Superoxide and Non-ionotropic Signaling in Neuronal Excitotoxicity

Abstract

Excitotoxicity is classically attributed to Ca²⁺ influx through NMDA receptors (NMDAr), leading to production of nitric oxide by neuronal nitric oxide synthase and superoxide by mitochondria, which react to form highly cytotoxic peroxynitrite. More recent observations warrant revision of the classic view and help to explain some otherwise puzzling aspects of excitotoxic cell injury. Studies using pharmacological and genetic approaches show that superoxide produced by NMDAr activation originates primarily from NADPH oxidase rather than from mitochondria. As NADPH oxidase is localized to the plasma membrane, this also provides an explanation for the extracellular release of superoxide and cell-to-cell "spread" of excitotoxic injury observed in vitro and in vivo. The signaling pathway linking NMDAr to NADPH oxidase involves Ca²⁺ influx, phosphoinositol-3-kinase, and protein kinase Cζ, and interventions at any of these steps can prevent superoxide production and excitotoxic injury. Ca²⁺ influx specifically through NMDAr is normally required to induce excitotoxicity, through a mechanism presumed to involve privileged Ca²⁺ access to local signaling domains. However, experiments using selective blockade of the NMDAr ion channel and artificial reconstitution of Ca²⁺ by other routes indicate that the special effects of NMDAr activation are attributable instead to concurrent non-ionotropic NMDAr signaling by agonist binding to NMDAr. The non-ionotropic signaling driving NADPH oxidase activation is mediated in part by phosphoinositol-3-kinase binding to the C-terminal domain of GluN2B receptor subunits. These more recently identified aspects of excitotoxicity expand our appreciation of the complexity of excitotoxic processes and suggest novel approaches for limiting neuronal injury.

Keywords: Glun2B; NADPH oxidase; calcium; glucose; glutamate; metabotropic; peroxynitrite; phosphoinositol-3-kinase.

FIGURE 1

Structure and glucose dependence of NADPH oxidase. (A) NADPH oxidase is a multi-subunit enzyme that uses electrons derived from NADPH on one side of a membrane to generate superoxide on the other side. The NOX2 isoform of NADPH oxidase diagrammed here is the dominant form expressed by neurons. NOX2 requires activation of both the GTPase rac and the p47^phox subunit to be catalytically functional. p47^phox functions as an organizing subunit that bring the cytosolic subunits together with the membrane-bound subunits. In neurons, p47^phox is activated by phosphorylation by PKCzeta (reprinted from Bedard and Krause, 2007, with permission). (B) Production of superoxide by NADPH oxidase requires glucose because glucose is the requisite substrate for the pentose phosphate shunt that regenerates NADPH from NADP⁺. By contrast, superoxide production from mitochondria can be fueled by pyruvate or other non-glucose substrates. (C) Neurons exposed to NMDA exhibit very attenuated superoxide production when deprived of glucose, indicating NADPH oxidase rather than mitochondria as the primary source. Data are means ± SEM; *p < 0.05 vs. no glucose. Redrawn from Brennan et al. (2009).

FIGURE 2

Extracellular release of superoxide by NMDA-induced NADPH oxidase activation. (A) Superoxide release from mitochondria will first encounter intracellular constituents of the cell in which it is enclosed, and can reach the extracellular space only be eluding cytosolic superoxide dismutase and crossing the lipid plasma membrane. By contrast, NADPH oxidase releases superoxide directly into the cytosol, where it can more readily interact with neighboring cells. (B) Cell-to-cell transmission of oxidative stress in neuronal cultures in which only a small subset of neurons (labeled green) contain functional NADPH oxidase. Application of NMDA to these cultures induces oxidative stress in neighboring neurons, as detected by the lipid peroxidation marker 4-hydroxynonenal (red). Cell nuclei are counterstained with DAPI (blue). Reprinted from Reyes et al. (2012). (C) Known steps in the signaling pathway linking NADPH oxidase to NMDAr activation. Reprinted from Minnella et al. (2018).

FIGURE 3

Non-ionotropic signaling in excitotoxicity. (A) Binding of the agonists glycine and NMDA to an NMDAr containing a GluN2B subunit triggers Ca²⁺ influx through the NMDAr channel and subsequent superoxide production by NOX2. (B) If the Ca²⁺ influx is blocked by the NMDAr glycine site antagonist 7-chlorokynurenic acid (7CK), there is no superoxide formation despite NMDA binding. (C) If, in addition, ionomycin is used to reconstitute the Ca²⁺ influx, superoxide is again produced by NOX2. (D) Ionomycin does not induce superoxide production when NMDA binding to GluN2B is blocked by (2R)-amino-5-phosphonopentanoate (AP5). Note the association of PI3K with the GluN2 subunit of the NMDAr requires ligand binding to the GluN2 subunit, but does not require Ca²⁺ influx. (E,F) Quantified measure of intracellular Ca²⁺ elevations and superoxide production under the conditions diagrammed in (A–D). Data are means ± SEM; *p < 0.05 vs. control. Reprinted from Minnella et al. (2018), with modifications.