NADPH oxidase-2: linking glucose, acidosis, and excitotoxicity in stroke

Abstract

Significance: Neuronal superoxide production contributes to cell death in both glutamate excitotoxicity and brain ischemia (stroke). NADPH oxidase-2 (NOX2) is the major source of neuronal superoxide production in these settings, and regulation of NOX2 activity can thereby influence outcome in stroke.

Recent advances: Reduced NOX2 activity can rescue cells from oxidative stress and cell death that otherwise occur in excitotoxicity and ischemia. NOX2 activity is regulated by several factors previously shown to affect outcome in stroke, including glucose availability, intracellular pH, protein kinase ζ/δ, casein kinase 2, phosphoinositide-3-kinase, Rac1/2, and phospholipase A2. The newly identified functions of these factors as regulators of NOX2 activity suggest alternative mechanisms for their effects on ischemic brain injury.

Critical issues: Key aspects of these regulatory influences remain unresolved, including the mechanisms by which rac1 and phospholipase activities are coupled to N-methyl-D-aspartate (NMDA) receptors, and whether superoxide production by NOX2 triggers subsequent superoxide production by mitochondria.

Future directions: It will be important to establish whether interventions targeting the signaling pathways linking NMDA receptors to NOX2 in brain ischemia can provide a greater neuroprotective efficacy or a longer time window to treatment than provided by NMDA receptor blockade alone. It will likewise be important to determine whether dissociating superoxide production from the other signaling events initiated by NMDA receptors can mitigate the deleterious effects of NMDA receptor blockade.

FIG. 1.

NADPH oxidase (NOX). The NADPH oxidase-2 (NOX2) complex generates superoxide on one side of the membrane, while oxidizing NADPH to NADP and H⁺ on the other side. Activation of the enzymatic complex requires translocation of the cytosolic subunits (light blue) p47^phox, p67^phox, and p40^phox to the membrane along with Rac1 and phospholipase A2 (PLA2), where the membrane-bound subunits (dark blue) gp91 and p22^phox are located. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

Neuronal NOX2 activation leads to oxidative stress in neighboring neurons. p47^phox−/− neurons, which cannot assemble a functional NOX2 complex, were transfected at low efficiency with GFP-labeled p47^phox (green) to reconstitute the NOX2 activity in a small fraction of cultured neurons. The cultures were treated with 100 μM N-methyl-D-aspartate (NMDA) for 30 min. 4-hydroxynonenal (4HNE) formation (red) identifies oxidative stress in many nontransfected neurons contiguous with processes of the two NOX2-competent neurons in the field (green). Neuronal soma and processes are labeled blue by immunostaining for MAP2. Reprinted from Reyes et al. (150). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

Coupling of NOX2 activation to NMDA receptor activation in neurons. Calcium influx via NR2B-containing NMDA-type glutamate receptors induces phosphoinositide-3-kinase (PI3K) to form phosphatidylinositol (3,4,5) trisphosphate (PIP3). PIP3 activates protein kinase C (zeta) (PKCζ), which in turn phosphorylates the p47^phox organizing subunit of NOX2. Phosphorylated p47^phox induces assembly of the NOX2 complex at the cell surface. The active NOX2 complex additionally requires binding to an activated GTPase, Rac1. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

Intracellular H⁺ inhibits NOX2 activity. The oxidation of NADPH during the enzymatic activation of NOX also produces H⁺, and removal of these H⁺ is required for sustained NOX activity. In most cells types, this is accomplished by the proton channel, Hv1, and/or the Na⁺/H⁺ exchanger, NHE1. In addition, to production of H⁺ by NOX, several other pathways may contribute to intracellular acidification during ischemia, and thus indirectly inhibit NOX2 activity. These include anaerobic metabolism of glucose to lactic acid, mitochondrial depolarization, and extrusion of Ca²⁺ by Ca²⁺/H⁺ exchange. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

Glucose is required for NOX2 activity. NOX2 oxidizes NADPH to NADP⁺, and continuous NOX2 function requires a continuous NADPH supply. NADPH is regenerated by the pentose phosphate pathway, which uses glucose as an obligate substrate. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

Hyperglycemia promotes reperfusion injury and hemorrhage by fueling NOX2. Neuronal superoxide production is low under basal conditions. It remains low after arterial occlusion because of limited oxygen supply and lactic acidosis. Upon reperfusion, the oxygen supply is restored and lactic acid is washed out. NOX2 then produces high levels of superoxide in response to extracellular glutamate acting at NMDA-type glutamate receptors. Glucose availability can be rate limiting for NOX2 superoxide production, and under hyperglycemic conditions, the increased glucose supply leads to increased superoxide production. This in turn can increase damage to blood vessel walls and promote reperfusion hemorrhage. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars