The Dichotomous Role of N-Methyl-D-Aspartate Receptors In

Holmes AR, Castellino FJ, Balsara RD. The Dichotomous Role of N-methyl-D-Aspartate Receptors in Ischemic Stroke. J Cardiol and Cardiovasc Sciences (2018) 2(4): 23-32

Mini Review Article Open Access

The Dichotomous Role of N-methyl- D-Aspartate Receptors in Ischemic Stroke Adam R. Holmes1, Francis J. Castellino1,2, Rashna D. Balsara1,2* 1W. M. Keck Center for Transgene Research, University of Notre Dame, Indiana 46556, USA 2Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA

Article Info Abstract

Article Notes Ischemia-mediated glutamate elevation causes activation of the N-methyl- Received: May 15, 2018 D-aspartate receptor (NMDAR) and consequent excitotoxicity. This triggers Accepted: August 11, 2018 a cascade of pathological events, including aberrant NMDAR ion channel 2+ *Correspondence: kinetics, large neuronal Ca influx, and activation of pro-death signaling Dr. Rashna D. Balsara, Department of Chemistry and pathways. Previous studies have shown that functional outcomes of post- Biochemistry, University of Notre Dame, Indiana 46556, ischemia are influenced by the type of GluN2 subunit assembled in the NMDAR USA; Telephone No: (574) 631-2958; Fax No: (574) 631- (GluN2A, GluN2B, GluN2C, or GluN2D), as well as its cellular location. GluN2A- 4048; Email: [email protected]. containing synaptic NMDAR’s activate pro-survival pathways, whereas, © 2018 Balsara RD. This article is distributed under the terms of activation of GluN2B-containing extrasynaptic NMDARs results in cell death. the Creative Commons Attribution 4.0 International License. However, there is no consensus omnium on the individual role of the GluN2 subunits in ischemia. Published studies suggest that the GluN2A, GluN2B, and Keywords: GluN2C subunits can promote either neuronal death or survival, depending Ischemia on the experimental model employed and the CNS region investigated. In this N-methyl- D- aspartate receptor mini-review, we aim to succinctly outline the mechanisms that underlie the GluN2 subunits Calcium influx dichotomous role of the NMDAR in ischemic stroke and possible NMDAR- Synaptic directed therapeutic approaches. Extrasynaptic Signaling Introduction The N-methyl- D-aspartate receptor (NMDAR) is a ligand and voltage gated ionotropic neuroreceptor that is co-agonized by

of Ca2+, the result of which induces neuronal signaling events and propagationglutamate and of action glycine. potentials. Activation These of this phenomena receptor allowsare requisite influx for the long-term potentiation and depression of pathways that

the post-synaptic membrane of neurons, although studies have also notedmediate their higher- presencelevel inexecutive non-neuronal functions. cells, The including NMDAR astrocytes is present and on oligodendrocytes. At resting potentials, these ligand-gated channels are blocked by Mg2+. Interaction of the NMDAR with glutamate in the synaptic cleft results in depolarization of post-synaptic neurons that 2+ and Na+ +1. However,

isthe accompanied synaptic bouton by influx accompanied of Ca by ,a and surfeit efflux of of post-K synaptic duringCa2+ neuropathologies, an excess of glutamate is released from processes that lead to neuronal death2. Thus, NMDARs play a critical role in influx, not only resulting maintaining in a multitude neuronal- ofrelated unfavorable physiology, downstream resulting from synaptic plasticity, but also in pathophysiological diseases, including ischemic stroke, Alzheimer’s disease, Parkinson’s disease, and alcohol dependence3.

