Abstract
Ionotropic activation of NMDA receptors (NMDARs) requires agonist glutamate and co-agonist glycine. Here we show that glycine enhances the activation of cell survival-promoting kinase Akt in cultured cortical neurons in which both the channel activity of NMDARs and the glycine receptors are pre-inhibited. The effect of glycine is reduced by shRNA-mediated knockdown of GluN2A subunit-containing NMDARs (GluN2ARs), suggesting that a non-ionotropic activity of GluN2ARs mediates glycine-induced Akt activation. In support of this finding, glycine enhances Akt activation in HEK293 cells over-expressing GluN2ARs. The effect of glycine on Akt activation is sensitive to the antagonist of glycine-GluN1 binding site. As a functional consequence, glycine protects against excitotoxicity-induced neuronal death through the non-ionotropic activity of GluN2ARs and the neuroprotective effect is attenuated by Akt inhibition. Thus, this study reveals an unexpected role of glycine in eliciting a non-ionotropic activity of GluN2ARs to confer neuroprotection via Akt activation.
Highlights
The N-methyl-D-aspartate receptor (NMDAR) is a subtype of ionotropic glutamate receptors that mediate the vast majority of excitatory neurotransmission in the mammalian central nervous system (CNS)[1]
Our results showed that treatment of glycine (100 μM) for 30 min increased Akt phosphorylation in the cortical neurons in which there were no Ca2+ influx into the NMDAR channels (Fig. 1A)
To exclude the possibility that residual Ca2+ in the extracellular solution might pass through NMDAR channels, we treated the neurons with the extracellular solution described above but with the addition of non-competitive NMDAR antagonist MK-80128,29
Summary
Glycine increases Akt phosphorylation independent of Ca2+ influx through NMDAR channels. We showed that after the channel activities of NMDARs were inhibited, L-689560 (50 μM) blocked glycine-induced Akt phosphorylation in the cultured neurons and in the HEK293 cells transfected with GluN1 +GluN2A (Fig. 5A,B). These data suggest that the glycine-GluN1 binding is required for the non-ionotropic activation of GluN2ARs. As a control study, we tested the effect GluN2B antagonist Ro 25-6981 (5.0 μM)[42,43]. As a further support for the role of glycine-GluN1 binding in mediating the effect of non-ionotropic GluN2ARs, we tested the role of D-serine in Akt phosphorylation in the cortical neurons and the HEK293 cells transfected with GluN1 +GluN2A or GluN1 +GluN2B following NMDAR channel inactivation procedure. These results provide functional evidence for the role of non-ionotropic activity of GluN2ARs in mediating the neuroprotective effect of glycine
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