Key Event Title
Key Event Component
|ionotropic glutamate receptor activity||ionotropic glutamate receptor complex||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment.||MolecularInitiatingEvent|
Level of Biological Organization
|Drosophila melanogaster||Drosophila melanogaster||Strong||NCBI|
|Rattus norvegicus||Rattus norvegicus||Strong||NCBI|
|Primates sp. BOLD:AAA0001||Primates sp. BOLD:AAA0001||Strong||NCBI|
How This Key Event Works
The MIE of this AOP can be triggered by direct binding of an agonist to NMDARs or indirectly through initial activation of KA/AMPARs. Indeed, binding of agonist to KA/AMPARs results in ion influx (Na+ and a small efflux of K+) and glutamate release from excitatory synaptic vesicles causing depolarization of the postsynaptic neuron (Dingledine et al. 1999). Upon this depolarization the Mg2+ block is removed from the pore of NMDARs, allowing sodium, potassium, and importantly, calcium ions to enter into a cell. At positive potentials NMDARs then show maximal permeability (i.e., large outward currents can be observed under these circumstances). Due to the time needed for the Mg2+ removal, NMDARs activate more slowly, having a peak conductance long after the KA/AMPAR peak conductance takes place. It is important to note that NMDARs conduct currents only when Mg2+ block is relieved, glutamate is bound, and the postsynaptic neuron is depolarized. For this reason the NMDA receptors act as “coincidence detectors” and play a fundamental role in the establishment of Hebbian synaptic plasticity which is considered the physiological correlate of associative learning (Daoudal and Debanne, 2003; Glanzman, 2005). Post-synaptic membrane depolarization happens almost always through activation of KA/AMPARs (Luscher and Malenka, 2012). Therefore, a MIE of this AOP is defined as binding of an agonist to these three types of ionotropic receptors (KA/AMPA and NMDA) that can result in a prolonged overactivation of NMDARs through (a) direct binding of an agonist or (b) indirect, mediated through initial KA/AMPARs activation. The excitotoxic neuronal cell death, triggered by sustained NMDARs overactivation in the hippocampus and/or cortex leads to the impaired learning and memory, defined as the adverse outcome (AO) of this AOP.
Biological state: L-glutamate (Glu) is a neurotransmitter with important role in the regulation of brain development and maturation processes. Two major classes of Glu receptors, ionotropic and metabotropic, have been identified. Due to its physiological and pharmacological properties, Glu activates three classes of ionotropic receptors named: α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA receptors), 2-carboxy-3-carboxymethyl-4-isopropenylpyrrolidine (kainate receptors) and N-methyl-D-aspartate (NMDA receptors, NMDARs), which transduce the postsynaptic signal. Ionotropic glutamate receptors are integral membrane proteins formed by four large subunits that compose a central ion channel pore. In case of NMDA receptors, two NR1 subunits are combined with either two NR2 (NR2A, NR2B, NR2C, NR2D) subunits and less commonly are assembled together with a combination of NR2 and NR3 (A, B) subunits (reviewed in Traynelis et al., 2010). To be activated NMDA receptors require simultaneous binding of both glutamate to NR2 subunits and of glycine to either NR1 or NR3 subunits that provide the specific binding sites named extracellular ligand-binding domains (LBDs). Apart from LBDs, NMDA receptor subunits contain three more domains that are considered semiautonomous: 1) the extracellular amino-terminal domain that plays important role in assembly and trafficking of these receptors; 2) the transmembrane domain that is linked with LBD and contributes to the formation of the core of the ion channel and 3) the intracellular carboxyl-terminal domain that influences membrane targeting, stabilization, degradation and post-translation modifications.
Biological compartments: The genes of the NMDAR subunits are expressed in various tissues and are not only restricted to the nervous system. The level of expression of these receptors in neuronal and non-neuronal cells depends on: transcription, chromatin remodelling, mRNA levels, translation, stabilization of the protein, receptor assembly and trafficking, energy metabolism and numerous environmental stimuli (reviewed in Traynelis et al., 2010). In hippocampus region of the brain, NR2A and NR2B are the most abundant NR2 family subunits. NR2A-containing NMDARs are mostly expressed synaptically, while NR2B-containing NMDARs are found both synaptically and extrasynaptically (Tovar and Westbrook, 1999).
