Mitochondrial localization of Sirt3 plays a role in various mitochondrial functions, such as maintaining basal ATP level and regulating apoptosis. Sirt3 has been shown to regulate energy homeostasis 
. Recent studies have suggested the protective role of Sirt3 in cells under stress condition. In this study, we i) identified the presence of Sirt3 in neurons, and ii) demonstrated that Sirt3 plays a role as a prosurvival factor in neurons against excitotoxic injury. We found that under excitotoxic injury, intracellular depletion of NAD with induced increased expression of Sirt3 in mitochondria. NAD depletion was induced either by PARP-1 activation, or by protein transfection of NADase, both of which have been shown by our group to selectively decrease cytosolic NAD 
. ROS produced following PARP-1 mediated NAD depletion was involved in the increased expression of Sirt3. Overexpression of Sirt3 reduced NMDA-induced ROS generation, thereby preventing neuronal death, whereas knockdown of Sirt3 enhanced neuronal death. This study is the first evidence demonstrating that the increase in mitochondrial Sirt3 contributes to neuroprotection from excitotoxicity.
Excessive activation of PARP-1 induces neuronal death under stress conditions such as DNA damage, excitotoxicity, or oxidative stress 
. PAR has been shown to induce mitochondrial release of AIF and cell death 
. However, our previous studies have shown that NAD depletion is the cause of PARP-1-mediated cell death and exogenous addition of NAD effectively inhibited cell death 
. Moreover, we have shown that activated PARP-1 depleted most of the cytosolic NAD and then consumed the mitochondrial NAD only after MPT permits mitochondrial NAD to exit into the cytosol 
. In accordance with our previous findings, this study demonstrated that PARP-1- mediated NAD depletion is responsible for neuronal death with chronic exposure to low-dose excitotoxic agents 
. Addition of exogenous NAD is a very novel concept, yet proving to be very effective 
. We demonstrated that NAD uptake into cells is mediated by P2X7 receptor-gated channels 
. Activation of P2X7 receptors occurs under basal conditions, and P2X7 receptors are further activated in injury conditions and allows for an increase in permeability. Therefore, it is not surprising that NAD levels are further increased under excitotoxic conditions. In addition, we have unpublished data suggesting that NAD uptake into cells is increased when cells are exposed to oxidative injury. In this study, intracellular NAD level was higher in neurons treated with NAD in the presence of NMDA than those treated with NAD alone ().
In the present study, we demonstrated that cytosolic NAD decreased rapidly in neurons within 4 hr after NMDA treatment, yet mitochondrial NAD levels remained unchanged. This is consistent with previous findings that PARP-1 activation selectively depletes cytosolic NAD prior to mitochondrial NAD release and utilization for PAR formation 
. In support of this, it was found that Sirt3 functioned as a mitochondrial NAD -dependent deacetylase 
, and the results from our findings imply that the NAD -dependent deacetylase activity of Sirt3 in mitochondria is maintained due to the preservation of mitochondrial NAD, whereas Sirt1 deacetylase activity is impaired due to depletion of cytosolic NAD. Consistent with our study, it was shown that maintenance of mitochondrial NAD under stress condition can promote cell viability even though cytosolic and nuclear NAD is depleted 
. Our studies support this finding, since we also demonstrate that conditions that promote intact mitochondria (and therefore intact mitochondrial NAD levels) prevents neuron death, which we similarly reported in MNNG-treated neurons 
as well as in ischemia/reperfusion injury in cardiac myocytes 
. However, one major difference may lie in the hypothesis. Yang et al demonstrated that mitochondrial NAD levels regulate cell survival (independent of changes in cytosolic NAD), whereas we demonstrated that cytosolic NAD depletion preceded mitochondrial NAD depletion, and that mitochondrial NAD depletion was mediated by opening of the mitochondrial permeability transition pore and release of the mitochondrial NAD pool 
. Interestingly, our data shows that mitochondrial NAD levels increase after NMDA treatment. The reason for this is still unclear, and remains to be further studied. One possible explanation is that the increase in mitochondrial NAD level plays a significant role in promoting cell viability, and this hypothesis supports the findings by Yang et al 
. Cellular NAD levels were restored by addition of exogenous NAD, which prevented excitotoxic neuronal death in spite of an enhanced generation of PAR polymers. Our results show clear evidence that cytosolic NAD depletion directly contribute to excitotoxic neuronal death, and we outline our working hypothesis as a schematic diagram in .
