Elsevier

Neurobiology of Disease

Volume 43, Issue 2, August 2011, Pages 507-515
Neurobiology of Disease

Misplaced NMDA receptors in epileptogenesis contribute to excitotoxicity

https://doi.org/10.1016/j.nbd.2011.04.024Get rights and content

Abstract

Pharmacological blockade of NR2B-containing N-methyl-d-aspartate receptors (NMDARs) during epileptogenesis reduces neurodegeneration provoked in the rodent hippocampus by status epilepticus. The functional consequences of NMDAR activation are crucially influenced by their synaptic vs extrasynaptic localization, and both NMDAR function and localization are dependent on the presence of the NR2B subunit and its phosphorylation state.

We investigated whether changes in NR2B subunit phosphorylation, and alterations in its neuronal membrane localization and cellular expression occur during epileptogenesis, and if these changes are involved in neuronal cell loss. We also explored NR2B subunit changes both in the acute phase of status epilepticus and in the chronic phase of spontaneous seizures which encompass the epileptogenesis phase.

Levels of Tyr1472 phosphorylated NR2B subunit decreased in the post-synaptic membranes from rat hippocampus during epileptogenesis induced by electrical status epilepticus. This effect was concomitant with a reduced interaction between NR2B and post-synaptic density (PSD)-95 protein, and was associated with decreased CREB phosphorylation. This evidence suggests an extra-synaptic localization of NR2B subunit in epileptogenesis. Accordingly, electron microscopy showed increased NR2B both in extra-synaptic and pre-synaptic neuronal compartments, and a concomitant decrease of this subunit in PSD, thus indicating a shift in NR2B membrane localization. De novo expression of NR2B in activated astrocytes was also found in epileptogenesis indicating ectopic receptor expression in glia. The NR2B phosphorylation changes detected at completion of status epilepticus, and interictally in the chronic phase of spontaneous seizures, are predictive of receptor translocation from synaptic to extrasynaptic sites.

Pharmacological blockade of NR2B-containing NMDARs by ifenprodil administration during epileptogenesis significantly reduced pyramidal cell loss in the hippocampus, showing that the observed post-translational and cellular changes of NR2B subunit contribute to excitotoxicity. Therefore, pharmacological targeting of misplaced NR2B-containing NMDARs, or prevention of these NMDAR changes, should be considered to block excitotoxicity which develops after various pro-epileptogenic brain injuries.

Highlights

► Phosphorylation changes and NR2B membrane redistribution are found in epileptogenesis. ► Ectopic astrocytic expression of NR2B occurs during epileptogenesis. ► Redistribution and ectopic NR2B expression mediate excitotoxicity.

Introduction

N-methyl-d-aspartate receptors (NMDARs) play a key role in synaptic transmission, long-term potentiation (Bliss and Collingridge, 1993), excitotoxic neuronal damage (Choi and Rothman, 1990, Mody and MacDonald, 1995) and seizures (Dingledine et al., 1990). NMDARs are heterotetrameric complexes of two constitutive glycine-binding NR1 subunits combined with two regulatory glutamate-binding NR2 subunits (i.e. A, B, C, D). NR3 subunits can assemble with NR1 and NR2 subunits to decrease NMDAR current amplitudes, or with the NR1 subunit alone to form glycine-activated receptors (Chatterton et al., 2002, Dingledine et al., 1999).