General role in biology: NMDA receptors, when compared to the other Glu receptors, are characterized by higher affinity for Glu, slower activation and desensitisation kinetics, higher permeability for calcium (Ca2+) and susceptibility to potential-dependent blockage by magnesium ions (Mg2+). NMDA receptors are involved in fast excitatory synaptic transmission and neuronal plasticity in the central nervous system (CNS). Functions of NMDA receptors:
1. They are involved in cell signalling events converting environmental stimuli to genetic changes by regulating gene transcription and epigenetic modifications in neuronal cells (Cohen and Greenberg, 2008).
2. In NMDA receptors, the ion channel is blocked by extracellular Mg2+ and Zn2+ ions, allowing the flow of Na+ and Ca2+ ions into the cell and K+ out of the cell which is voltage-dependent. Ca2+ flux through the NMDA receptor is considered to play a critical role in pre- and post-synaptic plasticity, a cellular mechanism important for learning and memory (Barria and Malinow, 2002).
3. The NMDA receptors have been shown to play an essential role in the strengthening of synapses and neuronal differentiation, through long-term potentiation (LTP), and the weakening of synapses, through long-term depression (LTD). All these processes are implicated in the memory and learning function (Barria and Malinow, 2002).
How It Is Measured or Detected
Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?
1. Ex vivo: The most common assay used is the NMDA receptor (MK801 site) radioligand competition binding assay (Reynolds and Palmer, 1991; Subramaniam and McGonigle, 1991; http://pdsp.med.unc.edu/UNC-CH%20Protocol%20Book.pdf; http://www.currentprotocols.com/WileyCDA/CPUnit/refId-ph0120.html). This assay is based on the use of the most potent and specific antagonist of this receptor, MK801 that is used to detect and differentiate agonists and antagonists (competitive and non-competitive) that bind to this specific site of the receptor. Also radioligand competition binding assay can be performed using D, L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity selective antagonist at the glutamate site of NMDA receptor, which is a quantitative autoradiography technique (Mugnaini et al., 1996). D-AP5, a selective N-methyl-D-aspartate (NMDA) receptor antagonist that competitively inhibits the glutamate binding site of NMDA receptors, can be studied by evoked electrical activity measurements. AP5 has been widely used to study the activity of NMDA receptors particularly with regard to researching synaptic plasticity, learning, and memory (Evans et al.,1982; Morris, 1989). The saturation binding of radioligands are used to measure the affinity (Kd) and density (Bmax) of kainate and AMPA receptors in striatum, cortex and hippocampus (Kürschner et al., 1998).
2. In silico: The prediction of NMDA receptor targeting is achievable by combining database mining, molecular docking, structure-based pharmacophore searching, and chemical similarity searching methods together (Neville and Lytton, 1999; Mazumder Borah, 2014)
Evidence Supporting Taxonomic Applicability
The major determinants for ligand e.g. for both co-agonist glycine binding and L-glutamate binding are well conserved between species from lower organism to mammals (reviewed in Xia and Chiang, 2009). PCR analysis, cloning and subsequent sequencing of the seal lion NMDA receptors showed 80% homology to those from rats, but more than 95% homologus to those from dogs (Gill et al., 2010).
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
Evidence for Chemical Initiation of this Molecular Initiating Event
L-Glutamate and glycine (or D-serine) are endogenous agonists that bind to the LBD of specific NMDA receptor subunits. Here listed some known agonists for NMDA receptor, some of them are specific to the NR1 subunit and some others to the NR2 subunit (reviewed in Traynelis et al., 2010):
Specific to NR1
Glycine, l-Serine, d-Serine, l-Alanine, d-Alanine, d-Cycloserine, HA 966, (+)-(1-hydroxy-3-aminopyrrolidine-2-one,) β-Cl-d-Alanine, β-F-dl-Alanine, tri-F-dl-Alanine, ACPC, 1-aminocyclopropane-1-carboxylic acid, ACBC, 1-aminocyclobutane-1-carboxylic acid, GLYX-13.
