD+ is an important co-enzyme for hydride transfer enzymes essential to multiple metabolic processes including glycolysis, pyruvate dehydrogenase complex, the TCA cycle and oxidative phosphorylation. The enzymes using NAD+ in hydride-transfer are known as dehydrogenases or oxidoreductases, which catalyze reduction of NAD+ into NADH (Examples shown in Figure 1). Mitochondrial NADH is then utilized by the electron transport chain and therein participates as a substrate in mitochondrial ATP production through oxidative phosphorylation (Figure 1).
The first enzyme utilizing NAD+ in glycolysis is glyceraldehyde 3-phosphate dehydrogenase [22]. It is the sixth step in glycolysis, wherein glyceraldehyde 3-phosphate is oxidized to D-glycerate 1,3-bisphosphate in the cytosol (See Figure 1) via transfer of a hydride equivalent to NAD+. As one glucose can be converted into two glyceraldehyde 3-phosphate equivalents, one mole of glucose can generate 2 moles of NADH. Maintenance of high NAD+/NADH in cytoplasm is sustained by two NADH utilization pathways, which combine to maintain a flux of hydride removal from hydride rich carbon substrates inherent to cellular energy metabolism. NADH equivalents generated in the cytoplasm via glycolysis are transferred to the mitochondria by shuttling mechanisms such as the malate-aspartate shuttle, in which NADH in the cytosol is oxidized to NAD+ and NAD+ in mitochondria is reduced to NADH. Alternatively, NADH is oxidized back to NAD+ in the cytoplasm via lactate dehydrogenase, correspondingly producing lactate from pyruvate.

The products of glycolysis are two moles of pyruvate, 2 moles of NADH and two moles of ATP. Pyruvate has multiple possible fates. In lactate dehydrogenase reaction pyruvate is reduced to lactate. (See Figure 1) [23]. For maximal energy yield pyruvate is alternatively acted upon by the pyruvate dehydrogenase complex to form acetylCoA with concomitant NAD+ reduction to NADH [24]. AcetylCoA can then enter the TCA cycle, where NAD+ equivalents are reduced to NADH moieties in several key steps by isocitrate dehydrogenase (IDH), oxoglutarate dehydrogenase (OGD) and malate dehydrogenase.

IDH is a key step in the TCA cycle, which
oxidizes isocitrate to oxalosuccinate, which is then decarboxylated to form α-ketoglutarate. IDH exists in three isoforms [25], with IDH3 located in mitochondria and used to support the TCA cycle. IDH1 and IDH2 catalyze the same reaction and use NADP+ as cofactor. The next enzyme in the cycle, OGD, catalyzes the reaction from α-ketoglutarate to succinyl CoA, with reduction of NAD+ to NADH. OGD is a key regulatory point in the TCA cycle and is inhibited by its product succinylCoA and NADH. ADP and calcium are allosteric activators of the enzyme [26]. OGD is considered to be a redox sensor in the mitochondria. Increased NADH/NAD+ ratio is associated with increased ROS production and inhibited OGD activity. Once ROS levels are removed, OGD activity can be restored [27] . Malate dehydrogenase completes the TCA cycle and produces the third equivalent of NAD+ reduction to NADH from one mole of acetyl-CoA that enters the cycle.

NADH formed from glycolysis (via the malate-aspartate shuttle) or the TCA cycle can react at Complex I, also known as the NADH/coenzyme Q reductase in the mitochondrial electron transport chain [28]. Each NADH consumed by the mitochondria results in the net production of 3 ATP molecules (Figure 1). The complete oxidation of one glucose molecule generates 2 NADH equivalents in cytosol and 8 NADH molecules in mitochondria, enabling production of 30 ATP equivalents from NADH of the total of 36 ATP equivalents derived from the whole process of catabolizing glucose to CO2 and H2O.

The NAD+/NADH ratio plays a crucial role in regulating the intracellular redox state, especially in the mitochondria and nucleus. Free NAD+/NADH ratio varies between 60 to 700 in eukaryotic cells, although the estimated mitochondrial NAD+/NADH ratios are possibly maintained at closer to 7-10 [29, 30]. Total NAD+ levels in mammalian cells appear to be maintained at 200-500 M under most conditions.

Mitochondrial NAD+ content appears to be relatively more abundant than cytosolic NAD+ in metabolically active cells and tissues, e.g. cardiac myocytes and neurons, probably because of the needs of these tissues for significant energy and ATP production [31]. Interestingly, the mitochondrial NAD+ pool is likely more stable compared to the cytosolic pool, possibly to preserve oxidative phosphorylation and to maintain cell survival even in stressed cells [32]. Under cytoplasmic NAD+ depletion, mitochondrial NAD+ levels can still be maintained for up to 1 day [33]. Remarkably, the mitochondria pool of NAD+ can provide the threshold parameter for the survival of the cell [32].

Inhibition of mitochondrial electron transport chain activity decreases mitochondrial conversion of NADH to NAD+ and reduces the mitochondrial NAD+/NADH ratio. Complex I/III inhibitors can decrease the intracellular NAD+/NADH ratio by more than 10 fold. This decreased ratio changes the ratio of α-ketoglutarate/citrate ratio and limits acetyl-CoA entry into the TCA cycle [27]. Thus feedback of NADH into metabolism is a key factor determining the rate of catabolism and energy production. Overall oxidative metabolism is decreased when mitochondrial NADH/NAD+ level is elevated. NAD+ within the nucleus also plays significant signaling roles in
controlling and regulating metabolic pathways [34, 35]. For example, nuclear NAD+ alters sirtuin 1 (SIRT1) activity [34, 35], which in turn regulates the activity of the downstream transcriptional regulators such as Forkhead box O (FOXOs) that play important role in metabolism, stress resistance and cell death [19, 36].

A decline in nuclear NAD+ level and elevation of NADH results in the accumulation of hypoxia-inducible factor 1 alpha (HIF-1α) stimulating the Warburg effect, a hallmark of cancer cell metabolism called aerobic glycolysis [37]. Aging has been shown to promote the decline of nuclear and mitochondrial NAD+ levels, and the risk of cancer development may be increased by this phenomenon [37], although the role of dinucleotides in cancer risk is currently being vigorously investigated.

3. NADPH/NADP+ roles
A structural analogue to NAD+ is NADP+ which incorporates a 2’-phosphate on the adenosine ribose moiety absent in NAD+. This structural and chemical difference enables a distinctive role for NADP+ and its reduced form NADPH in cells [38]. For example, intracellular levels of NADP+ and NADPH are maintained at significantly lesser amounts than NAD+ and NADH. In addition, the NADP+/NADPH ratio is preferentially maintained to favor the reduced form, very unlike the corresponding NAD+/NADH ratio [38, 39]. NADPH is essential for survival, and is important for detoxification of oxidative stress. For example, NADPH donates a hydride equivalent to generate antioxidant molecules, such as reduced glutathione, reduced thioredoxin and reduced peroxiredoxins [40-42], which help eliminate cellular reactive oxygen species (ROS) [43]. NADPH is also required in the activity of detoxifying enzymes, such cytochrome P450 that function in xenobiotic metabolism [44]. In inflammatory pathways, NADPH acts as a substrate for NADPH oxidase in neutrophils and phagocytes, which use these enzymes to kill pathogens by generating superoxides [45]. NADPH serves as a key electron donor in the synthesis of fatty acid, steroid and DNA molecules [38]. NADP+ is utilized in the pentose phosphate pathway to regenerate NADPH in a pathway which can ultimately produce ribose-5- phosphate for nucleotide synthesis [46]. Interestingly, in a non-redox capacity, NADP+ serves as a precursor for nicotinic acid adenine dinucleotide phosphate (NAADP), a potent calcium mobilizing messenger which regulates calcium homeostasis [47]. Numerous studies indicate that maintenance of NADP+ and NADPH levels are vital to ensure the survival of cells especially in oxidative stress [38].

NADP+ is generated from NAD+ in cells, by action of the enzyme NADK. A well-studied cytosolic NADK preferentially uses NAD+ (Km = 1.07 mM for NAD+) as a substrate over NADH in human cells, and the NADP+ is rapidly converted to NADPH by transdehydrogenase activity [48]. Interestingly, the overexpression or down- regulation of cytoplasmic NADK influences exclusively NADPH level without altering the level of NADP+, NAD+ and NADH [48]. The activity of NADK can be activated by oxidative stress and calcium/calmodulin [49] and inhibited by high NADPH levels [50]. A putative mitochondrial human NADK has been reported [51-53]. The reported Km for NAD+ is 22 μM [53], suggesting it is typically saturated with NAD+ substrate, unlike the cytoplasmic counterpart. This may indicate greater demand for NADP+ maintenance in mitochondria, although the lifetimes of the NADP+/(NADPH) moiety in cells or in subcellular compartments are unknown.

Robust NADP+ reduction to NADPH is key to maintenance of high NADPH/NADP+ ratio [38]. In the cytosol, NADPH is generated from NADP+ by glucose-6-phosphate dehydrogenase, 6-gluconate phosphate dehydrogenase, IDH1, 2 or cytosolic NADP+-dependent malate dehydrogenase enzymes. The NADPH generated in the cytoplasm is believed to be responsible for NADPH oxidase-dependent ROS generation. In mitochondria, NADPH is generated from NADP+ by IDH3, mitochondrial malate dehydrogenase or transhydrogenases. NADP+ and NADPH concentration changes and how they mediate downstream biological effects are of current interest, and these concentration changes may be influenced by NAD+ responsive mediators, such as SIRT3, which upregulates IDH3 activity and thereby affects mitochondrial NADPH/NADP+ ratio [54]. More research in this area is clearly needed, as the cross talks between NAD+ metabolism and NADPH/NADP+ dynamics are still poorly understood. Moreover, how cellular, hormonal or nutritional stimuli affect NADPH/NADP+ dynamics are still generally poorly understood as well.

4. Roles of NAD+ in signaling pathways
The realization that NAD+ could be directly involved in the regulation of cell biological processes through changes in global signaling events was revealed by two sets of studies made in the 1960s. The first of these were pioneered by Chambon, who published two papers in 1963, and 1966, investigating if ATP could become incorporated into nuclear proteins. He initially reported an nicotinamide mononucleotide (NMN) stimulated activity [55], which turned out to be the incorporation of the ADP-ribose moiety of NAD+ into acid-precipitated proteins [56]. Further analysis of the protein adducts by digestion with snake venom phosphodiesterase indicated a polymer, later determined to be poly-ADPribose [56]. This insightful and brilliant investigative work laid the foundation for decades of subsequent biochemical and biological investigations into the roles of ADP- ribosyltransfer in modulating protein activity in mammalian cells.

Interestingly, at nearly the same time, it became apparent that selected bacterial toxins might act via a similar kind of mechanism. In 1964, Collier and Pappenheimer determined that a key protein virulence factor of the organism Corynebacterium Diphtheria (the cause of the human disease Diphtheria) caused inhibition of protein synthesis in mammalian cells in an NAD+-dependent manner [57]. Honjo et al. later determined that Diphtheria toxin ADP-ribosylates the elongation factor Ef-2, which is required to complete protein synthesis within mammalian cells [58]. This covalent modification of a mammalian protein by ADPribosylation clearly altered protein function, and provided the prototype biochemical event upon which to postulate that NAD+ can serve as a protein modifying agent, with a direct consequence to a cellular protein activity.

Stemming from these seminal studies emanating from the 1960s, and with the advent of modern proteomics and bioinformatics approaches, it has become apparent that there are a variety of signaling enzymes that harness NAD+ as a substrate, and transfer the ADPribose unit to proteins by mono-ADP-ribosylation mechanisms, or by poly-ADP-ribosylation mechanisms thereby altering protein functions (For a recent comprehensive review on this topic, see Hassa et al.[59]). For example, the parent poly-ADPRibosyl polymerase (PARP), PARP1, along with other family members (PARP2, 5a and 5b), are responsible for conferring poly-ADP-ribosylpolymerase activity [60, 61], whereas another 11 PARPs are mono-ADP- ribosyltransferases. Surprisingly, although a number of these are likely to have key functions in NAD+ mediated signaling processes, only a subset of functions have been firmly described for family-members in this group.

In addition to PARP-related ADPribosyltransferases, there is another major group of ADP-ribosyltransferases, which are called sirtuins [62, 63]. Technically, sirtuins are ADPribosyltransferases because they use this mechanism chemically to effect deacylation of substrates. The connection of sirtuins to the ADP-ribosylating toxins and PARPs was not immediately apparent, as the sirtuins are distinct in structure, sequence and chemistry from known NAD+-consuming enzymes, responsible for ADPribosyltransfer [63]. However, sirtuins have been shown to react NAD+ with acyl-lysine modified protein substrates, leading to formation of transiently-ADP-ribosylated acyl-groups, causing subsequent deacylation of these substrates [63]. This protein deacylation activity leads to regulated changes in cell behaviors, linked to these deacylation events. Most remarkable of these are at the level of chromatin, where histone and transcription factor deacylations are responsible for regulation of a whole spectrum of gene regulatory changes (as reviewed recently by Kraus and co-workers [35]).

The activities of some ADPribosylating enzymes are regulated partially in a manner independent of the availability of NAD+ substrate. A case in point is the regulation of PARP1 and PARP2 by the formation of DNA strand breaks[59] . However, a key emerging premise in understanding the activities and roles of NAD+ utilizing enzymes, is that these enzymes are regulated dynamically by cellular NAD+ metabolism. Specifically, the idea that nicotinamide and NAD+ concentrations act as inhibitors or drivers, respectively, of ADPribosylating enzymes has become a key concept, supported by biochemical measurements which determine the apparent steady-state Km enzyme parameters for NAD+ (e.g. sirtuins) in the 100-200 μM range [64, 65], squarely within the range of physiological NAD+ concentrations. In fact, recent work examining the effects of supraphysiological NAD+ concentrations, obtainable by genetic modifications (CD38 [66] and PARP1 [67] knockouts for example), confirm that increased NAD+ causes activation of signaling programs linked to and requiring sirtuins, leading to downstream events such as increased mitochondrial biogenesis [67, 68].

Naturally, there are some caveats associated with these knockouts, since they also eliminate key biochemical activities from cells and tissues. However, evidence indicates that SIRT1 activity is enhanced in these high NAD conditions to produce deacetylation of peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), activating this co-transcriptional regulator, to enhance transcription of a broad subset of genes responsible for formation of new mitochondria [67]. Indeed, low calorie diets, fasting [32] and exercise [69] appear to enhance NAD+ synthesis [32], which leads to activation of sirtuins [70] and presumably PARP family members.

It is notable that PARP1 deletion leads to significant upregulation of NAD+ levels [67], indicating that baseline PARP activity is a key factor in establishing normal tissue NAD+ homeostasis, and implying that significant crosstalk exists between NAD+ metabolism, sirtuins and PARP enzymes [71].

5. Overall view of mammalian NAD+ metabolism
Consistent with the centrality of NAD+ to cellular bioenergetics, and supportive of the relatively high concentrations of NAD+ metabolites in cells (200-500 μM typically in mammalian cells), several distinct pathways are involved in the biosynthesis of NAD+. In humans this includes de novo pathways from the amino acid precursor tryptophan, and additional pathways, including from different nicotinoyl precursors, such as nicotinic acid, nicotinamide as well as nucleosides nicotinamide riboside (NR) and nicotinic acid riboside (NAR).

Broadly, NAD+ metabolism can be viewed in four main categories, 1. De Novo Synthesis, 2. Scavenging Pathways from preformed precursors (nicotinic acid, nicotinamide riboside and nicotinic acid riboside), 3. Core Recycling Pathway through nicotinamide. Finally, 4. ADPR-transfer/NAD hydrolysis, which occurs through a variety of enzymatic pathways, leading to cleavage of the N-glycosidic bond of nicotinamide to the ribose ring, thereby liberating nicotinamide and providing an ADPR-nucleophile product. Thus, three general types of synthesis pathways converge to produce NAD+, while consumption pathways comprised of several types of NAD+ consumers deplete NAD+. Steady-state NAD+ levels are set where the magnitude of the rate of turnover of NAD+ is equalized by the net formation rate contributed by the separate synthetic pathways.

Evidence indicates the entire NAD+ pool is consumed and resynthesized in mammals several times a day [72]. Under normal cellular and tissue conditions, synthesis of NAD+ is affected by the availability of possible precursors, so that availabilities of nicotinic acid, nicotinamide riboside, nicotinamide and tryptophan can alter NAD+ synthetic rates, thus affecting NAD+ level. Not each of these precursors is bioequivalent in this respect. Strikingly, nicotinamide is not limiting in many tissues, except possibly in liver, so availability of nicotinamide is not crucially tied to NAD+ formation rate, and even when administered at fairly substantial doses via diet [73]. The identity and not necessarily only quantity of NAD+ precursor is important to the biological NAD+ level. The importance of increased NAD+ levels to signaling pathways emphasizes the relevance of the different NAD biosynthetic pathways, which are examined in greater detail in the following section.

6. Pathway and enzymes to make NAD+ from tryptophan
Tryptophan (Trp) is a substrate for the de novo synthesis of NAD+ in humans, much of which is believed to take place in the liver [74]. Trp is one of the essential amino acids required for protein synthesis and metabolic functions, with primary producers including bacteria, fungi and plants [75]. After proteins are hydrolyzed into amino acids in the GI tract, Trp is available for protein synthesis, and the majority of Trp catabolism occurs through the kynurenine (KYN) pathway [76, 77]. In the brain, in particular, Trp is the building block for several essential molecules, for instance the neurotransmitter serotonin and the sleeping hormone melatonin [78]. These metabolites are synthesized in a pathway independent of the kynurenine pathway (Figure 2). Trp is also considered as a nutritionally relevant precursor to NAD+. In a niacin deficient diets, Trp can be a key source for NAD+ and NADP+ synthesis. However, due to other metabolic fates of this amino acid, it has been estimated that 60 mg of Trp converts to only 1 mg niacin [79].

Trp can be converted to NAD+ through an eight-step biosynthesis pathway. The first and rate-limiting step in this pathway is the conversion of Trp to N-formylkynurenine by indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO)[80]. TDO exists primarily in liver [81] and can be activated by Trp [76] or corticosteroids [82], whereas IDO is found in numerous cell types, such as microglia, astrocytes, neurons [83] and macrophages [84] that reside in extra-hepatic tissues and is activated by pro-inflammatory signals [85, 86]. The first stable intermediate in the pathway, KYN, can be metabolized through two distinct pathways to form kynurenic acid or NAD+ [86]. With the enzymatic reaction of kynurenine mono-oxygenase, and kynureninase KYN is metabolized into 3-hydroxyanthranillic acid, which is then converted by 3-hydroxyanthranilate-3,4- dioxygenase into 2-amino-3-carboxymuconate semialdehyde. This metabolic intermediate can be acted upon by 2-amino-3-carboxymuconate semialdehyde decarboxylase (ACMSD) (formally termed picolinic carboxylase) to provide picolinic acid, or non-enzymatically converted into quinolinic acid (QA) [87]. QA is metabolized by quinolinate phosphoribosyltransferase (QPRT) to form nicotinic acid mononucleotide (NAMN) and enter the salvage pathway for NAD+ synthesis in liver [88]. Trp will only be diverted to NAD+ synthesis when the substrate supply far exceeds the enzymatic capacity of ACMSD [89], which helps explain the weak niacin equivalence of 60 mg trp per mg niacin [79].

The daily recommendation of tryptophan intake is 4 mg/kg body weight for adults and 8.5-6 mg/kg body weight for infants to adolescents [90]. Being an alternative but weak source for NAD+, it is an interesting question why the Trp pathway to NAD is evolutionarily well-retained in humans (Cats do not use Trp efficiently to make NAD+ [89]). Studies have shown that the de novo biosynthesis rate of NAD+ from Trp is unchanged by the absence or presence of nicotinamide in the diet, as the urinary excretion of intermediates in the Trp pathway are not altered with 0 or up to 68.6 mg/day nicotinamide [91]. Upregulated Trp derived NAD+ biosynthesis pathway does not appear to compensate for a deficiency of other NAD+ precursors. Oral intake of Trp as high as 15 g/day renders low acute toxicity, with side effects such as drowsiness and headache [92]. Overconsumption of tryptophan causes toxicity in certain animal species. The LD50 value is 1.6 g/kg body weight in rats and 2 g/kg body weight in mice and rabbits with i.p. or i.v. administration. Oral LD50 is around 3 times that of i.p. dose [90]. Increases in Trp catabolism may result in adverse effects in the human body. Several intermediates and products in the KYN pathway, including QA, 3-hydroxyl-L-kynurenine and kynurenic acid, are neurotransmitters and display key roles in central nervous system [87]. High levels of QA in brain have been associated with neurodegenerative conditions such as Huntington’s disease [93] or seizure [94]. QA and KYN have also been shown to induce anxiety in mice [95, 96]. Therefore, it may not be ideal to use Trp as a primary dietary or pharmacologic source to enrich NAD+ level.

7. Pathway and enzymes to make NAD+ from nicotinic acid
In humans and mammals, nicotinamide and nicotinic acid are routed in non-overlapping pathways to NAD+. These separate paths stand in contrast to the NAD+ metabolism observed in flies, worms, yeast and many bacteria. In flies, yeast, worms and many bacteria the breakdown product of NAD+, nicotinamide, is directly converted to nicotinic acid by a highly conserved enzyme called nicotinamidase [97]. This key transformation connects the pathways of nicotinamide and nicotinic acid derived NAD+ biosynthesis in these organisms, as they represent consecutive metabolites in NAD+ production. On the other hand, humans and mammals lack nicotinamidase activity [19]. Consequently, nicotinamide is directly metabolized to NAD+ independent of nicotinic acid. Nicotinic acid is converted to the intermediate nicotinic acid phosphoribosyltransferase (NaMN) by action of the enzyme nicotinate phosphoribosyltransferase (Npt) (Figure 3). NaMN is common to the nicotinic acid salvage pathway and the tryptophan quinolinate pathway. Adenylation of NaMN can be accomplished by NaMN adenylyltransferase (nmnat). This enzyme has 3 isoforms nmnat-1, nmnat-2 and nmnat-3. Compartmentalization of these in cells are known, with the nmnat-1 nuclear, nmnat-2 golgi associated, and nmnat-3 mitochondrial. These enzymes can accept either NaMN or NMN as nucleotide substrates, with the NaMN being formed to nicotinic acid adenine dinucleotide (NaAD+). The terminal enzyme in this pathway is NAD+ synthetase. It converts the NaAD+ to NAD+ in an ammonia and ATP-dependent process. NAD+ synthetase in humans also combines a glutaminase activity which provides a source of the ammonia to complete the reaction. This basic set of transformations is found in nearly all organisms that can recycle nicotinic acid, and has become famously called the Preiss-Handler pathway, after Preiss and Handler who first described it over 60 years ago [16, 17, 98].

8. Core recycling pathway from nicotinamide
For mammalian cells the central challenge in NAD+ homeostasis is successful recycling of nicotinamide, released from NAD+ consuming processes, back to NAD+. Published data for NAD+ turnover in vivo indicate halflives of as little as 4-10 hours [72]. For resynthesis that equates to a minimal need to recycle 200-600 umol/kg per day of tissue in rats [73]. Assuming comparable numbers for a 75 kg human, 3 g of nicotinamide is required to be resynthesized to NAD+ up to several times per day (assuming 300 μmoles NAD/kg wet tissue)[99]. These levels are far below the amounts available from food intakes (1 lb tuna provides 100 mg vitamin B3, and 1 lb beef provides 30 mg B3, whereas 4 cups of broccoli provide only 4 mg of B3). These facts implicate efficient nicotinamide recycling as the basis for effective NAD+ maintenance in humans, and a consistent lack of pellagra in the developed world.

The nicotinamide recycling reaction is catalyzed by an enzyme called nicotinamide phosphoribosyltransferase (nampt). Nampt couples nicotinamide with PRPP to form nicotinamide mononucleotide (NMN) (Figure 4). Kinetic studies indicate that ATPase activity is also coupled to this process, which drives the equilibrium toward NMN formation[100, 101]. A valuable pharmacologic tool has been developed to inhibit this enzyme, called FK866. This inhibitor, which has a binding constant of 0.3 nM [102] can be applied to mammalian cells and leads to rapid depletion of cellular NAD+, causing levels to reach 30% within 4-8 hours. In vivo, this inhibitor has been evaluated as a possible anti-cancer agent, since it limits the abilities of tissues to recycle nicotinamide caused by anti-cancer genotoxins. Recently Nuncioni and coworkers showed that NAD+ contents can be significantly reduced in several tissues, including blood, spleen and heart with systemic administration of FK866 alone to mice [103].

Experiments to assess the role of Nampt in setting the NAD+ level in cells confirms that the level of the enzyme, and not nicotinamide concentrations themselves, have the largest effect on setting NAD+ level. For instance, Sinclair and Sauve and co-workers [32] used overexpression of Nampt or knockdown of Nampt in mammalian cultured cells to establish that Nampt levels regulate how much cellular NAD+ is available in cells. As expected, knockdown of Nampt caused reduced NAD+ levels in cells and also within the mitochondrial compartment. Conversely, overexpression caused increased cellular NAD contents and increased mitochondrial NAD+ levels. An interesting effect of overexpression of Nampt was noted on cell resistance to genotoxic stress, in that Nampt overexpressors proved resistant to apoptosis [32]. These protection effects required mitochondrial sirtuin activities, notably SIRT3 and possibly SIRT4 [32]. Interestingly, Nampt levels appear to be upregulated by dietary intake and exercise. Increased levels in liver were observed for fasted rats [32], and exercised rats [69]. Nampt levels also increase in humans in exercised legs as compared with matched unexercised legs [104].
Although comprehensive data on Nampt activity in humans is unclear, due to lack of published data, the efficiency of this nicotinamide recycling appears to be very high. For example, there is a relatively low Vitamin B3 requirement published by the Food and Nutrition Board at the Institute of Medicine which states that only 16 mg day Vitamin B3 is required for male adults above 16 and 14 mg day for females above 14[105]. This
12

implies a net loss of not greater than 0.5% total NAD+ per day, to maintain niacin homeostasis, if no additional input is available from outside sources. This is remarkable, if one considers the possibility that the entire NAD+ pool is being replaced 2-4 times per day, suggesting that only 0.1-0.2 % nicotinamide is lost per turnover cycle. This result implicates a highly efficient NAD+ resynthesis capacity in humans, made more impressive if one considers that nicotinamide is neutral, small (MW = 122) and a polar hydrophobic (LogP = -0.4), likely to passively diffuse through cell membranes, a phenomenon demonstrated in experimental studies [106]. The existence of nicotinamide methyltransferases and downstream catabolic enzymes that degrade nicotinamide to pyridone-related catabolites are a likely source for lost nicotinamide, and the amount of nicotinamide degradation products have been noted in some neurodegenerative diseases, suggesting enhanced NAD+ breakdown [21].

9. Pathways and enzymes to make NAD+ from nucleosides.
Additional relevant pathways to NAD+ are from nucleosides, including nicotinamide riboside (NR) and nicotinic acid riboside (NaR)(Figure 5). These pathways are facilitated by action of nicotinamide riboside kinases (Nrk1 and Nrk2) [107]. These enzymes are encoded by the human genome, and by genomes of other mammalian organisms and have been shown to enzymatically convert NR and NaR to NMN and NaMN respectively [108, 109].

X-ray crystallographic evidence, as well as biochemical data, show that these enzymes bind NR and NaR into an active site that discriminates against purines and pyrimidine nucleosides, thus making these enzymes preferentially specific for NR derivatives, as well as able to accommodate the antimetabolite drugs tiazofurin (TZ) and benzamide riboside (BR) [108]. NR and NaR raise NAD+ levels dose-dependently and up to 2.7 fold in mammalian cells [110] in a manner unlike the behavior of nicotinamide or nicotinic acid at the same concentrations. It is proposed that a transporter can convey NR and NaR into mammalian cells, and an NR transporter (Nrt1) in yeast has been identified [111]. Evidence for such a transporter is provided by studies of transport of BR and TZ [112]. Human concentrative nucleoside transporter 3 was most efficacious as a nucleoside transporter of both BR and TZ in transfected xenopus oocytes [112].

Recent data suggests that both NR and NaR could be produced in mammalian cells and released extracellularly, and could thereby be produced in mammals as NAD+ precursors [113], suggesting possible intercellular metabolic networks involving NR and NaR creation, release and transport into other cells. In addition, an NR degrading and possibly NaR degradative pathway may also help explain some of the effects of NR and NaR on cells and tissues, as first described by Kornberg [114]. The potency of NR in increasing cellular NAD+ has led to investigations to determine if it can treat diseases such as neurodegenerative disorders, or metabolic syndromes, based on the idea that reduced NAD+ level might be a risk factor in these conditions [32, 68, 70].

10. NAD+ metabolism as a target of therapy
Figure 6 provides a comprehensive view of NAD+ metabolic pathways as found in humans and how these pathways intersect and converge on the central metabolite NAD+. This figure also conveys the fact that different NAD+ precursors can enter NAD+ biosynthetic pathways and can converge to NAD+ via independent and also overlapping pathways. This distinctiveness in how different NAD+ precursors are metabolized is now clearly recognized with the understanding that these distinct NAD+ precursors have different properties and have distinct biological effects.