The presence of the NR2B subunit critically influences not only the pharmacological and electrophysiological properties of the NMDAR but also its cellular membrane distribution (Dingledine et al., 1999). NMDARs are mainly localized at the post-synaptic densities (PSD), where they are anchored to scaffolding proteins (e.g. PSD-95, SAP-102, SAP-93), while NMDARs containing the NR2B subunit have also been identified extrasynaptically (Petralia et al., 2010, Tovar and Westbrook, 2002) or presynaptically (Jourdain et al., 2007, Woodhall et al., 2001). The localization of NMDARs is an important factor that determines the functional consequences of receptor activation. For instance, post-synaptic NMDARs are activated by synaptically-released glutamate and mediate long-term potentiation and long-term depression of synaptic transmission (Bear and Malenka, 1994). The activation of these receptors induces CREB-dependent transcription of genes which are responsible for neuroprotection against different types of insults (e.g. apoptotic, excitotoxic, necrotic or oxidative) (Papadia and Hardingham, 2007, Hardingham et al., 2002; Sattler et al., 2000). However, NMDAR over-activation also can mediate excitotoxic effects due to excessive neuronal Ca2+ influx (Forder and Tymianski, 2009). Conversely, extra-synaptic NR2B-containing NMDARs are predominantly activated by glutamate released by astrocytes (Jourdain et al., 2007) or spilled over from the synaptic cleft during episodes of high frequency synaptic activity (Conti and Weinberg, 1999). Activation of these receptors causes CREB de-phosphorylation and contributes to the mechanisms of neuronal cell death (Fellin et al., 2004, Papadia and Hardingham, 2007, Hardingham et al., 2002; Sattler et al., 2000). Pre-synaptic NMDARs have been described in the hippocampus and entorhinal cortex where they promote glutamate release (Langer, 2008, Martin et al., 1991, Jourdain et al., 2007, Yang et al., 2006). These pre-synaptic receptors facilitate glutamate release in the entorhinal cortex of epileptic rats (Yang et al., 2006), thus promoting excitotoxicity and reinforcing seizures via an increase in glutamatergic neurotransmission.

In addition to composition and localization, the Tyr1472 phosphorylation of NR2B subunit of the NMDAR by the Src tyrosine kinase family is a key factor for determining receptor function, by increasing channel permeability to Ca2+ (Ali and Salter, 2001) and stabilizing the receptor at the PSD (Collingridge et al., 2004, Salter and Kalia, 2004). It was demonstrated that Ca2+ overload via activated post-synaptic, as well as via extra-synaptic NMDARs contributes to neuronal hyperexcitability (Kohl and Dannhardt, 2001, Rice and DeLorenzo, 1998) and excitotoxicity in seizure models (Araujo et al., 2008, Fellin et al., 2004, Sierra-Paredes and Sierra-Marcuno, 2007, Yang et al., 2006).

Some information exists on changes in NR2B subunit during seizures or in chronic epileptic tissue: increased NR2B subunit phosphorylation was reported in post-synaptic membranes of rat forebrain during the first 24 h after the onset of status epilepticus (Huo et al., 2006, Moussa et al., 2001, Niimura et al., 2005); a decrease of NR1 and NR2B protein levels was shown in human neocortical epilepsy specimens (Wyneken et al., 2003), and in cortical post-synaptic membranes or hippocampal homogenates in rats with either provoked (Auzmendi et al., 2009) or spontaneous seizures (Sun et al., 2009, Wyneken et al., 2003). Decreased NR2B mRNA levels were also described in pyramidal neurons of temporal lobe epilepsy (TLE) patients with hippocampal sclerosis (Mathern et al., 1998). Conversely, an upregulation of NR2B mRNA was found in pyramidal cells of non-sclerotic hippocampi from epileptic patients (Mathern et al., 1998) and NR2B protein levels were increased in post-synaptic membranes of dysplastic neurons in epileptic foci from focal cortical dysplasia (Mikuni et al., 1999, Colciaghi et al., 2011). These NR2B changes highlight both NMDAR receptor loss in degenerating neurons and adaptive modifications in response to seizures or to neuropathology.

However, no studies are available regarding NR2B subunit levels, its phosphorylation state or its membrane and cellular localization during epileptogenesis, the crucial post-injury phase prodromal to epilepsy development. The main focus of our study was therefore to study the NR2B subunit in epileptogenesis using a multidisciplinary approach applied to a rat model of TLE, one of the most drug-resistant forms of human epilepsy (Majores et al., 2007). As secondary endpoints, we also examined the NR2B subunit in the two phases encompassing epileptogenesis, i.e. the acute status epilepticus and the chronic phase of spontaneous seizures (these results are presented in the Supplementary Material) to unify in the same epilepsy model the scattered literature information.