Page 23 of 32 Holmes AR, Castellino FJ, Balsara RD. The Dichotomous Role of N-methyl-D-Aspartate Receptors in Ischemic Stroke. J Cardiol and Cardiovasc Sciences (2018) 2(4): 23-32 Journal of Cardiology and Cardiovascular Sciences

Expression, Structural, and Functional Properties biochemical, pharmacological, and electrophysiological of NMDAR properties to the ion channel in accordance with their 10 (Figure 1). NMDARs can Understanding the various properties of the specificdifferent physiologicaltypes of GluN2 rolessubunits, which possess functional physiological and pathological conditions. The NMDAR also exist as triheteromers composed of GluN1 and two NMDAR is integral to defining its functional role under and gating characteristics distinct from the diheteromeric NMDAR11,12 (Figure 2). the functional ion channel is regulated developmentally, temporally,is a heterotetrameric and spatially. complex, The channel and theis composed composition of two of glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. The mandatory GluN1 subunit has eight GluN1Expression subunit begins of the at GluNembryonic subunits day 14 is and spatially is present and isoforms (GluN1-1a to 4a and GluN1–1b to 4b) that arise throughouttemporally regulated. the CNS, theAlthough, distribution expression pattern of the of itsubiquitous various from alternative splicing4. There are four different GluN2 isoforms changes depending on the region of the brain, and subunits (GluN2A-D) that are products of independent genes5-7 . Most commonly, the NMDARs found in the forebrain In rodents, the GluN1-1 isoform is found concentrated in are composed of either two GluN2A or two GluN2B subunits. is based on the alternate splicing of exons 5, 21, and 22. GluN2C-containing NMDARs are ample in the cerebellum, is predominantly found in the thalamus and cerebellum. hippocampus, and amygdala. GluN2D subunits are mainly the cortex and hippocampus, whereas, the GluN1-4 subunit associated with NMDARs of the hippocampus, brain stem, the dentate gyrus and CA1-3 layer of the hippocampus. diencephalon, and basal ganglia8. A GluN3 (A/B) subunit The GluN1-1a isoform lacking exon 5 is found mainly in that binds to glycine and co-assembles with GluN1/GluN2 highly charged amino acids in the N-terminal region is subunits has been more recently discovered9. It is well concentratedThe GluN1-1b in the isoform CA3 hippocampal containing region. exon The 5 encodedGluN1-3 known that the GluN2 subunits impart unique biophysical, 13,14. High levels of GluN2B and

subunit is poorly expressed

Figure 1: A schematic representation of the heterotetrameric NMDAR that consists of two GluN1 subunits and two GluN2(A/B) subunits. The GluN1 subunit can bind to glycine, D-serine, 7-Chlorokynurenic acid (antagonist), and extracellular protons (H+), and shows the splicing sites on exons 5, 21, and 22. The GluN2(A/B) subunit has the glutamate binding site, but can also bind to various pharmacological agents in a subunit-dependent manner. Ifenprodil and conantokin-G/RlB are selective antagonists for the GluN2B subunit, whereas, NVP-AAM077 and TCN-201 are GluN2A-specific antagonists. Extracellular Zn2+ binds to the GluN2A subunit with high affinity (nM) and to

the GluN2B subunit with low affinity (µM). The TMD (1-4) topology that are connected by linker regions, the amino terminal end (NH2) and the C-terminal domain (CTD) are shown. Upon binding of the glutamate/glycine co-agonists, the Mg2+ block is removed allowing influx of Ca2+ and Na+ ions, and efflux of K+ ions. The open channel blockers MK-801, ketamine, and phencyclidine are some of the few uncompetitive NMDAR antagonists.

Page 24 of 32 Holmes AR, Castellino FJ, Balsara RD. The Dichotomous Role of N-methyl-D-Aspartate Receptors in Ischemic Stroke. J Cardiol and Cardiovasc Sciences (2018) 2(4): 23-32 Journal of Cardiology and Cardiovascular Sciences

Figure 2: A schematic representation of various combinations of the NMDAR subunits present in the CNS. The functional NMDAR ion channel mostly exists as a diheteromer consisting of GluN1/GluN2A or GluN1/GluN2B subunits. Triheteromeric NMDARs can be made up of GluN1/GluN2A/GluN2B, GluN1/GluN2C/GluN2D, or GluN1/GluN2A/GluN3A/B subunits. The NMDAR ion channels consisting of GluN1/GluN3A/B subunits preferentially bind to glycine over glutamate.