Specific to NR2
l-Glutamate, d-Glutamate, l-Aspartate, d-Aspartate, N-Methyl-l-aspartate, N-Methyl-d-aspartate, SYM208,1 l-Homocysteinsulfinate, d-Homocysteinsulfinate, l-Homocysteate, d-Homocysteate, l-Cysteinesulfinate, l-Cysteate, d-Cysteate, Homoquinolinate, Ibotenate, (R,S)-(Tetrazol-5-yl)glycine, L-CCG-IV, (2S,3R,4S)-2-(carboxycyclopropyl)glycine, trans-ACBD, trans-1-aminocyclobutane-1,3-dicarboxylate, cis-ADA, cis-azetidine-2,4-dicarboxylic acid, trans-ADC, azetidine-2,4-dicarboxylic acid, cis-ACPD, (1R,3R)-aminocyclopentane-cis-dicarboxylate, cis-2,3-Piperidinedicarboxylic acid, (R)-NHP4G, 2-(N-hydroxylpyrazol-4-yl)glycine, (R,S)-Ethyl-NHP5G, 2-(N-hydroxypyrazol-5-yl)glycine, (R)-Propyl-NHP5G, 2-(N-hydroxypyrazol-5-yl)glycine.
Domoic acid (DomA) is structurally similar to kainic acid (KA) and both of them are analogues of the excitatory neurotransmitter L-glutamate. DomA induces excitotoxicity by an integrative action on ionotropic glutamate receptors at pre- and post-synaptic sides. DomA directly activates KA/AMPARs receptors followed by indirect activation of the NMDARs. Indeed, indirect activation of NMDARs by DomA is linked to the fact that KA and AMPA receptors activated by DomA induce increased levels of intracellular Ca2+ and Na+ which, in turn, causes endogenous glutamate release that subsequently potentiates activation of NMDARs (Berman and Murray, 1997; Berman et al., 2002; Watanabe et al., 2011). DomA has been demonstrated through both in vitro and in vivo approaches to indirectly activate the NMDARs (reviewed in Pulido et al., 2008).
Glufosinate (GLF)((RS)-2-amino-4-(hydroxy(methyl)phosphonoyl)butanoic acid, phosphinothricin) is a phosphorus containing amino acid herbicide that is naturally occurring as a component of the bacteria-derived bactericidal and fungicidal tripeptides bialaphos and phosalacine (Lanz et al., 2014). There are studies suggesting that convulsive and amnesic effects of GLF are mediated through direct binding and activation of NMDAR (Lantz et al., 2014; Matsumura et al., 2001). GLF agonist action at the NMDAR is expected to occur through direct interaction with the glutamate binding site and requires binding of the glycine co-agonist as well as release of the magnesium block from the channel pore.
(for Abstract and MIE)
Barenberg P, Strahlendorf H, Strahlendorf., Hypoxia induces an excitotoxic-type of dark cell degeneration in cerebellar Purkinje neurons. J. Neurosci Res. 2001, 40(3): 245-54.
Barria A, Malinow R. (2002) Subunit-specific NMDA receptor trafficking to synapses. Neuron 35: 345-353. Cohen S, Greenberg ME. (2008) Communication between the synapse and the nucleus in neuronal development, plasticity, and disease. Ann Rev Cell Dev Biol 24: 183-209.
Berman F.W. and T. F. Murray, “Domoic acid neurotoxicity in cultured cerebellar granule neurons is mediated predominantly by NMDA receptors that are activated as a consequence of excitatory amino acid release,” Journal of Neurochemistry, 1997, 69: 693–703.
Berman W.F., K. T. LePage, and T. F. Murray, Domoic acid neurotoxicity in cultured cerebellar granule neurons is controlled preferentially by the NMDA receptor Ca2+ influx pathway,” Brain Research, 2002, 924: 20–29.
Cohen S, Greenberg ME. (2008) Communication between the synapse and the nucleus in neuronal development, plasticity, and disease. Ann Rev Cell Dev Biol 24: 183-209.
Daoudal G, Debanne D, Long-term plasticity of intrinsic excitability: learning rules and mechanisms. Learn Mem., 2003, 10(6):456-65.
Dingledine R, Borges K, Bowie D, Traynelis SF., The glutamate receptor ion channels. Pharmacol Rev., 1999, 51: 7–61.
Evans, R.H., Francis, A.A., Jones, A.W., et al., The Effects of a Series of ω-Phosphonic α-Carboxylic Amino Acids on Electrically Evoked and Excitant Amino Acid-Induced Responses in Isolated Spinal Cord Preparations. Br J Pharmac., 1982, 75: 65-75.