The unique properties of NAD+ precursors was first appreciated by the determination that high doses of nicotinic acid could cause lowering of serum lipids, including free fatty acids and low density lipoprotein (LDL)- cholesterol, and could increase the “good” cholesterol form called high density lipoprotein (HDL)- cholesterol [115]. Kirkland and co-workers explored effects of high doses of nicotinamide or nicotinic acid on tissue NAD+ levels in rats, and concluded that these precursors had different abilities to raise NAD+ levels in different tissues [73]. Nicotinamide had ability to increase NAD+ level in liver (47%), but was weaker in kidney (2%), heart (20%), blood (43%) or lungs (17%). Nicotinic acid raised NAD+ in liver (47%), and impressively raised kidney (88%), heart (62%), blood (43%) and lungs (11%) [73] [both nicotinamide and nicotinic acid were administered at 1000 mg/kg diet].
The reinvestigation of less-studied Vitamin B3 forms such as nicotinamide riboside and NMN for their possible NAD+ enhancing benefits became important with the recognition that NAD+ may act as a key signaling molecule in cellular physiology. The Brenner laboratory showed that NR raises NAD+ levels substantially in yeast [116]. Our laboratory then explored the ability of NR to increase NAD+ in mammalian cell lines and determined that cellular NAD+ levels increased as much as 270% above controls in several different cell lines [110]. This potency of NR to increase cellular NAD+ levels implied a novel mechanism of metabolism for this compound, possibly through action of the newly characterized Nrk enzymes. Our laboratory similarly showed that the NR relative, NaR was also able to increase NAD+ level in mammalian cells by as much as 1.9 fold [110], possibly through direct synthesis of NaMN.

11. NAD+ increases as a novel modality to treat diseases

The hypothesis that augmentation of NAD+ level could stimulate adaptive changes in cellular bioenergetic and survival adaptation has been experimentally examined. This hypothesis is anchored by biochemical demonstrations that human NAD+ consuming enzymes, such as sirtuins (and other mammalian sirtuins), have kinetic parameters (Km) which make them intrinsically sensitive to changes in NAD+ concentrations in the physiologic range found in cells. Key additional support for this idea is that NAD+ is an intrinsic regulator of cell bioenergetics as revealed by studies showing that the Nampt level is upregulated by dietary stress or by exercise. This suggests that some of the health beneficial effects of diet and exercise could derive in part from upregulated NAD+ production [69, 117, 118]. If so, this raises additional interest in the therapeutic prospects of raising NAD+ levels as a possible intervention to effect beneficial changes in human physiology.

Metabolic Syndrome The NAD+ enhancing effects of the compound NR were explored for potential therapeutic effects in a mouse model of metabolic syndrome. The Auwerx and Sauve laboratories found that NR enhanced NAD+ contents in several mammalian tissues, and induced mitochondrial biogenesis as determined by increased cristae content and increased expression of mitochondrial proteins, such as Complex V [119]. This data provided a pharmacologic mirroring of effects found in at least two genetic models where NAD+ levels were increased, where animals were protected from weight gain caused by high fat diets. For example, PARP1-/- animals displayed overexpression of mitochondrial proteins in skeletal muscle [67]. CD38-/- animals were protected from weight gain also showed impressive mitochondrial biogenesis in skeletal muscle [66].

NAD+ Signaling by activation of SIRT1 and PGC1α to promote mitochondrial biogenesis The mechanisms by which NAD+ increases can lead to mitochondrial biogenesis are still being examined, but one fundamental mechanism of action is through activation of SIRT1, and stimulated activity of the co-transcriptional activator PGC1α [20, 120]. SIRT1 deacetylation of PGC1α leads to activation and possibly stabilization of this protein, whereby it can coordinate with nuclear transcription factors that control mitochondrial biogenesis genes [121].

Thus, increases in NAD+ levels caused by genetic modifications (CD38-/- or PARP1-/-) or pharmacologic interventions such as NR administration lead to increased PGC1α deacetylation, increased transcription of genes in the mitochondrial biogenesis pathway, and increased oxidative activity as determined by assays of mitochondrial activity [119]. Similar consequences are associated with activation of SIRT1 and PGC1α signaling applying the putative SIRT1 activator resveratrol [122, 123]. Resveratrol has since been appreciated to have complex effects, including activation of AMPK [124] as well as potentiation of cAMP signaling [125]. Since these additional pathways are known to stimulate mitochondrial biogenesis it remains to be determined if and how these pathways contribute to the observed effects of NAD+ enhancement on mitochondrial biogenesis.

Mitochondrial Disorders Proof of concept studies establishing that increased NAD+ level stimulates mitochondrial biogenesis, place NAD+ squarely in the center of key signaling pathways with major impact for bioenergetic and survival physiology. Translationally directed followups to these provocative studies addressed the possibility that enhanced NAD+ production could provide a stimulative and ameliorative benefit in mitochondrial disorders.

Three independent studies in animal models of mitochondrial disease largely supported the initial findings and showed that increased NAD+ production achieved by one of the following: 1) by administration of NAD+ precursors (NR), 2) by PARP inhibition or 3) by PARP genetic knockout could improve mitochondrial function [67], improve exercise intolerance [68], and could improve mitochondrial protein levels [68, 126, 127]. In addition, sirtuins have been shown to be protect mitochondria from stress.

SIRT3, known to be influenced by NAD+ level, can reduce oxidative stresses through SOD activation [128, 129] and increased SIRT7 activity can alleviate mitochondrial protein folding stress [130]. It can be proposed that combinations of NAD+ concentration increases and SIRT7 induction can suppress mitochondrial stress and promote mitochondrial integrity. Collectively, these results have stimulated interest in the potential benefits of enhancing tissue NAD+ as a means to treat mitochondrial diseases [131].

DNA Repair Syndromes Cockayne’s Syndrome is an accelerated aging disease involving mutations in either Cockayne syndrome group A (CSA) or CSB proteins, involved in DNA repair, leading to progressive neurodegeneration [132, 133]. Bohr and coworkers verified that one feature of deficiency in CSB mutant animals is activation of PARP1, and increased PAR levels in CSBm/m cells[134]. Accompanying this increase in NAD+ turnover is a severe metabolic disruption including defects in weight gain due to a hypermetabolic phenotype and increased levels of lactate in brain tissues, such as the cerebellum [134]. Application of PARP inhibitors as a means to reverse this metabolic effect proved successful, in that the inhibitor PJ34 could increase oxygen consumption rate/extracellular acidification (OCR/ECAR) ratio, a measure of improvement in the normalization of catabolism [134]. Administration of NR for one week as an NAD+ repletion agent enabled improvements in NAD+ level in cerebellum of WT and CSBm/m animals. Moreover, ATP homeostasis was also substantially improved in this tissue. NR treated CSBm/m mice had cerebellum mitochondria that had corrected defects in membrane potential and ROS production [134].

Alzheimer’s Disease The ability of NR to penetrate into brain was recently verified, where it was then shown to provide improvements in Alzheimer’s Disease (AD) neuropathology in the Tg2576 mouse model of this disease [135]. Previous work had shown that the effects of calorie-restriction (CR) in animal models of AD provided reduced neuropathology, as determined by plaque burden, and improvements in behavioral scores measuring memory and cognition [135]. This was shown to be accompanied by increased NAD+ levels and increased NAD+/nicotinamide ratios in CR treated animals as compared to ad libitum fed Tg2576 transgenic mice [70]. These results suggested the possibility that increased NAD+ levels could provide a component in the protective mechanisms that are produced by CR. Administration of NR was shown to increase NAD+ levels in brain and caused reduced production of A1-42.

NR was shown to promote PGC1α levels in transgenic animals. In parallel, the authors showed that PGC1α-/- animals exhibited markedly worse neuropathology in the Tg2576 background [135]. Taken together, NR promotes NAD+ levels, increases PGC1α and improves behavioral and molecular markers indicative of resistance to AD progression.
Fatty Liver Disease Recent investigations into the ability of NAD+ to potentiate oxidative metabolism and to improve mitochondrial function and density led several collaborating groups, including ourselves, to investigate the effects of NR administration in models of liver disease, such as fatty liver disease [136]. These studies provided evidence that NAD+ elevation is protective of this disease in at least two disease models, such as high fat combined with high sucrose (HFHS), as well as in apolipoprotein E (ApoE)-/- animals fed a high fat high cholesterol (HFHC) diet. NR administered at 500 mg/kg protected from fatty liver induced by HFHS as determined by Oil-Red O staining, as well as fibrosis and lipogenesis markers [136].

In an anti-inflammatory effect, plasma TNFα was reduced to control levels in NR fed animals but was elevated 2 fold in HFHC fed animals. As hypothesized, NR administration led to increased oxidative metabolism as measured in isolated liver tissue; for example O2 consumption was elevated, as was citrate synthase activity. Activation of the mitochondrial unfolded protein response was also observed, suggesting that activation of mitochondrial protein stress may be a key to the benefits observed with NR, and therefore NAD+ increase [136]. Similar findings were obtained in ApoE-/- animals challenged with HFHC [136]. At this stage, more mechanistic studies are needed to understand how NR-induced NAD+ increases exert these protective effects. For one possible link to nuclear signaling, overexpression of SIRT7 can prevent the spontaneous development of fatty liver disease [137, 138] and SIRT7 suppresses mitochondrial protein folding stress by repressing NRF1 activity [130]. The full set of molecular players that participate to produce the protective effects of NR in prevention of fatty liver disease may belong to several signaling pathways.

Hepatic Carcinoma An illuminating study in the area of cancer biology in liver examined effects of NAD+ and unconventional prefoldin RPB5 interactor (URI) driven dysregulation of hepatocellular mTOR/S6K1 signaling pathways on development of hepatocellular carcinoma (HCC). Using overexpression of an oncogenic protein called URI in liver, Tummala et al.[139] found that livers became fibrotic and developed progressive hepatocellular dysplasia, resembling transformative cell phenotypes common to early stage human hepatocarcinoma. Diethylnitrosoamine induced accelerated hepatocellular carcinoma in heterozygous URI+/- and homozygous URI+/+ transgenic animals, with increased gene dosage of URI providing shortened induction to HCC in liver.

Interestingly, NAD+ levels in URI+/+ homozygous livers were found to be substantially lower (35-50% of control) at 3 weeks of life. To investigate if NAD+ repletion could mitigate the adverse effects of URI overexpression on liver phenotype, the authors administered NR via diet, and determined that this increased NAD+ levels back to near normal levels, and completely prevented the development of hepatocellular dysplasia observed in untreated controls. Treatment of 12 week old mice with fully developed hepatocellular carcinoma with NR for 48 weeks produced regression of HCC tumors [139].

Inflammatory Conditions The NLRP3 inflammasome complex, a component of innate immune surveillance, is known to be activated in a number of disease associated conditions, and is known to be overactivated in the physiology of obesity and is involved in disease states such as insulin resistance [140]. Overactivation of innate immunity can be triggered by pathogen-associated molecular patterns or damage associated molecular patterns (DAMPs), such as those derived from mitochondrial dysfunction [141]. DAMPs can stimulate assembly of the inflammasome, and lead to production of cytokines such as pro IL-1β or pro IL-18, which can
amplify inflammation [140]. Sack and coworkers [142] determined that nutritional intakes increase activation of the inflammasome, whereas fasting attenuates these activations. They found that NR treatment of human derived macrophages had attenuated inflammasome activation in a SIRT3 dependent manner, suggesting that NAD+ signaling can be a potential means to inhibit excessive inflammation triggered by nutritional inputs [142].

Cardiomyopathy Andrews and coworkers recently demonstrated that deletion of transferrin receptor 1 (Tfr1) leads to a profound defect in iron loading in the heart, causing early lethality in Tfr1-/- mice due to the iron deficit [143]. This impairment in iron loading causes mitochondrial defects, and leads to cardiodilation and early death at post-natal day 10. Remarkably, administration at birth of NR causes up to 50% improvements in survival of mice (day 15) [143]. Although mechanistically the effect of NR was not fully elucidated, the presumptive effect may be through improvement in mitochondrial function and maintenance, as this effect of NR has been noted in other situations as discussed in this review. Notably, NR decreased accumulation of p62, suggesting improvements in mitochondrial mitophagy [143]. The effect of improving NAD+ status on improving survival outcomes in a model of heart disease is notable, given the broad impact of this disease in human populations, and its substantial mortality.

Noise Induced Hearing Loss Hearing loss affects 100s of millions of people worldwide, from a variety of causes. Nevertheless, new treatments to prevent hearing loss are largely unavailable. In a recent study Brown and co-workers [144] showed that high intensity wide spectrum sound could cause loss of hearing in a mouse model of hearing loss. Overexpression of the mitochondrial sirtuin SIRT3 or the WLDs mouse which encodes a triplicate repeat of the NAD+ biosynthetic gene NMNAT1, could largely prevent the loss of hearing. To investigate if increased NAD+ content could also prevent the noise induced hearing loss, these investigators administered NR before, before and after, or only after noise exposure and showed that all three treatments fully protected hearing in all frequencies [144]. This finding strongly hinted that approaches which can augment NAD+ levels in the neurons and tissues of the ear could provide protection from trauma induced hearing loss and could also be meaningful in progressive hearing loss syndromes. These ideas will require additional study to determine if this approach can be broadly applied in hearing loss treatment.
Aging and the Diseases of Aging Although there is not a firm understanding of all the factors that cause biological aging to occur, there are a number of factors that appear to be common to aging, particularly in mammals. Among these are loss of regenerative potential, defects in DNA repair and mitochondrial decline. Although there is not a clear consensus on the pathways and causes that lead to these effects, the findings that enhancements in cellular NAD+ can improve outcomes in DNA repair syndromes (i.e. Cockaynes Syndrome) and in mouse models featuring mitochondrial deficiency (mito-disorders and Tfr1-/-) suggests that NAD+ stimulated pathways as modify aging phenotypes toward more youthful conditions (See current review on this by Verdin [145]). Mechanistically NAD can elevate the activity level of sirtuins, such as SIRT3 and SIRT7, as SIRT3 and SIRT7 have been shown to be depleted in aged stem cells and reintroduction of SIRT3 or SIRT7 reverses stem cell aging by reducing mitochondrial stresses [130, 146].

This idea that NAD+ could be central to aging has been directly evaluated in a fascinating and timely study authored by Sinclair and co-workers [37]. Leaping off from the idea that mitochondrial decline and increased ROS could be a central cause of aging, as advocated by Harman [147] these workers proposed that the decline is linked to imbalances in mitochondrial proteinse encoded by nuclear and mitochondrial genomes.

Imbalanced mitochondrial and nuclear gene transcription for mitochondrial proteins impairs mitochondrial activity in aged animals. Mitochondrial encoded proteins, in particular, were found to beadversely affected by age. A key cellular factor in maintaining a correct balance is the sirtuin SIRT1, as well as the SIRT1 substrate NAD+. Intriguingly, NAD+ homeostasis was found to be significantly impaired in older animals (22 months), causing NAD+ levels to drop to 40% of the levels observed in young animals (6 months)[37]. This suggested that replenishing NAD+ might rebalance mitochondrial-derived and nuclear-derived mitochondrial protein production.

Thus, aged mice were fed the compound NMN for one week, and this treatment was able to restore NAD+ levels in 22 month animals to amounts at observed at 6 months. Several key mitochondrial fitness parameters improved, including increased mitochondrial-encoded transcripts and increased ATP levels. This reversal in key molecular phenotypes, by a relatively straightforward NAD+ enhancement strategy provided new insights into the role NAD+ might play in human aging, and in mitochondrial decline, and suggested new ways to intervene to mitigate these effects.

12. Conclusions
The centrality of energy metabolism in organisms, and the integration of key metabolic components into signaling pathways that modulate organism health and physiology, make it clear that some of the more abundant and central factors, such as NAD+ can have unexpected roles in maintaining healthy physiology and could be important in the development of pathology. As this review attempts to illuminate, current knowledge of NAD+ metabolic pathways and knowledge of the ways in which NAD+ regulates key processes in cells and tissues is undergoing a current reblossoming of interest. This has been bolstered by identification and investigation of “newer” Vitamin B3 forms, such as NMN and NR, which provide new opportunities to pharmacologically modulate NAD+ metabolism and to possibly alleviate disease conditions.

These newer NAD+ precursors have shown impressive effects in a number of proof of concept studies that favorably mitigate, prevent or cure animal models of disease including AD, cardiovascular disease, metabolic syndromes, mitochondrial disorders, cancer and even aging. These developments set the stage for deeper investigative understanding into the mechanisms by which some diseases are sensitive to NAD+ status, and may pinpoint details of how deterioration of NAD+ homeostasis increases the susceptibility to human disease conditions.

Targeting NAD+ in Metabolic Disease

Nicotinamide adenine dinucleotide (NAD+) was discovered more than 100 years ago by Sir Arthur Harden as a low-molecular-weight substance present in boiled yeast extracts [1]. In the late 1920s, Joseph Goldberger fed Brewer’s yeast to dogs with pellagra, a devastating disease characterized by dermatitis, diarrhea, dementia, and death, and their health improved.

At that time, pellagra was endemic in parts of the United States, and so the Red Cross supplemented Brewer’s yeast to its food rations in pellagra-endemic areas; within weeks the disease burden dissipated [2, 3]. The health significance of NAD+ was established in 1937, when Conrad Elvehjem and his colleagues made the major discovery that the factor that prevented and cured pellagra was the NAD+ precursor, nicotinic acid [4, 5].

NAD+ plays a central role in cellular respiration, the cascade of reactions that generate adenosine triphosphate (ATP) from nutrient breakdown, by acting as a coenzyme for oxidoreductases and dehydrogenases [6–9]. As coenzymes, NAD+ and its phosphorylated and reduced forms, including NADP+, NADH, and NADPH, are critical for the activities of cellular metabolism and energy production [1, 10, 11]. NAD+ most commonly functions in energy-generating catabolic reactions (such as glycolysis, fatty oxidation, and citric acid cycle), where it is reduced to NADH, which is then shuttled into the mitochondria to generate ATP.

This generates an NAD+/NADH ratio, which is useful to assess the health and energy charge of the cell. The phosphorylated form, NADP(H)+, participates in anabolic reactions, such as fatty acid and cholesterol synthesis [8, 9, 12].

More recently and as importantly, NAD+ has been studied as a rate-limiting substrate for three classes of enzymatic reactions involved in posttranslational modification (Fig. 1), all of which exhibit breaking of the glycoside bond between nicotinamide and the adenosine 5′-diphosphate (ADP)-ribose moiety, and the latter is then transferred onto an acceptor molecule [6–9, 11]. The first class includes mono- and poly-ADP ribose transferases, among which the poly-ADP ribose polymerases (PARPs) are the most studied and are classically described as DNA repair proteins [13, 14].

The second class is the cyclic ADP ribose synthases (CD38 and CD157), which are membrane-bound ectoenzymes that produce and hydrolyze the Ca2+-mobilizing second messenger cyclic ADP-ribose from NAD+ and are therefore key in calcium homeostasis and signaling [15]. The third and most important class in terms of cellular energy metabolism consists of the sirtuins, named for their similarity to the yeast Sir2 gene-silencing protein. Seven sirtuins exist in mammals (SIRT1 through SIRT7), with diverse enzymatic activities, expression patterns, cellular localizations, and biological functions [16]. Sirtuins have a host of metabolic targets, resulting in profound effects on various cellular processes, such as mitochondrial biogenesis, cellular stress response, lipid metabolism, insulin secretion and sensitivity, apoptosis, circadian clock dynamics, inflammation, and aging [17].

Through these targets, sirtuins translate changes in feeding status, DNA damage, and oxidative stress into metabolic adaptations [18–20]. SIRT1, the most-studied sirtuin, targets multiple transcriptional coactivators, such as the peroxisome proliferator-activated receptor γcoactivator-1α (PGC-1α) and transcription factors, such as the forkhead box protein O1. PGC-1α is a central regulator of energy metabolism and mitochondrial biogenesis [21–24], whereas forkhead box protein O1 regulates mitochondrial fatty acid metabolism and protects against oxidative stress [25–27].

As nutrients influence the NAD+/NADH pool, these NAD+-dependent signaling reactions are recognized as the sensors of metabolism owing to their decisive regulatory roles in cellular metabolism [17]. Appropriate regulation of these NAD+-dependent processes relies on the cellular ability to conserve their NAD+ content. Therefore, inadequate NAD+ homeostasis can be pathologic, linked to impaired cell signaling and mitochondrial function [19, 28, 29].

The dependency of sirtuins on NAD+ [30], and the finding that yeast Sir2 protein is required for the lifespan extension mediated by caloric restriction (CR) [31], led to a renascent interest in NAD+ metabolism research, centered on modifying NAD+ availability to support sirtuin-mediated cellular metabolism to mimic CR. This interest was enhanced by the discovery of contemporary NAD+ precursors that can circumvent issues with existing molecules, which can also increase NAD+ in vivo and human tissues [32–34]. As we review here, these key findings underline the prospect of targeting NAD+ biosynthetic pathways to increase mitochondrial function and sirtuin activity in the combat against metabolic disease. We also highlight the challenges and the knowledge gaps that require investigating before these compounds can find their way to the clinics.

1. NAD+ Biosynthesis and Metabolism

In humans, NAD+ can be synthesized via the de novo/kynurenine pathway from the amino acid tryptophan [35, 36]. However, tryptophan is a poor NAD+ precursor in vivo [37]. Most organisms have alternative NAD+ synthesis pathways (Fig. 2) from the dietary vitamin B3 precursors nicotinic acid (NA), nicotinamide (Nam), and nicotinamide riboside (NR), or from a salvage pathway where the Nam molecule split from NAD+-consuming reactions is recycled into nicotinamide mononucleotide (NMN) via the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT), and NAD+ is regenerated [9, 11, 35, 38–41]. In addition, a more recently described salvage pathway recycles NR to NMN via the nicotinamide riboside kinases (NRKs) [32]. In humans, these different routes to NAD+ synthesis converge at the NAD+ and nicotinic acid adenine dinucleotide formation step catalyzed by the nicotinamide mononucleotide adenylyltransferases. Nicotinic acid adenine dinucleotide is then amidated to form NAD+.

Schematic overview of human NAD+ biosynthesis. NAAD, nicotinic acid adenine dinucleotide; NADS, NAD+ synthase; NAPT, nicotinic acid phosphoribosyltransferase; NMNAT, nicotinamide mononucleotide adenylyltransferase; QAPT, quinolinic acid phosphoribosyltransferase. *NAMPT is the rate limiting step in NAD+ biosynthesis.

Nicotinic acid riboside is an NAD+ biosynthesis intermediate that can be converted in yeast and human cells by NRKs into nicotinic acid mononucleotide and then to NAD+ [42]. It is the least-studied NAD+ precursor and is therefore beyond the scope of this review.

The energy sensor adenosine monophosphate-activated protein kinase (AMPK), which adapts cells to low-energy states in the support of ATP production [43, 44], activates NAMPT, increases NAD+ recycling, and enhances SIRT1 activity [45, 46].

In mammals, the entire NAD+ pool is used and replenished several times a day, balanced by the distinct NAD+ biosynthetic pathways [47]. Owing to its constant utilization, the half-life of NAD+ in mammals is short (up to 10 hours) [36, 48–51], with intracellular levels believed to be 0.4 to 0.7 mM [41]; however, the accuracy of this level depends on the cell type and physiologic state being assessed. It is clear that NAD+ concentrations differ substantially between cellular compartments, with mitochondrial NAD+ concentration being the highest and representing 70% to 75% of cellular NAD+ (10- to 100-fold higher than those in the cytosol) [52, 53]. The NAD+/NADH levels vary to adjust cellular and tissue physiology in response to changes in nutrient availability and energy demand. For instance, NAD+ levels drop in response to high-fat diet (HFD) in mice [33, 54] and with aging, contributing to age-related disorders, such as diabetes, cardiovascular disease, cancer, and neurodegenerative disease [55–58]. Conversely, the renowned health adaptive beneficial effects of CR and exercise have been linked to NAMPT activation and the subsequent rise in NAD+, sirtuins, and mitochondrial activity [46, 59–61].

2. Therapeutic NAD+ Boosting

The recommended daily allowance (RDA) of niacin, a collective term for NA and Nam, is around 15 mg/d and can be met through the consumption of meat, fish, and dairy products [12, 62]. More recently, NR was also detected in milk and yeast [32, 63].

A plethora of evidence suggests that higher rates of NAD+ synthesis can positively affect pathways that require NAD+ as a cosubstrate. The NAD+ pools can be elevated via provision of precursors [33, 54, 64, 65], NAD+ biosynthesis augmentation [45, 46], and inhibition of NAD+ consumers [57, 66–68].

3. NAD+ Precursor Supplementation

The most tractable approach to increase NAD+ would be via the supplementation of the different precursors, all of which increase NAD+ levels in human and animal tissues. This approach is the focus of this review because NAD+ precursors are naturally occurring in food and are readily available in isolated forms, allowing nutritional approaches to be applied to modulate NAD+ metabolism in vivo.

A. Niacin

NA has been used for >50 years in the treatment of hyperlipidemia [69, 70]. Dietary niacin is not associated with side effects because the tolerable upper intake level is not exceeded [62], whereas pharmacologic NA dosing is commonly associated with undesirable effects, thereby decreasing treatment adherence. NA is a ligand for the G-protein–coupled receptor GPR109A and is coexpressed on the epidermal Langerhans cells mediating prostaglandin formation, which induces troublesome flushing and other vasodilatory effects, such as itching, hypotension, and headaches [12, 71–73]. To overcome these problems, the selective antagonist of prostaglandin D2 receptors, laropiprant, was introduced into clinical practice in combination with extended-release NA (extended-release NA-laropiprant) [74]. Extended-release NA-laropiprant failed to prove advantageous in clinical trials; safety concerns arose, and the agent was therefore withdrawn from all markets [75]. A long-acting NA analog, acipimox, is undergoing clinical trials [76–79]. However, acipimox remains a GPR109A receptor ligand [80], thus retaining the potential for undesirable side effects that will limit its clinical utility.

Although Nam is the predominant endogenous precursor of the NAD+ salvage pathway, early reports suggested that it may not be as effective as other biosynthesis precursors in increasing NAD+ levels [41]; however, this likely reflects the relatively small dose of Nam used. Additionally, Nam effects likely depend on cell/tissue type and the pathophysiologic state. For instance, in a nonstressed state, Nam is inferior to NA as an NAD+ precursor in the liver [81], whereas under HFD-induced metabolic challenge, Nam is a more powerful NAD+ precursor and SIRT1 activator than NA [82]. Nam has been used for many years for a variety of therapeutic applications (such as diabetes mellitus) at doses up to 3 g/d, with minimal side effects [83]. Unlike NA, Nam has no GPR109A agonist activity [80], thus escaping the prostaglandin-mediated vasodilatory side effects. Yet, at high doses Nam has a toxic potential (particularly hepatotoxicity), raising health concerns [83] and, as well as with long-term use, can cause negative feedback to inhibit sirtuins [84, 85].

B. NR and NMN

NR has been recognized since the 1950s as an NAD+ precursor in bacteria that lack the enzymes of the de novo and Preiss–Handler pathways [86–88]. This changed in 2004, when Bieganowski and Brenner [32] detected the presence of NR in milk and identified two human NRK enzymes capable of synthesizing NAD+ from NR. Subsequent human and animal studies confirmed that NR can increase intracellular NAD+ in a dose-dependent fashion [34, 89, 90]. Likewise, NMN is an intermediate in the NAD+ salvage pathway. Although less studied than NR, several studies proved that NMN increases NAD+ levels in vitro and in vivo [33, 56, 91–93]. Several recent studies using NR and NMN have attracted major research interest and are discussed later.

4. NAD+ Biosynthesis Augmentation

Several AMPK and NAMPT activators have been studied. Resveratrol is a nonflavonoid polyphenol that is present in red grapes, wine, and pomegranates; activates AMPK and SIRT1; and improves metabolic health status in humans [94–98]. However, conflicting outcomes from clinical studies have questioned the efficacy of resveratrol in treating human metabolic disease [99]. Nonetheless, it remains a compound of substantial interest to many [100].

Various AMPK activators exist [101]. Among them is metformin, which was introduced in the 1950s to treat diabetes, with a multitude of favorable metabolic outcomes that rely on AMPK [102]. Cantó et al. [45] reported that the AMPK activators metformin and 5-aminoimidazole-4-carboxamide ribonucleotide, increase NAD+ and sirtuin activity, thereby regulating energy expenditure .

Other compounds have also been reported to increase NAMPT activity. P7C3, a neuroprotective chemical that enhances neuron formation, can bind NAMPT and increase NAD+ levels [103–105]. Likewise, the antioxidant troxerutin, a trihydroxyethylated derivative of the natural bioflavonoid rutin, markedly increased NAD+ levels and potentiated SIRT1 via NAMPT activation and PARP1 inhibition in HFD-treated mouse liver [106]. Remarkably, leucine supplementation in obese mice also increased NAMPT expression and enhanced intracellular NAD+ levels [107]. Moreover, proanthocyanidins, the most abundant flavonoid polyphenols in human diet, can dose-dependently increase NAD+ levels in rat liver via the increased expression of the de novo pathway enzymes [108], and possibly NAMPT [109]. Targeting microRNA, such as antagonizing hepatic miR-34a, has also been reported to increase NAMPT expression and NAD+ and SIRT1 activity in vivo [110].