GluN2D are observed in rodent embryos, but within the GluN2B-containing NMDARs suggests this allosteric regulation is due to tight packing of the ATD and LBD. subunit increases. Embryonically, the GluN2B subunit is The TMD of the GluN1a/GluN2B heterotetramer forms a prevalentfirst two weeks in the of brain, birth tobut adulthood after birth levels its distributionof the GluN2A is pseudo-four- fold symmetry due to rearrangement of the M4 helices, an arrangement that aids in regulating the GluN2C subunit is delayed starting at post-natal day 10-11, gating properties22. Additionally, binding of glutamate restricted to regions of the forebrain. Expression of the agonists and competitive antagonists is modulated by GluN2D subunit continues from the embryo to adulthood the LBDs23. The strategically placed helical segments of inand the is mostlydiencephalon, confined mesencephalon,to the cerebellum. and Expression spinal cord of the15. the TMD forms the channel pore that gates the voltage- dependent Mg2+ block, thereby regulating Ca2+ electrophysiological conductance24,25. The cytoplasmic Similarly, the GluN3 subunits have contrasting expression CTD is largely known for partnering with various influx signaling and ofprofiles. the hippocampus, GluN3A expression olfactory is bulb,low in cerebellum the embryonic amygdala, stage thalamus,that peaks and at postnatal hypothalamus, day 8 andin the decreases neocortex, in adulthood CA1 region16. themolecules CTD of and the scaffolding GluN2A, GluN2B, proteins and that GluN2C form complexes subunits postnatal stages and increase with age till adulthood, and affectsinvolved gating in signaling kinetics and26. receptorTruncated trafficking. CTD of Additionally,the GluN2A areWhereas, ubiquitously GluN3B distributed expression levelsin the areCNS low17,18 during early subunit caused reduced Ca2+ charge transfer and impaired of the various subunits was found to be enhanced from spatial working memory in mice27. Structurally, the GluN3 midgestation to early childhood (20 – 400 postconceptional. Expression weeks) compared to adults in the human parieto-occipital GluN subunits, and bind to glycine or D-serine. Alternate white matter, with GluN2A levels peaking at midgestation, splicingsubunits of exhibit the Grin3a the same mRNA modular gives riseproperties to long as(GluN3A- the otherL) and GluN1 and GluN2B levels peaking at the preterm period. Levels of GluN1, GluN2B, GluN2C, GluN2D, and GluN3A were higher in the parieto-occipital gray matter insertionsand a short at (GluN3A-the N-terminalS) isoforms, or C- terminal whereas, domains five isoforms28, 29. during early postnatal period (20 – 400 postconceptional of the GluN3B subunit exist due to in-frame deletions or The NMDAR displays distinct slow activation and weeks) compared to adults. However, GluN2A levels were deactivation properties compared to other iGluRs, such as lower in this region compared to adults19. AMPA and kainite receptors30. Not only are the functional kinetics activated by binding of glutamate/glycine, but the terminal domain (ATD), a ligand-binding domain (LBD), NMDAR is also subject to modulation by endogenous Mg2+, a transmembraneThe GluN subunits domain contain (TMD), an and extracellular an intracellular amino- C- H+, and Zn2+. The composition of the GluN2 subunits of the terminal domain (CTD). The ATD allosterically regulates NMDAR governs its single channel conductance and Ca2+ agonist potency by binding to Zn2+, ifenprodil, and permeability. Typically, GluN2A- and GluN2B-containing diheteromers manifest large conductances (~50 pS), longer the NMDAR20,21. X-Ray crystallography studies of GluN1a/ channel open times (3-5 ms), and higher open probabilities polyamines, thereby influencing the gating properties of

Page 25 of 32 Holmes AR, Castellino FJ, Balsara RD. The Dichotomous Role of N-methyl-D-Aspartate Receptors in Ischemic Stroke. J Cardiol and Cardiovasc Sciences (2018) 2(4): 23-32 Journal of Cardiology and Cardiovascular Sciences

