Neural Cell Adhesion Molecule-associated Polysialic Acid Inhibits NR2B-containing N-Methyl-d-aspartate Receptors and Prevents Glutamate-induced Cell Death

Martin S.L Hammond,Catrina Sims,Kodeeswaran Parameshwaran,Vishnu Suppiramaniam,Melitta Schachner,Alexander Dityatev

doi:10.1074/jbc.m602568200

Martin S.L Hammond, Catrina Sims + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m602568200

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Abstract

The neural cell adhesion molecule (NCAM) and its associated glycan polysialic acid play important roles in the development of the nervous system and N-methyl-D-aspartate(NMDA)receptor-dependent synaptic plasticity in the adult. Here, we investigated the influence of polysialic acid on NMDA receptor activity. We found that glutamate-elicited NMDA receptor currents in cultured hippocampal neurons were reduced by approximately 30% with the application of polysialic acid or polysialylated NCAM but not by the sialic acid monomer, chondroitin sulfate, or non-polysialylated NCAM. Polysialic acid inhibited NMDA receptor currents elicited by 3 microm glutamate but not by 30 microm glutamate, suggesting that polysialic acid acts as a competitive antagonist, possibly at the glutamate binding site. The polysialic acid induced effects were mimicked and fully occluded by the NR2B subunit specific antagonist, ifenprodil. Recordings from single synaptosomal NMDA receptors reconstituted in lipid bilayers revealed that polysialic acid reduced open probability but not the conductance of NR2B-containing NMDA receptors in a polysialic acid and glutamate concentration-dependent manner. The activity of single NR2B-lacking synaptosomal NMDA receptors was not affected by polysialic acid. Application of polysialic acid to hippocampal cultures reduced excitotoxic cell death induced by low micromolar concentration of glutamate via activation of NR2B-containing NMDA receptors, whereas enzymatic removal of polysialic acid resulted in increased cell death that occluded glutamate-induced excitotoxicity. These observations indicate that the cell adhesion molecule-associated glycan polysialic acid is able to prevent excitotoxicity via inhibition of NR2B subunit-containing NMDA receptors.

Highlights

The NMDARs are a subtype of ionotropic glutamate receptors that are found widely throughout the brain
Our study shows that both bacterially produced PSA and eukaryotically produced PSA-neural cell adhesion molecule (NCAM) inhibit the NR2B subunit-containing NMDARs of cultured hippocampal neurons
PSA inhibits the activity of single synaptosomal NR2B-containing NMDARs reconstituted in lipid bilayers

Summary

EXPERIMENTAL PROCEDURES

Chemicals—Polymers containing 25–50 sialic acid residues were purified from colominic acid (Fluka, Buchs, Switzerland) using anion exchange chromatography on a Hamilton PRPX column dissolved in 20 mM Tris, pH 7.4, with 0 –500 mM NaCl and detected at 214 nm [24]. Electrophysiological Recordings in Cultured Neurons—All recordings were performed in the whole-cell patch clamp mode at room temperature in extracellular solution containing 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2, 10 mM HEPES, 30 mM D-glucose, 12 mM D-sucrose, adjusted to pH 7.3 with NaOH. To isolate NMDAR currents in neurons, tetrodotoxin (1 ␮M, Alomone Labs, Jerusalem, Israel), tetraethylammonium chloride (10 mM, Sigma-Aldrich), 1,2,3,4-tetrahydrobenzofquinoxaline-7-sulfonamide (5 ␮M, Tocris, Bristol, UK), (RS)-methyl-4-carboxyphenylglycine (100 ␮M, Tocris), and picrotoxin (100 ␮M, Tocris) were added to the extracellular solution. These compounds were applied to block Naϩ and Kϩ voltage-dependent channels and ␥-aminobutyric acid, type A, glycine, and non-NMDA glutamate receptors.

Incorporation of synaptosomal

NMDAR currents at

Because experiments with hippocampal neurons revealed that

Findings

DISCUSSION