Schematic Diagram of NMDA-induced increase in mitochondrial Sirt3.
Since the identification and characterization of sirtuins in several age-related diseases including neurodegenerative diseases, modulation of sirtuin activity as a therapeutic target for prevention and cure of such diseases has received lots of interest. Sirt1 has been extensively studied relative to the other Sirt isoforms. Resveratrol is known to be an activator of Sirt1 in vitro
, and the mechanism of activation has been demonstrated 
. Although the role of sirtuins in neuronal survival has been recently documented, the exact mechanism of neuroprotection by sirtuins is still unclear and somewhat controversial. A recent report has shown that Sirt1 is upregulated in mouse models for Alzheimer's disease and amyotrophic lateral sclerosis protecting against neurodegeneration 
, whereas another report showed that Sirt1 increasingly expressed in neurons by excitotoxic injury induces NAD depletion enhancing neuronal death 
. Further, it was shown that Sirt1 protected neurons from low potassium-induced apoptosis 
, while Sirt2, Sirt3 and Sirt6 induced apoptosis and Sirt5 had protective effect depending on the subcellular localization. In the present study, we reported that only Sirt3 was markedly increased in neurons upon NMDA induced excitotoxicity, whereas no changes in Sirt1 or other mitochondrial Sirts (Sirt4 or 5) were detected with excitotoxic injury.
The exact sequence of events involved in Sirt3 processing and translocation to mitochondria is still unclear because of conflicting data in the literature. For example, it was reported that Sirt3 is processed in the nucleus prior to translocation to mitochondria 
, whereas another group reported that the long form is processed in the mitochondria to the short form 
. In this study, immunofluorescence of Sirt3 detected by an antibody specific to short Sirt3 was increased in both the nucleus and mitochondria in NMDA-treated neurons, suggesting that the increase in Sirt3 by excitotoxic conditions may occur both within and outside of mitochondria. Also, what is unclear is our reported Sirt3 long form size of 44 kD, which is different from the long form size (35 kD) reported by others 
. Wherever Sirt3 may be processed, our finding presented here demonstrated that excitotoxicity induces the increased expression and translocation of Sirt3 into mitochondria in neurons.
Recent evidence demonstrated the crucial role of NAD as a determining factor in cell survival under energetic stress, and the role of Sirt3 as a metabolic sensor is gaining further interest. Sirt3 was reported either in mitochondria 
or was shown to translocate from the nucleus to mitochondria 
, and Sirt3 expression and/or processing has been shown to increase in various tissues during stress conditions 
. Prior studies showed the role of Sirt3 in maintaining energy metabolism. The role of Sirt3 in the maintenance of ATP level and in regulating mitochondrial electron transport was shown using Sirt3 knockout mice 
. It was reported that constitutive expression of Sirt3 promoted the expression of mitochondrial genes, leading to enhanced mitochondrial electron transport activity 
. Interestingly, we found that NAD depletion by NADase protein delivery increased expression of mitochondrial Sirt3, whereas exogenous treatment with NAD prevented the increase in Sirt3 and rescued neurons. Our finding seems contradictory to a recent report that showed the activation of Sirt3 by exogenously added NAD 
. However, both studies demonstrated that exogenously added NAD rescued cells. It is likely that cytosolic NAD depletion induced mitochondrial Sirt3 to protect neurons from further energetic stress. Similar to our findings, Hirschey et. al.
showed that Sirt3 expression was upregulated during fasting in liver and brown adipose tissue and modulated mitochondrial fatty acid use 
. Our study suggested that Sirt3 could play a pivotal role in protecting neurons from excitotoxicity-mediated energetic stress by showing that Sirt3 was increased and activated in mitochondria, another pool of NAD, upon cytosolic NAD level depletion.
