NADPH in Cell Death

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Roles of NAD+ / NADH and NADP+ / NADPH in Cell Death Weiliang Xia1,2, Zheng Wang4, Qing Wang6, Jin Han5, Cuiping Zhao1,2, Yunyi Hong5, Lili Zeng3, Le Tang3 and Weihai Ying1,2,* 1

Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine; 2Med-X Research Institute, Shanghai Jiao Tong University; 3Department of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine; 4Shanghai Institute of Digestive DiseasesRenji Hospital, Shanghai Jiao Tong University School of Medicine; 5School of Life Science and Biotechnology, Shanghai Jiao Tong University and 6Shanghai, P.R. China; Neurobiology Research Centre, Graduate school of Medicine, Faculty of Health & Behavioral Sciences, University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia Abstract: A rapidly growing body of information has suggested that NAD (including NAD+ and NADH) and NADP (including NADP+ and NADPH) could be new fundamental factors in cell death: Many studies have indicated key roles of poly (ADP-ribose) polymerases and sirtuins --- two families of NAD-dependent enzymes --- in cell death; and NAD may also affect cell survival by influencing mitochondrial permeability transition, apoptosis-inducing factor and GAPDH. NAD may further influence cell survival by its effects on calcium homeostasis, gene expression and immunological functions. Due to the crucial roles of oxidative stress in cell death, NADPH may mediate cell death by its major effects on oxidative stress: NADPH is a key factor in cellular antioxidation systems; and NADPH oxidase is also a major generator of oxidative stress. With growing information about the novel biological properties of NAD and NADP, it is likely that new roles of NAD and NADP in cell death and various diseases will be elucidated. The elucidation may not only improve our understanding about the fundamental mechanisms of cell death, but also suggest new therapeutic targets for a variety of diseases.

Key Words: NAD, NADP, cell death, oxidative stress, mitochondria. 1. INTRODUCTION

2. ROLES OF NAD IN CELL DEATH

NAD, including nicotinamide adenine dinucleotide (NAD+) and reduced nicotinamide adenine dinucleotide (NADH), have been long known as key molecules in energy metabolism and mitochondrial functions. NADP, including nicotinamide adenine dinucleotide phosphate (NADP+) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), have been known as critical molecules in antioxidation and reductive biosynthesis [1-3]. However, a rapidly growing body of information has suggested that NAD and NADP also play crucial roles in various biological processes, including calcium homeostasis, gene expression, immunological functions and aging [1-5].

2.1. Poly(ADP-Ribose) Polymerases (PARPs) in Cell Death

Due to its pivotal roles in a number of major diseases, cell death has been a research topic of intensive interest. With the increasing evidence suggesting that NAD and NADP could belong to the fundamental factors in cell death, it is warranted to generalize the information regarding the roles of NAD and NADP in cell death. Through this generalization, we may not only obtain improved understanding regarding NAD/NADP on cell death, but also identify future research directions on this topic.

*Address correspondence to this author at the Med-X Research Institute, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai, 200032, P.R. China; Tel: 86-21-62932302; Fax: 86-21-62932302; E-mail: [email protected]

1381-6128/09 $55.00+.00

PARPs are a family of enzymes that consume NAD+ to produce poly(ADP-ribose) (PAR) on target proteins [6]. PARP-1 has been the most intensively studied member of PARP family, which plays important roles in a variety of biological processes, including DNA repair, gene expression, genomic stability, cell cycle, and cell death [4, 6, 7]. A number of studies have demonstrated that excessive PARP-1 activation mediates cell death induced by oxidative stress under many conditions [8, 9]. In animal model studies, PARP-1 has been indicated to play critical roles in many diseases, including ischemic brain damage [10, 11], Parkinson’s disease (PD) [12-14] and diabetes [15-19]. For examples, both pharmacological and genetic inhibition of PARP-1 can profoundly decrease infarct formation in animal models of brain ischemia [9, 20]; PARP-1 activation has also been shown to mediate the neuronal death induced by MPTP, a model toxin for PD, both in vitro [21, 22] and in vivo [2325]; and PARP-1 activation mediates -amyloid-induced neuronal death, which is an in vitro model of Alzheimer’s disease (AD) [26, 27]. A number of studies have further suggested that PARP-1 activation plays important roles in traumatic brain injury [28], diabetes [29], hypoglycemic brain injury [30], and shock and inflammation [6, 31]. Due to the-