Gill S, Goldstein T, Situ D, Zabka TS, Gulland FM, Mueller RW., Cloning and characterization of glutamate receptors in Californian sea lions (Zalophus californianus). Mar Drugs, 2010, 8: 1637-1649.
Glanzman DL., Associative learning: Hebbian flies. Curr Biol., 2005, 7: 15(11):R416-9.
Kürschner VC, Petruzzi RL, Golden GT, Berrettini WH, Ferraro TN., Kainate and AMPA receptor binding in seizure-prone and seizure-resistant inbred mouse strains. Brain Res. 1998, 5: 780-788.
Lantz SR, Mack CM, Wallace K, Key EF, Shafer TJ, Casida JE., Glufosinate binds N-methyl-D-aspartate receptors and increases neuronal network activity in vitro. Neurotoxicology, 2014, 45: 38-47.
Lefebvre KA, Robertson A., Domoic acid and human exposure risks: a review.Toxicon. 2010, 56: 218-30.
Luscher C. and Robert C. Malenka., NMDA Receptor-Dependent Long-Term Potentiation and Long-Term Depression (LTP/LTD). Cold Spring Harb Perspect Biol 2012, 4: a005710.
Matsumura N, Takeuchi C., Hishikawa K, Fujii T, Nakaki T., Glufosinate ammonium induces convulsion through N-methyl-D-aspartate receptors in mice. Neurosci Lett. 2001, 304: 123-5.
Mazumder MK, Borah A. Piroxicam inhibits NMDA receptor-mediated excitotoxicity through allosteric inhibition of the GluN2B subunit: an in silico study elucidating a novel mechanism of action of the drug. Med Hypotheses. 2014, 83(6): 740-6.
Mehta A, Prabhakar M, Kumar P, Deshmukh R, Sharma PL. Excitotoxicity: bridge to various triggers in neurodegenerative disorders. Eur J Pharmacol. 2013, 698: 6-18.
Morris, RJ. Synaptic Plasticity and Learning: Selective Impairment of Learning in Rats and Blockade of Long-Term Potentiation in vivo by the N-Methyl-D-Aspartate Receptor Antagonist AP5. J Neurosci., 1989, 9: 3040-3057.
Mugnaini M, van Amsterdam FT, Ratti E, Trist DG, Bowery NG. Regionally different N-methyl-D-aspartate receptors distinguished by ligand binding and quantitative autoradiography of [3H]-CGP 39653 in rat brain.British Journal of Pharmacology, 1996, 119: 819–828.
Neville KR, Lytton WW. Potentiation of Ca2+ influx through NMDA channels by action potentials: a computer model. Neuroreport., 1999, 10(17): 3711-6.
Pulido OM., Domoic acid toxicologic pathology: a review. Mar Drugs., 2008, 6: 180-219.
Reynolds IJ, Palmer AM. Regional variations in [3H]MK801 binding to rat brain N-methyl-D-aspartate receptors. J Neurochem. 1991, 56(5):1731-40.
Subramaniam S, McGonigle P. Quantitative autoradiographic characterization of the binding of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine ([3H]MK-801) in rat brain: regional effects of polyamines. J Pharmacol Exp Ther. 1991, 256(2): 811-9.
Schrattenholz A, Soskic V., NMDA receptors are not alone: dynamic regulation of NMDA receptor structure and function by neuregulins and transient cholesterol-rich membrane domains leads to disease-specific nuances of glutamate-signalling.Curr Top Med Chem., 2006, 6(7):663-86.
Tovar KR, Westbrook GL. (1999) The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J Neurosci. 19: 4180–4188.
Traynelis S, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, Hansen KB, Yuan H, Myers SJ, Dingledine R. Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev., 2010, 62: 405-496.
Watanabe KH, Andersen ME, Basu N, Carvan MJ 3rd, Crofton KM, King KA, Suñol C, Tiffany-Castiglioni E, Schultz IR. Defining and modeling known adverse outcome pathways: Domoic acid and neuronal signaling as a case study. Environ Toxicol Chem., 2011, 30: 9-21.
Xia S, Chiang AS. NMDA Receptors in Drosophila. In: Van Dongen AM, editor. Biology of the NMDA Receptor. Boca Raton (FL): CRC Press; 2009. Chapter 10. Available from: http://www.ncbi.nlm.nih.gov/books/NBK5286/
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