Excitotoxicity induced by excessive calcium influx into neurons initiates ionic imbalance, oxidative stress, and energy failure. Exogenously added NAD inhibited ROS generation in neurons treated with NMDA, whereas antioxidant agents such as trolox or 7-NI could not reduce NAD depletion. Once NAD turnover is enhanced by NMDA receptor activation, it seems that the treatment with antioxidants is not sufficient to prevent NAD depletion. However, BAPTA-AM, a cell-permeable Ca2+
chelator, significantly prevented NAD depletion (data not shown), indicating that Ca2+
-dependent intracellular signal transduction pathways may play an important role in this proposed mechanism. This data further suggests that other events following excessive deregulated [Ca2+
increase may also contribute to NAD depletion. For example, we previously reported that NMDA receptor activation promoted MPT induction presumably through excessive mitochondrial Ca2+
uptake, and MPT induction promoted release of mitochondrial Ca2+
and contributed to deregulated [Ca2+
. In addition to reducing ROS generation, exogenous addition of NAD almost completely prevented neuronal death, suggesting the definitive role of NAD in inhibiting the above events by excitotoxicity. Interestingly, we found that Trolox or 7-NI reduced Sirt3 expression. Consistent with this result, we observed that Sirt3 was increased in neurons treated with the peroxynitrite generator. These observations suggest that in our excitotoxicity model, NAD depletion is an upstream event of oxidative stress, and seems to be primarily responsible for the increased expression of Sirt3 in mitochondria.
What we posit is that the normal cell response to pathological insults is to reverse this stress. In this model, excitotoxic injury through opening of NMDA receptor-gated calcium channels increases ROS formation through PARP-1 activation and NAD depletion, which promotes an increase in mitochondrial Sirt3 to reduce further ROS formation (such as ROS-induced ROS formation). An increase in mitochondrial Sirt3 sufficient to counter further ROS formation will prevent injury, and inadequate mSirt3 will promote pathological ROS formation and subsequent cellular demise. Thus, a delicate balance exists to promote either cell survival (increase in mSirt3) or cell death (increase in ROS), depending on the degree of response. This is described in our schematic diagram ().
The role of Sirt3 in regulating ROS production has been demonstrated in recent studies. Constitutively expressed Sirt3 was shown to reduce ROS in adipocytes 
, and the increased expression of Sirt3 protected myocytes from genotoxic and oxidative stress and blocked cardiac hypertrophy by activating antioxidant enzymes such as MnSOD and catalase 
. Recently, Sirt3 knock out mouse embryonic fibroblast was shown to exhibit increased superoxide level 
. In support of these findings, we found that increased expression of mitochondrial Sirt3 significantly reduced ROS production following excitotoxic injury, whereas Sirt3 silencing increased ROS production. Neurons transfected with Sirt3-overexpressing plasmid showed less mitochondrial ROS production following excitotoxic injury. Our finding implies that the expression of Sirt3 in mitochondria was increased in response to oxidative stress following excitotoxic injury, which is required for neuroprotection against oxidative stress-mediated death. Moreover, the overexpression of Sirt3 inhibited excitotoxicity-mediated neuronal death, whereas Sirt3 siRNA transfection exacerbated neuronal death. The exact mechanism of how Sirt3 decreases mitochondrial ROS production in neurons is unclear and remains to be further investigated. Since the role of Sirt3 in neuronal survival has not been established, our study provides the first evidence that Sirt3 plays a pivotal role in neuronal survival under stress condition such as excitotoxic injury.
In conclusion, we have shown that increased Sirt3 in mitochondria plays a pivotal role in neuroprotection against NMDA induced excitotoxicity, and sheds light into the effects of Sirt3 on the regulation of neuronal survival. We demonstrated that NAD depletion and subsequent oxidative stress induced the increase of Sirt3 in neurons under NMDA-mediated oxidative stress. Since oxidative stress and energetic failure contribute to neurodegeneration, the roles of Sirt3 in controlling ROS generation and maintaining energy homeostasis have profound implications for intervention of neuronal death by neurodegenerative diseases.