Weihai Ying

Astrocyte Influences on Ischemic Neuronal Death

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.

NAD+ depletion is necessary and sufficient for poly(ADP-ribose) polymerase-1-mediated neuronal death.

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+ 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+ to restore neuronal NAD+ levels after PARP-1 activation. Exogenous NAD+ was found to enter neurons through P2X7-gated channels. Restoration of cytosolic NAD+ 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+ with NAD+ glycohydrolase produced a block in glycolysis inhibition, mitochondrial depolarization, AIF translocation, and neuron death, independent of PARP-1 activation. These results establish NAD+ depletion as a causal event in PARP-1-mediated cell death and place NAD+ depletion and glycolytic failure upstream of mitochondrial AIF release.

Poly(ADP-ribose) glycohydrolase mediates oxidative and excitotoxic neuronal death

Excessive activation of poly(ADP-ribose) polymerase 1 (PARP1) leads to NAD+ depletion and cell death during ischemia and other conditions that generate extensive DNA damage. When activated by DNA strand breaks, PARP1 uses NAD+ as substrate to form ADP-ribose polymers on specific acceptor proteins. These polymers are in turn rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG), a ubiquitously expressed exo- and endoglycohydrolase. In this study, we examined the role of PARG in the PARP1-mediated cell death pathway. Mouse neuron and astrocyte cultures were exposed to hydrogen peroxide, N-methyl-d-aspartate (NMDA), or the DNA alkylating agent, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Cell death in each condition was markedly reduced by the PARP1 inhibitor benzamide and equally reduced by the PARG inhibitors gallotannin and nobotanin B. The PARP1 inhibitor benzamide and the PARG inhibitor gallotannin both prevented the NAD+ depletion that otherwise results from PARP1 activation by MNNG or H2O2. However, these agents had opposite effects on protein poly(ADP-ribosyl)ation. Immunostaining for poly(ADP-ribose) on Western blots and neuron cultures showed benzamide to decrease and gallotannin to increase poly(ADP-ribose) accumulation during MNNG exposure. These results suggest that PARG inhibitors do not inhibit PARP1 directly, but instead prevent PARP1-mediated cell death by slowing the turnover of poly(ADP-ribose) and thus slowing NAD+ consumption. PARG appears to be a necessary component of the PARP-mediated cell death pathway, and PARG inhibitors may have promise as neuroprotective agents.

Astrocytes protect neurons from nitric oxide toxicity by a glutathione-dependent mechanism

Nitric oxide (NO) contributes to neuronal death in cerebral ischemia and other conditions. Astrocytes are anatomically well positioned to shield neurons from NO because astrocyte processes surround most neurons. In this study, the capacity of astrocytes to limit NO neurotoxicity was examined using a cortical co‐culture system. Astrocyte‐coated dialysis membranes were placed directly on top of neuronal cultures to provide a removable astrocyte layer between the neurons and the culture medium. The utility of this system was tested by comparing neuronal death produced by glutamate, which is rapidly cleared by astrocytes, and N‐methyl‐d‐aspartate (NMDA), which is not. The presence of an astrocyte layer increased the LD50 for glutamate by approximately four‐fold, but had no effect on NMDA toxicity. Astrocyte effects on neuronal death produced by the NO donors S‐nitroso‐N‐acetyl penicillamine and spermine NONOate were examined by placing these compounds into the medium of co‐cultures containing either a control astrocyte layer or an astrocyte layer depleted of glutathione by prior exposure to buthionine sulfoximine. Neurons in culture with the glutathione‐depleted astrocytes exhibited a two‐fold increase in cell death over a range of NO donor concentrations. These findings suggest that astrocytes protect neurons from NO toxicity by a glutathione‐dependent mechanism.