(Po, ~0.4-0.5), when compared to GluN2C- or GluN2D- evoked ischemic long-term potentiation (iLTP) that blocks physiological LTP and is thought to be the basis of aberrant (~37 pS), shorter channel open times (0.5-1 ms), and synaptic plasticity that alters neuronal connectivity47. Post- lowercontaining open diheteromersprobabilities (Po, that ~0.01) exhibit31 . lowAlso, conductancesGluN2A- and ischemic LTP can be mitigated by application of ifenprodil, GluN2B- containing NMDAR channels are more sensitive 48. The molecular outcome of to voltage-dependent Mg2+ inhibition compared to GluN2C- and GluN2D-containing NMDAR channels32,33. Moreover, duean GluN2B- to calpainspecific activation antagonist49 Ca2+ ischemiaspecies50, excitotoxicityand mitochondrial is neuronal damage necrosis51. These or apoptosisischemia- GluN2B subunits compared to receptors that incorporate , generation of reactive oxygen GluN2C influx or isGluN2D higher throughsubunits channels34,35. Mutagenesis containing studies GluN2A have or symptoms of stroke. shown that TMD sites Ser632 and Leu657 mediate unique driven excitotoxic events are manifested into clinical The composition and interaction of the GluN1 subunit channel properties of GluN2A and GluN2D, respectively, that is dependent on the interaction of GluN2 and GluN1 in regulating NMDAR-derived currents and Ca2+ subunits36. Crystal structure analysis showed that residues witha process the specificintegral GluN2to maintaining subunit playsphysiological a principal activity role 658Asp-Arg-Pro-Glu-Glu-Arg663 in the linker region in the CNS. It has been well established that the NMDAR influx, of LBD-TMD of GluN1 subunit is responsible for high plays opposing roles in eliciting distinct signaling pathways permeation of Ca2+. 15 depending on their cellular location. Ca2+/Calmodulin The GluN3 subtypes bind to glycine/D-serine, and in activates synaptically-located NMDARs, which transduces particular, GluN3A confers a dominant negative function to the ion channel by countervailing the canonical synaptic various signaling molecules and subsequently promoting and structural plasticity of GluN2-containing NMDARs by the PI3K/Akt pathway by serially complexing with destabilizing synapses by tagging them for elimination. and genes (BTG2, BCL6, BDNF)52-54. Inhibition of pro- activation of pro-survival pathways (ERK1/2, CREB)55,56. connections promoting synaptic stabilization which is vital Since the GluN2A subunits are abundantly found in the duringIn this postnatal manner, development GluN3A subunits37. The GluN3 fine-tunes subunits synaptic form apoptoticsynaptic NMDAR molecules channel, (JNK, this GSK3ß, subunit BAD) is associated also occurs with pro-survival pathways57. Conversely, GluN2B subunits are GluN2A (in neurons and hippocampus), GluN2B (neurons), ora functional GluN2C triheteromeric(in oligodendrocytes) complex withsubunits GluN1 andimparting either biochemical/electrophysiological properties that are enriched in extrasynaptic NMDAR channels which partner 38-40 unique from the GluN2-containing receptors . The ischemia.with different These proteins, interactions such lead as PSD-to further95, DAPK1, upregulation PTEN, diheteromeric GluN3 subunit-containing ion channels are STEP, and SFK/Panx1, to form pro-death complexes during 2+ refractory to blockage by Mg and SREBP1, within 1-12 hours of stroke onset, causing 41 801 , but recently a series of small molecule compounds, neuronalof death apoptosis target proteins,58 (Figure including 3). calpains, MAPK/JNK, , AP-5, memantine, and MK- the GluN3 subunit42. The GluN3A-containing triheteromeric Since, ischemia-induced damage is multifactorial, NMDARTK13, TK30, channels and TK80 have were lower found single- to specificallychannel conductance antagonize several molecular events involving various signaling of ~28 pS, lower open probability (Po, ~0.03), and low Ca2+ pathways occur during ischemia that contributes towards permeability43,44.

