D-serine is the dominant endogenous coagonist for NMDA receptor neurotoxicity in organotypic hippocampal slices

Abstract

D-serine occurs at high levels in the brain, where it is an endogenous coagonist at the "glycine site" of NMDA receptors. However, D-serine action has not been previously compared with that of endogenous glycine, and the relative importance of the two coagonists remains unclear. We now investigated the efficiencies of the two coagonists in mediating NMDA receptor neurotoxicity in organotypic hippocampal slices. Removal of endogenous D-serine from slices was achieved by pretreating the tissue with recombinant D-serine deaminase enzyme. This enzyme is several orders of magnitude more efficient than previous methods to remove D-serine. We report that complete removal of D-serine virtually abolished NMDA-elicited neurotoxicity but did not protect against kainate. Although levels of glycine were 10-fold higher than D-serine, endogenous glycine was ineffective in mediating NMDA receptor neurotoxicity. The effect of endogenous glycine could be observed only after simultaneous removal of endogenous D-serine and blockage of the glycine transporter GlyT1. Our data indicate that D-serine is the dominant coagonist for NMDA receptor-elicited neurotoxicity, mediating all cell death elicited by NMDA in organotypic slices. The results suggest an essential role for this unusual D-amino acid, with implications for the mechanism of neuronal death in the nervous system.

Figure 1.

Properties of recombinant DsdA and specific depletion of endogenous d-serine in organotypic hippocampal slices. A, SDS-PAGE analysis of 3 μg of recombinant DsdA. B, Kinetic analysis of DsdA and d-amino acid oxidase enzyme activities. C, HPLC analysis reveals specific depletion of endogenous d-serine in hippocampal organotypic slices by treatment with DsdA (10 μg/ml) for 90 min. D, Specific depletion of endogenous d-serine from conditioned culture medium by DsdA as revealed by HPLC analysis. DAAOX, d-amino acid oxidase; d-ala, d-alanine; d-ser, d-serine; L-gln, l-glutamine; L-thr, l-threonine. The results are representative of three to four experiments.

Figure 2.

Endogenous d-serine is essential for NMDA-elicited cell death in organotypic hippocampal slices. A, Control (Ctl). B, NMDA (500 μm) elicited robust cell death in all hippocampal areas as measured by PI uptake. C, Control treated with DsdA (10 μg/ml for 90 min). D, Destruction of d-serine by DsdA protected against NMDA-elicited cell death. E, NMDA plus MK-801 (30 μm) added 10 min before and during the NMDA insult. F, NMDA plus DNQX (100 μm) added 10 min before and during the NMDA insult. G, Densitometric analysis of PI uptake and reversal of DsdA effect by addition of 1 mm glycine (Gly). The values were expressed as a percentage PI uptake relative to that observed with excess l-glutamate (10 mm). The results are average ± SEM of 4-12 experiments done in quadruplicates. ^***p < 0.001, different from NMDA insult (n = 12).

Figure 3.

Endogenous d-serine is not required for kainate-elicited cell death. Conditions were as in Figure 2, except that neurotoxicity was induced by incubation with kainate (100 μm) instead of NMDA. A, Kainate insult. B, Kainate plus DsdA. C, Kainate plus 100 μm DNQX. D, Kainate plus 30 μm MK-801. E, Densitometric analysis of kainate-elicited neurotoxicity. Ctl, Control. The results are average ± SEM of six experiments done in triplicates from a different slice preparation. ^***p < 0.001, different from kainate insult.

Figure 4.

Glycine dependence for NMDA neurotoxicity in the absence of d-serine and potentiation by GlyT1 inhibitor. A-D, Effect of glycine (Gly) in NMDA-elicited neurotoxicity in the absence of endogenous d-serine. Slices were pretreated with DsdA (10 μg/ml for 90 min) and exposed to 500 μm NMDA as described in Figure 2. Basal glycine (A) accounted for ∼20 μm, as measured by HPLC. Exogenous glycine (20, 60, and 130 μm) were added in B, C, and D, giving the total glycine values of 40, 80, and 150 μm, respectively. E, Percentage of maximal PI uptake as a function of medium glycine concentration in slices pretreated with DsdA and exposed to NMDA. F, Effect of GlyT1 inhibitor on NMDA-elicited neurotoxicity in the absence or presence of DsdA. NFPS (1 μm) was present 30 min before and during NMDA insult. Ctl, Control. ^**p < 0.01, significant difference.