Neuronal NMDA receptor (NMDAR) activation leads to the formation of superoxide, which normally acts in cell signaling. With extensive NMDAR activation, the resulting superoxide production leads to neuronal death. It is widely held that NMDA-induced superoxide production originates from the mitochondria, but definitive evidence for this is lacking. We evaluated the role of the cytoplasmic enzyme NADPH oxidase in NMDA-induced superoxide production. Neurons in culture and in mouse hippocampus responded to NMDA with a rapid increase in superoxide production, followed by neuronal death. These events were blocked by the NADPH oxidase inhibitor apocynin and in neurons lacking the p47 phox subunit, which is required for NADPH oxidase assembly. Superoxide production was also blocked by inhibiting the hexose monophosphate shunt, which regenerates the NADPH substrate, and by inhibiting protein kinase C zeta, which activates the NADPH oxidase complex. These findings identify NADPH oxidase as the primary source of NMDA-induced superoxide production.Activation of the neuronal NMDAR initiates several downstream events, including cation influx, activation of nitric oxide synthase and formation of superoxide [1][2][3] . Superoxide functions as an inter-cellular messenger in long-term potentiation 4,5 and participates in redox inhibition of NMDAR channel function 6 ; however, superoxide can also promote neuronal death when NMDAR activation is sustained 1,7 . Notably, the primary source of superoxide induced by NMDAR activation remains unresolved.Initial studies suggested a mechanism in which Ca 2+ influx through NMDAR channels leads to mitochondrial depolarization 8,9 and subsequent mitochondrial production of superoxide 10,11 . However, a biochemical mechanism linking these events has not been identified and evidence supporting mitochondria as the primary source of neuronal superoxide production remains indirect 12,13 . Calcium was shown to induce superoxide production in isolated © 2009 Nature America, Inc. All rights reserved.Correspondence should be addressed to R.A.S. (Raymond.swanson@ucsf.edu). AUTHOR CONTRIBUTIONS A.M.B. carried out the cell culture studies and data analysis and prepared the manuscript drafts. S.W.S. supervised the mouse surgical studies and analyzed these data. S.J.W. performed mouse surgery studies and mouse brain histology. P.N. maintained the Sod2 + mouse colony and prepared the Sod2 + cell cultures. T.M.K. and Y.E. assisted with the p47 phox translocation studies and data analysis. H.L. assisted in the analysis of the cell culture ethidium fluorescence results. P.H.C. assisted with the studies involving Sod2 + neurons. R.A.S. organized the studies and prepared the final manuscript.Note: Supplementary information is available on the Nature Neuroscience website. 14 , but more recent studies indicate that this effect is highly dependent on experimental conditions, especially the presence of bovine serum albumin in the medium and succinate as a metabolic substrate 13 . The important question is w...

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.

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.

Clearing cellular debris after brain injury represents an important mechanism in regaining tissue homeostasis and promoting functional recovery. Triggering receptor expressed on myeloid cells-2 (TREM2) is a newly identified receptor expressed on microglia and is thought to phagocytose damaged brain cells. The precise role of TREM2 during ischemic stroke has not been fully understood. We explore TREM2 in both in vitro and in vivo stroke models and identify a potential endogenous TREM2 ligand. TREM2 knockdown in microglia reduced microglial activation to an amoeboid phenotype and decreased the phagocytosis of injured neurons. Phagocytosis and infarcted brain tissue resorption was reduced in TREM2 knock-out (KO) mice compared with wild-type (WT) mice. TREM2 KO mice also had worsened neurological recovery and decreased viable brain tissue in the ipsilateral hemisphere. The numbers of activated microglia and phagocytes in TREM2 KO mice were decreased compared with WT mice, and foamy macrophages were nearly absent in the TREM2 KO mice. Postischemia, TREM2 was highly expressed on microglia and TREM2-Fc fusion protein (used as a probe to identify potential TREM2 binding partners) bound to an unknown TREM2 ligand that colocalized to neurons. Oxygen glucose deprivation-exposed neuronal media, or cellular fractions containing nuclei or purified DNA, but not cytosolic fractions, stimulated signaling through TREM2. TREM2-Fc fusion protein pulled down nucleic acids from ischemic brain lysate. These findings establish the relevance of TREM2 in the phagocytosis of the infarcted brain and emphasize its role in influencing neurological outcomes following stroke. Further, nucleic acids may be one potential ligand of TREM2 in brain ischemia.