Acidosis potentiates oxidative neuronal death by multiple mechanisms

Abstract : Both acidosis and oxidative stress contribute to ischemic brain injury. The present study examines interactions between acidosis and oxidative stress in murine cortical cultures. Acidosis (pH 6.2) was found to potentiate markedly neuronal death induced by H2O2 exposure. To determine if this effect was mediated by decreased antioxidant capacity at low pH, the activities of several antioxidant enzymes were measured. Acidosis was found to reduce the activities of glutathione peroxidase and glutathione S‐transferase by 50‐60% (p < 0.001) and the activity of glutathione reductase by 20% (p < 0.01) in lysates of the cortical cultures. Like acidosis, direct inhibition of glutathione peroxidase with mercaptosuccinate also potentiated H2O2 toxicity. Because acidosis may accelerate hydroxyl radical production by the Fenton reaction, the effect of iron chelators was also examined. Both desferrioxamine and N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine, two structurally different iron chelators, significantly reduced H2O2‐induced neuronal death under both pH 7.2 and pH 6.2 conditions. These results suggest that the increased cell death produced by severe acidosis during cerebral ischemia may result in part from excerbation of oxidative injury. This exacerbation may result from both impaired antioxidant enzyme functions and increased intracellular free iron levels.

NAD+ as a metabolic link between DNA damage and cell death

DNA damage occurs in ischemia, excitotoxicity, inflammation, and other disorders that affect the central nervous system (CNS). Extensive DNA damage triggers cell death and in the mature CNS, this occurs primarily through activation of the poly(ADP‐ribose) polymerase‐1 (PARP‐1) cell death pathway. PARP‐1 is an abundant nuclear enzyme that, when activated by DNA damage, consumes nicotinamide adenine dinucleotide (NAD)+ to form poly(ADP‐ribose) on acceptor proteins. The mechanisms by which PARP‐1 activation leads to cell death are not understood fully. We used mouse astrocyte cultures to explore the bioenergetic effects of NAD+ depletion by PARP‐1 and the role of NAD+ depletion in this cell death program. PARP‐1 activation was induced by the DNA alkylating agent, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), using medium in which glucose was the only exogenous energy substrate. PARP‐1 activation led to a rapid but incomplete depletion of astrocyte NAD+, a near‐complete block in glycolysis, and eventual cell death. Repletion of intracellular NAD+ restored glycolytic function and prevented cell death. The addition of non‐glucose substrates to the medium, pyruvate, glutamate, or glutamine, also prevented astrocyte death after PARP‐1 activation. These studies suggest PARP‐1 activation leads to rapid depletion of the cytosolic but not the mitochondrial NAD+ pool. Depletion of the cytosolic NAD+ pool renders the cells unable to utilize glucose as a metabolic substrate. Under conditions where glucose is the only available metabolic substrate, this leads to cell death. This cell death pathway is particularly germane to brain because glucose is normally the only metabolic substrate that is transported rapidly across the blood–brain barrier.

Tricarboxylic Acid Cycle Substrates Prevent PARP-Mediated Death of Neurons and Astrocytes

The DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP1), contributes to cell death during ischemia/reperfusion when extensively activated by DNA damage. The cell death resulting from PARP1 activation is linked to NAD+ depletion and energy failure, but the intervening steps are not well understood. Because glycolysis requires cytosolic NAD+, the authors tested whether PARP1 activation impairs glycolytic flux and whether substrates that bypass glycolysis can rescue cells after PARP1 activation. PARP1 was activated in mouse cortical astrocyte and astrocyte-neuron cocultures with the DNA alkylating agent, N-methyl-N ′-nitro-N-nitrosoguanidine (MNNG). Studies using the 2-deoxyglucose method confirmed that glycolytic flux was reduced by more than 90% in MNNG-treated cultures. The addition of 5 mmol/L of α-ketoglutarate, 5 mmol/L pyruvate, or other mitochondrial substrates to the cultures after MNNG treatment reduced cell death from approximately 70% to near basal levels, while PARP inhibitors and excess glucose had negligible effects. The mitochondrial substrates significantly reduced cell death, with delivery delayed up to 2 hours after MNNG washout. The findings suggest that impaired glycolytic flux is an important factor contributing to PARP1-mediated cell death. Delivery of alternative substrates may be a promising strategy for delayed treatment of PARP1-mediated cell death in ischemia and other disorders.

