Neuronal Death by Glutamate Excitotoxicity: Protein Mediators & Strategies for Inhibition

Date 10/10/12. Updated 11/5/12.

Neuronal Cell Death Induced by Ischemic and Hemorrhagic Strokes

Commonly known as cerebral vascular events, strokes are a significant source of morbidity and mortality worldwide. Ischemic strokes account for approximately 80% of all strokes and are caused by obstructed arteries that deprive the brain of oxygen and other nutrients. In contrast, hemorrhagic strokes stem from ruptured blood vessels that increase intracranial pressure and reduce blood flow to the surrounding tissue. While the mechanisms of pathology differ between ischemic and hemorrhagic strokes, both induce neuronal cell death.1,2 Depending on the area of the brain affected, neuronal death can either be fatal or can impair speech, vision, or other sensorimotor functions.

Neuronal death following ischemic stroke is primarily attributed to dysfunction in the homeostasis of glutamate. Under physiological conditions, glutamate acts as the primary excitatory neurotransmitter in the nervous system.3 The release of glutamate into the synaptic space stimulates glutamate receptors of the NMDA subtype, which causes an influx of calcium and sodium and depolarization of the postsynaptic neuron. NMDA receptors (NMDA R) revert to the inactive state as transporters sequester glutamate into cells. However, ischemia causes ATP levels to decrease as cellular respiration is compromised. Decreased ATP production impairs glutamate transporters, results in neuronal depolarization, and leads to an unregulated accumulation of glutamate in the synaptic cleft. Excess glutamate over-activates NMDA R causing an influx of calcium, the production of reactive nitrogen species (RNS), mitochondrial dysfunction, and the generation of reactive oxygen species (ROS), all of which contribute to cell death. A secondary mechanism underlying neuronal cell death is inflammation.4,5 The ROS and cytokines released by dying neurons activate microglia, which secrete ROS and other cytotoxic factors that exacerbate neuronal damage.6

Molecular Targets for the Prevention of Stroke-induced Neuronal Cell Death

Tissue-Plasminogen Activator (tPA), the main treatment for ischemic strokes, activates the clot dissolving enzyme Plasmin, which restores  blood flow to ischemic areas of the brain.7 When administered within three hours of symptom onset, tPA improves neurological function in humans. However, it is also associated with an increased risk of intracerebral hemorrhage. The limitations of tPA treatment emphasize the need for new ischemic stroke therapies. Recently, several proteins that mediate ischemia-induced neuronal cell death have been identified as potential pharmacological targets for stroke therapy. For example, Ofengeim et al. recently demonstrated that cleavage of the anti-apoptotic protein Bcl-XL contributed to ischemia-induced apoptosis in a rat model.8 Neuronal cell death was reduced in transgenic mice expressing a cleavage-resistant form of Bcl-xL indicating that inhibition of Bcl-xL cleavage may have potential for stroke treatment. Recent reports indicate that the voltage-gated proton channel, Hv1 may be another pharmacological target for stroke treatment.9 Wu and colleagues showed that mice lacking Hv1 had lower stroke-induced infarct volumes relative to wild-type mice.9

Stroke-induced Neuronal Cell Death
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Stroke-induced Neuronal Cell Death. Under hypoxic conditions, glutamate accumulates in the synaptic cleft and over-activates NMDA receptors (NMDA R). Over-activated NMDA R trigger an influx of calcium and sodium, which stimulates the production of reactive oxygen species (ROS) such as superoxide (O2-), and hydrogen peroxide (H2O2) as well as reactive nitrogen species (RNS) such as nitric oxide (NO) and peroxinitrite (ONOO-). High concentrations of intracellular calcium, ROS, and RNS induce cell death by: 1) activating proteases that damage cellular architecture, 2) peroxidizing lipids, which disrupt membrane integrity, 3) stimulating microglia to produce cytotoxic factors, 4) disrupting mitochondrial function, and 5) inducing pyknosis (chromatin condensation). The red arrows indicate steps in ischemic stroke-induced cell death that may be targets for stroke therapies as discussed in the text.8,9,10,11,12
Abbreviations: NADPH Oxidase (NOX), Nitric Oxide Synthase (NOS).

Tat-NR2B9c is an Effective Neuroprotectant in Cynolomogus Macaques

Although rodent models of ischemic stroke have provided many lead compounds for clinical trials, few have translated to effective treatments in humans. However, Cook et al. recently demonstrated the efficacy of a small peptide inhibitor, Tat-NR2B9c, using a non-human primate stroke model.10 Tat-NR2B9c binds to Postsynaptic Density-95 kDa (PSD-95), a scaffolding protein that forms a ternary complex with NMDA R and Nitric Oxide Synthase (NOS). Tat-NR2B9c binds to PSD-95, which decreases ternary complex formation and inhibits NMDA R-induced activation of NOS, thereby reducing the production of Nitric Oxide (NO). When administered one or three hours after stroke initiation, Tat-NR2B9c significantly reduced infarct volumes and improved neurological function in macaques as determined by MRI and neurological assessments, respectively. Cook et al. also examined transcriptional activity in brain tissue near the infarct area as an indicator of cell functionality. Treatment with Tat-NR2B9c reduced the proportion of downregulated genes, indicating that inhibition of the PSD-95/NMDA R/NOS complex improved cell functionality in animals treated with Tat-NR2B9c compared to vehicle-treated animals.

These results coupled with the lack of toxicity and changes in transcriptional activity are encouraging indicators that Tat-NR2B9c may be effective in humans. This is further supported by the finding that the binding affinity of Tat-NR2B9c can be increased 1,000-fold by creating a dimeric form of the inhibitor.11 Human clinical trials with Tat-NR2B9c are ongoing.

Tat-NR2B9c Effectively Decreases Stroke Number in Humans

Since this article was originally published, the efficacy of Tat-NR2B9c in humans was examined as part of a phase II clinical trial. Tat-NR2B9c, referred to as NA-1 in the study, was administered to patients who were at risk of ischemic stroke after undergoing a procedure to repair an intracranial aneurysm.13 After the procedure, 92 patients received a ten minute infusion of NA-1 and 93 patients received a ten minute infusion of saline. At 2-4 days post-infusion, patients were monitored by MRI to assess the ability of NA-1 to reduce the number and volume of stroke lesions that developed as a result of the procedure. The results show that while stroke volumes did not differ between the two groups, patients treated with NA-1 had significantly fewer lesions compared to those treated with saline. Diffusion-weighted MRI demonstrated that the NA-1-treated group had an average of 4.1 lesions while the saline-treated control group had an average of 7.3 lesions, a difference that was statistically significant. NA-1 was not associated with any serious adverse reactions. The ability of NA-1 to reduce the number of stroke lesions without adverse effects is an encouraging indicator that the PSD-95/NMDA R/NOS complex is a valid pharmacological target for stroke treatment. The safety and efficacy of NA-1 will be investigated further in a larger clinical trial.


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