Activation of NMDA receptors and L-type voltage-gated calcium channels mediates enhanced formation of Fyn–PSD95–NR2A complex after transient brain ischemia

doi:10.1016/S0006-8993(02)03376-0

Brain Research

Volume 955, Issues 1–2, 15 November 2002, Pages 123-132

https://doi.org/10.1016/S0006-8993(02)03376-0 Get rights and content

Abstract

Recent studies have indicated that tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A) by Src family kinases (Src, Fyn, etc.) up-regulates NMDA receptors activity and postsynaptic density protein 95 kDa (PSD95) may mediate the regulation. To investigate whether the above processes are involved in brain ischemia-induced enhancement of NMDA receptors function, we examined the effects of transient (15 min) brain ischemia followed by reperfusion on interactions involving Fyn, NR2A and PSD95 in rat hippocampus by co-immunoprecipitation. Transient brain ischemia was induced by the method of four-vessel occlusion in Sprague–Dawley rats. Association between Fyn and NR2A increased immediately after brain ischemia and the increase was maintained for at least 24 h during followed reperfusion, up to about 1.7–1.8-fold relative to sham-groups. The 15-min reperfusion after brain ischemia induced enhanced co-immunoprecipitation of PSD95, Fyn and NR2A with one another. The associations of PSD95 with Fyn and NR2A increased at 0–24 h, 0–1 h of reperfusion, up to 6.9- and 2.1-fold relative to sham groups, respectively. Inhibiting activation of NMDA receptors or L-type voltage-gated calcium channels (L-VGCC) by ketamine or nifedipine attenuated the above increases of associations. These results suggest that stimulation of NMDA receptors and L-VGCC facilitates formation of a ternary complex: Fyn–PSD95–NR2A during transient brain ischemia followed by reperfusion, which may result in potentiation of NMDA receptor function and contribute to ischemic neuronal cell death.

Introduction

N-Methyl-d-aspartate (NMDA) receptors have been implicated in the pathogenesis of neurodegenerative disorders resulting from brain ischemia. Excessive release of glutamate causes over-activation of glutamate receptors mainly NMDA receptors and subsequent calcium overload, which is known as excitotoxicity, and then activates intracellular calcium-dependent signaling cascades that eventually lead to neuronal cell death several days after an ischemic episode [10], [17], [23]. Hippocampus is particularly vulnerable to such insults. However, the precise molecular mechanisms underling amplified up-regulation of NMDA receptors function induced by brain ischemia are still unclear.

Accumulating evidence has indicated that tyrosine phosphorylation of NMDA receptors up-regulates function of NMDA receptors [8], [32] and that the up-regulation is mediated principally by Src family protein tyrosine kinases (PTK) [2], [14], [35]. Three kinds of NMDA receptor subunits: NR1, NR2(A–D) and NR3 have been identified over the past decade. Multiple NR1 and at least one type of NR2 co-assemble to form functional channels. NR3 may combine with NR1–NR2 complexes and play a modulatory role [3]. NMDA receptors are phosphorylated on tyrosine residues of NR2 subunits particularly NR2A and NR2B [9], [16]. It has been shown that Src family PTK-mediated tyrosine phosphorylation of NR2A subunits serves a more important effect than that of NR2B on up-regulation of NMDA receptors activity in some physiological and ischemic cases [6], [12], [27], [29]. How is NR2A tyrosine phosphorylation by the Src family regulated?

Recently, a protein named PSD95 (postsynaptic density protein 95 kDa, also known as SAP90, synapse associated protein 90 kDa) has been reported to link NMDA receptors with downstream signaling proteins such as neuronal nitric oxide synthase (nNOS) and Fyn, one of five members of Src family in the CNS, in addition to clustering and anchoring of NMDA receptors [5], [22], [24], [30]. PSD95 is a member of membrane-associated guanylate kinase family. Like other members of the family, PSD95 consists of three N-terminal PDZ domains followed by one SH3 (Src-homology 3) domain and one C-terminal guanylate kinase (GK) domain. PSD95 proteins associate with NMDA receptors by binding of the first and second PDZ domains with C-terminal domain of NR2 subunits. Fyn binds to the third PDZ domain of PSD95 by its SH2 domain [4], [31] and the binding may result in activation of Fyn [33], [34]. Therefore, PSD95 may serve an essential function in controlling modulation of NMDA receptors activity by Src family [11], [22], [30].