5. Inhibition of NAD+ Consumers

Inhibiting the nonsirtuin NAD+ consumers also increases NAD+ levels and favors sirtuin activity. Inhibitors of PARPs or CD38 induce NAD+ levels, upregulate sirtuins, and enhance mitochondrial gene expression [67, 68, 89, 111]. PARP inhibitors are effective anticancer agents through DNA damage repair and improved oxidative metabolism (opposing the Warburg effect) in which the NAD+-sirtuin axis may be implicated [112–115]. The first PARP inhibitor, olaparib, is now licensed in the United States and Europe for the treatment of ovarian cancer [116, 117]. Therefore, PARP inhibitors may undergo further studies as NAD+-sparing agents to improve adaptive metabolism [118]. Interestingly, troxerutin and proanthocyanidins also inhibit PARPs in mice, thereby contributing to increased NAD+ [106, 108].

6. Type 2 Diabetes Mellitus

The global burden of obesity, insulin resistance, and type 2 diabetes mellitus (T2DM) continues to limit population health through increased cardiovascular disease risk and premature death [119].

Several studies support the notion that defective mitochondrial structure and function are strongly linked to insulin resistance and T2DM [120–128]. The most described mechanism is via defective mitochondrial fatty acid oxidation and the resultant accumulation of intracellular fatty acid metabolites and reactive oxygen species decreasing insulin sensitivity [129–133]. In addition, perturbed oxidative phosphorylation (OXPHOS) may be a direct cause of insulin resistance [134]. Supporting this, obesity reduces mitochondrial enzymatic activities [135, 136] and engenders metabolic inflexibility [137]; the inability to limit fatty oxidation and switch to carbohydrate oxidation in response to diet (and therefore insulin stimulation) [138–141].

Impaired NAD+-mediated sirtuin signaling is also implicated in insulin resistance and T2DM. In particular, defective SIRT1 activity is thought to be a factor in impaired insulin sensitivity [142–148]. This is endorsed by the finding that metformin acts through hepatic SIRT1 activation as part of its diabetes ameliorating effects [149]; results similarly observed with resveratrol [150].

Lifestyle manipulations, such as CR and exercise, can reverse insulin resistance and T2DM and share common mechanistic pathways of AMPK activation leading to elevated NAMPT-mediated NAD+ generation and SIRT1 activity to enhance mitochondrial function [46, 61, 151, 152]. Corroborating the link to NAD+, adipocyte-specific NAMPT deletion in mice decreased adiponectin production and resulted in severe multiorgan insulin resistance [92]. Aside from insulin sensitization, NAD+ and SIRT1 regulate glucose-stimulated insulin secretion in pancreatic β cells [153–155]. NAMPT inhibition and the lack of SIRT1 resulted in pancreatic β cell dysfunction [93, 156–159]. Interestingly, SIRT1 regulates the key components of the circadian clock, CLOCK and BMAL1 [160, 161], and when circadian misalignment is induced in mice, reduced hepatic BMAL1 and SIRT1 levels and insulin resistance ensue [150].

These lines of evidence suggest that an alternate strategy is to increase the level of NAD+ available to affected cells and tissues. Indeed, the NAD+ precursors used to enhance target tissue NAD+ availability have demonstrated efficacy to improve insulin sensitivity and reduce diabetic burden and associated metabolic derangements in preclinical models [33, 162].

NMN administration restored β cell glucose-stimulated insulin secretion and hepatic and muscle insulin sensitivity in mouse models of induced glucose intolerance [33, 92, 93]. Furthermore, Nam treatment in obese rats with T2DM promoted sirtuin-induced mitochondrial biogenesis and improved insulin sensitivity [82]. Similarly, NR supplementation attenuated HFD-induced obesity in mice, improved insulin sensitivity and glucose tolerance, and ameliorated the adverse lipid profile [54, 162]. Moreover, leucine supplementation in obese mice increased NAD+, mitochondrial biogenesis, insulin sensitivity, and lipid disposal [107].

Thus far, clinical data are limited to acipimox and resveratrol. Acipimox increased tissue insulin sensitivity in T2DM [79, 163–168] and improved β cell function when combined with dapagliflozin [76]. However, the results have been inconsistent at times. For instance, acipimox treatment in obese nondiabetic persons alleviated free fatty acids and fasting glucose with a trend toward reduced fasting insulin and homeostatic model assessment of insulin resistance [77], whereas van de Weijer et al.[78] did not report similar benefits in individuals with T2DM by using euglycemic hyperinsulinemic clamp studies. However, in the later study, this may have been related to the rebound increase in fatty acids after short-term acipimox administration [169]. Similarly, many describe that resveratrol decreases glucose and insulin levels in patients with impaired glucose tolerance and diabetes [95, 96, 170, 171], whereas others have not observed these findings [172]. The conflicting results among these studies may be explained by the heterogeneity in the selection of study population, dose and duration of treatment, and the methods of assessing insulin sensitivity.

7. Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease in the Western world, encompassing the spectrum of liver diseases, including simple steatosis, nonalcoholic steatohepatitis, cirrhosis, liver failure, and hepatocellular carcinoma [173]. Hepatic lipid accumulation, which leads to cellular dysfunction, termed lipotoxicity, forms the basis for the development of NAFLD [174–176]. Consequently, a set of metabolic adaptations supervene, such as increased βoxidation. This adaptation induces metabolic inflexibility and drives the oxidative stress and mitochondrial dysfunction that are apparent in NAFLD [177–180].

Sufficient NAD+ levels are essential for adequate mitochondrial fatty acid oxidation [181, 182], and lipid caloric overload in mice reduces hepatic NAD+ levels and triggers lipotoxicity [183]. Zhou et al. [184] demonstrated that hepatic NAD+ levels decline with age in humans and rodents, which may contribute to NAFLD susceptibility during aging. Likewise, ample evidence suggests that impaired hepatic SIRT1 and SIRT3 signaling contributes to NAFLD [183, 185–188] and that SIRT1overexpression reverses hepatic steatosis [189, 190]. Stressing the significance of adequate hepatic NAD+ homeostasis, aberrant NAD+ metabolism is also implicated in alcoholic hepatic steatosis [191, 192] and hepatocellular carcinoma [193].

Several strategies targeting NAD+ metabolism to enhance sirtuin signaling have proved beneficial in the context of NAFLD. Nam and resveratrol protected hepatocytes in vitro against palmitate-induced endoplasmic reticulum stress [64, 194]. NR attenuated the severe mitochondrial dysfunction present in fatty liver of mice on HFD via NAD+-mediated sirtuin activation [54, 195]. Remarkably, NR was able to target many of the molecular aspects of NAFLD pathogenesis, including decreasing hepatic expression of inflammatory genes, blood tumor necrosis factor-α levels, and the hepatic infiltration by CD45 leukocytes [196]. PARP inhibition in mice with NAFLD can correct NAD+ deficiency, augmenting mitochondrial function and insulin sensitivity and allaying hepatic lipid accumulation and transaminitis [197]. Considering the current data, and in the absence of licensed therapies for NAFLD, replenishing the hepatic NAD+ pool to activate sirtuins and tackle mitochondrial dysfunction is staged for assessment in human clinical studies.

8. Aging and Metabolic Decline

By the year 2050, it is projected that the US population aged ≥65 years will be 83.7 million [198], with other low-mortality countries displaying similar population proportions [199].

Sarcopenia, Greek for “poverty of flesh,” is a consistent manifestation of aging, associated with frailty, metabolic disease, cardiovascular morbidity and mortality, and substantial health care costs [200, 201]. Needless to say, strategies aimed at treating sarcopenia and age-related diseases are needed.

A decline in NAD+ homeostasis contributes to the aging process [202, 203]. Indeed, NAD+ and sirtuins regulate diverse pathways that control aging and longevity [31, 57, 204–206], converging on the ability to defend mitochondrial function [207]. Certainly, mitochondrial dysfunction and defective cellular energy signaling have emerged as critical in aging and age-related metabolic diseases, such as T2DM, NAFLD, and sarcopenia [55]. Specifically, altered mitochondrial homeostasis, through reduced NAD+ and SIRT1 activity, is advocated as a hallmark of muscle aging [56]. In addition, limiting NAD+ in mouse skeletal muscle induced the loss of muscle mass and function (i.e., sarcopenia) [208].

Age-related decline in NAD+ results from several mechanisms, which include accumulating DNA damage (and, consequently, chronic PARPs activation) [209, 210] and increased expression of CD38, clearing NAD+ and inducing mitochondrial dysfunction [211]. Additionally, chronic inflammation [212], a common feature in aging, reduces NAMPT expression and the ability to regenerate adequate NAD+ in multiple tissues [154].

The potential of NAD+ supplementation to support healthy aging is supported by several recent studies. NAMPT overexpression in aged mice matched the NAD+ levels and muscle phenotype of young mice [208]. Furthermore, SIRT1overexpressing mice were protected against the age-related development of diabetes and had a lower incidence of cancer [213]. NMN administration in aged mice restored NAD+ levels and the markers of mitochondrial function that decline with age [56].

Looking from a different angle, NR supplementation enhanced the expression of PGC-1α in the brain of a mouse model of Alzheimer’s disease, significantly attenuating the cognitive decline [214]. These findings affirm that decreased NAD+ levels contribute to the aging process and that NAD+ supplementation may prevent and even treat age-related diseases.

9. Discussion and Future Challenges

It is now well established that NAD+ is involved in metabolic regulation via redox and cell signaling reactions and that insufficient NAD+ is linked to a variety of metabolic and age-related diseases. The evidence reviewed here highlights that NAD+ levels can be therapeutically increased to potentiate sirtuins and mitochondrial function. This is a great opportunity in metabolic research that could conceivably lead to clinical utility.

The long-known lipid-lowering effects of NA may, at least partly, be NAD+ mediated. This hypothesis is favored because the half maximal effective concentration for the GPR109A receptor is in the nanomolar range [215, 216]; however, the therapeutic doses of NA are greatly in excess of this amount [71, 217]. Moreover, NR ameliorated hypercholesterolemia in mice without activating the GPR109A receptor [54]. Additionally, the liver lacks GPR109A receptors [218] but expresses liver X receptors, which regulate whole-body lipid homeostasis, that are upregulated by SIRT1 [219].

Although we have described the different pathways to NAD+ biosynthesis, it must be emphasized that not all tissues are capable of converting each precursor to NAD+ with equal efficacy, owing to the differences in the cell- and tissue-specific enzyme expression. For instance, cells must express the kynurenine pathway for de novoNAD+ synthesis, clearly active in the liver and brain [12], and must possess the Preiss–Handler pathway to use NA, which is active in most organs but less prominent in skeletal muscle. In contrast, the salvage pathways are crucial in all tissues to conserve NAD+ sufficiency [220]. Supporting this notion, the recommended daily allowance for NA is in milligrams, whereas an estimated 6 to 9 g of NAD+ are required daily to match turnover [58]. This is facilitated by the high affinity of NAMPT for Nam; thus, even small amounts of Nam are effectively converted to NMN and then NAD+ [221].

In the absence of head-to-head studies comparing the different compounds under defined conditions, it is currently not possible to identify the optimal NAD+ augmenting agent. The ubiquitous expression of NRKs, makes NR a precursor that can affect whole-body metabolism [162]. The inability of NR to activate the GPR109A receptor mitigates the undesirable NA side effects, and, unlike Nam, NR does not inhibit sirtuins. Furthermore, NAD+ generated from NR can target both nuclear and mitochondrial NAD+ pools, activating the respective compartmental sirtuins (i.e., nuclear SIRT1 and mitochondrial SIRT3) [54]. This may be an advantage over other molecules, such as PARP inhibitors, with effects confined to the nucleus [67]. Similar to NR, NMN metabolism into NAD+ is governed by the salvage pathway. However, NMN availability has not been characterized in the diet [93, 222], unlike the naturally available NR.

In major proof-of-concept studies, therapeutically increasing NAD+ has been used to treat mouse models of mitochondrial diseases. Treatment of cytochrome C oxidase deficiency in mice with NR, PARP inhibition, and the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide reversed the mitochondrial dysfunction and improved muscle performance [223–225], effects attributed to NAD+ and sirtuins activation. Treatment of patients with T2DM by using acipimox resulted in improved skeletal muscle oxidative metabolism and mitochondrial function, measured by high-resolution respirometry [78]. However, this acipimox effect was not observed in obese persons without T2DM when assessed by phosphocreatine recovery magnetic resonance spectroscopy, mitochondrial biogenesis gene expression, and mitochondrial density on electron microscopy [77]. Two differences between these studies may explain the observed discrepancy. First, high-resolution respirometry is the current gold standard for ex vivo assessment of mitochondrial respiration if increased oxidative phosphorylation is the question [226]. Second, whereas mitochondrial dysfunction is evident in patients with T2DM, this is not prominent in obese persons without diabetes. Thus, the effects of NAD+ precursor supplementation may vary depending on the intervention and specific pathophysiologic conditions. Nam acts as an NAD+ precursor, increasing SIRT1 activity (below a threshold of sirtuin inhibition), or, conversely, a SIRT1 inhibitor, depending on the specific pathophysiologic state [84, 85].

We still have a limited understanding of the molecular interconversions of the administered NAD+ precursors. Illustrating this, administered NR is converted to Nam in the circulation before entering the cell [208, 227], whereas NMN is transformed extracellularly into NR, which then enters the cell and converts into NAD+ [227].

Knowledge gaps still persist in the role of sirtuins in different contexts. Some reports suggest that not all beneficial SIRT1 activation is through NAD+ and that cyclic adenosine monophosphate plays a role, independent of NAD+, in low-energy states [228, 229]. Upon pharmacologic NAMPT inhibition, Nam failed to increase NAD+; however, this did not prevent SIRT1 upregulation, which was secondary to Nam-induced increase in intracellular cyclic adenosine monophosphate [64].

An important question is whether amplifying NAD+ and sirtuin activity is always desirable. SIRT1 upregulates T helper 17 cells that contribute to autoimmune disease when hyperactivated [230]. Correspondingly, SIRT1 inhibition supports the development of the regulatory T cells that protect against autoimmunity [231, 232]. Therefore, it is possible that SIRT1 activation places susceptible individuals at increased risk for autoimmune diseases. In the same way, whereas NR supplementation increased muscle stem cell number in aged mice, thereby enhancing mitochondrial function and muscle strength, it reduced the expression of cell senescence and apoptosis markers [233]; the state of senescence is important to protect against carcinogenesis [234]. Also, increased NAMPT expression is reported in some malignancies, calling into question whether increasing NAD+ might support aspects of the tumorigenic process [235].

Given the effect of the NAD+-sirtuin pathway on mitochondrial and metabolic homeostasis, novel supplementation strategies (e.g., using NR or NMN) may be exploited to increase endogenous NAD+ availability in the treatment of metabolic and age-related diseases. This is the time for carefully designed human clinical studies to further examine these compounds before we can propose them as being useful nutraceuticals to counteract metabolic disease.

NAD catabolism, GTP and mitochondrial dynamics

Mitochondrial NUDIX hydrolases: A metabolic link between NAD catabolism, GTP and mitochondrial dynamics

2. Pathology of NAD+ catabolism and downstream metabolic products
NAD+ is an important cofactor involved in multiple metabolic reactions that have a central role in cellular metabolism and energy production (Belenky et al., 2007; Frederick et al., 2016; Mouchiroud et al., 2013). Normally, NAD+ levels decline with age (CamachoPereira et al., 2016; Massudi et al., 2012; Verdin, 2015; Zhu et al., 2015) and NAD+ pools have been shown to decrease during neurodegenerative diseases and after ischemia-reperfusion or TBI (Kauppinen and Swanson, 2007; Martire et al., 2015; Park et al., 2016; Verdin, 2015; Zhou et al., 2015). This decline could be the result of the increased activity of several enzymes that use NAD+ as their substrate; these include: sirtuins, ADP-ribosyl transferases (ARTs), and the cyclic ADP-ribose synthases/ADP-ribosyl cyclases (CD38 and CD157) (Belenky et al., 2007; Feijs et al., 2013; Jesko et al., 2016; Malavasi et al., 2008; Mayo et al., 2008). These NAD+ dependent enzymes hydrolyze NAD+ to ADP-ribose (ADPr) or an ADPr variation (e.g. cyclic ADPr), and nicotinamide (Nam). The resulting Nam can function in a negative feedback fashion by inhibiting the activity of NAD+ dependent enzymes (Avalos et al., 2005; Long et al., 2016; Suzuki et al., 2010), however levels of Nam are usually too low to inhibit sirtuins activity under physiological conditions (Liu et al., 2013). Nam is also the precursor for nicotinamide mononucleotide (NMN), the immediate precursor to NAD+ via the salvage pathway (Imai and Guarente, 2014). The conversion of NMN to NAD+ is ATP dependent and catalyzed by the NMN adenyl transferases (NMNATs): NMNAT1 (nucleus), NMNAT2 (Golgi apparatus/endosomes), and NMNAT3 (mitochondria)

Additionally, commonly in acidic environments, NAD+ levels can also be depleted by NAD+ kinase, by phosphorylating NAD+ to NADPþ (Zhang et al., 2016a).

3. NAD+/NADH
Other then being a substrate, NAD+ plays a key role in cellular bioenergetic metabolism by reversibly being reduced to NADH and ultimately contributing to ATP generation via the glycolytic pathway and in the mitochondria through oxidative phosphorylation (Srivastava, 2016). The reduction of NAD+ is most apparent in the mitochondria, with liver mitochondrial NAD+/NADH ratios being tightly controlled around 7 to 8, while cytoplasmic ratios being much higher, ranging from 60 to 700 in most cells (Stein and Imai, 2012). Techniques to get more accurate measurements are relatively new but suggest that NAD+ pools and NAD+/NADH ratios can vary based on cell type (Cambronne et al., 2016; Christensen et al., 2014). The reduction of NAD+ to NADH is essential for the glyceraldehyde 3-phosphate (GAPDH) step of glycolysis and multiple steps in the tricarboxylic acid cycle (TCA) (Akram, 2014; Sirover, 1999). NADH generated in the mitochondria will be oxidized to NAD+ and donate electrons to complex I of the electron transport chain (Sazanov, 2015).

4. ARTs/PARP1/mtPARP1
There are 22 known human genes that encode proteins with an ADP-ribosyltransferase (ART) catalytic domain. These proteins transfer ADPr from NAD+ onto targeted amino acid residues of proteins. To generate the ADPr chains, ART’s release Nam from NAD+, and then form an a(1e2)O-glycosidic bond between two ADPr molecules. This post-translational modification is named either monoor poly-ADP-ribosylation (MARylation or PARylation), depending if the ART’s are transferring a single ADPr or are generating a chain (Gibson and Kraus, 2012; Hottiger et al., 2010). In vitro, these chains of APD-ribose can be around 200 residues, with the length and branching being dependent on the concentration of available NAD+ (Alvarez-Gonzalez and Jacobson, 1987; Alvarez-Gonzalez and Mendoza-Alvarez, 1995). This process of PARylation is reversible and is constantly being regulated by poly (ADP-ribose) glycohydrolase (PARG) and the mainly mitochondrial, ADP-ribosyl hydrolase-3 (ARH3) (Di Meglio et al., 2003; Mashimo et al., 2013; Niere et al., 2012). PARG and ARH3 associated degradation of PAR can result in releasing intact PAR chains (endoglycohydrolase) or by releasing free ADR-ribose (exoglycohydrolase) (Gibson and Kraus, 2012).
Poly (ADP-ribose) polymerase 1 (PARP1), also known as ADPribosyltransferase diphtheria toxin-like one (ARTD1), is responsible for the majority of PARylation and is involved in the repair of moderate single stranded DNA damage.

Historically, PARP1 has been reported to be exclusively nuclear, although there is growing evidence for PARP1 associated mitochondrial functions. Several studies report PARP1 can modulate mitochondria from the nucleus through: PAR translocation from the nucleus to the mitochondria, depletion of cellular NAD+ pools, and epigenetic regulating of nuclear genes that are involved in mitochondrial DNA transcription and repair (Alano et al., 2010; Fang et al., 2014; Fatokun et al., 2014; Lapucci et al., 2011). Additionally, several studies report that PARP1 can directly interact with mitochondria and that there is an intramitochondrial localized PARP1 (mtPARP1) (Lai et al., 2008; Rossi et al., 2009; Szczesny et al., 2014; Zhang et al., 2016b). Brunyanszki and colleagues, reviewed the growing body of work suggesting there is a mitochondrial PARP1, in which they hypothesize mtPARP1 is activated prior to nuclear PARP1 in the event of oxidative stress (Brunyanszki et al., 2016).

When discussing the possibility of mtPARP1 it is important to note that mitochondrial and cytosolic NAD+ pools are mostly distinct from each other, with studies showing mitochondrial NAD+ pools are maintained for over 24 h after cytoplasmic NAD+ depletion (Alano et al., 2007; Pittelli et al., 2010; Stein and Imai, 2012). Furthermore, the mechanism of mammalian mitochondrial NAD+ regulation is still unclear and it is unknown whether there is a mitochondrial membrane transporter to shuttle NAD+, NMN, or nicotinamide riboside (NR) between the mitochondria and cytosol (Stein and Imai, 2012). Using a novel NAD+ biosensor, Cambronne and colleagues determined that mitochondrial NAD+ pools appear to be regulated differently in various cell types. They found NMNAT3 depletion caused a significant decrease in mitochondrial NAD+ in HEK293T cells but not HeLa cells (Cambronne et al., 2016).

Additionally, Nikiforov and colleagues, reported using stably transfected 293mitoPARP cells that mitochondrial NAD+ pools are regulated by cytosolic NMN being transported to the mitochondria and then being converted to NAD+ by NMNAT3 (Nikiforov et al., 2011). These studies suggest NMNAT3 is essential for mitochondrial NAD+ production in some cells while other cells might use a yet to be discovered mitochondrial NAD+ shuttle. Furthermore, mitochondrial NAD+ pools can vary greatly depending on the cell type: cardiac myocytes (10.02 ± 1.82 nmol/mg protein), neurons (4.66 ± 0.37 nmol/mg protein), and astrocytes (3.20 ± 1.02 nmol/ mg protein). The study also reported cytoplasmic NAD+ pools were most similar to those in neuronal mitochondria. This suggests NAD+ pools are split between the cytoplasm and mitochondria differently depending on the cell type (Alano et al., 2007). Therefore, it is reasonable to suggest that the pathologic outcome of PARP1-dependent depletion of mitochondrial or cytosolic NAD+ could vary greatly depending on the cell type.

For example, Modis and colleagues reported PARP1 depleted mitochondrial NAD+ pools at a much faster rate than cytosolic. They used PARP1 (shPARP1) silencing in A549 cells, which resulted in a four-fold increase in mitochondria NAD+, while showing no change in total cellular NAD+ (Modis et al., 2012).

Several studies have shown substantial amounts of mitochondrial targeted proteins that are subjected to PARylation (Brunyanszki et al., 2016; Gagne et al., 2012). It has also been reported that mtPARP1 interacts with mitochondrial specific DNA base excision repair enzymes. Interestingly, nuclear PARP1 has a positive effect on DNA repair, while mtPARP1 was observed to negatively affect base excision repair enzymes EXOG and DNA polymerase gamma (Polg) (Rossi et al., 2009; Szczesny et al., 2014). In contrast, Rossi and colleagues reported that mtPARP1 is important for normal mitochondrial DNA ligase III function (Rossi et al., 2009). The same study also showed that mitofilin, a mitochondrial inner membrane (IM) protein, plays an important role in regulating the mitochondrial localization of PARP1 (Rossi et al., 2009).

Normally, mitofilin is involved in keeping the cristae membranes connected to the inner boundary membrane and promoting protein import through the mitochondrial intermembrane space (IMS) assembly pathway via being coupled to the outer membrane (OM) (von der Malsburg et al., 2011). Interestingly, mtPARP1 and DNA ligase III co-occupy the D-loop region in mtDNA, and mitofilin depletion results in decreased mtPARP1 and impaired binding of DNA ligase III to mtDNA (Rossi et al., 2009). These studies have led to Brunyanszki and colleagues hypothesizing that mtPARP1 is normally bound to mitofilin but under oxidative stress is released, consuming mitochondrial NAD+, and PARylating mitochondrial proteins, including the electron transport chain complexes (Brunyanszki et al., 2016).

Conversely, parthanatos is cell death induced by nuclear PARP1 overactivation, as a result of extensive nuclear DNA damage (Fatokun et al., 2014). Excessive PARP1-dependent PARylation causes some PAR chains to translocate to the cytoplasm and bind to apoptosis inducing factor (AIF) on the mitochondrial OM (Wang et al., 2011). AIF is predominately located in the mitochondrial IMS and attached to the IM; although it has been reported that about 30% of AIF is loosely associated with the cytosolic side of the OM (Yu et al., 2009). The binding of PAR to AIF helps induce the release of AIF from the mitochondria and it’s translocation to the nucleus (Fatokun et al., 2014; Wang et al., 2011). This translocation of AIF to the nucleus causes chromatin condensation, and large scale DNA fragmentation resulting in cell death (Yu et al., 2002).

PARP1 overactivation is also associated with NAD+ and ATP depletion (Gerace et al., 2014). PARP1 has been reported to inhibit ATP generation by glycolysis via PAR translocating from the nucleus into the cytoplasm and binding to hexokinase 1 (HK1) on the mitochondrial OM (Andrabi et al., 2014; Fouquerel et al., 2014). The binding of PAR to HK1 can allosterically decrease HK1 activity and cause HK1 to migrate to the cytoplasm, resulting in the inhibition of glycolysis and the reduction of mitochondrial ATP production (Fouquerel et al., 2014).

5. Sirtuins/OAADPR
The sirtuins are class III histone deacetylases (HDACs) and consist of seven isoforms SIRT1-SIRT7, with SIRT3SIRT5 being localized in the mitochondria. Sirtuins require NAD+ for their activity and modulate protein signaling and function by removing acetyl groups attached to lysine residues (Jesko and Strosznajder, 2016; Jesko et al., 2016). In the mitochondria, SIRT3 is the major lysine deacetylase; reports show SIRT3 Knockout (KO) mice and cells have hyperacetylation of mitochondrial proteins, while the same effect was not seen in SIRT4 or SIRT5 KO animals (Lombard et al., 2007; Sol et al., 2012). Sirtuins remove acetyl groups by first cleaving Nam from NAD+ (forming ADPr) and then transferring the acetyl group from the substrate to ADPr, resulting in the formation of O-acetyl-ADP-ribose (OAADPr) (Borra et al., 2004). Uniquely, SIRT5 hydrolyzes malonyl and succinyl lysines, forming O-malonyl-ADPr and O-succinyl-ADPr (Du et al., 2011).

Macro domain (macroD) proteins have been reported to hydrolyze OAADPr to ADPr (Chen et al., 2011). Normally these proteins bind to NAD+ metabolites (e.g. PAR), and are involved in a broad range of biological functions, including DNA repair (Han et al., 2011). ARH3, which normally plays a role in degrading PAR to ADPr, was also found to hydrolyze OAADPr to ADPr in a Mg2þ dependent mechanism in cells (Ono et al., 2006). This finding suggests ARH3 could play a role in both OAADPr and ADPr regulation. Interestingly, compared to PARP1, SIRT1 has a lower affinity (higher Km) for NAD+ and is less effective at breaking down NAD+ (lower Km/Kcat). This suggests PARP1 activation could deplete NAD+ pools at a faster rate then SIRT1 and consequently reduce the function of the NAD+ dependent sirtuins (Canto et al., 2013).

6. CD38/CD157
The membrane proteins CD38 and the less abundant CD157 are multifunctional ecto-enzymes that use NAD+ as a substrate to form cyclic ADP-ribose (cADPr), or under acidic conditions use NADPþ as a substrate to form nicotinamide acid ADP (NAADP). Additionally, CD38 and CD157 can act as glycohydrolases and hydrolyze cADPr to ADPr and under acidic conditions convert NAADP to ADPrphosphate (ADPrP) (Malavasi et al., 2008; Quarona et al., 2013). Normally, cADPr, ADPr, ADPrP, and NAADP play roles in cell signaling pathways and regulating cytoplasmic Ca2þ fluxes (Malavasi et al., 2008; Quarona et al., 2013; Sumoza-Toledo and Penner, 2011). CD38 and CD157 are commonly located on the membranes of immune cells but can be found throughout the body; playing a role in the immune response, hormone secretion, cell activation, egg fertilization, and muscle contraction (Malavasi et al., 2008).
In CD38KO mice, brain tissue NAD+ levels have been observed to be up to 10 fold higher than in wild type mice, although the rate of NAD+ consumption by CD38 may vary greatly depending on the brain region (Aksoy et al., 2006; Long et al., 2016). Interestingly, Camacho-Pereia and colleagues showed CD38 levels not only increase with age but also correlate with a decline of NAD+, suggesting CD38 might be the major cause of the decline of NAD+ during normal aging (Camacho-Pereira et al., 2016). Surprisingly, our lab found CD38KO mice to have dramatically higher PAR levels (particularly in neurons) and decreased PARG activity when compared to WT mice. These findings suggest CD38KO mouse studies might be difficult to interpret due to complexities derived from alterations in several enzymes involved in NAD+ catabolism as a result of knocking out the constitutively expressed CD38 gene (Long et al., 2016).

