Glutamate excitotoxicity is a cell death mechanism triggered by excessive glutamate release from neurons and glial cells. Glutamate-mediated excitotoxicity is an important mechanism leading to injury after ischemic stroke. Exploring the role of glutamate excitotoxicity in ischemic stroke, including glutamate release, reuptake mechanisms, N-methyl-d-Aspartate (NMDA) receptor, and its downstream cell signaling pathways, may provide potential therapeutic targets for ischemic stroke.

At Ace Therapeutics, we specialize in providing comprehensive analysis services to help clients investigate ischemia-triggered glutamate excitotoxicity. We aim to help clients develop potential neuroprotective drugs and therapeutic approaches for the treatment of ischemic stroke.

Related Services

• Analysis of Ischemia-Triggered Glutamate Excitotoxicity
• Glutamate Receptor Antagonist Development for Stroke

Glutamate

Glutamate is the primary excitatory neurotransmitter in the central nervous system (CNS) and plays a crucial role in learning and memory. Even though most glutamate comes from the diet, it can't pass through the BBB and must be produced by CNS cells from alpha-ketoglutarate, a Krebs cycle intermediate. Also recirculated from glutamine is glutamate through a glial cell cycle. During normal synaptic communication, glutamate is released into the synaptic cleft and activates postsynaptic receptors; however, its levels must be tightly regulated to prevent excitotoxicity. Glutamate is the most excitatory neurotransmitter in the CNS and it's involved in learning and memory.

Overview of Glutamate Receptors

After glutamate is released, its postsynaptic receptors fire. They are ionotropic and metabotropic receptors. Activation of these receptors supports excitatory synaptic communication, as well as plasticity processes such as LTP and LTD, which are critical for learning and memory.

Ionotropic Glutamate Receptors (iGluRs)

These receptors act as ion channels for fast excitatory neurotransmission and flow Na⁺ and K⁺ ions that produce action potentials. When activated, excitatory amino acid transporters (EAATs) flush glutamate from the synaptic cleft. The three classes of iGluRs that differ in agonist sensitivity are NMDA, -amino acid-3-hydroxy-5-methyl-4-isoxazole (AMPA), and kainic acid (KA) receptors.

• NMDA Receptors (NMDARs): These receptors have high cation permeability, especially for Ca²⁺, and are typically blocked by Mg²⁺ under resting conditions. Activation requires both glutamate and glycine. NMDARs consist of various subunits (GluN1, GluN2, GluN3), with GluN3 acting as a negative regulator of synapse maturation.
• AMPA Receptors (AMPARs): Formed from GluA subunits (GluA1–4), AMPARs are crucial for fast synaptic transmission. The GluA2 subunit undergoes RNA editing, rendering the receptors impermeable to Ca²⁺.
• Kainic Acid Receptors (KARs): These are tetrameric channels made of GluK subunits (GluK1–5), with specific combinations required for functionality.

Metabotropic Glutamate Receptors (mGluRs)

These monomeric receptors are comprised of an extracellular binding domain for glutamate and an intracellular binding domain for G-proteins. By binding glutamate, G-protein is triggered, it is separated from the receptor and its effectors are altered, such as enzymes, transcription factors, and other ion channels. They are monomeric, involving eight isoforms (mGluR1–8) split into three classes (I, II, and III), varying in sequence identity, pharmacology, and effects inside cells.

Glutamate Excitotoxicity in Ischemic Stroke

The brain needs a lot of oxygen and glucose, and it's susceptible to reduced circulation (i.e., in ischemic stroke). When neurons have less ATP in response to ischemia and hypoxia, their energy stores become depleted and essential ion pumps such as Na⁺/K⁺-ATPase are destroyed. It is a perturbation that boosts glutamate release and suppresses glutamate reuptake leading to dysfunctional glutamate metabolism and intracellular calcium overproduction. Too much glutamate rouses the NMDAR on postsynaptic membranes to the point of cell death.

There are multiple mechanisms and pathways lead to excitotoxic cell damage including pro-death signaling cascade events downstream of glutamate receptors, calcium overload, oxidative stress, mitochondrial impairment, excessive glutamate in the synaptic cleft as well as altered energy metabolism.

Fig.1. Molecular mechanisms of ischemia and glutamate excitotoxicity. (Neves, et al., 2023)

Ca²⁺ Overload

Under normal conditions, the sodium-potassium ATPase maintains the action potential by exchanging Na⁺ and K⁺ ions, while other pumps like the Na⁺/Ca²⁺ exchanger (NCX) and Ca²⁺-ATPase regulate calcium levels. During ischemic conditions, energy depletion disrupts these pumps, leading to an influx of Na+ and Ca²⁺ and a loss of K⁺, which causes a rise in intracellular calcium. This calcium overload triggers excessive glutamate release, and the impaired glutamate transport further increases extracellular glutamate levels. This glutamate accumulation contributes to excitotoxicity and neuronal death. Stabilizing ion pump function early in ischemic stroke could help protect against this excitotoxic damage.

Fig.2. The balance of ion and glutamate concentration at neuro synapses. (Shen, et al., 2022)

Impaired Glutamate Release and Reuptake

In normal brain function, glutamate is synthesized in neurons and astrocytes, released into the synaptic space, and then cleared by astrocytes for reuptake. This process involves energy-consuming pathways and specific transporter proteins. However, during ischemic stroke, energy depletion disrupts glutamate metabolism, leading to uncontrolled extracellular glutamate concentrations, which contribute to excitotoxicity and cell death. Studies show that high glutamate concentrations can cause significant neuronal damage. Interventions that inhibit glutamate synthesis and release, or promote its clearance by astrocytes, may help protect against excitotoxic damage.

Dual Role of the NMDAR in Death and Survival

Normal activation of NMDAR by glutamate supports various neural functions and promotes neuronal survival. However, excessive activation during pathological conditions like ischemia triggers neurotoxic cascades leading to neuronal death. NMDAR antagonists can protect neurons from excitotoxic damage but may interfere with normal receptor activity, limiting their effectiveness.

NMDAR is composed of GluN1 and GluN2 subunits, playing distinct roles. In the non-excitotoxic brain, synaptic NMDARs containing the GluN2A subunit are critical for neuronal survival and development, while extrasynaptic NMDARs containing GluN2B are associated with neuronal death.

Fig.3. Distinct subpopulations of the NMDAR mediate neuronal death and survival. (Lai, et al., 2014)

1. Neves, D., et al. (2023). Molecular mechanisms of ischemia and glutamate excitotoxicity. Life sciences, 328, 121814.
2. Shen, Z., et al. (2022). Glutamate excitotoxicity: Potential therapeutic target for ischemic stroke. Biomedicine & Pharmacotherapy, 151, 113125.
3. Lai, T. W., et al. (2014). Excitotoxicity and stroke: identifying novel targets for neuroprotection. Progress in neurobiology, 115, 157-188.