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Neuronal gap junctions are required for NMDA receptor-mediated excitotoxicity: implications in ischemic stroke.

ABSTRACT: N-methyl-D-aspartate receptors (NMDARs) play an important role in cell survival versus cell death decisions during neuronal development, ischemia, trauma, and epilepsy. Coupling of neurons by electrical synapses (gap junctions) is high or increases in neuronal networks during all these conditions. In the developing CNS, neuronal gap junctions are critical for two different types of NMDAR-dependent cell death. However, whether neuronal gap junctions play a role in NMDAR-dependent neuronal death in the mature CNS was not known. Using Fluoro-Jade B staining, we show that a single intraperitoneal administration of NMDA (100 mg/kg) to adult wild-type mice induces neurodegeneration in three forebrain regions, including rostral dentate gyrus. However, the NMDAR-mediated neuronal death is prevented by pharmacological blockade of neuronal gap junctions (with mefloquine, 30 mg/kg) and does not occur in mice lacking neuronal gap junction protein, connexin 36. Using Western blots, electrophysiology, calcium imaging, and gas chromatography-mass spectrometry in wild-type and connexin 36 knockout mice, we show that the reduced level of neuronal death in knockout animals is not caused by the reduced expression of NMDARs, activity of NMDARs, or permeability of the blood-brain barrier to NMDA. In wild-type animals, this neuronal death is not caused by upregulation of connexin 36 by NMDA. Finally, pharmacological and genetic inactivation of neuronal gap junctions in mice also dramatically reduces neuronal death caused by photothrombotic focal cerebral ischemia. The results indicate that neuronal gap junctions are required for NMDAR-dependent excitotoxicity and play a critical role in ischemic neuronal death.

SUBMITTER: Wang Y

PROVIDER: S-EPMC3007655 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Neuronal gap junctions are required for NMDA receptor-mediated excitotoxicity: implications in ischemic stroke.

Wang Yongfu Y Denisova Janna V JV Kang Ki Sung KS Fontes Joseph D JD Zhu Bao Ting BT Belousov Andrei B AB

Journal of neurophysiology 20101013 6

N-methyl-D-aspartate receptors (NMDARs) play an important role in cell survival versus cell death decisions during neuronal development, ischemia, trauma, and epilepsy. Coupling of neurons by electrical synapses (gap junctions) is high or increases in neuronal networks during all these conditions. In the developing CNS, neuronal gap junctions are critical for two different types of NMDAR-dependent cell death. However, whether neuronal gap junctions play a role in NMDAR-dependent neuronal death i ...[more]

PMID: 20943940

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Project description:Local cerebral hypoperfusion causes ischemic stroke while driving multiple cell-specific responses including inflammation, glutamate-NMDAR-mediated neurotoxicity, edema formation and angiogenesis. Despite the relevance of these pathophysiological mechanisms for disease progression and outcome, molecular determinants controlling the onset of these processes are only partially understood. In this context, our study intended to investigate the functional role of EphB2 – a receptor tyrosine kinase that is crucial for synapse function and binds to membrane-associated ephrin-B ligands. Cerebral ischemia was induced in EphB2-deficient mice by transient middle cerebral artery occlusion followed by different times (6, 12, 24 and 48 h) of reperfusion. Histological, neurofunctional and transcriptome analyses indicated an increase in EphB2 phosphorylation under these conditions and attenuated progression of stroke in EphB2-deficient mice. Moreover, while infiltration of microglia/macrophages and astrocytes into the peri-infarct region was not altered, expression of the pro-inflammatory mediators MCP-1 or IL-6 was decreased in these mice. In fact, in vitro analyses indicated that binding of EphB2 to astrocytic ephrin-B ligands stimulates NF-κB-mediated cytokine expression via the MAPK pathway. Further magnetic resonance imaging of the EphB2-deficient ischemic brain revealed a lower level of neuronal cytotoxic edema formation within 6 h upon onset of reperfusion. On the mechanistic level, absence of neuronal EphB2 decreased the mitochondrial Ca2+ load upon activation of NMDAR but not AMPAR. Moreover, activation of EphB2 by ephrin-B2 enhanced the NMDA-evoked excitotoxic neuronal cell death in vitro while neuron-specific loss of ephrin-B2 reduced the extent of cerebral tissue damage in the acute phase of ischemic stroke. Collectively, EphB2 may promote the immediate response to an ischemia-reperfusion event in the central nervous system by (i) pro-inflammatory activation of astrocytes via ephrin-B-dependent signalling and (ii) amplification of the NMDA-evoked neuronal excitotoxicity.