Our data show that NR2B subunit is increased in pre-synaptic and extra-synaptic neuronal compartments during epileptogenesis, in concomitance with its decreased phosphorylation and post-synaptic localization. Moreover, ectopic expression of both NR2B and NR1 occurred in activated astrocytes. The time-course changes in NR2B phosphorylation during and after seizures support that the NMDAR receptor translocation to extrasynaptic sites arises at the end of status epilepticus, and likely occurs also in the interictal phase of spontaneous seizures. Blockade of NR2B-containing NMDAR with ifenprodil during epileptogenesis significantly reduced excitotoxicity, thus suggesting that therapeutic interventions targeting misplaced NR2B-containing NMDAR could afford neuroprotection after pro-epileptogenic injuries.

Section snippets

Experimental animals

Adult male Sprague–Dawley rats (225–250 g) were purchased from Charles River (Calco, Italy) and were housed at constant temperature (23 °C) and relative humidity (60 ± 5%) with free access to food and water and a fixed 12 h light/dark cycle. All experimental procedures were conducted in conformity with institutional guidelines that are in compliance with national (D.L.n.116, G.U., Suppl 40, February 18, 1992) and international guidelines and laws (EEC Council Directive 86/609, OJ L 358, 1, December

The level and phosphorylation of the NR2B subunit are decreased in PSD during epileptogenesis

The hippocampal levels of Tyr1472 phosphorylated NR2B subunit (P-NR2B) were decreased in PSD-enriched fraction by 37 ± 13% below control values 96 h after SE onset (p < 0.05); at the same time, the total levels of NR2B, NR1 and PSD-95 were significantly reduced by 25 to 44% (p < 0.05 and p < 0.01) as assessed by Western blot (Fig. 1A).

Supplementary Fig. 1A depicts the changes in P-NR2B during the acute phases of seizures and in chronic epileptic rats: 2 h after SE onset or after the occurrence of a

Discussion

In this study, we provide new findings on the changes in the phosphorylation and localization of the NR2B subunit of the NMDAR in the rat hippocampus during epileptogenesis.

Using a rat model of electrically induced status epilepticus evolving to spontaneous seizures, we obtained the following evidence during the epileptogenesis phase: 1. There is a significant reduction in the levels of P-NR2B in the post-synaptic compartment; 2. The total levels of NR2B, NR1 and PSD-95 are concomitantly

Conclusion

The present study shows changes in the phosphorylation and localization of the NR2B subunit of the NMDAR in the rat hippocampus during the epileptogenesis triggered by status epilepticus. Our data indicate that the NR2B subunit redistributes in neuronal membranes with an increased localization in extra-synaptic and pre-synaptic compartments, and a concomitant decrease at post-synaptic sites. Moreover, we observed NR2B ectopic expression in activated astrocytes. Pharmacological blockade of

Fundings

This work was supported by Fondazione Monzino and Fondazione Cariplo (to AV).

Acknowledgments

The authors are grateful to Prof. M. De Baets for his constructive comments. We thank Dr. A. Möller (Abbott, Ludwigshafen, Germany) for his generous supply of A-705253 and for the helpful discussion on the treatment protocol. We also thank Dr. D. Francon (Sanofi-Aventis, Bagneux, France) for generously supplying ifenprodil for our pharmacological studies.

References (70)

  • G.E. Hardingham et al.

    Coupling of extrasynaptic NMDA receptors to a CREB shut-off pathway is developmentally regulated

    Biochim. Biophys. Acta

    (2002)
  • J.L. Hellier et al.