Discerning the Role of GluN2 Subunits in Stroke (nNOS)excitotoxicity. via its PDZ1/PDZ2The PSD-95 domains scaffolding causing protein over specifically activation interacts with GluN2B and neuronal nitric oxide synthase59. causing depolarization of neuronal cells resulting in Pathological levels of NO precipitate a chain of protein accrualLack of glucose/oxygenpathological levels results of inglutamate. loss of ionic Ischemia homeostasis also of nNOS and hence increased levels of nitric oxide (NO) hinders clearance of glutamate resulting in glutamate causing DNA fragmentation and neuronal death60. Disrupting oxidationthe PSD-95/GluN2B and lipid interaction peroxidation by using reactions the Tat- eventuallyNR2B9c or NA-1 peptide successfully attenuated infarct size with NMDARspillover ion in thechannel extrasynaptic with an spaces.overload Downstream, of cellular Ca this2+ ischemia-mediated excitotoxicity hyperactivates the primate models of stroke61-63. Another repercussion of 45. Furthermore, perturbations in improvementpathological Ca in2+ neurological deficits in rat and non- human theinflux ionic making balance it the causes underlying spreading mechanism depolarization of ischemia- and edemainduced in excitotoxicitythe grey matter accompanied by depression of the cytoplasmic neuronaltail of GluN2B influx isthereby the activation stimulating of death- the spontaneous activity also known as nonspreading silence, pro-associatedapoptotic protein activity kinase of DAP 1 (DAPK) protein2+ /calmodulin/that binds to which can be negated by the NMDAR agonist, ketamine46. Acut interaction with the Tat-GluN2BK1CT1292- by the1304 peptide Ca mitigated calcinerin phosphatase axis. Disrupting the GluN2B/DAPK1 e deprivation of oxygen/glucose also induces NMDAR- Page 26 of 32 Holmes AR, Castellino FJ, Balsara RD. The Dichotomous Role of N-methyl-D-Aspartate Receptors in Ischemic Stroke. J Cardiol and Cardiovasc Sciences (2018) 2(4): 23-32 Journal of Cardiology and Cardiovascular Sciences

Figure 3: Signaling pathways activated by synaptic or extrasynaptic NMDARs. Physiological levels of Ca2+ influx due to stimulation of the NMDAR by glutamate at the synaptic cleft activates ERK signaling, which in turn phosphorylates CREB at Ser133 resulting in transcription of pro-survival molecules like Brain-derived Neurotrophic Factor (BDNF). During an ischemic event, glutamate spill over activates extrasynaptic NMDARs that inhibits the ERK/CREB signaling axis. Additionally, several pro-death pathways are also activated, of which, the death- associated protein kinase 1 (DAPK1)- and calpain (Cal)-mediated pathways are shown. Pathological levels of Ca2+ influx stimulates calcineurin (C) to dephosphorylate DAPK1 at Ser308 to promote excitotoxicity. Caplain mediates neuronal death by cleaving striatal-enriched protein tyrosine phoshatase (STEP) 61 to inactive STEP 33.

64. The Akt signaling GluN2 subunits in ischemia is lacking due to the differential molecule is also negatively affected by ischemia-induced roles played by the GluN2A and GluN2B subunits during Cathe2+ excitotoxicity due to ischemia and tensin homolog deleted on chromosome 10 (PTEN) GluN2A/GluN2B triheteromeric NMDAR’s in the adult influx due to interaction of the upstream phosphatase ischemia, as well as due to the extensive presence of GluN1/ ion channels. The direct interaction between GluN1/PTEN abrogated ischemia-induced brain injury compared to WT promotesmolecule withischemia- GluN1/GluN2B-mediated neuronalcontaining death, extrasynaptic whereas mice.forebrain. In contrast, Mice deficient selectively in theblocking GluN2A the subunitGluN2A showedsubunit down regulating PTEN levels reduced neuronal injury in with the glutamate antagonist NVP-AAM077 resulted in a vitro and in vivo by increasing phosphorylation of Akt and larger infarct size, indicating that GluN2A was essential for BAD forging pro-survival activity and decreasing neuronal ischemic neuroprotection in vivo67. The pro-apoptotic effect death65. Also, reduction of PTEN levels decreased NMDAR- of GluN2A was attributed to phosphorylation of Ser1232 by cyclin-dependent kinase in a model of ischemia, whereas, the NMDAR ion channels, thus obliquely regulating GluN2B decreased tyrosine phosphorylation was associated with activityevoked 65extrasynaptic. Uncoupling current,the PTEN/NMDAR but not synaptic interaction activity byof decreased ischemia-induced neuronal death68. accumulation of PTEN and conferred neuroprotection with peptide Tat-K13 abrogated 66 ischemia-induced nuclear . of stroke.Inhibition It has ofbeen the demonstrated GluN2B subunit that administration with specific of antagonists has proved to be beneficial in animal models improvedA thorough neurological understanding deficits of the role of individual GluN2B-specific inhibitors, ifenprodil and conantokin-G,