7. PARG/AHR3
Uncontrolled PARP1 activation has been proposed to deplete intracellular NAD+ and ATP (Fouquerel et al., 2014; Szabo and Dawson, 1998). To regulate PARP1 activation, PARG (nuclear/cytoplasmic) and AHR3 (predominantly mitochondrial) degrade PAR, which can have toxic effects (Dumitriu et al., 2004; Mashimo et al., 2013). It has been suggested that the free PAR polymers is the major toxic molecule generated by the combined activity of PARP1 and PARG (Andrabi et al., 2006). Several reports suggested that many types of PAR structures could play a crucial role in stress-dependent signaling processes in vivo (Dawson and Dawson, 2004; Hong et al., 2004). However, most free or protein-associated PAR polymers are rapidly degraded in vivo to ADPr (Jacobson et al., 1983). In the nucleus and cytoplasm, PARG is the predominant PAR glycohydrolase, reportedly acting as an endoglycohydrolase, hydrolyzing off chunks of PAR from the protein, as well as an exoglycohydrolase, releasing ADPr (Wang et al., 2014).
Recently, Niere and colleagues discovered that ARH3 and not PARG is responsible for degrading PAR to ADPr in the mitochondrial matrix (Niere et al., 2012). ARH3 has also been postulated to play a protective role against parthanatos in the cytoplasm and nucleus (Mashimo et al., 2013). In this theory, PARG acts as an endoglycohydrolase, releasing PAR fragments from proteins in the nucleus, allowing translocation of PAR from the nucleus to the cytoplasm. To counteract this, ARH3 hydrolyzes the detached PAR, releasing ADPr, preventing PAR induced AIF translocation to the nucleus and subsequent cell death via parthanatos (Mashimo et al., 2013).

8. NUDIX hydrolase, enzyme controlling cellular and mitochondrial ADP-ribose levels
The intracellular levels of ADPr are tightly controlled by specific ADPr hydrolases, which hydrolyze ADPr to adenosine monophosphate (AMP) and D-ribose 5-phosphate; thereby, limiting free ADPr accumulation (Fernandez et al., 1996; Ribeiro et al., 1995). This family of enzymes catalyze the hydrolysis of a nucleoside
diphosphate linked to another moiety x, hence the acronym “NUDIX” (Mildvan et al., 2005). For NUDIX hydrolases to be active, Mg2þ or a similar cofactor (e.g. Zn2þ) must bind to NUDIX (Zha et al., 2006). Cloning and expression of human cDNA coding for proteins with NUDIX motifs have revealed two human ADPr hydrolases: NUDT5, an ADP-sugar pyrophosphatase (Gasmi et al., 1999), and NUDT9, an ADPr pyrophosphatase (Perraud et al., 2003). While NUDT9 is highly specific for ADPr, NUDT5 only has a preference for ADPr and can also hydrolyze other ADP-sugar conjugates (Zha et al., 2008).
The human NUDT9 gene gives rise to two alternatively spliced mRNAs, NUDT9a and NUDT9b (Li et al., 2002). NUDT9a possesses a putative mitochondrial targeting sequence and accumulates in mitochondria, while NUDT9b is a cytosolic enzyme (Lin et al., 2002; Perraud et al., 2003). Thus, NUDT9 can play an important role in cellular bioenergetic metabolism, particularly under stressed conditions that can lead to high levels of mitochondrial ADPr.

This is important since free ADPr has been reported to be a competitive inhibitor of NADH oxidation at complex I of the electron transport chain (Zharova and Vinogradov, 1997). Similarly, in rat ventricular myocytes ADPr was reported to inhibit ATP-sensitive Kþ channels (Kwak et al., 1996). Additionally, excessive cytosolic ADPr, cADPr, NAADP, OAADPr, and ADPrP are potential agonists along with ROS, and Ca2þ of the transient receptor potential melastatin 2 (TRPM2) ion channel by binding to the channels NUDT9-H domain. Opening of the TRPM2 ion channel is important for Ca2þ signaling; although, overactivation can cause an influx of Ca2þ, leading to inflammation through chemokine recruitment, and ROS (Grubisha et al., 2006; Shimizu et al., 2013; Toth et al., 2015; Yamamoto and Shimizu, 2016). Interestingly, TRPM2-mediated neuronal death in ischemic brain injury is dimorphic, with TRPM2 knockdown only protecting male brains (Shimizu et al., 2013).

NUDIX hydrolases have also been shown as potential metabolizing enzymes of the byproduct of sirtuin activity, OAADPr, producing AMP and O-acetylated ribose 5’-phosphate (Tong and Denu, 2010). This OAADPr hydrolysis was shown using the NUDIX hydrolases YSA1 (yeast), NUDT5 (mouse), and NUDT9 (human). The same study reported YSA1 and NUDT5 to have a similar affinity to hydrolyzing OAADPr or ADPr, while NUDT9 was 500 fold more efficient hydrolyzing ADPr compared to OAADPr (Rafty et al., 2002).

9. Mitochondrial NAD+ catabolism
The presence of NUDT9a in mitochondria suggests that enzymes generating ADPr are also localized in the intra-mitochondrial compartment. As discussed earlier, several studies have reported that PARP1 is localized not only in the nucleus, but also in the mitochondria (Du et al., 2003; Lai et al., 2008; Pankotai et al., 2009; Rossi et al., 2009). Furthermore, ARH3 has been reported to control mitochondrial PAR hydrolysis (Niere et al., 2012). Concerted activity of mitochondrial PARP1 and ARH3 results in increased levels of matrix ADPr. Intra-mitochondrial ADPr is then metabolized by the NUDT9a, generating AMP and D-ribose 5-phosphate (Perraud et al., 2003). This NUDT9 dependent intra-mitochondrial AMP generation from the hydrolysis of ADPr has been hypothesized to trigger an AMP-dependent mitochondrial failure due to inhibition of the adenine nucleotide translocase (ANT) (Formentini et al., 2009). The primary role for ANT is to exchange ADP/ATP across the mitochondrial IM, thus releasing ATP into the cytosol and importing ADP into the matrix to be phosphorylated via oxidative phosphorylation (Liu and Chen, 2013). In the cell, AMP is generally converted to ADP by adenylate kinases (AKs) (Panayiotou et al., 2014).

10. Adenylate kinases
Adenylate kinases (AKs) are found throughout the body and are involved in homeostasis of cellular adenine nucleotides by transferring a phosphate from a donor, usually ATP, to AMP, resulting in ADP. There are 9 known AKs (AK1-AK9), which differ by subcellular localization, tissue distribution, phosphate donor, and substrate. While AK2, AK3, and AK4 are all mitochondrial, only AK3 and AK4 are found in the brain (Panayiotou et al., 2014). Noma and colleagues reported AK3 was specific to the mitochondrial matrix, found in most tissues, and specifically can only use GTP or ITP as a phosphate donor; making AK3 a possible major consumer of mitochondrial GTP. AK4, is also mitochondrial but is found predominately in the kidney and has low enzymatic activity, with a slight preference to GTP over ATP as a phosphate donor (Noma et al., 2001; Panayiotou et al., 2010).

11. Mitochondrial dynamics and downstream NAD+ metabolites
Recently, it was recognized that an imbalance in mitochondrial fusion and fission could result in deterioration of mitochondrial bioenergetics (Knott et al., 2008; Stetler et al., 2013). Both fusion and fission have physiologic functions (Knott and Bossy-Wetzel, 2008). For example, mitochondrial fission can have a protective role by segregation of damaged and inactive mitochondria and through facilitating autophagic clearance (Barsoum et al., 2006). When mitochondria in cells are stressed, they undergo fission, a process that is reversible since small fragmented organelles can fuse and regain the normal pre-insult morphology (Barsoum et al., 2006; Knott and Bossy-Wetzel, 2008; Owens et al., 2015). The fission of mitochondria is frequently observed in neurons exposed to mitochondrial toxins or excitotoxic levels of glutamate (Barsoum et al., 2006).

Reduction of mitochondrial length compared to control tissue was observed in the brain following focal ischemia, suggesting post-insult mitochondrial fragmentation (Barsoum et al., 2006). We detected extensive mitochondrial fission following global ischemic insult in both hippocampal neurons and astrocytes (Owens et al., 2015). Fission and fusion are regulated by dynamin family proteins that act as GTPases. Fusion is controlled by mitofusin-1 and -2 (MFN1 and MFN2), which are localized in the mitochondrial OM, and by the mitochondrial IM optic atrophy protein (OPA1) (Hoppins et al., 2007; Song et al., 2009). Since these enzymes are GTP binding proteins the efficiency of fission and fusion depends on the cytosolic and mitochondrial GTP levels. Therefore, Intra-mitochondrial GTP depletion could inhibit OPA1, while cytosolic GTP depletion could inhibit the MFNs and Drp1. Interestingly IM fusion requires high GTP levels while OM fusion has less demanding GTP requirements (Escobar-Henriques and Anton, 2013). Our previous studies suggests that there is dramatic fragmentation of mitochondria following ischemic insult and only mitochondria in ischemia resistant cells re-fuse at a later recovery time (Owens et al., 2015). Furthermore, genotoxic stress-induced mitochondrial NAD+ catabolism leads to significant reduction of GTP pools in mitochondrial matrix, suggesting a causal link between downstream NAD+ degradation products and GTP metabolism (unpublished data). Similarly, Dagher reported using cell culture that cellular GTP levels are severely depleted during chemical anoxia (Dagher, 2000).

12. Intra-mitochondrial GTP metabolism
GTP molecules in mitochondria are generated by nucleoside diphosphate kinase (NDPK) or succinyl-CoA synthetase (ligase)
(SCS) in the citric acid cycle (Sanadi et al., 1954) (Fig. 1). NDPK converts GDP to GTP by using ATP as a phosphate donor. SCS generates succinate and CoA from succinyl-CoA, and GDP, as a cofactor, is phosphorylated to GTP in the presence of inorganic phosphate. GTP is involved mainly in energy transfer within the cell. In mitochondria, GTP with ATP is utilized during protein translocation into the mitochondrial matrix (Sepuri et al., 1998). As mentioned above, AK3 is located in the mitochondrial matrix and uniquely uses GTP instead of ATP as a phosphate donor to phosphorylate AMP (Noma et al., 2001). Downstream NAD+ catabolism leads to generation of intra-mitochondrial AMP due to ADPr hydrolysis by NUDT9a. AMP is then converted to ADP by transferring high-energy phosphate from intra-mitochondrial GTP by AK3. Concurrently, low levels of intra-mitochondrial NAD+ also compromise TCA cycle enzyme activities, including a-ketoglutarate dehydrogenase that produces succinyl-CoA. Reduced succinyl-CoA compromises GTP production by SCS, which further depletes mitochondrial GTP and can inhibit mitochondrial fusion (Fig. 1).

13. AMPK/MFF/Drp1 mitochondrial fission
AMP-activated protein kinase (AMPK) is a serine/threonine kinase that is activated by the depletion of energy levels and is important in cellular metabolism by phosphorylating numerous cellular targets. AMPK is predominately located in the cytosol and is heterotrimeric, consisting of three subunits, abg; a is catalytic, b is scaffolding, and g is regulatory. AMPK is activated by phosphorylation of Thr172 in the a subunit by liver kinase B1 (LKB1) or calmodulin-dependent protein kinase kinase-b (CaMKKb) (Kim et al., 2016; Li et al., 2015). Cellular AMP: ATP ratios are reported to be the main regulator of AMPK, either with inhibition of mitochondrial ATP production or high levels of AMP acting as activators (Hardie et al., 2012).

Ekholm and company reported that after 15 min of ischemia AMP levels increase 25 fold, while concurrently ATP levels decrease 25 fold (Ekholm et al., 1993). AMP is able to allosterically activate AMPK by binding to the g subunit, which stimulates LKB1 phosphorylation of Thr172 on the a subunit (Fig. 2). Conversely, ATP reduces AMPK activation by competing with AMP for g subunit binding (Gowans et al., 2013). Furthermore, AMPK can also be activated in a Ca2þ dependent pathway by CaMKKb phosphorylation of Thr172 on the a subunit (Woods et al., 2005).

Interestingly, AMPK activation has recently been directly linked to mitochondrial fission (Fig. 1). Toyama and colleagues reported activated AMPK is able to phosphorylate mitochondrial fission factor (MFF) at Ser155 and Ser172 on the mitochondrial OM. MFF is a dominant receptor for the mitochondrial fission protein DRP1 (Toyama et al., 2016). The phosphorylation of MFF by AMPK has been shown to catalyze mitochondrial fission by causing the translocation of DRP1 from the cytoplasm to the mitochondrial OM. Drp1 wraps around constriction sites on the mitochondrial OM, causing fission, and can eventually lead to mitophagy (Toyama et al., 2016; Wang and Youle, 2016).

14. Conclusion
Pathophysiology of neurodegenerative diseases and acute brain injury encompass DNA damage with subsequent increases in nuclear and mitochondrial PARP1 activation and the depletion of cellular and mitochondrial NAD+ pools. Mitochondrial NAD+ depletion can cause inhibition of oxidative phosphorylation and TCA cycle enzymes activity. PAR is degraded in the nucleus and cytoplasm by PARG/AHR3 and by ARH3 in the mitochondria. Consequently, high PARylation will significantly increase in ADPr levels, which could inhibit complex I in the mitochondria or bind the NUDT9-H domain on TRPM2 receptors, causing influxes of Ca2þ
A. Long et al. / Neurochemistry International xxx (2017) 1e9 5

6 A. Long et al. / Neurochemistry International xxx (2017) 1e9
Fig. 1. Schematic diagram linking mitochondrial NAD+ catabolism with the depletion of mitochondrial GTP and mitochondrial fragmentation in a neurodegenerative disease/acute brain injury model. Localization of enzymatic reactions are color-coded: Blue (mitochondrial), red (cytoplasmic), green (nuclear and mitochondrial), purple (insult or outcome). Red arrows show changes in enzymatic activity or substrate levels as a result of the disease/injury insult. Mitochondrial and nuclear poly (ADP-ribose) polymerase 1 (PARP1) leads to generation of Poly (ADP-ribose) (PAR) as a result of disease or insult. ADP-ribose (ADPr) is generated by hydrolyzing PAR by NAD+ glycohydrolases; ADP-ribosyl hydrolase-3 (ARH3) and poly (ADP-ribose) glycohydrolase (PARG), and then is further degraded to AMP by NUDIX hydrolases NUDT9a and NUDT9b.

Accumulated AMP is then phosphorylated by adenylate kinse 3 (AK3) to ADP by transferring high-energy phosphate from GTP. Depletion of NAD+ will inhibit the TCA cycle enzyme succinyl-CoA synthetase (SCS)-linked GTP generation. GTP can also be generated by nucleoside diphosphate kinase (NDPK), which phosphorylates GDP by using ATP as phosphate donor. However, since the TCA cycle and mitochondrial respiration is inhibited due to low NAD+ levels, the NDPK activity will be limited.

This depletion of mitochondrial GTP can inhibit fusion by reducing the functionality of GTP dependent mitochondrial fusion enzymes, such as optic atrophy protein (OPA1). Elevated cytosolic AMP can also shift the cellular AMP: ATP ratio, which leads to liver kinase B1 (LKB1) phosphorylation and activation of AMP-activated protein kinase (AMPK). Once activated, AMPK can phosphorylate mitochondria fission factor (MFF), which causes an increase in cytosolic Drp1 migration to the mitochondrial OM and subsequent fission. Thus, depletion of NAD+, reduction of mitochondrial GTP, and elevated AMP can lead to excessively fragmented mitochondria.

Fig. 2. Simplified schematic diagram of the overall mechanism linking NAD+ catabolism to mitochondrial fragmentation. Insult caused from disease or acute injury causes NAD+ depletion and PARP1 activation. This leads to high levels of ADPr and AMP in the cell and mitochondria. Mitochondrial AMP phosphorylation and low NAD+ levels cause depletion in mitochondrial GTP and inhibition of GTP dependent mitochondrial fusion proteins. High levels of AMP also increase AMP: ATP ratios, which leads to the activation of AMPK and subsequent mitochondrial fission.

Additionally, mitochondrial NUDT9a and cytosolic NUDT9b/NUDT5 regulate ADPr levels by hydrolysis, producing AMP and D-ribose-5-phosphatate.
We propose a possible mechanism for elevated mitochondrial AMP levels to cause a shift to mitochondrial dynamics to fission (Fig. 2). First, high AMP levels have been reported to inhibit ATP production by inhibiting ANT. Decreasing ATP production along with the already high levels of AMP will result in a spike in the AMP: ATP ratio, thus activating AMPK. AMPK has been shown to regulate mitochondrial fission by phosphorylating MFF, resulting in the translocation of Drp1 to the mitochondria and Drp1 induced mitochondrial fission. Additionally, high AMP levels will increase AK3 activity, which consumes mitochondrial GTP. Depletion of mitochondrial GTP levels can disrupt GTPases, such as the mitochondrial fusion proteins.

Thus, in stress conditions, NAD+ depletion can significantly deplete mitochondrial GTP levels by inhibiting the TCA cycle and through high AK3 activity, resulting in excessively fragmented mitochondria (Fig. 1).
The link between NAD+ catabolism and mitochondrial fragmentation is an understudied area with translational potential. Understanding mitochondrial PARP1 activity and finding ways to regulate cellular ADPr and AMP levels in disease/acute injury states through studying the NUDIX hydrolases could be a target to reduce mitochondrial fragmentation. Additionally, better understanding of intra-mitochondrial GTP metabolism and the complexities of AMPK in relation to mitochondrial dynamics could be targets for future studies.

Mitochondrial transplantation: From animal models to clinical use in humans

1. Introduction
The importance of the mitochondrion in the maintenance and preservation of cellular homeostasis and function is well established and there is a sufficient body of evidence to show that mitochondrial injury or loss of function is deleterious (Durhuus et al., 2015). The mechanisms leading to mitochondrial dysfunction are varied and include genetic changes occurring at the nuclear or the mitochondrial genome, environmental insult or alterations in homeosis. In all cases, the end result of mitochondrial dysfunction is cellular dysfunction that can limit or severely modulate organ function and ultimately increase morbidity and mortality.

In our research, we have focused on the myocardium, a highly aerobic organ in which mitochondria comprise 30% of cellular volume (Faulk et al., 1995; Faulk et al., 1995a; Toyoda et al., 2000; Toyoda et al., 2001; McCully et al., 2003). The mitochondria supply the energy requirements of the myocardium. This energy is derived through oxidative phosphorylation in the myocardium and is dependent upon the coronary circulation. Under equilibrium conditions the mitochondria within the heart extract greater than 79% of arterial oxygen from the coronary arteries (Fillmore et al., 2013). As heart rate increases or if myocardial workload is increased the oxygen demand is increased and is dependent upon increased coronary flow. Thus, any interruption or impedance in coronary blood flow will significantly limit oxygen delivery to the heart and significantly decrease function and hemostasis (Akhmedov et al., 2015; Doenst et al., 2013; Kolwicz et al., 2013). It is generally accepted that the cessation of coronary blood flow, and thus oxygen delivery, is the initial step in the process leading to myocardial ischemic injury. The sequence of events and the mechanisms associated with this injury are many and are reviewed elsewhere (Lesnefsky et al., 2003; Kalogeris et al., 2012; Ong et al., 2015 Kalogeris et al., 2016 ; Lesnefsky et al., 2017). The end result of ischemia is loss of high energy synthesis and the depletion of high energy stores such that the heart is unable to support hemostasis and maintain function (Rosca et al., 2013 ) .

This reduction of high energy synthesis and stores is rapid. 31Pnuclear magnetic resonance studies have shown that following regional or global ischemia wherein the blood flow to the heart is temporarily ceased, high energy phosphate synthesis and stores are rapidly decreased within 6 minutes and that this decrease continues for at least 60 180 minutes after the restoration of blood flow and is associated with significantly decreased myocardial cellular viability and myocardial function (Tsukube et al., 1997).

Mitochondrial modulations induced by ischemia in the myocardium are many. We and others have demonstrated that following ischemia there are changes in mitochondrial morphology and structure (Rousou et al., 2004; Lesnefsky et al., 2004; McCully et al., 2007). Transmission electron microscopy and lightscattering spectrophotometry have shown that ischemia significantly increases mitochondrial matrix and cristae area and mitochondrial matrix volume (McCully et al., 2007). In addition, there is a decrease in mitochondrial complex activity, cytochrome oxidase I Vmax and a decrease in oxygen consumption and an increase in mitochondrial calcium accumulation (Faulk et al.,1995a).

These changes occur in concert with changes in mitochondrial transcriptomics, with downregulation of annotation clusters for mitochondrion function and energy production and the downregulation of cofactor catabolism, generation of precursor metabolites of energy, cellular carbohydrate metabolism, regulation of biosynthesis, regulation of transcription, and mitochondrial structure and function (enrichment score >2.0, P<.05) ( Black et al., 2012; Masuzawa et al., 2013). In addition, there are changes evident in overall protein synthesis. Proteomic analysis has shown that ischemia significantly alters mitochondrial proteins involved in fatty acid and glucose metabolism, ATP biosynthesis, and oxidoreductase activity (fold change >1.4, P < .05) ( Black et al., 2012; Masuzawa et al., 2013). All these changes are associated with decreased myocardial cellular viability and decreased myocardial function and suggest that the mitochondrion plays a key role in myocardial viability and function following ischemia and repercussion. In total, these data have provided a basis for continued mitochondrial associated investigations into the rescue and preservation of myocardial tissue and myocardial function (Suleiman et al., 2001). The methodologies for these investigations have been many and varied. In general, the approach to cardioprotection has been either associative or indirect with emphasis on a single mechanistic route or complex or the use of an additive or inhibitor, used either as a single therapy or in combination with others. These include, but are not limited to, the use of pharmaceuticals either before or after ischemia, such as antioxidants, the use of calcium channel antagonists, adenosine, adenosine deaminase inhibitors, adenosine transport inhibitors, or a combination of both , adenosine receptor agonists, mitochondrial ATPsensitive potassium channel openers, phosphodiesterase inhibitors, 5' AMPactivated protein kinase activators, metabolic modulators, antiinflammatory agents and procedural approaches including, preischemia, postischemia and remote ischemic preconditioning (Hsiao et al., 2015 ; Madonna et al., 2015; Hausenloy et al., 2016; Laskowski et al., 2016; OrenesPiñero et al., 2015). In some methodologies, therapeutic intervention is required days or months prior to the ischemic event. Unfortunately, clinical trials using these approaches, either alone or in combination, have for the most part been unsuccessful. 2. Mitochondrial transplantation
We rationalized that the therapeutic approach to cardioprotection should be comprehensive and rather than involving a single or multiple mechanistic pathways, intervention should be specific. To this end, we speculated that the replacement or augmentation of mitochondria damaged during ischemia and reperfusion should be the target for therapeutic intervention. We hypothesized that viable mitochondria isolated form the patient’s own body, from a nonischemic area, and then delivered by direct injection into the ischemic organ would replace or augment damaged mitochondria; thus, allowing for the rescue of myocardial cells and restoration of myocardial function. We have termed this therapeutic intervention; mitochondrial transplantation (McCully et al., 2009; Masuzawa et al., 2013; Cowan et al., 2016; Kaza et al., 2016).
2.1 Mitochondrial isolation

Mitochondrial transplantation is based on the delivery of isolated, viable mitochondria to the target organ. The isolation of mitochondria can be performed using a variety of techniques and methodologies. In our initial studies, we used a standard procedure requiring consecutive low and high speed centrifugation to isolate purified mitochondria. This procedure required approximately 90120 minutes to complete. The repetitive centrifugation steps increases the time for mitochondrial isolation and ultimately reduce mitochondrial viability (Graham 2001; Frezza et al., 2007; Pallotti and Lenaz 2007; Wieckowski et al., 2009; FernándezVizarra et al., 2010; Schmitt S, et al., 2013).

In cardiac surgery, and in many other surgical interventions, the interventional time is 4560 minutes and therefore mitochondrial isolation times of 90120 minutes are inappropriate for clinical usage. Mitochondrial isolation time must be short and efficient so that the therapeutic use of mitochondrial transplantation would not extend the surgical time and possibly add to patient morbidity or mortality. To meet the demands and requirements for clinical application, we have developed a rapid methodology for the isolation of autologous mitochondria (Preble et al., 2014; Preble et al., 2014a).

Firstly, two small pieces of autologous tissue are obtained from the patient’s own body during the surgical procedure. The tissue is dissected out using a #6 biopsy punch. The amount of tissue is less than 0.1 gram. The source of tissue is dependent upon the surgical entry point and access. The only requirement being that the tissue source must be free from ischemia and be viable.

In our studies, we have used viable, nonischemic skeletal muscle tissue as a source for isolated mitochondria. The muscle tissue was obtained from the pectoralis major or the rectus abdominis based on standardized minithoracotomy or sternotomy, respectively. Other tissue sources can also be used and we have used liver tissue with excellent results. The tissue, once obtained is immediately used for mitochondria isolation.

The methodology for the isolation of mitochondria for use in mitochondrial transplantation is simple and rapid and can be performed in under 30 minutes. The freshly isolated tissue is homogenized using a commercial automated homogenizer. For our uses we have used the Miletenyi gentleMACS Dissociator (Miltenyi Biotec Inc., San Diego, CA).

This homogenizer was chosen over the more standardized PotterElvehjem homogenizer (glass with Teflon pestle) or bead based homogenizers as it provides a sterilized, disposable unit for homogenization with fixed plastic wings for homogenization and an automated programable homogenization protocol allowing for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization methods.

The other systems require cleaning and sterilization for each usage and allow for variance in homogenization based on the operator. Bead homogenization systems were not considered due to the possibility of contamination of the mitochondria by bead fragments. Once the tissue is obtained homogenization can be performed in 90120 seconds.

The homogenized tissue is then subjected to brief digestion (10 minutes on ice) with subtilisin A (Protease from Bacillus licheniformis, Type VIII, Sigma, Aldrich, St. Louis, MO) and the digested homogenate filtered through a series of disposable sterile mesh filters. The filtration can all be performed in 23 minutes.

The mitochondria can then be used for direct application or can be concentrated by centrifugation (9000 rpm at 4 oC for 10 minutes). The mitochondrial yield using this methodology is approximately 1 x 109 to 1 x 1010 mitochondria, using the two biopsy tissue samples (< 0.1 g) and provides sufficient mitochondria for quality assurance and quality control assessment ( Preble et al., 2014a). The isolated mitochondria are of the correct size and shape as assessed by electron microscopy and have normal cristae and membranes and show no damage or injury. The isolated mitochondria are pure and have no detectable cytosolic, nuclear or microsomal components. Functional analysis of isolated mitochondria shows that the isolated mitochondria maintain membrane potential and viability and that oxygen consumption and respiratory control index for malateinduced complex I and succinateinduced complex II are equal to that of mitochondria isolated by other methodologies (Rousou et al., 2004; McCully et al., 2009). Once isolated, the mitochondria are immediately used for mitochondrial transplantation. We have found that the isolated mitochondria can be stored on ice for approximately 1 hour but storage beyond this time point greatly reduces efficacy. This is in agreement with previous reports that have shown that mitochondrial bioenergetic function is decreased to <10–15% of normal after the mitochondria were frozen, even when preservatives are used (Wechsler, 1961; Olson et al., 1967). In all our studies, we have used total mitochondria for mitochondrial transplantation. The bioenergetic function of this population includes that of subsarcolemmal and intrafibrillar mitochondria. Previous studies have shown that these mitochondrial subpopulations have differing oxygen consumption and metabolism (Riva et al., 2005; Chen et al., 2008; Kurian et al., 2012). In our studies, we have examined the cardioprotective efficacy of intrafibrillar, subsarcolemmal and total mitochondria. We have found that total mitochondria provide for cardioprotection and that no added cardioprotection is provided using either subsarcolemmal or interfibrillar mitochondrial subpopulations (McCully et al., 2009). It is important that the isolated mitochondria be intact and viable. The use of dead, nonviable mitochondria, mitochondrial proteins or complexes, mitochondrial DNA/RNA or high energy phosphates alone or in combination do not provide cardioprotection (McCully et al., 2009). It has been previously demonstrated that exogenous ATP supplementation and/or ATP synthesis promoters do not restore highenergy phosphate stores and have no beneficial effects on postischemic functional recovery in the heart (McCully et al., 2009). Following determination of mitochondrial number and viability the isolated mitochondria are suspended in 1 ml of respiration buffer containing 250 mmol/l sucrose, 2 mmol/l KH2PO4, 10 mmol/l MgCl2, 20 mmol/l K+HEPES buffer, pH 7.2, 0.5 mmol/l K+EGTA, pH 8.0, 5 mmol/l glutamate, 5 mmol/l malate, 8 mmol/l succinate and 1 mmol/l ADP. Quality control and assurance parameters have been previously described by us (Preble et al., 2014). The isolated mitochondria are then directly injected into the ischemic zone of the heart just prior to reperfusion using a 1 mL tuberculin syringe with a 28or 32gauge needle. The injection volumes are 0.1 mL and contain approximately 1 x 107 mitochondria at each injection site. This volume is optimal and allows for mitochondrial uptake within the myocardium with no backflow leakage of the injected mitochondria. In our studies, we have found that 810 individual injections are sufficient to cover the areaat–risk, although the absolute number of injection sites can be increased. 2.2 Mitochondrial delivery
The direct injection of mitochondria is simple and allows for focal concentration of the injected mitochondria. In our studies the number of mitochondria used for direct injection is 13 x 107 mitochondria. The mitochondria are suspended in 1 mL respiration buffer and injected at 810 sites within the area at risk using a 1 mL tuberculin syringe with a 2832 gage needle (McCully et al., 2009; Masuzawa et al., 2013; Cowan et al., 2016; Kaza et al., 2016). Mitochondrial concentrations > 2 x 108 are not fully suspended in 1 mL of respiration buffer and are therefore not advised for use for mitochondrial transplantation by direct injection.