The poly(ADP-ribose) glycohydrolase inhibitor gallotannin blocks oxidative astrocyte death.

&NA; Poly(ADP‐ribose) polymerase (PARP) is now recognized as an important mediator of cell death, but a role for poly(ADP‐ribose) glycohydrolase (PARG) in cell death has not previously been described. PARG is the key enzyme degrading ADP‐ribose polymers produced by PARP. Here we report effects of the PARG inhibitor gallotannin on oxidative cell death. Pre‐incubation of cultured murine astrocytes with as little as 100 nM gallotannin produced significant reductions in H2O2‐induced cell death assessed both 24 and 72 h after H2O2 exposure. Gallotannin was more than 10‐fold more potent than the PARP inhibitor benzamide in preventing H2O2‐induced cell death. These results provide the first evidence that PARG inhibitors could be used to prevent oxidative cell death.

Intranasal administration with NAD+ profoundly decreases brain injury in a rat model of transient focal ischemia.

Excessive poly(ADP-ribose) polymerase-1 (PARP-1) activation plays a significant role in ischemic brain damage. Increasing evidence has supported the hypothesis that PARP-1 induces cell death by depleting intracellular NAD+. Based on our in vitro finding that NAD+ treatment can abolish PARP-1-mediated cell death, we hypothesized that NAD+ administration may decrease ischemic brain injury. In this study, we used a rat model of transient focal ischemia to test this hypothesis. We observed that intranasal NAD+ delivery significantly increased NAD+ contents in the brains. Intranasal delivery with 10 mg/kg NAD+ at 2 hours after ischemic onset profoundly decreased infarct formation when assessed either at 24 or 72 hours after ischemia. The NAD+ administration also significantly attenuated ischemia-induced neurological deficits. In contrast, intranasal administration with 10 mg/kg nicotinamide did not decrease ischemic brain damage. These results provide the first in vivo evidence that NAD+ metabolism is a new target for treating brain ischemia, and that NAD+ administration may be a novel strategy for decreasing brain damage in cerebral ischemia and possibly other PARP-1-associated neurological diseases.

Differences among cell types in NAD(+) compartmentalization: a comparison of neurons, astrocytes, and cardiac myocytes.

Activation of the nuclear enzyme poly(ADP‐ribose)‐1 leads to the death of neurons and other types of cells by a mechanism involving NAD+ depletion and mitochondrial permeability transition. It has been proposed that the mitochondrial permeability transition (MPT) is required for NAD+ to be released from mitochondria and subsequently consumed by PARP‐1. In the present study we used the MPT inhibitor cyclosporine‐A (CsA) to preserve mitochondrial NAD+ pools during PARP‐1 activation and thereby provide an estimate of mitochondrial NAD+ pool size in different cell types. Rat cardiac myocytes, mouse cardiac myocytes, mouse cortical neurons, and mouse cortical astrocytes were incubated with the genotoxin N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) in order to activate PARP‐1. In all four cell types MNNG caused a reduction in total NAD+ content that was blocked by the PARP inhibitor 3,4‐dihydro‐5‐[4‐(1‐piperidinyl)butoxy]‐1(2H)‐isoquinolinone. Inhibition of the mitochondrial permeability transition with cyclosporine‐A (CsA) prevented PARP‐1‐induced NAD+ depletion to a varying degree in the four cell types tested. CsA preserved 83.5% ± 5.2% of total cellular NAD+ in rat cardiac myocytes, 85.7% ± 8.9% in mouse cardiac myocytes, 55.9% ± 12.9% in mouse neurons, and 22.4% ± 7.3% in mouse astrocytes. CsA preserved nearly 100% of NAD+ content in mitochondria isolated from these cells. These results confirm that it is the cytosolic NAD+ pool that is consumed by PARP‐1 and that the mitochondrial NAD+ pool is consumed only after MPT permits mitochondrial NAD+ to exit into the cytosol. These results also suggest large differences in the mitochondrial and cytosolic compartmentalization of NAD+ in these cell types.