Accumulating evidence has suggested that NAD (including NAD+ and NADH) and NADP (including NADP+ and NADPH) could belong to the fundamental common mediators of various biological processes, including energy metabolism, mitochondrial functions, calcium homeostasis, antioxidation/generation of oxidative stress, gene expression, immunological functions, aging, and cell death: First, it is established that NAD mediates energy metabolism and mitochondrial functions; second, NADPH is a key component in cellular antioxidation systems; and NADH-dependent reactive oxygen species (ROS) generation from mitochondria and NADPH oxidase-dependent ROS generation are two critical mechanisms of ROS generation; third, cyclic ADP-ribose and several other molecules that are generated from NAD and NADP could mediate calcium homeostasis; fourth, NAD and NADP modulate multiple key factors in cell death, such as mitochondrial permeability transition, energy state, poly(ADP-ribose) polymerase-1, and apoptosis-inducing factor; and fifth, NAD and NADP profoundly affect aging-influencing factors such as oxidative stress and mitochondrial activities, and NAD-dependent sirtuins also mediate the aging process. Moreover, many recent studies have suggested novel paradigms of NAD and NADP metabolism. Future investigation into the metabolism and biological functions of NAD and NADP may expose fundamental properties of life, and suggest new strategies for treating diseases and slowing the aging process.

Poly(ADP-ribose)-1 (PARP-1) is a key mediator of cell death in excitotoxicity, ischemia, and oxidative stress. PARP-1 activation leads to cytosolic NAD ϩ depletion and mitochondrial release of apoptosis-inducing factor (AIF), but the causal relationships between these two events have been difficult to resolve. Here, we examined this issue by using extracellular NAD ϩ to restore neuronal NAD ϩ levels after PARP-1 activation. Exogenous NAD ϩ was found to enter neurons through P2X 7 -gated channels. Restoration of cytosolic NAD ϩ by this means prevented the glycolytic inhibition, mitochondrial failure, AIF translocation, and neuron death that otherwise results from extensive PARP-1 activation. Bypassing the glycolytic inhibition with the metabolic substrates pyruvate, acetoacetate, or hydroxybutyrate also prevented mitochondrial failure and neuron death. Conversely, depletion of cytosolic NAD ϩ with NAD ϩ glycohydrolase produced a block in glycolysis inhibition, mitochondrial depolarization, AIF translocation, and neuron death, independent of PARP-1 activation. These results establish NAD ϩ depletion as a causal event in PARP-1-mediated cell death and place NAD ϩ depletion and glycolytic failure upstream of mitochondrial AIF release.

Extensive activation of poly(ADP-ribose) polymerase-1 (PARP-1) by DNA damage is a major cause of caspase-independent cell death in ischemia and inflammation. Here we show that NAD ؉ depletion and mitochondrial permeability transition (MPT) are sequential and necessary steps in PARP-1-mediated cell death. Cultured mouse astrocytes were treated with the cytotoxic concentrations of N-methyl-N-nitro-N-nitrosoguanidine or 3-morpholinosydnonimine to induce DNA damage and PARP-1 activation. The resulting cell death was preceded by NAD ؉ depletion, mitochondrial membrane depolarization, and MPT. Sub-micromolar concentrations of cyclosporin A blocked MPT and cell death, suggesting that MPT is a necessary step linking PARP-1 activation to cell death. In astrocytes, extracellular NAD ؉ can raise intracellular NAD ؉ concentrations. To determine whether NAD ؉ depletion is necessary for PARP-1-induced MPT, NAD ؉ was restored to near-normal levels after PARP-1 activation. Restoration of NAD ؉ enabled the recovery of mitochondrial membrane potential and blocked both MPT and cell death. Furthermore, both cyclosporin A and NAD ؉ blocked translocation of the apoptosis-inducing factor from mitochondria to nuclei, a step previously shown necessary for PARP-1-induced cell death. These results suggest that NAD ؉ depletion and MPT are necessary intermediary steps linking PARP-1 activation to AIF translocation and cell death. PARP-1,1 the most abundant of the poly(ADP-ribose) polymerase family members, is rapidly activated by DNA damage. PARP-1 utilizes NADϩ to form poly(ADP-ribose) polymers on specific acceptor proteins. PARP-1 activation appears to facilitate DNA repair under moderate stress conditions (3), but extensive PARP-1 activation promotes cell death under conditions that cause extensive DNA damage such as excitotoxicity and ischemia (2, 4). Both genetic deletion and pharmacological inhibition of PARP-1 activity can markedly reduce cell death under these conditions (5-7).The sequence of events leading from PARP-1 activation to cell death has not been fully established. PARP-1-mediated cell death is caspase-independent, lacks many morphological hallmarks of classical apoptosis, and was long considered a direct result of energy failure (5, 8, 9). Extensive PARP-1 activation causes NAD ϩ depletion, which, in turn, can lead to ATP depletion (5, 8, 10). However, it has also been shown that translocation of the apoptosis-inducing factor (AIF) from mitochondria to the nucleus is required for PARP-1-mediated cell death (11). Thus, a fundamental unanswered question is whether NAD ϩ depletion is a necessary and causal event in PARP-1-mediated cell death, and, if so, what is the link between NAD ϩ depletion and mitochondrial release of AIF.Mitochondria permeability transition (MPT), like PARP-1 activation, is a significant factor leading to cell death after excitotoxicity, ischemia-reperfusion, and other disorders (12, 13). MPT results from the opening of a large conductance channel in the inner mitochondrial membrane (14, 15). Pharmacol...

Glutamate excitotoxicity, oxidative stress, and acidosis are primary mediators of neuronal death during ischemia and reperfusion. Astrocytes influence these processes in several ways. Glutamate uptake by astrocytes normally prevents excitotoxic glutamate elevations in brain extracellular space, and this process appears to be a critical determinant of neuronal survival in the ischemic penumbra. Conversely, glutamate efflux from astrocytes by reversal of glutamate uptake, volume sensitive organic ion channels, and other routes may contribute to extracellular glutamate elevations. Glutamate activation of neuronal N-methyl-D-aspartate (NMDA) receptors is modulated by glycine and D-serine: both of these neuromodulators are transported by astrocytes, and D-serine production is localized exclusively to astrocytes. Astrocytes influence neuronal antioxidant status through release of ascorbate and uptake of its oxidized form, dehydroascorbate, and by indirectly supporting neuronal glutathione metabolism. In addition, glutathione in astrocytes can serve as a sink for nitric oxide and thereby reduce neuronal oxidant stress during ischemia. Astrocytes probably also influence neuronal survival in the post-ischemic period. Reactive astrocytes secrete nitric oxide, TNFalpha, matrix metalloproteinases, and other factors that can contribute to delayed neuronal death, and facilitate brain edema via aquaporin-4 channels localized to the astrocyte endfoot-endothelial interface. On the other hand erythropoietin, a paracrine messenger in brain, is produced by astrocytes and upregulated after ischemia. Erythropoietin stimulates the Janus kinase-2 (JAK-2) and nuclear factor-kappaB (NF-kB) signaling pathways in neurons to prevent programmed cell death after ischemic or excitotoxic stress. Astrocytes also secrete several angiogenic and neurotrophic factors that are important for vascular and neuronal regeneration after stroke.

No abstract