    NMDA receptor-mediated long-term alterations in epileptiform activity in experimental chronic epilepsy

    Neuropharmacology

    (2009)
  • J.Z. Huo et al.

    Increase in tyrosine phosphorylation of the NMDA receptor following the induction of status epilepticus

    Neurosci. Lett.

    (2006)
  • S.Z. Langer

    Presynaptic autoreceptors regulating transmitter release

    Neurochem. Int.

    (2008)
  • G.W. Mathern et al.

    Hippocampal AMPA and NMDA mRNA levels and subunit immunoreactivity in human temporal lobe epilepsy patients and a rodent model of chronic mesial limbic epilepsy

    Epilepsy Res.

    (1998)
  • I. Mody et al.

    NMDA receptor-dependent excitotoxicity: the role of intracellular Ca2+ release

    Trends Pharmacol. Sci.

    (1995)
  • H. Monyer et al.

    Developmental and regional expression in the rat brain and functional properties of four NMDA receptors

    Neuron

    (1994)
  • R.C. Moussa et al.

    Seizure activity results in increased tyrosine phosphorylation of the N-methyl-d-aspartate receptor in the hippocampus

    Brain Res. Mol. Brain Res.

    (2001)
  • G. Perea et al.

    Tripartite synapses: astrocytes process and control synaptic information

    Trends Neurosci.

    (2009)
  • R.S. Petralia et al.

    Organization of NMDA receptors at extrasynaptic locations

    Neuroscience

    (2010)
  • T. Ravizza et al.

    Status epilepticus induces time-dependent neuronal and astrocytic expression of interleukin-1 receptor type I in the rat limbic system

    Neuroscience

    (2006)
  • T. Ravizza et al.

    Innate and adaptive immunity during epileptogenesis and spontaneous seizures: evidence from experimental models and human temporal lobe epilepsy

    Neurobiol. Dis.

    (2008)
  • A.C. Rice et al.

    NMDA receptor activation during status epilepticus is required for the development of epilepsy

    Brain Res.

    (1998)
  • C. Schmitz et al.

    Design-based stereology in neuroscience

    Neuroscience

    (2005)
  • Q.J. Sun et al.

    Alterations of NR2B and PSD-95 expression in hippocampus of kainic acid-exposed rats with behavioural deficits

    Behav. Brain Res.

    (2009)
  • K.R. Tovar et al.

    Mobile NMDA receptors at hippocampal synapses

    Neuron

    (2002)
  • W. Tu et al.

    DAPK1 interaction with NMDA receptor NR2B subunits mediates brain damage in stroke

    Cell

    (2010)
  • B. Viviani et al.

    Cytokines and neuronal ion channels in health and disease

    Int. Rev. Neurobiol.

    (2007)
  • X.M. Zha et al.

    Regulation of hippocampal synapse remodeling by epileptiform activity

    Mol. Cell. Neurosci.

    (2005)
  • I.M. Araujo et al.

    Calpain activation is involved in early caspase-independent neurodegeneration in the hippocampus following status epilepticus

    J. Neurochem.

    (2008)
  • J. Auzmendi et al.

    The NMDAR subunit NR2B expression is modified in hippocampus after repetitive seizures

    Neurochem. Res.

    (2009)
  • T.V. Bliss et al.

    A synaptic model of memory: long-term potentiation in the hippocampus

    Nature

    (1993)
  • J.E. Chatterton et al.

    Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits

    Nature

    (2002)
  • Q. Chen et al.

    Differential roles of NR2A- and NR2B-containing NMDA receptors in activity-dependent brain-derived neurotrophic factor gene regulation and limbic epileptogenesis

    J. Neurosci.

    (2007)
  • D.W. Choi et al.

    The role of glutamate neurotoxicity in hypoxic–ischemic neuronal death

    Annu. Rev. Neurosci.

    (1990)
  • Cited by (91)

    • Hippocampal circuits

      2022, Neurocircuitry of Addiction
    View all citing articles on Scopus
    View full text