Fluorescence microscopy has demonstrated that transplanted mitochondria delivered by direct injection are present and viable for at least 28 days following injection into the myocardium, the end point of our animal experiments (Masuzawa et al., 2013, Kaza et al., 2016).

The transplanted mitochondria are widely distributed from the epicardium to the subendocardium >2–3 mm from the injection site (McCully et al., 2009; Masuzawa et al., 2013). The majority of injected mitochondria are found initially within the interstitial spaces between cardiomyocytes. Within 1 hour postdelivery, the transplanted mitochondria are detectable within cardiomyocytes residing near the sarcolemma between Zlines of the sarcomeres and in clusters around endogenous damaged mitochondria as well as near the nucleus. Enumeration of injected mitochondria has shown that 43.52% ± 4.46 (mean ± SEM) of the injected mitochondria are attached to or found within cardiomyocytes (Cowan et al., 2016).

We have speculated that the mechanisms through which the transplanted mitochondria distribute within the myocardium following direct injection into the myocardium may be associated with alterations in myocardial structure that occur after myocardial ischemia. Our studies have demonstrated that following ischemia there is a significant increase in myocardial interfibrillar space that provides for both longitudinal and transverse myocardial interfibrillar separations (Tansey et al., 2006).

These structural changes are not associated with alterations in conduction velocity anisotropy or in tissue edema but occur coincident with significant decreases in postischemic functional recovery and increased myocardial apoptosis and necrosis. We have hypothesized that these interfibrillar separations allow for the distribution of injected mitochondria within the myocardium.

While the delivery of mitochondria by direct injection is practical for many applications, it does not allow for global distribution of the transplanted mitochondria.

Multiple injections are needed for global distribution within the heart and require organ manipulation to access posterior and lateral aspects. To allow for global distribution of mitochondria we have recently demonstrated that mitochondria can also be delivered to the target organ by vascular infusion (Cowan et al., 2016).

In preliminary investigations, we have found that for optimal distribution 1 x 109 mitochondria in 5 mL respiration buffer is efficacious and multiple injections can be performed. Using this protocol we have demonstrated that vascular delivery of mitochondria through the coronary arteries results in the rapid and widespread distribution of exogenous mitochondria throughout the heart, within 10 minutes and provides for cardioprotection.

To demonstrate uptake and distribution of the transplanted mitochondria we have labeled isolated mitochondria with 18Frhodamine 6G and magnetic iron oxide nanoparticles. The use of these labels allowed for image analysis using positron emission tomography, computed tomography, and magnetic resonance imaging. Our results show that the transplantation of mitochondria by vascular delivery is rapid and effective Decaycorrected measurements of 18Frhodamine 6G using a dose calibrator revealed most of the 18Frhodamine 6G labeled mitochondria remained contained within the injected hearts throughout reperfusion (77.3% ± 5.5, mean ± SEM).

Positron emission tomography and computed tomography revealed that the transplanted mitochondria were distributed from the heart apex to the base. Quantitative assessment of perfused mitochondrial position in the heart tissue demonstrated that 24.76% ± 2.50 (mean ± SEM) and 23.64% ± 2.42 (mean ± SEM) of the transplanted mitochondria were associated with cardiomyocytes and blood vessels, respectively (Cowan et al., 2016).

To demonstrate the efficacy of vascular delivery of mitochondria to the heart, we delivered unlabeled mitochondria to the regionally ischemic heart. In these experiments we demonstrated that vascular delivery of mitochondria through the coronary arteries following transient ischemia and reperfusion significantly decreased myocardial infarct size and significantly enhanced postischemic functional recovery. The reductions in infarct size and the enhancement of regional functional recovery achieved with vascular delivery of mitochondria are not significantly different from that obtained by direct injection of mitochondria (McCully et al., 2009; Masuzawa et al., 2013; Cowan et al., 2016). These data indicate that autologous mitochondrial transplantation is efficacious as a cardioprotective therapy whether these organelles ae delivered by directly injection or delivered by vascular infusion through the coronary arteries.

2.3 Localization to endorgan by vascular delivery
In all our studies, we have noted that the distribution of mitochondria following delivery by direct injection or by vascular infusion remains within the heart and is not detectable in other organs. This finding is important as the delivery of mitochondria by vascular infusion provides for localized therapy without crosscontamination to other endorgans. In the heart we deliver mitochondria by injection into the coronary arteries to avoid systemic distribution; however, we have also delivered mitochondria to the lung by vascular infusion through the pulmonary artery.

In these studies, the mitochondria were labeled with 18Frhodamine 6G as above. Positron emission tomography and computed tomography showed that the mitochondria were localized in the lung and were not detectable in any other areas of the body.

At present, we do not have a mechanism for this “endorgan homing”, where the transplanted mitochondria are retained by the immediate downstream organ, but suggest that this observation may play an important therapeutic role in future studies and applications using mitochondrial transplantation.

2.4 Mechanisms of mitochondrial uptake and internalization
In previous studies, we have investigated a variety of mechanisms that may be associated with mitochondrial uptake and internalization following mitochondrial. These studies were performed using well established pharmacological blockers of clathrin mediated endocytosis, actinmediated endocytosis, macropinocytosis, and tunneling nanotubes (Le et al., 2000; BereiterHahn Jet al., 2008; Lou et al., 2012; Islam et al., 2012; Huang et al., 2013; Kitani et al., 2014). Our studies demonstrated that autologous mitochondria delivered by direct injection are internalized by actindependent endocytosis (Pacak et al., 2015).

Mitochondrial uptake by vascular delivery appears to be more complex. The rapid and widespread uptake of mitochondria when delivered by vascular infusion would suggest that mechanisms allowing for the rapid passage of mitochondria through the vascular wall are involved. Previous studies support the concept that cells can routinely escape from the circulation. It has been previously shown that certain cardiac and mesenchymal stem cells appear to be actively expelled from the vasculature in a process different from diapedesis (Cheng et al., 2012; Allen et al., 2017).

Transmigration of stem cells through the vascular wall requires extensive remodeling of the endothelium (Allen et al., 2016). Cheng et al., 2012 have suggested that this occurs in three stages 1) adhesion of infused cells to the microvessel lining; 2) pocketing of infused cells by endothelial projections; 3) breakdown of the adjacent vascular wall, releasing cells into the interstitium.

The first two steps require integrindependent interactions between transplanted cells and host endothelium, while matrix metalloproteinases mediate the subsequent breakdown of the microvessel wall. These steps are unlikely to occur rapidly or to be associated with the mitochondrion.

Another possible mechanism for mitochondrial uptake may be diapedesislike. Previous studies have shown that some cells routinely escape from the circulation. For example, leukocyte extravasation (i.e. diapedesis) between venous endothelial cells is a wellunderstood process that involves cell adhesion proteins.

It is unlikely, however, that isolated, exogenous mitochondria use a similar mechanism as leukocytes to move through the wall of blood vessels as they do not express the array of proteins involved in diapedesis on their outer membrane (Pagliarini et al., 2008; Calvo et al., 2010).

Whether mitochondria pass between or through endothelial cells and the region of the vasculature at which this process occurs remain to be determined. The size of the isolated mitochondria and the rapidity of their uptake by vascular perfusion would not appear to support any currently defined mechanisms. At present, we are investigating several possible mechanisms of mitochondrial uptake by vascular infusion; but, have yet to conclusively identify a definitive mechanistic route.

3. Mitochondrial transplantation for cardioprotection

3.1 Animal models
In the isolated perfused rabbit heart model and subsequent studies in the in vivo rabbit and pig heart models, we have investigated the use of mitochondrial transplantation as a cardioprotective therapy (McCully et al., 2009; Masuzawa et al., 2013; Cowan et al., 2016; Kaza et al., 2016). In these studies, the myocardium was made temporarily ischemic by ligating the left anterior descending artery with a snare. This temporary ligation results in the attenuation or cessation of coronary blood flow such that oxygen delivery to the myocardium is insufficient to meet oxygen demand. The resulting injury is termed ischemia/reperfusion injury and is characterized by the loss of myocardial cell viability and decreased contractile function within the area at risk. In general, we subject 2530% of the left ventricular mass to ischemia, resulting in a loss in cell viability of approximately 30% and a reduction in contractile force of approximately 25% based on regional systolic shortening. These decrements in cell viability and function are reproducible between large animal models and mimic events occurring in human acute myocardial infarction or surgical intervention during cardiopulmonary bypass. Following 30 minutes of temporary ligation the snare is released and coronary blood flow to the region is reestablished. To show the efficacy of mitochondrial transplantation, we typically deliver autologous mitochondria by direct injection or by vascular delivery, just prior to or at the very start of reperfusion. Control hearts receive vehicle alone (respiration buffer with no mitochondria).

3.2 Effects of mitochondrial transplantation on arrhythmogenicity
The transplantation of mitochondria has similarities in methodology with stem cell transplantation. In stem cell transplantation using skeletal muscle myoblasts, it has been reported that skeletal muscle myoblasts, clustering of the cells can occur, resulting in arrhythmia with postoperative episodes of sustained tachycardia due to alterations in electrical coupling (Macia et al., 2009). To demonstrate that mitochondrial transplantation is not proarrhythmic we have used serial 12 lead electrocardiogram (ECG) analysis and optical mapping (Masuzawa et al., 2013). Our results show that there is no proarrhythmogenicity associated with mitochondrial transplantation. We observed no ventricular tachycardia, bradycardia, fibrillation, or conduction system defects or repolarization heterogeneity associated with mitochondrial transplantation. In addition, there were no changes in serial ECG, QRS duration or corrected QT interval. There was no evidence of any changes associated with ventricular wall motion disturbances, left ventricle hypertrophy, valve dysfunction, fibrosis, or pericardial effusion either at the time of injection, or at any time up to 4 weeks following transplantation of autogeneic mitochondria (Masuzawa et al., 2013).
To confirm these findings, we also performed optical mapping (Masuzawa et al., 2013). In these studies, a 400fold increase in mitochondria, 8.4 x 107/gram tissue wet weight as compared to 2 x 105/gram tissue wet weight, was directly injected into the heart. The number of mitochondria injected per site was significantly greater than that used in the in situ heart (4.2 × 108 vs. 1.2 × 106), so that any acute arrhythmogenic responses could be observed. Our results showed that even with this highly increased mitochondrial load, sequential isopotential maps from the left ventricles injected with mitochondria showed no detectable abnormal impulse propagation on the myocardial surface associated with mitochondrial transplantation.

3.3 Effects of mitochondrial transplantation on immune and autoimmune response
In all our studies, we have used autologous mitochondria isolated from the patient’s own body for cardioprotection. The use of autologous tissue was based on the assumption that this approach would not cause an immune response and avoid the need for antirejection therapy required for nonautologous cellbased therapies (Kofidis et al., 2005). To confirm these expectations serial analysis immune and inflammatory markers was performed. The effects of mitochondrial transplantation were also investigated using multiplex (42plex) analysis of cytokines and chemokines (Masuzawa et al., 2013). The results from these studies confirmed that there was no immune or inflammatory response associated with autologous mitochondrial transplantation (Masuzawa et al., 2013). In addition there was no upregulation of cytokines associated with the immune response that is seen in patients with acute heart transplantation rejection (Rose, 2011).
The possibility of autoimmune response due to the presence of increased mitochondrial number in the myocardium was also investigated. The need for these studies was based on previous studies indicating that oxidative modification of E2 subunits of mitochondria pyruvate dehydrogenase, branched chain 2oxoacid dehydrogenase, and 2oxoglutarate dehydrogenase is a critical step leading to the induction of an autoimmune response in the liver as demonstrated by the presence of antimitochondrial antibodies (Leung et al., 2007). Indirect immunofluorescence was used to test for the presence of antimitochondrial antibodies (AMA) in serial blood samples. AMAs were not detected in the serum of any animals treated with autologous mitochondrial transplantation indicating that the transplantation of mitochondria does not induce an autoimmune response (Masuzawa et al., 2013).
3.4 Effects of mitochondrial transplantation on cellular viability and function

Our results have demonstrated that direct injection or vascular delivery of mitochondria to the heart rescues cell function and myocardial contractile function following ischemia and reperfusion. Creatine kinaseMB isoenzyme (CKMB) and cardiac troponin–I (cTnI) are specific and sensitive markers of myocardial injury, and elevated levels indicate myocardial injury (Pourafkari et al., 2015). Mitochondrial transplantation significantly decreased serum CKMB and cTnI indicating that myocardial injury following 30 minutes of transient ischemia was decreased (Masuzawa et al., 2013; Kaza et al., 2016). These effects were confirmed by infarct analysis using triphenyl tetrazolium chloride analysis to determine necrosis and terminal deoxynucleotidyl transferasemediated dUTP nickend labeling (TUNEL) and caspase activity to determine apoptosis in hearts treated with mitochondrial transplantation and those treated with vehicle alone (Masuzawa et al., 2013). Our results demonstrated that mitochondrial transplantation significantly decreased myocardial injury, including both necrosis and apoptosis, resulting from transient ischemia.
Increased myocardial cellular viability would be expected to correlate with enhanced myocardial function and our studies confirm this assumption. Our results show that 10 minutes following mitochondrial transplantation myocardial function is significantly enhanced as compared to hearts receiving injection of respiration media (vehicle) alone and that this function remains enhanced for at least 28 days – the end point of our studies (Masuzawa et al., 2013; Kaza et al., 2016). This is in contrast to hearts receiving vehicle alone that had persistent left ventricular hypokinesis.
3.5 Mechanisms of mitochondrial transplantation
The mechanisms through which mitochondrial transplantation provides cardioprotection have yet to be fully elucidated. At present, our studies have shown that the transplanted mitochondria act both extraand intracellularly. Once transplanted, the mitochondria increase total tissue ATP content and ATP synthesis. This increase in high energy acts rapidly, as early

as 10 minutes following delivery of the mitochondria to the heart, to enhance cardiac function as determined by echocardiography and pressure volume measurement. The mitochondria then act to upregulate proteomic pathways for the mitochondrion and the generation of precursor metabolites for energy and cellular respiration (P < 0.05, Enrichment Score > 2.0) (Masuzawa et al., 2013).
At 10 minutes to one hour following transplantation the transplanted mitochondrial are internalized into cardiomyocytes, by actindependent endocytosis (Pacak et al., 2015). Once internalized, the transplanted mitochondria further increase cardiomyocyte ATP content and upregulate cardioprotective cytokines (Masuzawa et al., 2013). These cytokines have been shown to be associated with enhanced cardiac function by stimulating cell growth, proliferation, and migration, enhancing vascularization, providing protection against cardiomyocyte apoptosis and improving functional cardiac recovery and cardiac remodeling independent of cardiac myocyte regeneration (Masuzawa et al., 2013).
We have recently shown that transplanted mitochondria also act at the mitochondrial genomic level. In previous experiments, we have shown that following ischemia there is damage to mitochondrial DNA resulting in the reduction of ATP synthesis (Levitsky et al., 2003). These alterations were associated with poor recovery following cardiac surgery in humans. Our studies demonstrate that mitochondrial transplantation replaces damaged mitochondrial DNA with intact mitochondrial DNA and rescues myocardial cell function (Pacak et al., 2015).
The transplanted mitochondria maintain viability and function for at least 28 days, the limit of our studies (Masuzawa et al., 2013; Cowan et al., 2016; Kaza et al., 2016). This is in contrast to xenoand allotransplanted cells that are rapidly rejected leading to the loss of transplanted cells, despite the use of antirejection pharmaceuticals (Yau et al., 2003; Hamamoto et al., 2009).

4 Human application
Premised upon these in vivo studies demonstrating the efficacy of mitochondrial transplantation for cardioprotection we have recently performed the first clinical application of mitochondrial transplantation (Emani et al., 2017). The study was performed in pediatric patients who suffered myocardial ischemiareperfusion injury. All procedures were performed under an Innovative Therapies process developed by the Boston Children’s Hospital Institutional Review Board. An individual review of the proposed therapy for each patient was provided by two independent physicians, not involved in the patient’s care. Families were extensively counseled regarding the experimental nature of the procedure and a separate Innovative Therapies consent form was signed.
Five pediatric patients in critical condition who were unable to be weaned off extracorporeal membrane oxygenation (ECMO) support due to myocardial dysfunction related to ischemia and reperfusion were treated with autologous mitochondria. Patient diagnosis included dextrotransposition of the great arteries (4 days and 25 days of age ), hypoplastic left heart syndrome (6 days), left ventricular outflow tract obstruction (6 months of age) and tricuspid atresia 1B (2 years of age) (Emani et al., 2017).
The cause of ischemia was coronary artery obstruction that was relieved in 4 patients, and LV distension with subendocardial ischemia in one patient. The autologous mitochondria were isolated from the patient’s rectus abdominis muscle. The patients received 10 mitochondrial injections, 100 uL each containing 1 x 107 ± 1 x 104 mitochondria. The mitochondria were delivered to the myocardium by direct injection with a 1 mL tuberculin syringe (28gauge needle). All injections were delivered to the area affected by ischemiareperfusion that was identified by epicardial echocardiography as being hypokinetic. Following mitochondrial transplantation, all 5 patients had significant improvement in their myocardial systolic function. Epicardial echocardiography showed moderate to severe systolic function ventricular dysfunction with regional hypokinesis prior to treatment (Emani et al., 2017). Ventricular function was improved to mildmoderate to normal systolic function at 46 days following autologous mitochondrial transplantation and improved to mild dysfunction in one patient and normal systolic function with no regional hypokinesia detected in any patient at 10 days following autologous mitochondrial transplantation (Emani et al., 2017).

All but one patient were successfully weaned off ECMO support by the 2nd day post mitochondrial transplantation. The single patient who was unable to wean off ECMO support suffered irreversible multiorgan failure despite the recovery of myocardial function following mitochondrial transplantation. This patient was on ECMO support for 15 days prior to treatment with mitochondrial transplantation. There were no adverse complications such as arrhythmia, intramyocardial hematoma or scarring with mitochondrial transplantation, in agreement with our animal studies. This case study demonstrates for the first time the potential role mitochondrial transplantation to improve ventricular dysfunction following ischemiareperfusion injury in humans.

While this is the first clinical usage of mitochondrial transplantation in humans, the same protocol can be used in adults and in other settings of ischemiareperfusion injury (Emani et al., 2017). The ability to use mitochondrial transplantation for clinical intervention in situations such as the stunned myocardium is enhanced by the fact that mitochondrial harvest and isolation can be performed within 2030 minutes during the same procedure and involves minimal manipulation of muscle tissue.

5 The timing of mitochondrial transplantation delivery
Studies by us and by others have shown that the mechanisms associated with ischemic myocardial injury converge on the mitochondrion. Rapidly following the onset of ischemia there are alterations in mitochondrial structure, complex activity, oxygen consumption, high energy synthesis and changes in mitochondrial transcriptomics and proteomics.

All these changes occur during ischemia and persist following reestablishment of coronary blood flow (reperfusion). For practical efficacy we therefore deliver the transplanted mitochondria just prior to reperfusion or during early reperfusion in order to limit the effects of ischemia on the transplanted mitochondria. We reasoned that if the mitochondria were transplanted at the start of ischemia they too would be damaged. This approach has been shown to be efficacious in our animal studies. In the human trial described above the delivery of mitochondria was many days after the ischemic insult. In these cases, the patient’s heart was unable to provide sufficient contractile force to be weaned from ECMO (Emani et al., 2017).

We believe that the role of mitochondrial transplantation is the rescue of cells and cellular function. This is premised on our animal studies. The results obtained in the clinical studies in humans are most likely the result of reversal of myocardial stunning. Stunning “describes the mechanical dysfunction that persists after reperfusion despite the absence of myocellular damage and despite the return of normal or nearnormal perfusion (Kloner et al., 1998). Mentzer, 2011, has noted that myocardial stunning, is a frequent consequence after heart surgery and is characterized by a requirement for postoperative inotropic support despite a technically satisfactory heart operation. In the stunned heart the myocardial cells remain alive but there is a prolonged depression of cardiac contractility after reperfusion. This depression in contractility can last days but with extended time results in patient mortality.
In the human cases in which we have used mitochondrial transplantation it is likely that we have rescued the stunned myocardium. The mechanisms most likely involve those we have demonstrated in our animal models, namely, restoration of high energy synthesis and replacement of damaged mitochondrial DNA. The veracity of these mechanisms remains to be elucidated.

6 Mitochondrial transplantation for the rescue of other tissues
While our studies have focussed on the heart and ischemia and reperfusion, other groups have shown that mitochondrial transplantation can be used to enhance drug sensitivity in human breast cancer cells (Elliott et al., 2012); to rescue cell function in cells harboring the mitochondrial DNA mutation (Chang et al., 2013); Parkinson’s disease (Chang et al., 2016); liver ischemia/reperfusion injury (Lin et al., 2013); in cellular studies demonstrating that isolated mitochondria rescue mitochondrial respiratory function and improved the cellular viability in cardiomyocytes (Kitani et al., 2014) and neurorecovery after stroke ( Hayakawa et al., 2016).

These studies demonstrate the potential of mitochondrial transplantation in a variety of diseases. The utility in other organ related diseases and syndromes remains to be investigated. However, diabetes, Alzheimer’s disease and dementia, posttraumatic stress disorder, concussion and others have all been shown to be associated with alterations occurring at or affecting mitochondrial function. Included in these pathological disorders are ischemiareperfusion events affecting pulmonary, renal, hepatic, cerebral, ocular and skeletal muscles. We expect that the application and usage of mitochondrial transplantation will provide for a simple and efficacious therapeutic approach to many of these disease and will significantly ameliorate morbidity and mortality.

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NMNAT: It’s an NAD+ synthase, chaperone, AND neuroprotector

Nicotinamide mononucleotide adenylyl transferases (NMNATs) are a family of highly conserved proteins indispensable for cellular homeostasis. NMNATs are classically known for their enzymatic function of catalyzing NAD+ synthesis, but also have gained a reputation as essential neuronal maintenance factors. NMNAT deficiency has been associated with various human diseases with pronounced consequences on neural tissues, underscoring the importance of the neuronal maintenance and protective roles of these proteins.

New mechanistic studies have challenged the role of NMNAT-catalyzed NAD+ production in delaying Wallerian degeneration and have specified new mechanisms of NMNAT’s chaperone function critical for neuronal health. Progress in understanding the regulation of NMNAT has uncovered a neuronal stress response with great therapeutic promise for treating various neurodegenerative conditions under question [3]. Given the essential role for NAD+ in cellular metabolism, it is not surprising that the enzyme is required for the survival of all living organisms, from archaebacteria to humans.

The discovery of the remarkable neuroprotective function of NMNAT pro- teins sparked a burst of investigations on NMNAT in the nervous system. More recently, NMNAT mutations have been identified to cause a severe form of retinal degener- ation and NMNAT deficiency has been associated with complex neurological diseases. In this review, we focus on the function of NMNAT in the nervous system and discuss the recent advances in understanding the regulatory mechanisms of neuronal maintenance that are relevant for neuroprotective therapies against neuro- degenerative conditions. For previously published reviews that focus on the neuroprotective effects of NMNAT, particularly in axon degeneration and injury, we refer readers to [1,4–6].

Genetic links between NMNAT and diseases of the nervous system
The only known monogenetic disease associated with NMNAT proteins is Leber congenital amaurosis (LCA), one of the most common forms of inherited blindness in children. Compound heterozygous or homozygous muta- tions in NMNAT1 cause LCA9 (OMIM 608553), an auto- somal recessive condition characterized by severe early- onset and rapid progression of vision loss and retinal degeneration [7]. To date more than 30 mutations spread across the NMNAT1 gene have been reported, including missense, nonsense, and splicing mutations (Figure 1) [8–11]. Importantly, most LCA9 patients have reported normal physical and mental health, suggesting a specific requirement of NMNAT1 for maintenance of the neural tissue in the retina [9].

So far no human diseases have been shown to be directly caused by mutations in NMNAT2 or NMNAT3, though several studies have signified a putative contribution of NMNAT deficiency to the progression of complex neu- rological diseases. Microarray studies have shown that NMNAT2 mRNA levels are reduced in various neurode- generative diseases, including Alzheimer’s disease (AD), Parkinson’s disease, and Huntington’s disease [12–14]. In AD, NMNAT2 transcript levels negatively correlate with cognitive dysfunction and AD pathology [15 ]. Genome- wide association studies have indicated a SNP (rs952797) located 126 kb downstream of the NMNAT3 gene that is associated with late-onset AD [16]. Although these asso- ciation studies do not demonstrate a causal relationship,

 

Introduction

Humans have three NMNAT genes that produce three NMNAT protein isoforms with distinct tissue expression patterns and subcellular localizations [1]. NMNAT1 is ubiquitously expressed and is enriched in the nucleus. NMNAT2 is predominantly expressed in the brain and is localized to the cytosol and enriched in membrane com- partments. NMNAT3 is also widely expressed but is highest in liver, heart, skeletal muscles, and red blood cells [2]. NMNAT3 is reported to have two splice variants, encoding mitochondrial localized FKSG76 and cytosolic NMNAT3v1, though the expression and function of these endogenous protein variants are still they do recommend NMNAT as an important contributor to neuronal health.

Molecular functions of NMNAT necessary for neuronal maintenance and protection 

LCA9 and other neurological disease phenotypes associ- ated with NMNAT deficiency are consistent with its neural maintenance function, yet the neuroprotective capacity of NMNAT had already emerged with the serendipitous discovery of the slow Wallerian degenera- tion mutant (Wlds) mouse [17,18]. In the Wlds mouse, degeneration of the distal axon following axotomy (Wallerian degeneration) is remarkably delayed by a dominant mutation causing overexpression and redistri- bution of Nmnat1 to the cytoplasmic compartment of the axon [4]. The neuroprotective role of NMNAT is effica- cious against not only axonal injury, but also models of various neurodegenerative conditions such as toxic neuropathy, glaucoma, spinocerebellar ataxia, tauopathy, and Huntington’s disease [1,5,19–25]. Furthermore, NMNAT homologues across species, including archae- bacteria, yeast, fly, mouse, and human, exhibit a conserved cytoprotective function, even when expressed in different model organisms [1,19]. While these studies chronicle the potent and conserved protection NMNAT proteins confer against a variety of neuronal insults, the precise mechanism by which NMNAT engenders this protection has been hard to pinpoint.

NMNAT proteins have two distinct functions that can bestow neuronal maintenance and protection: NAD synthase activity and chaperone activity [26]. NMNAT catalyzes the reversible conversion of NMN (nicotin- amide mononucleotide) to NAD+ in the final step of both the de novo biosynthesis and salvage pathways. NAD+ is a vital coenzyme important for metabolism and redox biology, as well as a substrate in multiple signaling processes [27]. Thus, NMNAT enzymes play a straight- forward role in neuronal maintenance via balancing NAD+ consumption with production. A molecular chap- erone function has also been demonstrated by a number of NMNAT proteins tested, including Drosophila Nmnat, mouse Nmnat2, and human NMNAT3 [15,19,28]. In vitro these NMNAT proteins bind to client proteins and prevent thermal-stress induced unfolding [15,19]. Thus, NMNAT chaperones can contribute to neuronal protein homeostasis.