Taken together, we proposed that brain ischemia potentiates formation of a ternary complex: Fyn–PSD95–NR2A, which facilitates association between Fyn and NR2A and results in enhancement of NMDA receptors function. In the present paper, the hypothesis was examined by analyzing the effects of transient (15 min) brain ischemia followed by reperfusion on interactions of Fyn, NR2A and PSD95 with one another in rat hippocampus. Furthermore, to clarify the roles of NMDA receptors and L-type voltage-gated calcium channels (L-VGCC) activation in ischemia-induced amplified up-regulation of NMDA receptors activity, the effects of ketamine (a selective NMDA receptor antagonist) and nifedipine (a potent L-VGCC blocker) on these changed interactions induced by ischemia and reperfusion were examined.

Section snippets

Brain ischemia treatments

Adult male Sprague–Dawley rats (Shanghai Experimental Animal Center, Chinese Academy of Science), weighing 200–250 g, had free access to food and water and were maintained on a 12-h dark/light cycle before surgery. Transient brain ischemia was induced by the four-vessel occlusion method [20], as described previously [12], [18]. Rats that showed completely flat bitemporal electroencephalogram, maintenance of dilated pupils and absence of a corneal reflex during occlusion were selected for the

Effects of transient brain ischemia followed by reperfusion on interactions of Fyn with NR2A

To investigate whether Fyn is involved in enhanced NMDA receptors activity after ischemia, the interactions of Fyn with NR2A at various times of reperfusion (0 and 15 min, 1, 6, 24, and 72 h) after ischemia were determined by immunoprecipitating with antibody against Fyn and then blotting with antibody against NR2A. Immunoblotting with the two antibodies was used to determine the changes of protein levels. The association of Fyn with NR2A increased immediately after 15 min of ischemia, reached

Discussion

NMDA receptors function is modulated by tyrosine phosphorylation. Brain ischemia raises tyrosine phosphorylation levels of NMDA receptors [12], [18], [29], but the exact molecular mechanisms are still not known. Previously, we reported that transient brain ischemia followed by reperfusion induced rapid and sustained (15 min–24 h of reperfusion) elevation in tyrosine phosphorylation levels of NR2A subunits and Src was related to the elevation [12]. In this paper, we investigated the alteration

Acknowledgements

This study is supported by the National Natural Science Foundation of China (No. 30170220; 30070182).

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Research reportActivation of NMDA receptors and L-type voltage-gated calcium channels mediates enhanced formation of Fyn–PSD95–NR2A complex after transient brain ischemia

Abstract

Introduction

Section snippets

Brain ischemia treatments

Effects of transient brain ischemia followed by reperfusion on interactions of Fyn with NR2A

Discussion

Acknowledgements

Neuroscience

J. Biol. Chem.

Brain Res.

J. Biol. Chem.

Neurodegeneration

Biochem. Biophys. Res. Commun.

Mol. Cell

Tyrosine phosphorylation of the N-methyl-d-aspartate receptor by exogenous and postsynaptic density-associated Src-family kinases

J. Neurochem.

Increased NMDA current and spine density in mice lacking the NMDA receptor subunit NR3A

Nature

Roles of postsynaptic density-95/synapse-associated protein 90 and its interacting proteins in the organization of synapses

Cell Mol. Life Sci.

Signal-processing machines at the postsynaptic density

Science

Subtype-specific regulation of recombinant NMDA receptor-channels by protein tyrosine kinases of the src family

J. Physiol.

Cleavage of structural proteins during the assembly of head of bacteriophage T

Nature

A peptide activator of endogenous tyrosine kinase enhances synaptic currents mediated by NMDA receptors

Eur. J. Neurosci.

The changing landscape of ischaemic brain injury mechanisms

Nature

The postsynaptic density protein PSD-95 differentially regulates insulin- and Src-mediated current modulation of mouse NMDA receptors expressed in Xenopus oocytes

J. Neurochem.

G-protein-coupled receptors act via protein kinase C and Src to regulate NMDA receptors

Nature Neurosci.

Research report
Activation of NMDA receptors and L-type voltage-gated calcium channels mediates enhanced formation of Fyn–PSD95–NR2A complex after transient brain ischemia