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Roles of NAD + / NADH and NADP+ / NADPH in Cell Death

se lines of findings, PARP-1 has become a therapeutic target for various diseases [6, 31, 32]. We have provided direct evidence indicating that NAD+ depletion mediates PARP-1-induced cell death [33, 34]. It has also been indicated that MPT [34] and apoptosisinducing factor (AIF) translocation [35] link NAD+ depletion to cell death. New mechanisms regarding PARP-1 cytotoxicity have also been suggested by several lines of studies: First, ADP-ribose, that can be generated by PARP-1/PARG, is capable of causing TRPM2 opening, leading to elevated intracellular calcium concentrations and cell death [27, 36, 37]; however, it has also been suggested that PAR, instead ADP-ribose monomers, mediates PARP-1-induced AIF translocation and cell death [38, 39]. Second, it was suggested that NAD+ depletion causes cell death by decreasing SIRT1 activity [40]. Third, c-Jun N-terminal kinases (JNKs) could contribute to PARP-1-mediated cell death [41-44]; and extracellular signal-regulated kinases 1/2 (ERK1/2) can induce PARP-1 activation by directly phosphorylating the enzyme [41]. Cumulative evidence has suggested that other PARPs could also play significant roles in cell injury: It was reported that PARP-2 activity is detrimental in an animal model of focal brain ischemia, while it is beneficial in a model of global ischemia [45]; a recent study has suggested that PARP-2 mediates the survival of CD4+CD8+ doublepositive T cells during thymopoiesis [46]; and it has also been found that overexpression of tankyrase 2 can cause rapid cell death [47]. Future studies are warranted to further determine the interactions among PARPs under various physiological and pathological conditions. 2.2. Sirtuins in Cell Death It has been demonstrated that Sir2 is a key enzyme mediating the life span of yeast and C. elegans [48]: A decrease or increase in gene copy of Sir2 shortens or extends the replicative life span of yeast, respectively [49]; and increased gene copy of the Sir2 gene homolog in C. elegans also extends its life span [50]. Sir2 is a NAD+-dependent histone deacetylase, which produces protein deacetylation by consuming NAD+. Sirtuins are the mammalian homologs of Sir2, which include seven members, i.e., SIRT1-7. A number of studies have indicated that sirtuins are novel, key factors in cell death under many conditions. SIRT1 is the major subject in a majority of published studies regarding the roles of sirtuins in cell death. Most of these studies have suggested that SIRT1 activity is beneficial for cell survival. For example, it has been suggested the nicotinamide mononucleotide adenylyltransferases-1 (NMNAT-1) in the Wallerian degeneration slow (Wld(S)) protein could mediate the protective effects of the Wallerian mutation against axonal degeneration [51, 52]; both SIRT1 overexpression and administration of the SIRT1 activator resveratrol can decrease -amyloid-induced NF-B signaling and produce neuroprotective effects [53]; and SIRT1 deficiency can lead to significant increases in PARP-1 activity, causing AIF-mediated cell death [54]. Several mechanisms regarding the roles of SIRT1 in cell death have been suggested: First, SIRT1 may enhance cell survival by decreasing acetylation of p53 thus enhancing p53 degradation [55]; sec-