There is still considerable controversy surrounding the mechanism of NMNAT-mediated neuroprotection. The prevailing hypothesis is that NMNAT overexpression pro- vides continuous enzyme activity in injured neurons, thus preventing the consequent decrease in NAD+ and the accumulation of the precursor NMN [6]. However, recent work revealed that NAD+ depletion following axon injury is due to a dramatic increase in consumption by the pro- degenerative SARM1 (sterile alpha and TIR motif-con- taining 1), but that overexpression of cytoplasmic Nmnat1 blocks SARM1-dependent NAD+ consumption without increasing NAD+ synthesis [29 ]. Thus, NMNAT-medi- ated axon protection hinges on its ability to block pro- degenerative SARM1 signaling, but this does not rely on enzymatic conversion of NMN to NAD+. One alternative is that NMNAT provides neuroprotection via an enzyme- independent function, leading to the hypothesis that NMNAT mediated protection is chaperone-dependent. Recently, it has been shown that Nmnat2’s enzyme activity is dispensable for relieving the toxic phosphorylated tau burden in a model of fronto-temporal dementia and parkinsonism 17 (FTDP-17), and that Nmnat2 complexes with the classical HSP90 chaperone, possibly to promote refolding of toxic tau [15 ]. In another study, overexpres- sion of cytoplasmic Nmnat1 partially preserved neuronal function in a model of early-onset FTDP-17 by decreasing insoluble tau aggregates, without altering phosphorylated tau [30]. Similarly, overexpression of yeast homologs of NMNAT, NMA1 and NMA2, suppresses proteotoxicity in yeast models of polyglutamine- and a-synuclein-induced neurodegeneration also by enhancing clearance of mis- folded proteins [22].

Though the precise mechanism may vary by disease model, it is clear that NMNAT over- expression alleviates proteotoxic stress in neurodegenera- tive conditions associated with protein misfolding, consis- tent with a chaperone function. Further supporting the chaperone function of NMNAT is the identification of its endogenous ‘client’ protein in Drosophila synapses, where Nmnat maintains active zone structure by directly interacting with the active zone protein Bruchpilot (BRP) in an activity-dependent manner [28]. Perhaps a source of resistance to this hypothesis is that enzyme- inactive mouse Nmnat proteins fail to protect axons [31– 33], although enzyme-inactive Drosophila Nmnat is suffi- cient to protect against activity-induced retinal degenera- tion, Wallerian degeneration, and axonal degeneration induced by loss of JNK (c-Jun N-terminal kinase); in these conditions NMNAT chaperone-dependent protection is perhaps less implicit since here the client for such a chaperone role is not apparent [34–36]. Continued advances in NMNAT overexpression-mediated protection in neurodegenerative models will be invaluable for identi- fying not only the underlying mechanisms of NMNAT’s protection, but also by extension, the primary triggers of neurodegeneration.

Mouse models of NMNAT1-dependent LCA recapitu- late several aspects of human disease and further delin- eate disease progression; importantly, degeneration occurs after retinal development, is observed first within the photoreceptors, and is only observed in inner retinal neurons and RPE (retinal pigment epithelia) cells in advanced stages [37]. The sensitivity of the retina, there- fore, may reflect a twofold requirement of NMNAT: biosynthesis of NAD+ and chaperone activity for meeting the high metabolic and protein turnover demands of photoreceptors, respectively. Although LCA mutations are widely distributed across the NMNAT1 gene, most NMNAT1 mutant proteins characterized exhibit several common features. NAD+ synthase activity is reduced by most mutations in vitro, yet patient fibroblasts exhibit normal basal NAD+ levels, suggesting that enzyme deficiency is not a primary cause of disease [10,38 ]. Importantly, ectopic expression of several LCA9 NMNAT1 mutants is sufficient to protect cultured neu- rons from Wallerian degeneration, indicating that LCA9- causing mutations do not disrupt the qualitative neuro- protective properties of NMNAT proteins [38 ]. How- ever in vitro, NMNAT1 mutant proteins are more sus- ceptible to stress-induced unfolding compared to wild type protein, which may amplified in vivo to cause disease [38 ]. Interestingly, Drosophila photoreceptors are also particularly vulnerable to loss of Nmnat, but Nmnat’s enzyme activity is completely dispensable for mainte- nance of retinal health and function in this model [34]. So while LCA9 may be primarily attributed to a loss of NMNAT1 protein stability, more studies are needed to determine whether retinal degeneration arises from a consequential loss of NAD+ synthesis and/or loss of the molecular chaperone function (Figure 1).

Regulation of NMNAT during stress and disease
Because of the essential neuronal maintenance role of NMNAT and its capacity for neuroprotection, under- standing the regulation of NMNAT levels becomes key to unlocking its therapeutic potential. It is critical that neurons maintain sufficient levels of NMNAT2; NMNAT2 loss is considered an initiating event in Wal- lerian degeneration and also appears to be consistent in progression of neurodegenerative conditions [39]. At the protein level, NMNAT2 is constitutively degraded by the ubiquitin-proteasome system, the exact players of which are beginning to emerge. Several ubiquitin ligases, including Phr1 (Highwire in Drosophila), Skp1a, and Fbxo45, have been identified, though direct evidence of NMNAT ubiquitination is lacking [40–42]. Genetic interference of these ligases increases NMNAT levels and subsequently enhances neuronal protection. In addi- tion to protein regulation, two functional cAMP-response elements (CREs) have been identified in the Nmnat2 promoter region that influence expression at the tran- scriptional level. In an FTDP-17 tauopathy model, a pathological reduction of phospho-CREB (CRE binding protein) levels and binding with the Nmnat2 promoter resulted in decreased Nmnat2 transcript and protein [24].

 

In contrast to mammals that use three NMNAT genes to produce three different NMNAT protein isoforms, Dro- sophila has only one Nmnat gene that is alternatively spliced to generate two variants: Nmnat-RA mRNA variant encodes the nuclear Nmnat-PC protein isoform and Nmnat-RB variant encodes the cytosolic Nmnat-PD isoform. In a Drosophila model of spinocerebellar ataxia 1 (SCA1), overexpression of Nmnat-PD significantly improved behavioral and morphological defects by reduc- ing neuronal mutant human Ataxin-1 (hAtx1-[82Q]) aggregates, yet surprisingly, overexpression of Nmnat- PC increased aggregate size and exacerbated neurode- generation [43 ]. Importantly, cytoplasmic targeted Nmnat-PC is also ineffective in relieving hAtx-[82Q] proteotoxicity. This study revealed a functional diver- gence underlying Drosophila Nmnat isoform-specific neuroprotection. Therefore, alternative splicing in Dro- sophila uses one gene to produce two functionally exclu- sive mRNA variants: one housekeeping variant, Nmnat- RA, that is stably expressed under normal conditions, and one stress response variant, Nmnat-RB, that can be quickly induced under stress conditions for neuronal protection (Figure 3). Taken together with the previous finding that the transcription of Drosophila Nmnat pre- mRNA is increased under various stress conditions [44], the observation that stress also drives post-transcriptional alternative splicing to preferentially generate Nmnat-RB sheds light on the complex neuronal stress response to achieve self-protection [43 ].

Regulation of Drosophila Nmnat under neuronal stress may provide insight into regulation of NMNAT2 in mammals in neurodegenerative conditions. Notably, the transcript level of the neuroprotective Drosophila variant Nmnat-RB is reduced in late stages of models of tauopathy and SCA1, however it is increased in the early stages, suggesting an upregulation to combat pathogenic protein aggregation during disease progression that is perhaps overwhelmed at more advanced stages of neuro- nal degeneration and cell death [43 ]. Similarly, although many studies show a decrease of NMNAT2 transcript postmortem in patients with neurodegenerative diseases, there is also evidence of increased levels at the early stages of disease. For example, in mild cognitive impairment and early stages of AD, NMNAT2 mRNA levels are higher in the frontal cortex of patients exhibit- ing high levels of oxidative stress-induced neuronal DNA damage, compared to those with lower levels of DNA damage [45]; at this stage the nervous system is mounting a stress response as HSP90 is also upregulated in patient brains with high oxidative damage. In a genome-wide gene-expression study of another mouse model of FTDP- 17, Nmnat2 transcript is significantly upregulated in the hippocampus during early disease progression, but is downregulated at end stages of disease when neurofibril- lary tangle burden is heavy [46]. Here the early rise of Nmnat2 is likely a proteotoxic stress response, while the late decrease is consistent with observed downregulation of synaptic genes corresponding to synaptic loss and upregulation of inflammatory and apoptotic genes indic- ative of neuronal cell death. Since endogenous upregula- tion of NMNAT under stress conditions seems to be insufficient to maintain neural integrity in the long term, NMNAT and its transcriptional and post-transcriptional regulation can serve as a therapeutic target for pharma- cological intervention to slow the progression of neuro- degenerative diseases.

Concluding remarks

The long life of neurons, up to a century in humans, makes neuronal maintenance an important challenge. It is conceivable that compromised maintenance would result in degeneration in an age-dependent manner. The emer- gence of the chaperone function of NMNAT, an NAD+ synthase, specifically in the nervous system for neuronal maintenance, exemplifies an evolutionarily conserved

strategy of ‘repurposing’ (or ‘moonlighting’) housekeep- ing enzymes. Neurons have developed transcriptional and post-transcriptional regulatory mechanisms to bal- ance the metabolic activity and stress response role of NMNAT. Thus, understanding neuronal requirements for NMNAT the NAD+ synthase and NMNAT the chaperone under various stress and disease conditions, in addition to dissecting the regulatory mechanisms that direct the activity of NMNAT, has huge implications for neuroprotective therpapies. Furthermore, identifying druggable targets to enhance expression and/or reduce protein degradation would be a pharmacologically approachable way to achieve higher NMNAT protein levels and therefore confer neuroprotection.

Nicotinamide mononucleotide inhibits JNK activation to reverse Alzheimer disease.

Study published here

Highlights

  •   NMN improved behavioral measures of cognitive impairments in AD-Tg mice.
  •   NMN decreased β-amyloid production, amyloid plaque burden, synaptic loss, and inflammatory responses in AD-Tg mice.
  •   NMN reduced JNK activation in AD-Tg mice.
  •   NMN regulated the expression of APP cleavage secretase in AD-Tg mice.

Abstract

Amyloid-β (Aβ) oligomers have been accepted as major neurotoxic agents in the therapy of Alzheimer’s disease (AD). It has been shown that the activity of nicotinamide adenine dinucleotide (NAD+) is related with the decline of Aβ toxicity in AD. Nicotinamide mononucleotide (NMN), the important precursor of NAD+, is produced during the reaction of nicotinamide phosphoribosyl transferase (Nampt). This study aimed to figure out the potential therapeutic effects of NMN and its underlying mechanisms in APPswe/PS1dE9 (AD-Tg) mice. We found that NMN gave rise to a substantial improvement in behavioral measures of cognitive impairments compared to control AD-Tg mice. In addition, NMN treatment significantly decreased β-amyloid production, amyloid plaque burden, synaptic loss, and inflammatory responses in transgenic animals. Mechanistically, NMN effectively controlled JNK activation. Furthermore, NMN potently progressed nonamyloidogenic amyloid precursor protein (APP) and suppressed amyloidogenic APP by mediating the expression of APP cleavage secretase in AD- Tg mice. Based on our findings, it was suggested that NMN substantially decreases multiple AD- associated pathological characteristically at least partially by the inhibition of JNK activation.

Introduction

As a chronic neurodegenerative disorder, Alzheimer’s disease (AD) is clinically featured by progressive pattern of cognitive deficits and memory impairment. Disturbed energy metabolism in the brain and oxidative stress are two potential factors leading to neural degeneration and cognitive impairments [1]. Aβ oligomers are found to be associated with the pathology of AD [2]. Recent studies indicates that Aβ oligomers inhibit synaptic transmission prior to neuronal cell death [3] and LTP (long-term potentiation), an experimental model for synaptic plasticity and memory [4]. In addition, Aβ oligomers are also found to be relevant to the producing of the free oxygen radical. So far, there is no curative treatment for AD [5]. Considering the varied and well-defined pathologies of AD, new therapies with the functions of reducing pathologies are needed to prevent or slow disease progression.

Nicotinamide adenine dinucleotide (NAD), oxidized (NAD+) or reduced (NADH), plays a key role in many metabolic reactions, for both forms of NAD regulate transfer of hydrogens metabolic reactions, oxidative or reductive [6], as well as mitochondrial morphological dynamics in brain [7]. Among these two forms, oxidized NAD is particularly important to mitochondrial enzyme reactions and cellular energy metabolism [8, 9]. In normal conditions, as people ages, the level of NAD+ drops [6], inhibiting cellular respiration and further causing decreased mitochondrial ATP and possibly cellular death. NAD+ serves a substrate for enzymes that depend on NAD+, such as ADP-ribosyl cyclase (CD38), poly(ADP-ribose) polymerase 1 (PARP1), and Sirtuin 1 (SIRT1) [10].

To treat neurodegenerative diseases, NAD+ depletion and cellular energy deficits need to be prevented for protecting nerves [10]. There are four pathways synthesizing NAD+ in mammals. The salvage pathway (primary route) way is to use nicotinamide, nicotinic acid, nicotinamide riboside, or the de novo pathway with tryptophan [11]. As an essential precursor of NAD+, Nicotinamide mononucleotide (NMN) is produced during the reaction of nicotinamide phosphoribosyltransferase (Nampt). Nampt is essential to regulating NAD+ synthesis [12], for it stimulates phosphoribosyl components to separate from phosphoribosyl pyrophosphates and to combine with nicotinamides. In this way, NMN is generated and with NMN adenylyltransferase, NMN is converted to NAD+. However, the potential therapeutic effects of NMN on AD remain unclear.

c-Jun N-terminal kinases (JNKs) are a family of protein kinases that play a central role in stress signaling pathways implicated in gene expression, neuronal plasticity, regeneration, cell death, and regulation of cellular senescence [13]. Activation of JNK has been identified as a key element responsible for the regulation of apoptosis signals and therefore, it is critical for pathological cell death associated with neurodegenerative diseases and, among them, with Alzheimer’s disease (AD) [14].

As suggested, NAD+ may be essential to brain metabolism and might influence memory and learning. According to recent studies, the stimulation of NAD level is relevant to the reduced amyloid toxicity in AD animal models [15]. Therefore, in this study, the potential therapeutic effects of NMN and the mechanisms of its action regulated in JNK in APPswe/PS1dE9 mice with AD were investigated.

Materials and Methods

Animals

The Institutional Animal Experiment Committee of Tongji University, China, approved all procedures conforming to the Animals’ Use and Care Policies. APPswe/PS1dE9 transgenic mice (6 months old) were purchased from Beijing Bio-technology, China. All animals were maintained in an environment that was pathogen-free. During the experimental period, water and food were accessible to all mice, and the body weight of mice and the intake of food and water were identified at the beginning of the study and then on a weekly basis. In addition, all mice that receive the treatment were observed for their general health. APPswe/PS1dE9 transgenic mice (AD-Tg) and their nontransgenic wild-type mice (NTG) were randomly assigned into four groups with six mice in each group, and each type was treated by NMN and vehicle, respectively Subcutaneous adiministration of NMN (100 mg/kg, Sigma N3501) in sterile (Phosphate Buffered Saline) PBS (200 μl) was applied to each mouse of NMN-treated groups every other day for 28 days. Each mouse with vehicle treatment subcutaneously received sterile PBS (200 μl) every other day for 28 days.

Behavioral Tests

Behavioral tests were carried out by 2 experimenters who were blinded to the treatments twelve weeks after the treatments.

Memory and spatial learning test

To evaluate the memory and spatial learning of all animals, a Morris water navigation task was performed as described previously [16]. Generally, a tracking system (Water 2020; HVS Image, Hampton, UK) was utilized to monitor the trajectory of all mice. During the training trials, a platform with the diameter of 5cm was hidden 1.5 cm below the surface of water and maintained at the same quadrant. In every trial, all mice had at most 1 minute to find the hidden platform and climb onto it. If one mouse cannot find the platform within 1 minute, the experimenters would manually guide the mouse to the platform and kept it there for 10 seconds. The trial was carried out 4 times daily for 6 days. The escape latency referring to the time that a mouse spent in finding the platform is considered as spatial learning score. Following the last training trial, the probe trial was carried out for spatial memories by allowing animals to take a free swim in the pool with the platform removed for 1 minute (swim speeds are equal). The time that each animal took to reach the previously platform-contained quadrant was measured for spatial memories.

Measurement of Passive Avoidance

To assess contextual memories, passive avoidance test was carried out, which was described in the previous studies [17]. Briefly speaking, a two-compartment apparatus with one brightly lit and one dimly lit was used. During the training trial, the animal was put into the light lit compartment. After 60 seconds, the door between the two compartments was opened. The acquisition latency refers to the first latency time of mice to ran into the dimly lit compartment. After coming into the lit compartment, mice were exposed to a mild foot shock (0.3mA) for 3 seconds with the door closed. After 5 seconds, the animals were taken out of the compartment. One day later after the acquisition trial, the mice underwent a retention test. Like in the acquisition test, the latency to go into the dark compartment without foot shock was regarded as retention latency to test retention memory. Longer latency indicates better retention.

Tissue Preparation

Following the two behavioral tests, 24 mice were first anesthetized and then infused with icy normal saline in a transcardial way. The brains were taken out and cut into 2 hemibrains along the midsagittal plane. One of the hemispheres was kept in PBS with 4% paraformaldehyde. Following the xylene treatment, the other fixed hemisphere was maintained in the paraffin for immunohistochemical tests. Then the cerebral cortex and the hippocampus were separated quickly from the hemisphere on the ice. For biochemical tests, they were maintained at −80°C following the separation. The hippocampus, brain cortex, and as well as the whole brain were weighed, respectively.

Immunohistochemistry

Immunohistochemical staining was carried out as described [16]. Briefly speaking, 10 μm brain slices were deparaffinized and rehydrated. To retrieve antigens, proteinase K (200μg/ml) was treated for the staining of Aβ, and sodium citrate (0.01M, pH 6.0) was for the staining of microglia and astrocyte. Sections were blocked through incubation with fetal bovine serum (2%) and Triton X-100 (0.1%) for nonspecific binding. For immunohistochemical analysis, the section was incubated at 4°C for a night with anti-Aβ1-16 monoclonal antibody (1:600; Cell Signaling Technology, Massachusetts, USA) and monoclonal antibody anti-Iba1 (1:1,000; Osaka Wako Pure Chemical Industries, Japan) for rabbits and also monoclonal anti-Aβ antibody (1:200; Billerica, MA) for mouse.

Olympus (Tokyo, Japan) microscope with a connection to a digital microscope camera was applied to capture the images for quantitative analyses. The plaques in μm2s and the proportion of area kept by plaques positive to Aβ1–16 respectively, microglia positive to Iba1 were obtained with imaging software (Bethesda Media Cybernetics, MD). The mean value of every parameter was obtained from 6 sections with an equidistant interval of 150μm through the hippocampal region of each mouse in all groups. All measurements were blindedly conducted.

Enzyme-Linked Immunosorbent Assay (ELISA)

As described before, soluble Aβ fractions and insoluble ones were obtained from both the cortex and hippocampi of brain homogenates of mouse using RIPA (Radioimmunoprecipitation assay buffer) buffer and formic acid, respectively [18]. The levels of both the insoluble and soluble Aβ were identified using the ELISA kits (Camarillo Invitrogen, CA). Besides, concentrations of oligomeric Aβ of brain homogenates treated with RIPA were obtained employing an ELISA kit for amyloid β oligomer (Gunma Immuno-Biochemical Laboratories, Japan).

Proinflammatory Cytokines Measurement

As described, mouse brain proinflammatory cytokine was evaluated [19]. The expressions of TNFα, IL-6, and IL-1β were identified with immunoassay kits (Minneapolis R&D Systems, Minnesota, USA) which is for measuring these factors in mouse.

Western blotting (WB) analysis

The cortex and hippocampus tissue was homogenized with icy PBS and the lysate was for Western blot. At first SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) was applied to divide the proteins on NuPage Bis-Tris gel (12%, Invitrogen). The separated protein was subsequently transferred to nitrocellulose membrane which was blocked with 5% nonfat milk and probed overnight at 4°C with anti-p-JNK, anti-JNK, anti-APP, anti-sAPPα, anti- sAPPβ, anti-phosphorylated APP (p-APP, Thr668), anti-ADAM10, anti-BACE1 (CA Santa

Cruz Biotechnology, USA), anti-CDK5, anti–p-CDK5, anti–p-GSK3β, anti-GSK3β, anti-SYP, anti–postsynaptic density-95, anti–β-actin Abcam (Cambridge, MA, USA). The membrane was cleaned with TBS/0.05% Tween-20 and incubated at room temperature with secondary antibodies conjugated with horseradish peroxidase for 60 minutes, following incubation with primary antibodies. Enhanced chemiluminescence reagents (Pierce, Rockford, IL, USA) were used for detecting signals.

Statistical Analysis

The data were expressed as mean ± SD. The comparisons in the speed of swimming and escape latency between the groups during the test of memory and spatial learning were made employing two-way ANOVA with repeated measures. Next, post hoc least significant difference (LSD) test was used for multiple comparison. Before post hoc LSD test or Student t test, 1-way ANOVA was employed for the rest data. Statistical analyses were carried out with Prism version 5. A P < 0.05 was considered statistically significant.

Results

NMN Treatment Rescues Cognitive impairments in AD-Tg Mice

The test of memory and spatial learning has shown that 1-year-old AD-Tg mice have experienced impairment in memory and spatial learning [16]. In our study, comparied to the vehicle-/NMN-treated wild-type (WT) mice, the vehicle-treated AD-Tg mice had a longer escape latency, which showed severe impairment of spatial learning in the test (Fig. 1A). However, with shorter escape latency, NMN treatment greatly improved the impairment of spatial learning in vehicle-treated AD-Tg animals (Fig. 1A). Besides, it was also identified that compared with two wild-type groups, vehicle-treated AD-Tg animals spent less time in the target quadrant during the probe trial (p < 0.01), suggesting severe spatial memory impairment. AD-Tg mice treated with NMN spent longer time in the target quadrant, which indicates marked alleviation of the spatial learning impairments present in AD-Tg mice treated with vehicle (p<0.01) (Fig. 1B).

To further identify the alleviation of memory deficits by NMN treatment in AD-Tg mouse, contextual memories were evaluated employing the measurement of passive avoidance [17]. As illustrated in Fig. 1C, retention latency was decreased compared with two wild-type mice groups (p < 0.01), suggesting impaired contextual memories in the AD-Tg animals treated by vehicle. In contrast, NMN-treated mice exhibited longer retention latency compared with those treated with vehicles (p < 0.01), demonstrating outstanding reversal of NMN in contextual memories. All these data indicate that NMN treatment markedly improves cognitive impairments in AD-Tg animals.

NMN Suppresses JNK Phosphorylation in AD-Tg Mice

JNK, also called a protein kinase activated by stress, is said to play a role in a couple of pathophysiological processes in AD [13]. Therefore, in this study, we tested the inhibitory effects of NMN on the activation of JNK through Western blotting. It was revealed by quantitative analysis that p-JNK level was significantly grown in hippocampus and cerebral cortex in the vehicle-treated AD-Tg mice when contrasting to two wild-type groups (Fig. 2A; p < 0.01), whereas NMN gave rise to a sharp decline in p-JNK in hippocampus and cerebral cortex with a comparison to the vehicle-treated AD-Tg mice (Fig. 2B; P< 0.01). Both reductions symbolized a reverse to the wild-type level. But the whole expression of JNK kept unchanged in all the 4 groups. Conclusively, all data indicate that NMN treatment has an inhibitory effects on JNK activation in AD-Tg mice.

NMN Treatment Decreases the Level of Aβ and Deposition in AD-Tg Mice

The role of reduced activation of JNK in the changes of the Aβ level and deposition was studied in AD-Tg mice through employing histological and biochemical analyses. As presented in Figs. 3A-D, it was found that NMN-treated AD-Tg mice had a sharp reduction in the levels of Aβ when comparing to the vehicle treatment group (p < 0.01). Comparing to the vehicle treatment group, NMN treatment gave rise to a marked decrease in Aβ oligomers (p < 0.01) (Fig. 3E). Immunohistochemical staining identified this observation, indicating the lessened diffuse plaques and also the shrinked area taken by diffuse plaques in AD-Tg mice treated by NMN compared to the vehicle treatment group (Figs. 4A-D). Thus, on the basis of the findings, it was demonstrated that the generation of Aβ in the brain of AD-Tg mice is effectively decreased by the inhibited activation of JNK with NMN treatment.

NMN Treatment Changes the Processing of APP in AD-Tg Mice

To study the mechanism of inhibition on the production of Aβ and deposition, the effects of NMN on the processing of APP were examined by Western blotting. As presented in Figs. 5A-C, the level of full-length APP expression was greatly increased in the brain of AD-Tg mouse treated with vehicle compared with wild-type ones (p < 0.01). However, they kept unaltered between the group treated with vehicle and that with NMN. Importantly, it was found that NMN treatment remarkably lowered the increased levels of p-APP in the AD-Tg mice treated by vehicle (p < 0.01). Besides, α-secretase cleaved sAPPα and β-secretase cleaved sAPPβ in the brain tissues of Tg mice were tested via Western blotting. It was shown by quantitative analyses that NMN treatment led to a remarkable elevation of sAPPα (p < 0.01) and a marked decline in sAPPβ (p < 0.01) compared with the transgenic mice treated by vehicle (Figs. 5D-F). Based on these data, it was indicated that NMN treatment is strongly effective in suppressing the phosphorylation of APP, improving cleaving of APP by α-secretase, and decreasing the cleaving of APP by β-secretase in AD-Tg mice brains.

NMN Treatment Improves Inflammatory Responses in AD-Tg Mice

Since JNK activation is indicated to play a role in the inflammatory response induced by Aβ in previous studies [20], whether reduced activation of JNK influences neural inflammation in AD- Tg animals was investigated. The role of NMN on the neural inflammation was identified by measuring proinflammatory cytokines that were in the lysates of cortical tissues. It was found that the level of IL-6, IL-1β, and TNFα were sharply declined in the AD-Tg mice treated by NMN relative to those by vehicle (Figs. 6A-C). According to these findings, NMN treatment is indicated to be potently effective in the amelioration of neural inflammation in AD-Tg mice brains.

NMN Treatment Ameliorates Synaptic Loss in AD-Tg Mice

The loss of synapse is an important pathological characteristic of AD and said to be relevant to the cognitive impairments of AD [21]. The changes in SYP (presynaptic marker) level and PSD- 95 level (postsynaptic marker) were investigated via Western blotting. It was showed by quantitative analysis that SYP levels and the levels of PSD-95 expression substantially reduced in hippocampus and brain cortex of AD-Tg mice treated with vehicle relative to WT ones

(p < 0.01), whereas NMN treatment significantly elevated SYP levels and the levels of PSD-95 expression in hippocampus and brain cortex relative to AD-Tg mice treated with vehicle (p < 0.01) (Fig. 6D-F). This finding suggest that NMN treatment sharply ameliorates the loss of synapse in AD-Tg mice brains.

Discussion

In the present study, it is mainly found that NMN treatment substantially improves primary pathological characteristics of the AD-modeled AD-Tg mice, including cognitive impairments, neuroinflammation, Aβ pathology, and synaptic loss, which consistent with a recent study [22]. It was also found that NMN treatment inhibited JNK activation and amyloidogenic processing of APP by mediating the expression of APP-cleavage secretase, and also facilitated APP processing in AD-Tg mice. The data prove that NMN treatment greatly reduces multiple AD-associated pathological characteristics, at least partially by the inhibition of JNK activation.

Numerous studies have reported the increase of abnormal activation of JNK in both the transgenic AD mice models and the AD patients [23-25]. Conforming to the above previous studies, we also found that the level of phosphorylated JNK in AD-Tg mice treated by vehicle was higher than that in the wild-type group, but NMN treatment in AD-Tg mice potently suppressed the phosphorylation of JNK to the basic level of WT groups. The controlled activation of JNK through NMN gave rise to a substantial decrease of Aβ pathology in AD-Tg animals. According to the studies before, active JNK is proved to engage in BACE1 expressions and PS1 expressions [26, 27]. In addition, the increased BACE1 and PS1 in AD-Tg mice treated by vehicle were found to be greatly suppressed by NMN to the basic level of WT groups (data not shown). More interestingly, it was also observed that NMN treatment led to substantially elevated sAPPα and reduced sAPPβ. It was notable that according to the previous studies, APP phosphorylation at the site of Thr668 is proved to promote the β-secretase cleavage of APP to grow Aβ generation in vitro [28]. In present study, we also found that the administration of NMN in AD-Tg mice significantly declined the elevated phosphorylation of APP to the primary level of WT controls, indicating an in vivo inhibition mechanism of Aβ pathology through NMN treatment. Collectively, all these findings indicate that the potent effects of NMN on the marked decrease in Aβ pathology in the brains of AD-Tg mice may be responsible for its enhancement of nonamyloidogenic APP processing. What we found is consistent to a recent study demonstrating that genetic depletion of JNK3 in 5XFAD mice is attributed to a significant decrease in the levels of Aβ and the total plaque loads [29]. Recently numerous studies suggested energy failure and accumulative intracellular waste also play a causal role in the pathogenesis of several neurodegenerative disorders and Alzheimer’s disease (AD) in particular regulated by potential role of several metabolic pathways Wnt signaling, 5′ adenosine monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), Sirtuin 1 (Sirt1, silent mating-type information regulator 2 homolog 1), and peroxisome proliferator-activated receptor gamma co- activator 1-α (PGC-1α) [30, 31]. It will be warrant to study if NMN also participate in regulation of these signaling pathways.