Page 27 of 32 Holmes AR, Castellino FJ, Balsara RD. The Dichotomous Role of N-methyl-D-Aspartate Receptors in Ischemic Stroke. J Cardiol and Cardiovasc Sciences (2018) 2(4): 23-32 Journal of Cardiology and Cardiovascular Sciences ameliorates ischemia-mediated infarct size and neuronal available to counter the detrimental effects of stroke are limited, especially at the neurovascular and neuroprotective level. Administration of the thrombolytic agent, alteplase pro-deathsurvival by decreasingCREB molecules neurological69,70 deficits, restoring (recombinant tissue type-Plasminogen Activator) is the cytoarchitecture,GluN2B-containing andNMDAR increasing channels the were expression treated with of most widely used drug to dissolve the clot and restore blood conantokin-G, increased neuronal survival. When was extrasynaptic, observed by narrow window of ~3.5 hours after onset of stroke during whichflow. However, it should the be effectivenessadministrated, of and alteplase it could is limitedalso increase to the conantokin-increased pro-T71survival. Perturbing levels the of interactionERK, CREB, ofand the enhanced GluN2B the risk of hemorrhage80. Another option of removing the mitochondrial viability, compared to a non-subunit-specific clot is by mechanical embolectomy that can be performed 81 mitigated the detrimental effects of O2/glucose deprivation as late as 8-12 hours after onset of stroke . Progression of CTDof cultured with PSD95, cortical CMKIIα, neurons 72and. Although, clathrin the adaptor GluN2B protein- subunit2 plays a prominent role in cell death induced by ischemia, of stroke leading to neuronal death is a relentless process. it is not involved in cell death caused by cardiac arrest and Therefore,stroke is linear the andoptimal the NMDAR-plan fordriven stroke excitotoxic therapy effectswould cardiopulmonary resuscitation73. Post-ischemia, mRNA primarily involve removal of clot followed by secondary neuroprotective strategies. To this end several preclinical hoursexpression following levels stroke, of the mRNA GluN subunitslevels of GluN1 changes increases, temporally but the NMDAR. decreasescausing ischemic- by day mediated7. Whereas, apoptosis. the mRNA Within levels the of GluN2Afirst 24 studies had developed inhibitors that specifically targeted Although blockade of the NMDAR by various antagonists and GluN2B decreases initially, but start to increase at 48 was effective in attenuating ischemic damage in murine hours post-ischemia74. and rodent models, the clinical availability of NMDAR- The role of the anatomically restricted GluN2C subunit directed antagonists remains elusive. Some non-competitive in ischemia is ambiguous. GluN2C plays a neuroprotective NMDAR inhibitors, such as, dizocipline, selfotel, aptiganel role in a murine model of global cerebral ischemia in the hydrochloride, and gavistinel, were investigated but hippocampus due to upregulation of the GluN2C subunit suspended at various stages of clinical trials because and decreased Ca2+ 75. This contrasts with another of adverse side-effects that included psychotic and/or neurological complications82 in mice conferred influxneuroprotection, as observed by class, ifenprodil and its analogues, were well-tolerated in smallerstudy that infarct reported size and that less a deficiency cerebral edema of GluN2C in a subunitmiddle clinical trials, yet were not .effective The GluN2B- comparedspecific to inhibitor placebo carotid artery occlusion model of ischemia, compared administrations83. Treatment with various neuroprotective 76 to WT mice . Furthermore, it has been shown that post- compounds, e.g., MgSO4 and glycine-site inhibitors, have also ischemic GluN2C-/- proved to be ineffective. Conantokins, which are neuroactive peptides derived from the Conus species of marine snails, and Tauopathy, as well mice as exhibitdecreased enhanced levels of neurological Fyn kinase which effectively inhibit NMDAR-evoked currents and Ca2+ andrecovery diminished associated phosphorylation with less cytoarchitecturalof Tyr1336 of the GluN2B deficits 77. epilepsy and pain, but globally affect other receptors84. The direct role of GluN2D in ischemia has not as yet been influx in a subunit-specific manner, have been evaluated for Several groups have reported that memantine which is subunitelucidated. in theThe cerebralneonates cortex and adults compared of naked to WT mole mice rats a non-competitive antagonist of the NMDAR used to treat have an unusually high proportion of GluN2D subunits that impedes Ca2+ 78 . Alzheimer’s disease was also found to be efficacious for influx, thus rendering these animals highly accompanied by diminished astrogliosis and enhanced treatment of stroke. Improvement in neurological deficits tolerantNMDAR to living channels in hypoxic containing conditions the GluN3A subunits have vascular density in the penumbra region of the core infarct low Ca2+ permeability and sensitivity to Mg2+, and play a was observed in mice treated with memantine administered protective role during ischemic insult, both in vivo and in after induction of stroke. However, this recovery was vitro. The GluN3A subunit dampens ischemia-mediated non-neuroprotective as reduction in core infarct size was not observed, although increase in brain derived GluN2A- and GluN2B-containing ion channels. GluN3A-/- neurotrophic factor and phospho-tyrosine was observed miceexcitotoxicity showed impaired by counteracting neurological the recovery, hyperactivation larger infarct of in the peri-infarct area indicating memantine-stimulated volume, and elevated cell death compared to WT mice79. improvement in plasticity85. Prophylactic administration NMDAR as a Therapeutic Target of Ischemia decreased infarct size with improved behavioral outcomes, Although occurrence of ischemic stroke is prevalent butof memantine administration at a of low higher dosage doses of of 0.2 memantine mg/kg significantly (2-10 mg/ (~800,000 strokes annually in the USA), treatment plans kg) was counterproductive and potentiated ischemic injury

