Misplaced NMDA receptors in epileptogenesis contribute to excitotoxicity
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)
- et al.
NMDA receptor regulation by Src kinase signalling in excitatory synaptic transmission and plasticity
Curr. Opin. Neurobiol.
(2001) - et al.
Synaptic plasticity: LTP and LTD
Curr. Opin. Neurobiol.
(1994) - et al.
A neuron–glia signalling network in the active brain
Curr. Opin. Neurobiol.
(2001) Synaptic patterns on different cell types in the different laminae of the cat visual cortex. An electron microscope study
Brain Res.
(1968)- et al.
Shaping excitation at glutamatergic synapses
Trends Neurosci.
(1999) - et al.
Excitatory amino acid receptors in epilepsy
Trends Pharmacol. Sci.
(1990) - et al.
Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors
Neuron
(2004) - et al.
Postsynaptic mechanisms of excitotoxicity: involvement of postsynaptic density proteins, radicals, and oxidant molecules
Neuroscience
(2009) - et al.
Surface trafficking of N-methyl-d-aspartate receptors: physiological and pathological perspectives
Neuroscience
(2009) - et al.
The tripartite synapse: roles for gliotransmission in health and disease
Trends Mol. Med.
(2007)
Coupling of extrasynaptic NMDA receptors to a CREB shut-off pathway is developmentally regulated
Biochim. Biophys. Acta
NMDA receptor-mediated long-term alterations in epileptiform activity in experimental chronic epilepsy
Neuropharmacology
Increase in tyrosine phosphorylation of the NMDA receptor following the induction of status epilepticus
Neurosci. Lett.
Presynaptic autoreceptors regulating transmitter release
Neurochem. Int.
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.
NMDA receptor-dependent excitotoxicity: the role of intracellular Ca2+ release
Trends Pharmacol. Sci.
Developmental and regional expression in the rat brain and functional properties of four NMDA receptors
Neuron
Seizure activity results in increased tyrosine phosphorylation of the N-methyl-d-aspartate receptor in the hippocampus
Brain Res. Mol. Brain Res.
Tripartite synapses: astrocytes process and control synaptic information
Trends Neurosci.
Organization of NMDA receptors at extrasynaptic locations
Neuroscience
Status epilepticus induces time-dependent neuronal and astrocytic expression of interleukin-1 receptor type I in the rat limbic system
Neuroscience
Innate and adaptive immunity during epileptogenesis and spontaneous seizures: evidence from experimental models and human temporal lobe epilepsy
Neurobiol. Dis.
NMDA receptor activation during status epilepticus is required for the development of epilepsy
Brain Res.
Design-based stereology in neuroscience
Neuroscience
Alterations of NR2B and PSD-95 expression in hippocampus of kainic acid-exposed rats with behavioural deficits
Behav. Brain Res.
Mobile NMDA receptors at hippocampal synapses
Neuron
DAPK1 interaction with NMDA receptor NR2B subunits mediates brain damage in stroke
Cell
Cytokines and neuronal ion channels in health and disease
Int. Rev. Neurobiol.
Regulation of hippocampal synapse remodeling by epileptiform activity
Mol. Cell. Neurosci.
Calpain activation is involved in early caspase-independent neurodegeneration in the hippocampus following status epilepticus
J. Neurochem.
The NMDAR subunit NR2B expression is modified in hippocampus after repetitive seizures
Neurochem. Res.
A synaptic model of memory: long-term potentiation in the hippocampus
Nature
Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits
Nature
Differential roles of NR2A- and NR2B-containing NMDA receptors in activity-dependent brain-derived neurotrophic factor gene regulation and limbic epileptogenesis
J. Neurosci.
The role of glutamate neurotoxicity in hypoxic–ischemic neuronal death
Annu. Rev. Neurosci.
Cited by (91)
Epileptiform GluN2B–driven excitation in hippocampus as a therapeutic target against temporal lobe epilepsy
2022, Experimental NeurologyHippocampal circuits
2022, Neurocircuitry of AddictionProgenitor Cells Play a Role in Reinstatement of Ethanol Seeking in Adult Male and Female Ethanol Dependent Rats
2023, International Journal of Molecular SciencesIn the fast lane: Receptor trafficking during status epilepticus
2023, Epilepsia Open