Some recent studies indicate that enhanced neuroinflammation is essential to the development of AD [32-34]. In our study, a marked decline in the proinflammatory cytokines levels (IL-6, IL-1β, and TNFα) proved that NMN treatment effectively controlled the neuroinflammatory responses of the brain of AD-Tg mouse. Considering the key role of oligomeric and fibrillar Aβ for activation of microglia cells and astrocytes with the subsequent generation of proinflammatory cytokines [34], the reduction in neuroinflammatory responses may be less important to the substantial reduction in Aβ pathologies presented in the AD-Tg mice treated by NMN. Several previous studies had proved that JNK represents an important mediator for activation of glial cell and proinflammatory cytokines [35, 36]. Thus, the favorable effects of NMN on lowered inflammatory responses in AD-Tg groups can be largely responsible for its direct control of inflammation by inhibiting JNK activation. Based on the previous reports, it was proved that some proinflammatory cytokines (ie, IL-1β, interferon gamma, and TNFα) may elevate the expression of β-secretase and γ-secretase to ameliorate amyloidogenic APP processing and Amyloid-β production by an in vitro JNK-mediated pathway [33]. Hence, we have reasons to believe that the reduced proinflammatory cytokines through NMN treatment may be effective in reducing the production of Aβ in vivo. Moreover, in our study, it was demonstrated that NMN treatment ameliorates cognitive impairments in AD-Tg mouse models. An increasing evidence has proved that grown Aβ levels, neuroinflammation, synaptic dysfunction and loss are closely related to the cognitive dysfunction in AD [37]. In addition, our data confirms the finding of a recent research, which revealed that genetic down-regulation of JNK3 gives rise to a remarkable amelioration of cognitive impairments in 5XFAD mice [29]. Collectively, our findings, along with all the previous research, demonstrate that the inhibited JNK activaty by NMN is potently effective in ameliorating AD-associated cognitive deficits.

Synaptic loss is a major pathological change of AD and is tightly associated with AD-related cognitive impairments [37]. It was presented that PSD-95, a biomarker of postsynaptic density, is essential to synapse maturation and synaptic plasticity [38], and that SYP, a presynaptic protein, also acts as an integral membrane protein in the synapse and it plays a key role in plasticity of synapses [39]. Therefore, it can be soundly supposed that the greatly lowered expression of PSD- 95 and SYN presented in the study may suggest the impairment of synaptic integrity and  plasticity in AD-Tg mice treated by vehicle. Intriguingly, the treatment of NMN in AD-Tg animals substantially elevated the lowered PSD-95 and SYN expression level back to the primary level of WT controls. Since it was demonstrated by several studies that Amyloid-β- induced synaptic loss and dysfunction are regulated through the JNK activation [40, 41], the possible mechanisms behind NMN treatment leading to the elevated expression of PSD-95 and SYN in AD-Tg animals may be responsible for its inhibitory effects on JNK activation. Thus, it is possible that the treatment of NMN may ameliorate the impaired synaptic plasticity which is caused by toxic Aβ species in AD-Tg mice.

In summary, this study provides essential preclinical evidences that NMN takes effects in reversing cognitive deficits and substantially lowering the burden of amyloid plaque, neuroinflammation, cerebral amyloid-β concentrations, and loss of synapse in middle-aged AD- Tg mice, at least partially by the inhibition of JNK activation. According to our findings, NMN could be a new target for disease-modifying treatments of AD.

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Mitochondrial dysfunction and the inflammatory response

The .pdf version of this research can be found at https://www.researchgate.net/

1. Introduction
Inflammation, a basic biological response against invading pathogens, contributes to repairing damage and preventing further tissue or cell injury (Medzhitov, 2008). However, when the inflammatory response cannot be appropriately attenuated, multi-organ failure or chronic inflammation can develop (Medzhitov, 2008).

In this sense, increasing levels of pro-inflammatory cytokines have been associated with disease evolution and unfavorable outcomes in several pathological scenarios, such as chronic heart failure, peritoneal dialysis, pancreatitis, and cancer (Berres et al., 2011; Gravante et al., 2009; Hiller et al., 2011; Nevado et al., 2006; Yndestad et al., 2012).

While each of these disorders has a specific pathogenesis and disease evolution, they all involve inflammatory processes. In addition, the inflammatory process also has a role in mediating the aging process via changes in redox status and oxidative stress-induced inflammatory responses (Gilca et al., 2007); this process has been called inflammaging (Salminen et al., 2012). In fact, aging and low-grade inflammation involves many of the same molecular pathways, including oxidative stress and DNA damage pathways.

The molecular inflammation hypothesis of aging postulates that chronic low-grade inflammation in aging causes cumulative damage to biological macromolecules through the formation of oxygen radicals (Chung et al., 2009).

This process would eventually render cells more susceptible to damage and more sensitive to catabolism, processes associated with an exacerbated response to age-related diseases (Chung et al., 2009; Jian et al., 2011; Loeser, 2009). Hence, the risk of degenerative diseases and poor outcomes of acute processes rise exponentially with age.

This is also supported by studies showing that healthy centenarians exhibit low levels of inflammatory biomarkers (Franceschi, 2007).

Altered mitochondrial function is linked to several acute and chronic inflammatory diseases (Naik and Dixit, 2011). Additionally, mitochondria have long been proposed to play a key role in aging (Green et al., 2011).

As a consequence of their central role in ATP formation via the mitochondrial respiratory chain (MRC), mitochondria are the major source of reactive oxygen species (ROS) and are thus highly involved in oxidative stress processes (Brookes et al., 2004). However, mitochondria are also targets of these molecules (Brookes et al., 2004).

Under physiological conditions, about 1–3% of molecular oxygen is incompletely reduced during redox reactions in the MRC, and this in turn leads to production of the ROS superoxide anion (O2−) as a by-product of these reactions. In this scenario, complex interactions in antioxidant defense systems repress oxidative stress within mitochondria (Kienhöfer et al., 2009).

However, under pathological conditions, an excess of O2− ions are produced, which causes the activation of redox-sensitive transcription factors, such as the key regulator of tissue inflammation, nuclear factor-κB (NF-κB), and a subsequent increase in the expression of cytokines, chemokines, eicosanoids, inducible nitric oxide synthase (iNOS), and adhesion molecules (Chung et al., 2009).

In addition to alteration of signaling pathways regulated by ROS, the excess of ROS also drives oxidation of membrane lipids, proteins, and mitochondrial DNA (mtDNA) (Henze and Martin, 2003).

In particular, mtDNA seems to be especially sensitive to oxidative damage, and therefore susceptible to mutations, because of its close proximity to the site of ROS production and the lack of protective histones. In fact, aged individuals, whose cells have accumulated a high level of oxidative damage, exhibit an increased rate of mutagenesis in their mtDNA (Kujoth et al., 2005).

Moreover, superoxide anions can combine with nitric oxide (NO) to produce peroxynitrite (ONOO-), a powerful oxidant capable of affecting mitochondrial integrity (Brown, 2003; Escames et al., 2011).

Mitochondrial dysfunction drives mitochondrial genome mutagenesis, affecting genes encoding respiratory chain complexes and compromising the efficiency of oxidative phosphorylation (OXPHOS), which may lead to further mtDNA mutations and even mutations in the nuclear genome, thereby causing increased cell damage (Escames et al., 2011).

In this review, we aim to highlight the significance of mitochondrial dysfunction and to discuss the contribution of mitochondria to the development of inflammatory human diseases and the aging process.

2. Mitochondrial dysfunction may modulate inflammatory processes

It is well known that mitochondria are not only the primary source of ATP, but also participate in many other cell signaling events, such as regulation of calcium (Ca2 +) homeostasis, orchestration of apoptosis, and production of ROS, as mentioned above (Brookes et al., 2004).

In this sense, ROS are the major host defense mechanism against infection and harmful agents (Chen et al., 2012), and mitochondria are the principal mediators of inflammation (Tschopp, 2011).

During viral infection the pattern recognition receptors RIG-I and MDA5 attach to viral RNA allowing its interaction with a mitochondrial polypeptide adaptor, MAVS, which finally drives the production of type I interferon (Saitoh and Akira, 2010).

Interestingly, pathogens have recently been shown to have developed their own reciprocal defense systems; for example, Leishmania infection has been shown to cause marked upregulation of uncoupling protein 2 (UCP2), a negative regulator of mitochondrial ROS generation, potentially preventing ROS-mediated inactivation to suppress macrophage defense mechanisms and facilitate parasite survival (Basu Ball et al., 2011).

However, under dysregulated processes, when mitochondria are compromised by damage or mutations, excess O2− ions are produced, and cellular stress cannot be effectively resolved. Thus, mitochondria are the principal arbitrators of the pro-inflammatory status; they act through modulating innate immunity via redox-sensitive inflammatory pathways or direct activation of the inflammasome, a group of protein complexes whose activation results in immediate activation of caspase-1, thereby allowing for cleavage and subsequent activation of the inactive precursors IL-1β and IL-18 (Strowig et al., 2012).

Additionally, both of these pathways, i.e., the redox-sensitive inflammatory pathway and the inflammasome pathway, may work together to activate inflammatory cytokines, leading to an overstimulation of the inflammatory response (Escames et al., 2011).

Taken together, these recent studies have suggested the unexpected supplementary role of mitochondria as drivers of the inflammatory process.

2.1. Activation of redox-sensitive inflammatory pathways by mitochondrial impairment
Cellular systems that protect against oxidants involve antioxidative defense enzymes (superoxide dismutase [SOD], glutathione peroxidase [GPx], and catalase) (Kienhöfer et al., 2009), oxidant scavengers (vitamin E, vitamin C, carotenoids, uric acid, and polyphenols) and mechanisms to repair oxidant-induced damage to lipids, proteins, or DNA (Karger et al., 2012; Ugarte et al., 2010).

Despite these protective mechanisms, uncontrolled generation of ROS can overwhelm the capacity of cellular antioxidant protection causing mitochondrial dysfunction. In this sense, in vivo studies in transgenic mice showed that the overexpression of catalase targeted to mitochondria reduces age-associated pathologies and the mice have an extension in lifespan (Schriner et al., 2005).

Additionally, several studies have demonstrated that mitochondrial dysfunction may generate low-grade inflammatory and matrix degradation responses in several cell types via mitochondrial Ca2 + exchange, ROS generation, and NF-κB activation (Amma et al., 2005; Cillero-Pastor et al., 2008; Ichimura et al., 2003).

Thus, the flow of mitochondrial Ca2 + plays a crucial role in the evolution of cell signals and in the regulation of mitochondrial activity. Moreover, excessive mitochondrial Ca2+ accumulation has been shown to be critical for disease development (Dada and Sznajder, 2011; Tanaka et al., 2004).

For example, increased mitochondrial Ca2+ levels in cardiomyocytes contribute to cardiac inflammation and dysfunction after burn injury or sepsis (Maass et al., 2005).

Additionally, when cells are treated with inhibitors of mitochondrial Ca2 + exchange, ROS levels decrease significantly, leading to subsequent reductions in the levels of pro-inflammatory mediators. The rise in mitochondrial Ca2 +, a consequence of importing cytosolic Ca2 +, augments the mitochondrial production of ROS by different mechanisms, including 1) Ca2+ stimulation of the tricarboxylic acid cycle, which would enhance electron flow into the respiratory chain; 2) Ca2 + stimulation of NO synthase, elevating NO levels, which would in turn inhibit respiration at complex IV and enhance ROS generation; and 3) Ca2+ binding to cardiolipin (Brookes et al., 2004).

Thus, ROS production by mitochondrial electron transport could represent an intermediate step in pro-inflammatory gene expression/NF-κB signaling.
Mitochondrial dysfunction has also been shown to increase the responsiveness of several types of cells to cytokine-induced inflammatory responses, resulting in a significant amplification of the inflammatory response through ROS generation and NF-κB activation (Vaamonde-García et al., 2012; Nakahira et al., 2011).

When mitochondrial dysfunction was induced in normal synovial fibroblasts by an inhibitor of mitochondrial ATP synthase, oligomycin, an otherwise less-efficient concentration of IL-1β was as effective as a 10-times greater concentration of IL-1β would be in the absence of pretreatment with the mitochondrial inhibitor (Valcárcel-Ares et al., 2010).

In lung epithelial cells, pre-existing mitochondrial dysfunction induced by antisense oligonucleotides to complex III increased mitochondrial ROS generation, resulting in marked potentiation of ragweed pollen extract-induced accumulation of inflammatory cells in the airways (Aguilera-Aguirre et al., 2009).

In other cell types, mitochondrial impairment also increased the generation of ROS, resulting in potentiation of cytotoxicity or inflammatory cell accumulation (Bulua et al., 2011; Schulze-Osthoff et al., 1993).

Moreover, several studies have reported enhanced sensitivity to NF-κB activation in the context of mitochondrial dysfunction, suggesting that NF-κB binding sites may be essential for inflammatory gene expression (Ungvari et al., 2007; Vaamonde-García et al., 2012).

As expected, this inflammatory response is modulated by antioxidants. Additionally, these in vitro studies are supported by in vivo results. Thus, the effects of in vivo NF-kB inhibition (with pyrrolidinedithiocarbamate, PDTC or resveratrol) in aged rats significantly attenuated inflammatory gene expression and inhibited monocyte adhesiveness in vessels (Ungvari et al., 2007).

2.2. Mitochondrial modulation of the NLRP3 inflammasome.
Recent studies have shown mitochondria as important players in NLRP3 inflammasome activation (Green et al., 2011; Kepp et al., 2011; Nakahira et al., 2011; Shimada et al., 2012; Zhou et al., 2011).

Cell damage can initiate the accumulation of damage-associated molecular patterns (DAMPs), molecules recognized by the innate immune system and able to develop an inflammatory response (Krysko et al., 2011b) through the framework of the inflammasome and subsequent caspase-1 activation and pro-inflammatory cytokines release. Mitochondria have been recently identified as key sources of DAMPs (mito-DAMPs) playing a role in DAMP-modulated inflammation in different disorders, such as systemic inflammatory response syndrome (SIRS), rheumatoid arthritis (RA), cirrhosis, cancer, and heart diseases (Green et al., 2011; Zhang et al., 2010; Zitvogel et al., 2012), as well as the aging process (Salminen et al., 2012).

DAMPs activate the same receptors that detect pathogen-associated molecular patterns (PAMPs) (Krysko et al., 2011), such as Toll-like membrane receptors (TLRs) and cytoplasmic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs).

One of the best-studied NLRs, because of its solid association with several inflammatory diseases, is NLRP3. NLRP3, a cytosolic receptor, is one of 22 described human NLR family members; it functions to monitor the cytosol for stressful situations and has a crucial role in regulating immune responses (Kepp et al., 2011; Schroder and Tschopp, 2010; Shimada et al., 2012; Tschopp, 2011).

Once activated, the NLRP3 receptor induces NLRP3 oligomerization and recruitment of the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and procaspase-1, generating a multiprotein platform called the NLRP3 inflammasome. Then, NLRP3 activates caspase-1 and in turn drives the maturation of pro-inflammatory cytokines, such as IL-1β and IL-18. The NLRP3 inflammasome also conducts caspase-1-mediated cleavage of other substrates such as cytoskeletal proteins, glycolytic enzymes and caspase-7 (Tschopp, 2011; Zitvogel et al., 2012).

Mitochondria through ROS generation act as integrators across different stimuli activating the NLRP3 inflammasome (Kepp et al., 2011).

Increased levels of ROS are essential for NLRP3 activation; therefore, scavenging ROS with different chemicals suppresses inflammasome activation (Tschopp, 2011). Initially, Tschopp’s group, performing experiments in the human acute monocyte leukemia cell line, THP-1, suggested that NLRP3 inflammasome-activating ROS were generated by an NADPH oxidase stimulated upon particle phagocytosis, since in their model, ROS production was inhibited by NADPH oxidase inhibitors (Dostert et al., 2008).

However, later experiments performed by the same group (Zhou et al., 2011) and others showed that this result was ambiguous since immune cells from chronic granulomatous disease (CGD) patients, which have defective NADPH activity and thereby lack NADPH-dependent ROS generation, achieved normal levels of active caspase-1 and, in turn, active IL-1β.

Thus, these studies provided evidence of the existence of an alternative source of ROS (Meissner et al., 2010; van Bruggen et al., 2010; van de Veerdonk et al., 2010).

Since mitochondria are the main source of ROS, it is not surprising that mitochondria could also be the principal initiator of NLRP3 inflammasome activation. For example, the subcellular localization of NLRP3 suggests an important link between mitochondria and NLRP3. Under unstimulated conditions, most NLRP3 and ASC proteins are associated with the endoplasmic reticulum (ER).

Once the inflammasome is activated, NLRP3 redistributes to the perinuclear space and colocalizes with mitochondria (Zhou et al., 2011). Additionally, inhibition of voltage-dependent anion channels (VDACs), which are regulated by Bcl-2 family members through a decrease in mitochondrial Ca2 + levels and the resulting ROS production, significantly decreases NLRP3 inflammasome activation (Zhou et al., 2011).

Intriguingly, VDAC is also required for pro-apoptotic activity by Bax.
Other danger signals, specifically mtDNA and ATP, have been implicated in NLRP3 inflammasome activation; these signals can be released following different types of cell death (discussed below).

Indeed, it has been reported that mtDNA in the plasma of patients with femur trauma reaches values several thousand times higher than that of healthy volunteers, with functionally important immune repercussions (Zhang et al., 2010).

Additionally, mtDNA has been detected in RA synovial fluid and induces in vivo and in vitro inflammatory responses (Collins et al., 2004). Also, recent results have identified degraded mtDNA as a new DAMP subtype and a possible trigger of neurodegeneration (Mathew et al., 2012).

With regard to ATP, studies have shown that shedding of ATP from necrotic hepatocytes activates the NLRP3 inflammasome to generate an inflammatory microenvironment, alerting circulating neutrophils to adhere within liver sinusoids (McDonald et al., 2010).

Also, it has been shown that the release of extracellular ATP during apoptosis could be required for the successful immunogenic tumor cell death elicited by cancer treatment (Hahn et al., 2012).

Moreover, ATP can decrease intracellular K+ concentrations, a necessary event for NLRP3inflammasome activation, and mitochondria could also have a crucial role in modulating this process since they possess several K+ channels and can modulate intracellular K+ levels (Heinen et al., 2007).

Finally, NFPs is also considered a crucial mito-DAMP in several pathologies, including SIRS; this molecule can attract and stimulate neutrophils through high-affinity formyl peptide receptors (Zhang et al., 2010).

Interestingly, DAMPs and mito-DAMPs can activate antigenpresenting cells (APCs), as well as other nonimmune cell types, such as mesenchymal stem cells and astrocytes (Mathew et al., 2012; Pistoia and Raffaghello, 2011). In one study, degraded mtDNA strongly induced pro-inflammatory IL-1β, IL-6, MCP-1, and TNFα in mouse primary astrocytes, demonstrating the role of degraded mtDNA in inflammasome activation (Mathew et al., 2012).

The NLRP3 inflammasome and NF-κB pathways could work together to activate inflammatory cytokines, leading to overstimulation of the inflammatory response (Escames et al., 2011).

Once constituted, inflammasomes activate inflammatory caspases, mainly caspase-1, and subsequently induce IL-1β and IL-18 secretion. Then, pro-inflammatory cytokines can also activate NF-κB, generating a vicious cycle that increases the level and duration of the inflammatory response. The activation of inflammasomes may represent a possible explanation for this synergistic effect between mitochondrial impairment and cytokines.

Thus, studies investigating the role of mitochondria in inflammasome activation are revealing novel concepts in the development of inflammatory response, and these mechanisms may be of help in explaining the usual association of mitochondria with several inflammatory disorders and the aging process.

2.2.1. Role of mitochondria in the inflammatory response modulated by cell death.
Depending on the type of cell death, the dying cells could release and present at their surface specific signals that could modulate the type of immune response (Zitvogel et al., 2010).

Mitochondria have a key role in cell death (Kepp et al., 2011). Cell death can result from a variety of processes, including the apoptotic pathway and the necrotic pathway, 2 well-described extremes.

Apoptosis involves a series of orchestrated events requiring protein synthesis and ATP consumption. The dying cell forms vesicles that fragment into several apoptotic bodies; these are phagocytosed by macrophages or adjacent cells, thus avoiding the inflammatory reaction. Interestingly, contradictory recent data show that this paradigm is not always true, and depending on the inflammatory scenery, apoptosis could elicit a powerful inflammatory response (Shimada et al., 2012).

In particular, this study provides a link between apoptosis and inflammasome activation via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome. In this sense, Nakahira et al. also find that cyclosporine A, which modulates intrinsic apoptosis through inhibition of mitochondrial permeability transition, prevents the release of IL-1β by apoptotic stimuli (Nakahira et al., 2011).

Moreover, in vitro studies showed that silencing of antiapoptotic protein Bcl-2 increases IL-1β secretion while Bcl-2 overexpression resulted in the contrary (Shimada et al., 2012). In contrast, necrosis results from a rapid loss of cell homeostasis as intracellular ATP stores become depleted.

Cells increase their volume, leading to loss of cell membrane integrity and leakage of cellular components, thus inducing inflammation. In this study by Shimada above, necrotic stimuli did not secrete IL-1β (Shimada et al., 2012).

Mitochondrial DAMPs can also trigger autophagy. Autophagy is usually considered a type of cell death, although this process is thought to be an attempt to adapt and survive during periods of stress, such as nutrient deprivation, hypoxia, etc. (Edinger and Thompson, 2004).

Autophagy can control inflammation through macrophage-mediated clearing of apoptotic corpses or by inhibiting NLRP3 inflammasome activation by removing permeabilized or ROS-producing mitochondria (mitophagy). Thus, the inhibition of mitophagy results in spontaneous inflammasome activation (Goldman et al., 2010; Levine and Kroemer, 2008; Nakahira et al., 2011). Besides, mitochondria also play an important role in autophagia as they supply membranes for the biogenesis of autophagosomes (Hailey. 2010). Decreases in the expression of several autophagy-related genes, as well as the presence of mutations in these genes, have been found both in different inflammatory diseases, including neurodegenerative diseases, cardiovascular diseases, and Crohn’s disease (Gegg and Schapira, 2011; Travassos et al., 2010), and in the aging process (Salminen et al., 2012). For example, alterations in PINK1 and Parkin genes, which cooperate to identify and label damaged mitochondria for selective degradation via autophagy or mutations in ATG16 and IRGM, respectively, have been described. Additionally, the in vivo relevance of autophagic regulation of caspase-1 mediated inflammatory responses has been shown in two animal models of sepsis (the endotoxic shock model and the cecal ligation and puncture model of polymicrobial sepsis) (Nakahira et al., 2011). Furthermore, autophagy also declines during aging, generating the inflammatory condition via activation of inflammasomes, which in turn can actually accelerate the aging process (Salminen et al., 2012).

Mitochondria are triggers for the apoptotic pathway; several soluble pro-apoptotic proteins, including cytochrome c, are released from the mitochondrial intermembranous space after mitochondrial outer membrane permeabilization (MOMP). This leads to the formation of the apoptosome and the subsequent activation of the caspase cascade, which will trigger the execution phase of apoptosis. On the other hand, the mitochondrial permeability transition (MPT), which depends on the mitochondrial matrix protein cyclophilin D (Nakagawa et al., 2005), results in the instantaneous dissipation of the mitochondrial transmembrane potential and cessation of OXPHOS, thus rapidly increasing ROS, depleting ATP levels, and triggering necrotic cell death (Green et al., 2011; Hahn et al., 2012; Sasi et al., 2009).

As mentioned before, mtDNA can directly bind NLRP3; however, the current results do not rule out that mtDNA may bind to other members of the NLRP3 complex. ATP released from the dying cell constitutes a “find me” signal that acts as a chemo-attractant for monocytes and macrophages. Apoptosis releases much higher levels of cellular ATP related to necrosis (Hahn et al., 2012). It is therefore possible that ATP binding would be necessary for mtDNA binding (Shimada et al., 2012). DAMPs are often modified by processes of proteolysis and oxidation, which are related to cell death mechanisms. In this sense, HMGB1, a member of the high mobility group box family of DNA, can be released by necrotic cells, triggering a powerful immune response. During apoptosis, the generation of ROS by mitochondrial oxidized HMGB1 in turn enabled its immunostimulatory capacity (Hahn et al., 2012; Krysko et al., 2011).

Overall, depending on the specific circumstances and environment, cells can suffer from different lethal stress pathways promoting specific inflammatory responses, i.e., sterile inflammation or pathogen-induced inflammation. A better understanding of these processes is necessary for the development of new successful therapies to mitigate inflammatory diseases.

3. Pro-inflammatory mediators impair mitochondrial activity
A great number of inflammatory mediators, including the cytokines TNFα and IL-1β and the reactive nitrogen intermediate NO, may induce mitochondrial damage (López-Armada et al., 2006a; Maneiro et al., 2005; Stadler et al., 1992; Zell et al., 1997). TNFα and IL-1β decrease the activity of MRC complex I, ATP production, and mitochondrial membrane potential (Δψm). These mediators also induce the accumulation of significant amounts of ROS (Guidarelli et al., 2007; Kim et al., 2010). Interestingly, complex I together with complex III have been suggested to be major sources of ROS (Guzy et al., 2005), and the activities of complexes II and IV were also decreased in other cell types (Biniecka et al., 2011; Maneiro et al., 2003). Moreover, in adipocytes, the changes induced by TNFα cause pronounced morphological changes in the mitochondria, presumably due to variations in the levels of several mitofusion proteins (Chen et al., 2010). Another important study by Rowlands et al. showed that the severity of inflammation in mouselung microvessels is modulated by the inhibitory effect of TNFR1 ectodomain shedding by mitochondrial Ca2+ (Rowlands et al., 2011). Thus, mitochondrial Ca2+ and ROS are primary mediators of TNFαmediated inflammatory responses (Dada and Sznajder, 2011).

NO is another particularly important mediator in the pathophysiology of inflammatory processes. A variety of NO donors have been shown to suppress mitochondrial energy production in different cell types. Moreover, most of the catabolic effects of NO are potentially related to the ability of NO to combine with the superoxide anion to generate peroxynitrite, which, as mentioned earlier, is a powerful oxidant capable of inhibiting important enzymes and affecting mitochondrial integrity (Fermor et al., 2010; Johnson et al., 2000; Maneiro et al., 2005). In particular, NO induced a significant, reversible decrease in the activity of complex IV as well as a reduction in Δψm (Tomita et al., 2001). NO can also irreversibly inhibit respiration, most likely through ATP synthase and the strong oxidant peroxynitrite, which inactivates all respiratory complexes. Other studies demonstrated that, in intact U937 cells, peroxynitrite enhances the formation of superoxide ions in the mitochondria through a Ca2+-dependent mechanism that involves the inhibition of complex III, which then dismutates to H2O2 (Guidarelli et al., 2007).

Mitochondria play an important role during apoptosis. As mentioned above, severe mitochondrial damage can even lead to cell death by necrosis or by activation of the apoptotic signaling pathway and related caspases (Cillero-Pastor et al., 2011; Irrinki et al., 2011; López-Armada et al., 2006b; Maneiro et al., 2005). Indeed, the classic signs of cell death are preceded by mitochondrial alterations, which include changes in MRC activities, loss of Δψm, decreases in energy production, and/or induction of the mitochondrial permeability transition (MPT), and all of these alterations are modulated by both cytokines and NO. Thus, such pro-inflammatory mediators can modulate cell survival by exerting their effects on mitochondria. In addition, mito-DAMPs can be generated as consequence of these cell death processes.

To summarize, mitochondrial complexes could suffer permanent alterations caused by mutations in DNA or inflammatory mediators, such as reactive nitrogen species (RNS). Other stimuli, including cytokines and NO, induce functional and reversible modification of mitochondrial complexes at physiological concentrations. The combination of both effects (permanent and functional alteration of mitochondrial complexes) may cause a more substantial modification of mitochondrial activity, establishing continual, sublethal mitochondrial damage and thereby generating a vicious cycle of increasing inflammation that is difficult to break. Therefore, preserving mitochondrial function could reduce excessive oxidative stress and may represent a novel therapeutic advantage for patients with inflammatory diseases and chronic low-grade inflammation in aging.

4. Physiological function of ROS in the regulation of cell signaling
Although ROS has been traditionally linked to disease states, its crucial roles in normal physiological processes (vascular tone, oxygen sensing, and skeletal muscle physiology) and protective mechanisms, such as the above-mentioned host defense system and control of inflammation and immune responses, are becoming obvious. In this sense, there is a tight balance between appropriate redox states and oxidative stress. The final balance results from the magnitude and class of ROS, as well as the persistence of ROS production. Thus, high ROS concentrations may drive unspecific damage to nucleic acids, proteins, carbohydrates, and lipids or overtake the signaling pathways regulated by ROS.

In contrast, low to moderate levels of ROS lead to physiological regulation of cell signaling pathways (protein phosphorylation, ion channels, and transcription factors) and subsequently control cellular processes, such as differentiation, apoptosis, and migration.