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ond, SIRT1 may also reduce cell death by preventing PARP1 activation [54]; and third, SIRT1 may indirectly reduce microglia-induced neurotoxicity by producing deacetylation of NFB [53]. However, SIRT1 could also be detrimental to cell survival under certain conditions: SIRT1 can deacetylate NFB thus enhancing the susceptibility of cells to TNF toxicity [56]. SIRT2 is a tubulin deacetylase. It has been reported that SIRT2 inhibitors rescues alpha-synuclein-mediated neurotoxicity in a PD model [57]. Activation of SIRT2 by resveratrol was also shown to abolish the protective effects of Wld(s) protein against axonal degeneration [58]. Thus, in contrast to the mainly beneficial roles of SIRT1 activity in cell survival, current studies have suggested that SIRT2 produces deleterious effects on cell survival under some conditions. SIRT3 is a mitochondrial deacetylase [59]. It has been reported that SIRT3 can reduce stress-induced apoptosis of cardiomyocytes by the following pathway: SIRT3 deacetylates Ku70, which promotes the binding of Ku70 to Bax thus preventing Bax translocation into the mitochondria [60]. It has also been reported that fasting-induced increases in NMNAT expression require mitochondrial presence of SIRT3 and SIRT4 to decrease stress-induced cell apoptosis [61]. However, in contrast to these observations suggesting beneficial roles of SIRT3 activity in cell survival, it has been reported that SIRT3 is pro-apoptotic and contributes to the JNK2-mediated apoptosis [62]. In summary, sirtuins have rapidly emerged as critical factors in cell survival. These enzymes appear to be the key links among NAD, p53, NFB, aging and cell death. While SIRT1 appears to be largely beneficial to cell survival, SIRT2 seems to be mainly detrimental to cell survival. Just like such factors as nitric oxide and NFB, sirtuins can be either beneficial or detrimental to cell survival. It is expected that future studies on the roles of sirtuins in cell death would provide ample information that would profoundly deepen our understanding regarding the mechanisms of cell death. 2.3. NAD in Apoptosis There have been only small number of studies regarding the roles of NAD in apoptosis, which have suggested that NAD may be involved in apoptosis: NADH / NADPH depletion is an early event in apoptosis [63]; and selective inhibitors of NAD+ synthesis can induce apoptosis [64]. There are several potential mechanisms underlying the roles of NAD in apoptosis: 1) NADH / NAD+ ratio is a major index of cellular reducing power that affects MPT, which mediates apoptosis under many conditions [65]; 2) NAD plays a key role in energy metabolism that is a key factor determining cell death modes; 3) NAD+-dependent sirtuins may mediate apoptosis [56]; and 4) NAD+ levels significantly affect the activity of caspase-dependent endonuclease DFF40 --- an executioner of DNA fragmentation in certain apoptotic cascades [66]. 2.4. NAD, Oxidative Stress and Cell Death Due to the critical roles of oxidative stress in cell death, NAD may affect cell survival by influencing oxidative stress and antioxidation systems in cells: First, NAD+ can be converted by NAD kinases to NADP+ --- the precursor for

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NADPH formation [67]; second, NADH / NAD + ratio is an index of cellular reducing potential, since the redox couple plays key roles in numerous redox reactions; third, some studies have suggested that NADH can produce direct antioxidation effects [68-70]; and fourth, NAD+ can inhibit ROS generation from -ketoglutarate dehydrogenase and pyruvate dehydrogenase [71]. It is noteworthy that excessive intracellular NADH can produce ‘reductive stress’, which may result from the capacity of xanthine oxidase / xanthine dehydrogenase to generate ROS by oxidizing NADH [72], or from the capacity of NADH to induce release of ferrous iron from ferritin [66]. 2.5. AIF and GAPDH in Cell Death AIF is a NADH oxidase, which can act as both a prodeath factor and a pro-survival factor [73-75]. Nuclear translocation of AIF from mitochondria is a key step in both caspase-independent apoptosis [74] and PARP-1 cytotoxicity [35]. However, AIF also plays an important role in mitochondrial Complex I activity [76]; and genetic deletion of AIF can produce dilated cardiomyopathy, skeleton muscle atrophy, and neurodegeneration [74]. Because it has been indicated that AIF is a NADH oxidase and prevention of NAD+ depletion can block PARP-1-mediated nuclear translocation of AIF [34], it is highly likely that NAD mediates the activity of AIF. Our latest study has found that aurintricarboxylic acid --- a Ca2+-Mg2+-endonuclease inhibitor --can abolish DNA alkylating agent-induced nuclear condensation in astrocytes, despite nuclear translocation of AIF (unpublished finding). This observation suggests that at least under certain conditions, it may be insufficient for nuclear AIF translocation itself to induce nuclear condensation. NAD is the major co-factor of GAPDH. GAPDH has been established as a mediator of apoptosis under many conditions. It has been found that GAPDH binds Siah which is then translocated into nucleus to mediate apoptosis [77-80]. 2.6. NAD in Axonal Degeneration Axonal degeneration is a key pathological change in multiple neurological diseases [81]. Wallerian degeneration slow (Wld(S)) mouse model is a widely used model for studying the mechanisms of axonal degeneration [81]. The mutation of Wld(S) mice leads to overexpression of a chimeric protein --- Wld(S) protein that consists of the key NAD+-synthesizing enzyme NMNAT-1 and the ubiquitin assembly protein Ufd2a. The overexpression of Wld(S) protein can cause delay of injury-induced axonal degeneration. It has been suggested that the NMNAT-1 in the Wld(S) protein could mediate the protective effects of the Wallerian mutation by affecting SIRT1 --- a member of sirtuins [51], or by preventing NAD+ loss in degenerating axons [52]. However, there are also studies suggesting that NMNAT-1 itself may be insufficient to produce the protective effects of the Wld(S) protein [82, 83]. 3 ROLES OF NADP IN CELL DEATH Oxidative stress is a key factor in cell death. Because NADP metabolism plays critical roles in both antioxidation system and generation of oxidative stress, NADP could play important roles in cell death.