Page 28 of 32 Holmes AR, Castellino FJ, Balsara RD. The Dichotomous Role of N-methyl-D-Aspartate Receptors in Ischemic Stroke. J Cardiol and Cardiovasc Sciences (2018) 2(4): 23-32 Journal of Cardiology and Cardiovascular Sciences in mice86. Memantine is a well-tolerated drug widely used literature regarding the role of the different GluN2 subunits to treat patients with Alzheimer’s disease, and pre-clinical in ischemia. These opposing results may stem from post- research data supports the feasibility of memantine being utilized to treat patients with stroke. Lipton and colleagues GluN subunits in a spatio-temporal dependent manner. The synthesized analogs of memantine, the NitroMemantines typeischemic of ischemia changes model in expression employed, and and activity the duration of the various of the treating ischemia compared to memantine by effectively subunits. An ideal neuroprotective agent is one that would that proved to be significantly more neuroprotective in selectivelyinsult, also blockinfluences the deleterious the expression NMDAR- and activitydriven pathways of GluN2 a voltage-dependent manner and allowing sustained without globally affecting normal synaptic function. blockingsynaptic activity hyperactivation87. Thus, ofmaking extrasynaptic memantine NMDARs and its in References analogs attractive therapeutic candidates for treatment of ischemia. In fact, early phase 1 clinical trials are in progress 1. channels. Pharmacol Rev. 1999; 51: 7-‐‑61. Dingledine R, Borges K, Bowie D, et al. The glutamate receptor ion ischemic stroke. 2. Weilinger NL, Maslieieva V, Bialecki J, et al. Ionotropic receptors to evaluate the efficacy of memantine in patients with acute and ion channels in ischemic neuronal death and dysfunction. 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