A brief description of the most relevant physiological roles of ROS is as follows. ROS participate in immune system, at both innate and acquired levels. These molecules produced by phagocytes represent one of the early actions of defense against pathogen invasion. Notably, some pathogens have been shown to downregulate mitochondrial ROS generation and thereby prevent macrophage defense mechanisms, favoring pathogen persistence (Basu Ball et al., 2011). An example of the importance of ROS in immune system signaling is reflected in chronic granulomatous disease (CGD), which is caused by a lack of ROS-generating phagocytes. In this manner, CGD patients inefficiently kill invader bacteria by ROS, suffering recurrent infections (Mauch et al., 2007). Interestingly, CGD patients and mice defective for ROS production also show evidence of noninfectious inflammatory states, supporting the role of ROS in controlling inflammation and immune responses (van de Veerdonk et al., 2010). With respect to acquired immunity, ROS participates in signal transduction cascades within T lymphocytes (Alfadda and Sallam, 2012). Furthermore, as detailed earlier, recent evidence highlights a role for ROS in immune responses through NRPL3 activation (Nakahira et al., 2011; Shimada et al., 2012; Zhou et al., 2011).

Other illustrations of the crucial role of ROS in normal physiological processes are the key regulation of vascular system, the role of ROS in oxygen sensing through stabilization of hypoxia-inducible factor-1 (HIF-1) and the subsequent stimulation of angiogenesis and bioenergetic responses to rescue redox homeostasis, the role of ROS in skeletal muscle physiology regulation through glucose uptake during contraction, and the role of ROS in regulating gene stability, transcription, and signal transduction (Alfadda and Sallam, 2012).
Finally, increasing evidence that antioxidants may be harmful highlights the physiological role of ROS. Specifically, Ristow et al. reported that antioxidant supplementation may prevent increased insulin sensitivity by reducing the expression of ROS-sensitive transcriptional regulators of insulin sensitivity associated with exercise training in humans (Ristow et al., 2009). Also, the nuclear factor-erythroid 2-related factor 2 (Nrf2), a redox stress-sensitive transcription factor that induces several antioxidant and detoxification genes, is kept inactive in the absence of redox stress by its binding to Kelch-like ECH-protein 1 (Keap1). Thus, disruption of Nrf2 signaling impaired angiogenesis and microvascular rarefaction in aging (Valcarcel-Ares et al., 2012). In this sense, it is worth bearing in mind that antioxidant strategies will likely not be successful because their activity is too unspecific, too insufficient, and too delayed; however, it is also plausible that the ineffectiveness of antioxidant strategies may arise from their inhibition of essential cell functions that require ROS.

However, excessive levels of ROS cause not only oxidative damage to macromolecules, but can also affect the physiological signaling pathways regulated by ROS. Among other consequences, increased ROS production will lead to augmented activation of the transcription factors HIF-1, NF-κB, and activator protein-1 (AP-1) as well as the NLRP3 inflammasome. This will increase the release of pro-inflammatory cytokines that in turn will enhance ROS production, and hence, feed-back in a self-stimulatory manner, thereby amplifying the inflammatory response (Dröge, 2002).

5. Mitochondrial dysfunction and inflammatory responses in chronic human pathologies
Extensive research over the past decade has revealed that continued mitochondrial dysfunction can lead to chronic inflammation, which in turn may mediate most chronic diseases, including rheumatoid, cardiovascular, neurological, or metabolic diseases.

5.1. Rheumatoid diseases
Osteoarthritis (OA) and RA are the most common rheumatoid pathologies. In both of these conditions, the mitochondrial characteristics, including MRC activity, ATP synthesis, and Δψm, are adversely affected in several cell types. In particular, ex vivo studies have revealed that OA chondrocytes, as compared with normal chondrocytes, showed significant decreases in the activities of MRC complexes II and III and Δψm (Johnson et al., 2000; Liu et al., 2010; Maneiro et al., 2003). In RA and

systemic juvenile idiopathic arthritis, studies have demonstrated that there is a deficiency in a subunit of the MRC complex IV in the synovium (Biniecka et al., 2011; Ishikawa et al., 2009). Furthermore, human RA synoviocytes (Da Sylva et al., 2005; Ospelt and Gay, 2005) and normal synoviocytes under arthritis-like conditions, such as hypoxia (Biniecka et al., 2011), which causes a significant increase in Δψm in synovial cells, have been shown to exhibit significantly higher mutation rates than normal-aged or OA synoviocytes. These data support the conclusion that mitochondrial mutagenesis is correlated with the local inflammatory environment in arthritis (Harty et al., 2011). Other cells, such as T lymphocytes, show mitochondrial impairment in patients with RA and systemic lupus erythematosus (Gergely et al., 2002; Moodley et al., 2008).

Oxidative stress can contribute to the activated phenotype of synoviocytes in inflammatory arthritis and OA chondrocytes (Davies et al., 2008; Filippin et al., 2008; Henrotin et al., 2005). Interestingly, studies have shown that inhibition of complex III or V induces ROS production and NF-κB activation in human articular chondrocytes (CilleroPastor et al., 2008; Milner et al., 2007) and synovial cells (Valcárcel-Ares et al., 2010) in vitro. Additionally, inhibition of complex III or V in chondrocytes/synoviocytes induces low-grade inflammatory and matrix degradation processes through the synthesis of pro-inflammatory stimuli, including prostaglandin E2 (PGE2), the chemokines IL-8 and monocyte chemotactic protein-1, and several matrix metalloproteinases (MMPs) (Cillero-Pastor et al., 2010; Cillero-Pastor et al., 2008; VaamondeGarcía et al., 2012).

Specifically, it has been shown that the inhibition of MRC activity induces cyclooxygenase-2 (COX-2) expression and PGE2 production through redistribution of mitochondrial Ca2+, generation of ROS, and activation of the transcription factor NF-κB (Biniecka et al., 2010; Cillero-Pastor et al., 2008). Mitochondrial dysfunction can also induce PGE2 liberation in human OA chondrocytes through 4-hydroxy-2nonenal (4-HNE) (Vaillancourt et al., 2007), a lipid peroxidation byproduct that is associated with a higher frequency of mtDNA mutations, which are often increased in articular tissues of patients with inflammatory arthritis (Biniecka et al., 2010; Vaillancourt et al., 2007).

In addition to the above processes, the decline in mitochondrial function can also increase the inflammatory responsiveness of both normal human chondrocytes and synoviocytes to cytokines, promoting mechanisms that may contribute to joint destruction and pain (Valcárcel-Ares et al., 2010; Vaamonde-García et al., 2012). Interestingly, the hypoxic state of arthritic joints in vivo, which correlates with increased disease activity and mtDNA mutagenesis rates, exacerbates the inflammatory response in synoviocytes by increasing COX-2 expression and the release of MMPs in response to IL-1β (Demasi et al., 2004). In fact, inflammasome activation may explain these synergistic phenomena. NF-κB signaling and the NLRP3 inflammasome pathway could work together to activate inflammatory cytokines, thereby leading to overstimulation of the inflammatory response (Escames et al., 2011). As additional support for the critical role of the inflammasome in the pathogenesis of OA, the NLRP3 inflammasome could also mediate the pathological effects of hydroxyapatite crystals in vitro and in vivo (Jin et al., 2011).

Other DAMPs/mito-DAMPs are also highly secreted in tissues of rheumatoid patients. In fact, oxidized mtDNA, which has immunostimulatory properties and is capable of inducing arthritis in mice, can be detected in the synovial fluid of RA patients, but not in that of control subjects (Collins et al., 2004). Some studies have also considered cytochrome c a mitoDAMP that can induce arthritis (Pullerits et al., 2005); however, recent data suggest that this implication is not mediated by a direct role in NLRP3 activation (Shimada et al., 2012). Moreover, as mentioned earlier, autophagy prevents the accumulation of dysfunctional mitochondria. In this regard, OA cartilage has been shown to be deficient in autophagic processes, and the pharmacological activation of autophagy may be an effective therapeutic approach for OA (Caramés et al., 2012). Together, these findings support the critical role of mitochondrial dysfunction in rheumatoid disorders.

5.2. Cardiovascular diseases
Abnormalities in mitochondrial functions are increasingly recognized in association with cardiomyopathies. In fact, the myocardium is a highly energy-demanding tissue, with mitochondria supplying greater than 90% of ATP. Consequently, mitochondria are essential for cardiomyocyte function and viability (Dutta et al., 2012). Since ROS are constantly generated during mitochondrial respiration, it is not surprising that a dysregulation in the production of free radicals can cause oxidative damage in the heart. This oxidative damage may cause mtDNA mutations that result in mitochondrial dysfunction. Moreover, increased mitochondrial Ca2 + levels modulate myocardial inflammation and dysfunction in states of injury, such as sepsis and burn trauma (Maass et al., 2005). Another important study showed that escape of mtDNA from autophagy-mediated degradation leads to TLR9-mediated inflammatory responses in cardiomyocytes and may induce myocarditis and dilated cardiomyopathy (Oka et al., 2012).

With advanced age, mitochondrial ROS production significantly increases both in the heart (Judge et al., 2005) and vasculature (Ungvari et al., 2007), making the vascular endothelium and smooth muscle susceptible to ROS oxidation. In fact, mitochondria-derived ROS likely contribute to the development of chronic low-grade vascular inflammation in aging by activating redox signaling pathways (Dai et al., 2012). This low-grade vascular inflammation actives the redox-sensitive transcription factor NF-kB, stimulating endothelial activation by production and secretion of cytokines, growth factors, and proteases in the vascular wall, thereby promoting atherosclerosis. This damage is augmented by excessive ROS generation in the heart and the vasculature, as well as defective oxidant scavenging. These mechanisms disturb endothelial homeostasis (i.e., induce endothelial dysfunction), a characteristic feature of patients with coronary atherosclerosis (Munzel et al., 2010).

Traditionally, it has been hypothesized that cardiovascular diseases are precipitated by atherosclerosis due to arterial blockage from fatty deposits. However, in recent years, diagnostic and therapeutic strategies have been based not only on control of cholesterol levels, healthy diet, and smoking, but also taking into account other factors, such as inflammatory markers and mitochondria (Garg, 2011; Marchant et al., 2012). In particular, the roles of inflammasomes in the development of cardiovascular diseases are currently being studied in depth (Garg, 2011). Finally, pathways that improve mitochondrial function and attenuate mitochondrial oxidative stress (i.e., antioxidant therapies) show efficacy in various animal models and promising results for the treatment of cardiovascular diseases.

5.3. Neurological diseases
Increasing evidence has demonstrated the role of mitochondrial dysfunction in neurological and neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), dementia, multiple sclerosis, ataxia, and encephalomyopathies (Monje et al., 2003; Morán et al., 2012; Witte et al., 2010). A common feature in neurodegenerative diseases is the presence of neuroinflammation and oxidative stress. In both AD and PD, oxidative stress activates inflammatory signaling pathways through several mechanisms, including exacerbating the production of ROS, inducing mitochondrial dysfunction, activating microglia and astrocytes to release pro-inflammatory cytokines, and others (Schapira, 2010; von Bernhardi and Eugenín, 2012; Whitton, 2007). In fact, animal models of PD, induced by the mitochondrial inhibitor rotenone, exhibit increased production of IL-1β in the hypothalamus (Yi et al., 2007).

Furthermore, pro-inflammatory cytokines released in activated glia have been shown to stimulate increased ROS production in the brain, contributing to other mechanisms that amplify ROS production and lead to deterioration of mitochondrial function. In addition to exacerbating disease progression, this process may also suppress neurogenesis. Severe mitochondrial damage may even lead to cell death by necrosis or by activation of apoptotic signaling and activation of caspases (Voloboueva and Giffard, 2011).

Because neurodegenerative diseases are accompanied by chronic inflammation, the induction of mitophagy may have beneficial effects by removing permeabilized or ROS-producing mitochondria (Green et al., 2011). Moreover, as mentioned earlier, alterations in several genes involved in autophagy, such as PINK1 or Parkin, have been described in PD. Furthermore, downregulation of mitochondrial ROS may also decrease astrocyte activation (Voloboueva and Giffard, 2011). Finally, it has been recently described in mouse primary astrocytes how degraded mtDNA could be a new identified subtype of DAMP inducing inflammasome activation and ensuing neurodegeneration (Mathew et al., 2012).

5.4. Metabolic diseases
Insulin resistance, pancreatic islet β-cell failure, and elevated plasma free fatty acid (FFA) levels are features commonly associated with the development of several metabolic diseases, including obesity, type 2 diabetes mellitus, and metabolic syndrome. Accumulating evidence indicates that mitochondrial dysfunction is a central contributor or mediator of these processes (Coletta and Mandarino, 2011; Ma et al., 2012; Martins et al., 2012; Vial et al., 2010).

Diverse sources of inflammation can contribute to the insulin resistance observed in obesity and type 2 diabetes. Elevated plasma FFA levels play an important role in the development of skeletal muscle insulin resistance through different mechanisms, including oxidative stress, inflammation, and mitochondrial dysfunction (Coletta and Mandarino, 2011; Martins et al., 2012). The inflammation induced by FFAs may induce impairment in mitochondrial function in adipocytes (Ji et al., 2011; Youssef-Elabd et al., 2012). Furthermore, FFAs can activate these inflammatory signaling pathways directly through interactions with members of the TLR family and inflammasome activation (Reynolds et al., 2012; Wen et al., 2011; Youssef-Elabd et al., 2012).
Since mitochondrial dysfunction drives NLRP3 inflammasome activation, it is not surprising that this pathway can underlie cellular metabolic disorders. Specifically, a large body of evidence demonstrates a pathological role for the association of NLRP3 with a nonmitochondrial protein, thioredoxin-interacting protein (TXNIP), in type 2 diabetes and obesity. For example, TXNIP deficiency impairs activation of the NLRP3 inflammasome, improving glucose tolerance and insulin sensitivity in β-cells and adipocytes and decreasing fat deposits in the liver (Vandanmagsar et al., 2011; Zhou et al., 2010). Interestingly, high levels of oxidized DNA have been related with diabetes (Simone et al., 2008). It is possible that the elevated levels of damaged DNA detected in diabetes could be related with the activation of NLRP3 (Shimada et al., 2012). Consequently, mitochondria may represent novel pharmacological targets for therapeutic interventions in metabolic disorders.

5.5. Cancer and immunogenic cell death
Mitochondrial processes play an important role in tumor initiation and progression. Otto Warburg made the first association between mitochondrial dysfunction and cancer in the 1930s. He observed increased rates of aerobic glycolysis in a variety of tumor cell types, and he hypothesized that the cell’s choice of the glycolytic route to produce ATP instead of the more productive oxidative phosphorylation route may be due to the impaired respiratory capacity of these cells (Warburg, 1956). Years after this discovery, a number of cancer-related mitochondrial defects have been identified. In this sense, altered expression and activity of respiratory chain subunits as well as mtDNA mutations have been associated with human tumors. For example, MRC Complex I dysfunction has been associated with the pathogenesis of Hürtle cell tumors of the thyroid (Máximo et al., 2005). In the same way, low activity of Complex III has been found in breast cancer (Putignani et al., 2008) and Hürtle cell tumors (Bonora et al., 2006; Stankov et al.,
2006).

The aggressiveness of renal cell tumors is related to lower Complex II, III, and IV activities (Simonnet et al., 2002). As we know, dysfunction in MRC complexes is associated with mtDNA mutations. In fact, mutations in mtDNA have been reported in a variety of cancers, including ovarian, thyroid, salivary, kidney, liver, lung, colon, gastric, brain, bladder, head and neck, and breast cancers, as well as leukaemia (Modica-Napolitano and Singh, 2004).

In the context of mitochondrial dysfunction, we found that ROS is a key player in tumor formation and progression. It is now recognized that ROS plays an important role as a signaling molecule, mediating changes in cell proliferation, differentiation, migration, and invasiveness, as well as large-scale changes in gene transcription (Weinberg and Chandel, 2009). It is well known that mitochondrial dysfunction increases ROS levels, and tumor cells generally exhibit higher levels of ROS than normal cells (Weinberg and Chandel, 2009). One signaling pathway that may be particularly important to ROS-mediated tumorigenesis involves the activation and stabilization of HIF. HIF is a key transcription factor in cancer progression that regulates the enhancement of both glucose metabolism and angiogenesis. HIF activation occurs under hypoxic conditions, but ROS also appears to stabilize HIF under conditions of normal oxygen concentrations, leading to aberrant activation of HIF and promoting tumorigenesis (Chandel et al., 1998).

In addition, it has been demonstrated that tumors lacking the tumor-suppressor succinate dehydrogenase (Complex I) and fumarate hydratase exhibit impaired HIF degradation (King et al., 2006), once again linking mitochondrial impairment with oncongenesis.
As was discussed earlier, mitochondrial ROS production results in activation of the NLRP3 inflammasome with the consequent release of pro-inflammatory cytokines (Zitvogel et al., 2012). In vivo experiments suggest that inflammasome products, such as IL-1β, can directly drive oncogenesis or suppress immunosurvellaince mechanisms, thereby facilitating tumor development (Zitvogel et al., 2012). In the same way, many human cancers are etiologically linked to chronic inflammatory processes, such as those that occur with gastric cancer, hepatic cancer, and colorectal cancer (Coussens and Werb, 2002). However, the role of the inflammasome in carcinogenesis is controversial. Yet, in sharp contrast, the inflammasome or its products, depending on the specific tumor and its grade, can reduce tumorogenesis by causing cell death and promoting antitumor immune responses (Zitvogel et al., 2012).

In this context, recently developed therapies have been able to induce cell death by triggering ROS production. These include a specific group of mitochondrial-targeted anticancer drugs (mitocans) (Hahn et al., 2012). An example of these mitocans is the vitamin E succinate. This pro-oxidant selectively targets cancer cell mitochondria and is a potent inducer of ROS, leading to apoptosis in cancer cells but not in related normal cells. This is because cancer cells are under much more oxidative stress than normal cells and thus are more vulnerable to further damage by ROS-generating agents. Interestingly, these types of therapies have immune-enhancing properties, meaning that they are able to modulate antitumor immune responses, stimulating the immune system to cause immunogenic tumor cell death by upregulating the release of DAMP molecules. Of note, the activation or inhibition of the inflammasome as cancer therapy will likely depend on the specific cancer and its grade (Hahn et al., 2012).

6. Mitochondrial dysfunction and inflammatory responses in acute human pathologies
Generally, the acute inflammatory response to infection and tissue damage should be able to reduce injury to the organism, preventing further damage to the cells or tissues. However, as mentioned above, when the acute inflammatory responses cannot be effectively resolved, progressive damage and multi-organ failure can occur. Sepsis, a representative acute inflammatory disease, is unfortunately the main cause of death in critical care units. Mitochondrial dysfunction and impaired oxygen consumption play a role in sepsis, and the severity of sepsis has

been shown to correlate with mitochondrial damage in both humans and experimental models (Garrabou et al., 2012; Kung et al., 2012). Thus, changes to the ultrastructure of the mitochondria and significant inhibition of mitochondrial complex activities have been described in several cell types isolated from septic patients. Also, during overwhelming sepsis, increased Ca2+ influx into the mitochondria leads to mitochondrial dysfunction, resulting in the release of cytochrome c and cell death (Dada and Sznajder, 2011).

In this context, as mentioned before, mitochondrial damage may release several mito-DAMPs, including mtDNA. Consistent with this, septic plasma samples have shown significantly increased amounts of mtDNA and inflammatory cytokines, with corresponding NF-κB activation, and a correlation between adverse outcomes in sepsis and levels of extracellular mtDNA has been demonstrated (Garrabou et al., 2012). Since mtDNA can activate the NLRP3 inflammasome, its participation in the pathogenesis of sepsis is not surprising.

Interestingly, NLRP3 polymorphisms may be used as relevant risk estimates for the development of sepsis, which highlights the importance of NLRP3 in the pathogenesis of sepsis (Zhang et al., 2011). As expected, mitochondrial antioxidants have been shown to alleviate oxidative and nitrosative stress in several in vitro and in vivo model of sepsis (Apostolova et al., 2011; Zang et al., 2012). Taken together, these findings have led to a better understanding of the pathophysiological
processes of acute diseases and will be fundamental in improving the outcome of sepsis-related disorders.

7. Mitochondrial protection attenuates inflammation
Excessive mitochondrial oxidative stress plays a central role in triggering the deleterious cascade of events associated with inflammatory diseases and chronic low-grade inflammation in aging. In this regard, several approaches to preserve mitochondria are under investigation, including the use of antioxidant compounds, development of specific mitophagy-inducing therapies, and the pharmacological manipulation of mitochondrial sirtuins (SIRTs).

Strategies that control ROS production could limit either the redox-sensitive inflammatory pathway or the direct activation of the inflammasome. Thus, several meta-analyses have supported that dietary antioxidant intake is associated with a lower incidence of inflammatory diseases (Alissa and Ferns, 2012; Demetriou et al., 2012; Lahiri et al., 2012; Patelarou et al., 2011). In particular, resveratrol, a natural antioxidant found in grape skin and red wine, improves mitochondrial function by preventing oxidative stress and subsequent inflammation (Catalgol et al., 2012). In vitro, this molecule attenuates mitochondrial oxidative stress in several cell types, i.e., by restoring mitochondrial

complex III activity, which is considered to be the major source of mitochondrial oxidative stress (Xu et al., 2012). Moreover, resveratrol pretreatment efficiently prevents inhibition of mitochondrial membrane depolarization and ATP depletion, preserving mtDNA content and inhibiting COX-2 activity through a decrease in NF-κB activation in different cells (Dave et al., 2008; Lin et al., 2011; Ungvari et al., 2009; Xu et al., 2012). In animal models, resveratrol protects from developing age-related and acute diseases by improving mitochondrial function (Elmali et al., 2007; Jian et al., 2012; Lin et al., 2012; Ungvari et al., 2010).
As described earlier, an imbalance between mitophagy and mitochondrial biogenesis could be involved in inflammatory pathology. Autophagy/mitophagy decreases ROS production and mtDNA release after MPT opening, in turn affecting the NF-κB pathway and inflammasome activation. Thus, pharmacological induction of autophagy could mitigate inflammatory reactions. For example, activation of autophagy by rapamycin reduces the severity of experimental OA (Caramés et al., 2012). Interestingly, resveratrol also improves mitochondrial function through induction of mitochondrial biogenesis (Csiszar et al., 2009; Ungvari et al., 2009).

Recent studies revealed that SIRTs or class III histone deacetylases, have an essential role in mitochondrial protection against oxidative stress (Pereira et al., 2012). Three of the 7 SIRTs described in humans, SIRT3, SIRT4, and SIRT5, are localized inside mitochondria, which lends support to the potential role of SIRTs in mitochondrial biology (Hirschey et al., 2010). SIRTs can indirectly regulate the expression of several inflammatory genes through deacetylation of several signaling proteins, including the transcription factors FOXO (Salminen et al., 2008) and NF-κB (Natoli, 2009) and the peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) (Sugden et al., 2010). Deacetylation of FOXO by SIRT1 or SIRT3 upregulates the expression of SOD2 and catalase, thereby decreasing cellular ROS levels. Also, deacetylation of PGC-1α by both SIRT1 and SIRT3 stimulates mitochondrial biogenesis (Pereira et al., 2012). As mentioned above, several studies have demonstrated that resveratrol can protect mitochondrial functions through activation of SIRTs (Shinmura et al., 2011).

While the role of mitochondria in the development of the inflammatory response is now beginning to be understood, there is still a lot to be discovered. Understanding the mechanisms that induce inflammation through mitochondrial impairment may help to identify relevant therapeutic targets for the treatment of multiple degenerative and acute disorders as well as the aging process. For these reasons, it is plausible that, in few years, specific mitochondrial-targeting pharmacological treatments of inflammatory responses may become a reality. Indeed, some pharmaceutical companies have already begun designing new molecules that are the equivalent of super-antioxidants and have demonstrated exciting results in several preclinical studies in vitro and in vivo (McManus et al., 2011; Mukhopadhyay et al., 2012).

8. Limitations and future research directions
The use of effective strategies to increase our knowledge about the role of mitochondrial dysfunction in the inflammatory components of multiple degenerative and acute diseases, as well as in the aging process, is necessary for the development of new successful therapies aimed at improving human health. Thereby, an appropriate way to avoid contradictory results is the use of unmanipulated cells from healthy and diseased donors. Most in vitro studies are conducted in isolated cells kept in standard media with high glucose concentrations and under aerobic conditions, quite different from effects in tissues in a live organism, which could lead cells to obtain their energy predominantly from anaerobic glycolysis. Thus, the hypoxic nature of rheumatoid joints in vivo, which is correlated with increased mtDNA mutagenesis rate and disease activity, increases the inflammatory response in synoviocytes by increasing COX-2 expression and MMPs release in response to IL-1β (Demasi et al., 2004). For this reason, it is likely that the effects of mitochondrial impairment observed in cultures

maintained in normal atmosphere will even be more crucial in damaged tissue, where mitochondrial activity is probably lower due to genetic predisposition, somatic mutations in the mtDNA of the MRC as a consequence of the elevated levels of pro-inflammatory mediators (notably ROS), or the direct effects of pro-inflammatory mediators on the MRC. Future in vitro studies must take this into account. In addition, an important tool in these studies could be the transmitochondrial cybrid system, which allows the study of the effects of mitochondrial dysfunction and inflammatory responses with a common nuclear background, thereby avoiding the large number of inflammatory properties regulated by nuclear-encoded genes (Kaipparettu et al., 2010).

The majority of studies to date were conducted using in vitro or ex vivo strategies. Thus, it is necessary to translate the results to in vivo designs to support a clear role for mitochondrial dysfunction in the development of the inflammatory response. Animal models are also useful tools in medical science research. In this sense, different studies have reported that transgenic expression of mitochondrial-targeted catalase in mouse models of chronic diseases, such as cancer or Alzheimer’s disease, reduced primary tumor invasiveness and decreased metastasis incidence and burden (Goh et al., 2011) and several typical events of the disease (Mao et al., 2012).

Finally, in transgenic mouse lines, the overexpression of catalase targeted to mitochondria reduced age-associated pathologies and led to an extension in lifespan (Schriner et al., 2005). Overall, these in vivo studies support that mitochondria-targeted molecules may be an effective therapeutic approach to treat or prevent inflammation linked to numerous degenerative and acute diseases. In addition, other interesting models to be kept in mind are represented by mice with increased mtDNA mutations that exhibit pathology associated to human aging (Trifunovic, 2006) Analysis of the inflammasome has not been performed in these animals, but could be a useful model in which to study the role of mitochondria in NLRP3 inflammasome activation.

However, despite our increasing knowledge on the role of mitochondria in the development of the inflammatory response, there is still much to learn. Several key questions remain to be addressed. For example, which mechanisms determine whether mitochondria induce the inflammasome or apoptosome? Along these lines, recent research has suggested that oxidized mtDNA released during apoptosis results in activation of the NLRP3 inflammasome, providing a link between apoptosis and inflammasome activation (Shimada et al., 2012).

Interestingly, VDAC, whose activity is modulated by Bcl-2 family members, is decisive for inflammasome activation, and also in apoptosis induction by Bax (Yamagata et al., 2009; Zhou et al., 2011). However, what is the exact mechanism through which mtDNA activates the NLRP3 inflammasome? It also remains to be determined the importance of inflammasome in non-immune cell types.

Furthermore, other factors, such as tissue specificity, the effects of environmental factors, and age, may determine the final outcome. Clearly, more work is necessary to shed light on how mitochondrial dysfunction modulates the inflammatory response associated with distinct degenerative and acute diseases as well as the aging process.

9. Conclusions

Both acute and chronic inflammatory diseases, as well as the aging process, have been linked to accumulation of ROS and RNS, which could potentially be a main source of mitochondrial genomic instability leading to respiratory chain dysfunction. Indeed, mitochondrial impairment could modulate innate immunity through both redox-sensitive inflammatory pathways and direct activation of the inflammasome. Mitochondria could integrate these 2 pathways, leading to an overstimulation of the inflammatory response (Fig. 1). In conclusion, there is substantial evidence supporting the hypothesis that a decline in mitochondrial function is essential to the development of the inflammatory phenotype observed in multiple human degenerative or acute diseases as well as in advanced age.

All of these findings support mitochondria as new pharmacological targets. The preservation of mitochondrial function could reduce oxidative stress and may represent a novel therapeutic advantage for patients with degenerative or acute diseases as well as functional decline in aging. Despite these advances in our knowledge of inflammatory alterations, it is necessary to keep in mind that the best treatment is prevention; this is true for both inflammatory-related diseases and in healthy aging. Although we are becoming increasingly aware of the importance of a healthy diet, there is still a long way to go. Adequate nutrition could in fact be the best way to prevent aging declines and inflammatory degenerative or acute diseases. Thus, as Hippocrates said 25 centuries ago, “Let food be the medicine and medicine be the food”

Inflammation Part 3: resolving inflammation – resolvins, protectins, maresins and lipoxins

These articles by James Watson and Vince Giuliano are not actually research, but are such in depth background that I consider must reading to understand NAD+  This one is published here

Inflammation Part 2: The Tale of Three Stress Sensors and their Interactions: 1)Inflammation, 2)Genomic Instability (p53), and 3)Oxidative stress (Nrf2)

These articles by James Watson and Vince Giuliano are not actually research, but are such in depth background that I consider must reading to understand NAD+  This one is published here