Ying et al.

NADPH is a key factor in cellular antioxidation through the following mechanisms: First, GSH is essential for the functions of such key antioxidation enzymes as glutathione peroxidase [84], while NADPH is required for regeneration of GSH from GSSG; second, NADPH is a key component in another important antioxidation system --- thioredoxin system [85]; and third, in such cell types as red cells NADPH binds the important H2O2-disposing enzyme catalase [86], which can reactivate catalase when catalase is inactivated by H2O2. Cumulating evidence has demonstrated that NADPH can also significantly contribute to generation of oxidative stress through the activity of NADPH oxidase that generates superoxide from oxygen and NADPH. NADPH oxidase activity is present in not only phagocytes, but also various tissues and cell types [87, 88]. There are seven members of NOX family of NADPH oxidase, including phagocyte NADPH oxidase itself (NOX2/gp91(phox)) and six homologs of the cytochrome subunit of the phagocyte NADPH oxidase, i.e., NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2 [87]. These enzymes share the capacity to transport electrons across the plasma membrane, resulting in superoxide generation, which can be regulated by such factors as small guanosine triphosphatase Rac, protein kinase C and Ca2+ [87-90]. Many studies have indicated NADPH oxidase as a key factor in cell death under both in vitro and in vivo conditions [91]. For examples, NADPH oxidase appears to play a key role in the ROS generation in the neurons exposed to oxygen-glucose deprivation --- an in vitro model for brain ischemia [92]; both genetic and pharmacological inhibition of NADPH oxidase is protective against ischemic brain injury [93]; and the NADPH oxidase activation in astrocytes was shown to mediate -amyloid-induced neuronal death [94]. Interestingly, it has been reported that ischemia can induce NOX2 expression at the nucleus of cardiomyocytes, which appears to mediate ischemia-induced apoptosis [95]. Because numerous studies have indicated critical roles of oxidative stress in cell death [96], it is expected that many future studies would indicate important roles of NADPH oxidase and other NADPH-related enzymes in various diseases. 4. THERAPEUTIC POTENTIAL OF NAD AND NADP 4.1. Therapeutic Potential of NAD + Our cell culture studies have provided the first evidence indicating that NAD+ treatment can abolish PARP-1-induced astrocyte death [33, 34, 97, 98]. Recent in vitro studies have also shown that NAD + treatment can attenuate OGD-induced neuronal death [99] and PARP-1-mediated myocyte death [40]. These results suggest that NAD+ may be used in vivo to decrease PARP-1-mediated tissue injury. We have used a rat model of transient focal ischemia to test our hypothesis that NAD+ administration can decrease ischemic brain injury [100]: Intranasal delivery with NAD+ at 2 hrs after ischemic onset was shown to reduce infarct formation by nearly 90%, and to significantly decrease the ischemia/reperfusioninduced neurological deficits. These observations have provided the first in vivo evidence that that NAD+ administration may be a new strategy for treating stroke patients, and

Roles of NAD + / NADH and NADP+ / NADPH in Cell Death

NAD+ metabolism is a novel target for decreasing ischemic brain damage. NAD+ may have its unique merits as a cytoprotective agent: 1) NAD+ may reduce cell death not only by blocking PARP-1 toxicity, but also by enhancing energy metabolism and SIRT-1 activity; 2) NAD + is protective even when applied at 3-4 hrs after PARP-1 activation, suggesting that NAD+ administration may have long window of opportunity in decreasing tissue injury; and 3) compared with other cytoprotective factors as pyruvate, NAD+ can produce greatest protective effects against PARP-1 cytotoxicity in vitro [33, 34]. It is noteworthy that NAD+ administration may decrease the brain injury in not only cerebral ischemia, but also many other diseases [101] due to the following reasons: NAD + treatment can profoundly decrease PARP-1-mediated cell injury; and pathological roles of PARP-1 have been implicated in many diseases such as diabetes, PD and AD [5]. Several studies have reported that NADH administration can produce beneficial effects in treating PD [102, 103], which may be accounted for partially by the capacity of NADH to increase bioavailability of plasma levodopa. NADH administration may also be used for treating AD patients, since NADH can improve cognitive functions [104]. Our studies have provided direct evidence showing that NADH treatment can decrease the cell death induced by the DNA alkylating agent MNNG [98], thus providing certain explanations for the beneficial effects of NADH. However, our cell culture studies have also suggested that NADH treatment can be cytotoxic when cells are exposed to the peroxynitrite generator SIN-1 (data not shown), in contrast to the beneficial effects of NAD+ administration against SIN1 toxicity. These observations not only highlight the merits of NAD+ as a cytoprotective agent, but also suggest that great caution should be taken when applying NADH for treating diseases. 4.2. Therapeutic Potential of NAD + Precursors Nicotinamide administration can decrease tissue injury in the animal models of cerebral ischemia [105-108], spinal cord injury [109], PD [110] and multiple sclerosis [111]. Cell culture studies have also shown the cytoprotective effects of nicotinamide against oxidative stress and oxygen-