Project description:Local cerebral hypoperfusion causes ischemic stroke while driving multiple cell-specific responses including inflammation, glutamate-induced neurotoxicity mediated via NMDAR, edema formation and angiogenesis. Despite the relevance of these pathophysiological mechanisms for disease progression and outcome, molecular determinants controlling the onset of these processes are only partially understood. In this context, our study intended to investigate the functional role of EphB2, a receptor tyrosine kinase that is crucial for synapse function and binds to membrane-associated ephrin-B ligands.Cerebral ischemia was induced in Ephb2-/- mice by transient middle cerebral artery occlusion followed by different times (6, 12, 24 and 48?h) of reperfusion. Histological, neurofunctional and transcriptome analyses indicated an increase in EphB2 phosphorylation under these conditions and attenuated progression of stroke in Ephb2-/- mice. Moreover, while infiltration of microglia/macrophages and astrocytes into the peri-infarct region was not altered, expression of the pro-inflammatory mediators MCP-1 and IL-6 was decreased in these mice. In vitro analyses indicated that binding of EphB2 to astrocytic ephrin-B ligands stimulates NF-?B-mediated cytokine expression via the MAPK pathway. Further magnetic resonance imaging of the Ephb2-/- ischemic brain revealed a lower level of cytotoxic edema formation within 6?h upon onset of reperfusion. On the mechanistic level, absence of neuronal EphB2 decreased the mitochondrial Ca2+ load upon specific activation of NMDAR but not during synaptic activity. Furthermore, neuron-specific loss of ephrin-B2 reduced the extent of cerebral tissue damage in the acute phase of ischemic stroke.Collectively, EphB2 may promote the immediate response to an ischemia-reperfusion event in the central nervous system by (i) pro-inflammatory activation of astrocytes via ephrin-B-dependent signaling and (ii) amplification of NMDA-evoked neuronal excitotoxicity.

Project description:Hyperexcitability of neuronal networks can lead to excessive release of the excitatory neurotransmitter glutamate, which in turn can cause neuronal damage by overactivating NMDA-type glutamate receptors and related signaling pathways. This process (excitotoxicity) has been implicated in the pathogenesis of many neurological conditions, ranging from childhood epilepsies to stroke and neurodegenerative disorders such as Alzheimer's disease (AD). Reducing neuronal levels of the microtubule-associated protein tau counteracts network hyperexcitability of diverse causes, but whether this strategy can also diminish downstream excitotoxicity is less clear.We established a cell-based assay to quantify excitotoxicity in primary cultures of mouse hippocampal neurons and investigated the role of tau in exicitotoxicity by modulating neuronal tau expression through genetic ablation or transduction with lentiviral vectors expressing anti-tau shRNA or constructs encoding wildtype versus mutant mouse tau.We demonstrate that shRNA-mediated knockdown of tau reduces glutamate-induced, NMDA receptor-dependent Ca2+ influx and neurotoxicity in neurons from wildtype mice. Conversely, expression of wildtype mouse tau enhances Ca2+ influx and excitotoxicity in tau-deficient (Mapt -/-) neurons. Reconstituting tau expression in Mapt -/- neurons with mutant forms of tau reveals that the tau-related enhancement of Ca2+ influx and excitotoxicity depend on the phosphorylation of tau at tyrosine 18 (pY18), which is mediated by the tyrosine kinase Fyn. These effects are most evident at pathologically elevated concentrations of glutamate, do not involve GluN2B-containing NMDA receptors, and do not require binding of Fyn to tau's major interacting PxxP motif or of tau to microtubules.Although tau has been implicated in diverse neurological diseases, its most pathogenic forms remain to be defined. Our study suggests that reducing the formation or level of pY18-tau can counteract excitotoxicity by diminishing NMDA receptor-dependent Ca2+ influx.

Dataset Information

Neuronal gap junctions are required for NMDA receptor-mediated excitotoxicity: implications in ischemic stroke.

Publications

Neuronal gap junctions are required for NMDA receptor-mediated excitotoxicity: implications in ischemic stroke.

Similar Datasets

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