Fig. (1). The pathways from NAD isoforms to cell death are shown.

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glucose deprivation [112, 113]. The protective effects may result from the capacity of nicotinamide to restore NAD+ levels [105, 114], inhibit PARP-1 [6, 114], activate Akt1 [115] and block mitochondrial permeability transition [113, 116]. However, nicotinamide may also impair cell survival and longevity, due to its inhibitory effects on sirtuins [117]. Recent studies have also shown that nicotinamide riboside -- a new NAD+ precursor in eukaryotes --- can enhance Sir2dependent gene silencing and extend the replicative lifespan of yeast without calorie restriction [118]. The beneficial effects of nicotinamide riboside appear to result from the capacity of nicotinamide riboside to increase NAD + synthesis [118]. Increasing evidence has also suggested significant biological activities of nicotinic acid --- an important component in NAD+ metabolism: Nicotinic acid can significantly affect brain functions by such pathways as inducing glutamate release [119]. 4.3. Therapeutic Potential of NADPH Oxidase It appears to be increasingly important for cell survival to maintain NADPH as a ‘good guy’, since it can act as either a ‘good guy’ or a ‘bad guy’ in cellular antioxidation systems. It is of particular importance to modulate NADPH-related properties to reduce the detrimental effects of NADPH in diseases, because NADPH oxidase can play critical pathological roles in multiple illnesses. Several strategies may be used to modulate NADPH oxidase activity: 1) Directly inhibit NADPH oxidase by using NADPH oxidase inhibitors; 2) inhibit excessive NADPH generation by modulating the multiple NADPH-generating reactions; 3) maintain the activities of such NADPH-consuming enzymes as glutathione reductase, so as to ensure efficient NADPH flux through these pathways, resulting in prevention of excessive NADPH supply to NADPH oxidase; and 4) indirectly inhibit NADPH oxidase by manipulating such intracellular modulators of the enzyme as the small guanosine triphosphatase Rac [87, 88, 90]. 5. SUMMARY Based on the above discussion, it is increasingly clear that NAD and NADP play critical roles in cell death, which can affect cell survival through several pathways (Fig. 1). It


is expected that a growing number of pathways would be elucidated by future studies, which would further highlight the central roles of NAD and NADP in linking cell survival with various biological properties, including energy metabolism, mitochondrial functions, calcium homeostasais, oxidative stress/antioxidation and gene expression. Due to the pivotal roles of aberrant cell death in multiple major illnesses, including stroke, myocardial ischemia, cancer, Alzheimer’s disease, and diabetes, it is conceivable that NADand NADP-dependent pathways would become increasingly attractive therapeutic targets for the diseases.

Ying et al.

[10] [11] [12] [13] [14]

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ACKNOWLEDGMENTS This work is supported by a Key Grant for Basic Research of Shanghai Municipal Scientific Committee (Grant# 08JC1415400, to W.Ying), by a Key Grant of Shanghai Municipal Educational Committee (to W.Ying), by the Shanghai Med-X Engineering & Technology Research Center Grant for Physical Therapy and Diagnostics of Major Diseases (Grant# 08DZ2211200), by the ‘Start-Up Funds from 985 Project’ (to W.Ying), and by Shanghai Municipal Scientific Committee (06JC14052).

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ABBREVIATIONS AD

= Alzheimer’s disease

AIF

= Apoptosis-inducing factor

ERK

= Extracellular signal-regulated kinase

JNK

= c-Jun N-terminal kinase

NAD

= Nicotinamide adenine dinucleotide

NADP

= Nicotinamide adenine dinucleotide phosphate

PARP

= Poly(ADP-ribose) polymerases

PD

= Parkinson’s disease

NMNAT-1 = Nicotinamide mononucleotide adenylyltransferases-1

[20]

[21]

[22]

[23]

[24]

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