GLYX-13, a NMDA Receptor Glycine-Site Functional Partial Agonist, Attenuates Cerebral Ischemia Injury In Vivo and Vitro by Differential Modulations of NMDA Receptors Subunit Components at Different Post-Ischemia Stage in Mice

Abstract

Excessive activation of NMDA receptors (NMDARs) is implicated in pathological synaptic plasticity also known as post-ischemic long-term potentiation (i-LTP) which was produced by glutamate mediated excitotoxicity after stroke. In the past decades, many NMDARs inhibitors failed in clinical investigations due to severe psychotomimetic side effects. GLYX-13 is a NMDAR modulator with glycine site partial agonist properties and has potential protective effects on ischemic neuronal death. However, the underlying molecular mechanism of GLYX-13 attenuating the ischemic neuronal damage remains elusive. Our study was conducted to examine the molecular, cellular and behavioral actions of GLYX-13 in stroke, and further characterize the mechanism underlying the neuroprotective actions via modulation of the NMDAR subunit composition. In present study we found that in vitro oxygen-glucose deprivation (OGD) stroke model, GLYX-13 blocked i-LTP and restored the ratio of NR2A/NR2B subunit composition. The glycine site of NMDARs full coagonist D-serine completely blocked the effects of GLYX-13 on i-LTP. Besides, in vivo middle cerebral artery occlusion (MCAO) model, GLYX-13 decreased the cerebral infarct volume and reduced injury of hippocampus. Western analysis showed that GLYX-13 down-regulated the expression of phosphorylated NR2B (Tyr1472) and up-regulated phosphorylated NR2A (Tyr1325). Furthermore, GLYX-13 treatment along with NR2B specific antagonist (Ro256981) failed to exhibit any additional neuro-protective effects, whereas the application of NR2A antagonist (NVP-AAM007) abolished the neuroprotective effects of GLYX-13, which suggested that the protective action of GLYX-13 should be by its regulation of NMDAR subunit components. Our study provides important insights on the potential protective mechanism of GLYX-13 in ischemia and proposes the glycine site of NMDARs as a novel target for developing therapeutic strategies to store synaptic function in stroke.

Keywords: GLYX-13; ischemia; oxygen-glucose deprivation; pathological synaptic plasticity; transient middle cerebral artery occlusion.

Figure 1

Effect of GLYX-13 on Neurological function. Neurological score were evaluated 24 h after reperfusion. Statistical analysis was determined by Mann-Whitney U-test, n = 12, ***p < 0.001.

Figure 2

GLYX-13 treatment reduced ischemic infarct volume in vivo middle cerebral artery occlusion (MCAO) model. Cerebral infarct volume was assessed via triphenyltetrazolium chloride (TTC) staining of coronal sections 24 h after reperfusion in representative mice. The infarct volume was analyzed in each group. Data were expressed as mean ± SEM (n = 12 animals per group). One-way analysis of variance (ANOVA) and Newman-Keuls post test, ***p < 0.001, vs. Sham; ^###p < 0.001, vs. vehicle.

Figure 3

Administration of GLYX-13 attenuating post-ischemic neuronal injury. Representative hematoxylin and eosin (HE) staining coronal brain sections showed the hippocampal CA1, CA3, dentate gyrus (DG) areas, respectively.

Figure 4

Administration of GLYX-13 dramatically decreased the number of Fluoro Jade C (FJC) positive neurons in hippocampus. Three regions (CA1, CA3 and DG) were further evaluated to compare the FJC fluorescent signals between sham, vehicle and GLYX-13 treated group. Photomicrographs of FJC-stained three regions of interest were shown in each group. GLYX-13 treatment dramatically reduced the number of FJC positive neurons in DG and CA3 region (^###p < 0.001) and also reduced the number of FJC positive neurons in CA1 (^#p < 0.05). The numbers of FJC positive neurons were expressed as the mean number per field of view. Data were expressed as mean ± SEM (n = 5); one-way ANOVA and Newman-Keuls post test, ***p < 0.001, vs. Sham; ^###p < 0.001, ^#p < 0.05, vs. vehicle.

Figure 5

GLYX-13 reduced the expression of pY1472 NR2B 4 h after reperfusion. (A) Representative images of western blot. The intensity of each band was quantified by image j and normalized in relation to β-actin. (B–D) Statistical analysis of densitometric data from indicated experimental groups. The values are expressed as mean ± SEM, n = 3 independent experiments. One-way ANOVA and Newman-Keuls post test, *p < 0.05, **p < 0.01, vs. Sham; ^#p < 0.05, vs. Vehicle.

Figure 6

GLYX-13 increased the expression of pY1325 NR2A 4 h after reperfusion. (A) Representative images of western blot. The intensity of each band was quantified by image j and normalized in relation to β-actin. (B–D) Statistical analysis of densitometric data from indicated experimental groups. The values are expressed as mean ± SEM, n = 3 independent experiments. One-way ANOVA and Newman-Keuls post test, *p < 0.05, vs. Sham; ^#p < 0.05, vs. Vehicle.

Figure 7

GLYX-13 ameliorated the loss of NR2A after 24 h reperfusion. (A) Representative images of western blot. The intensity of each band was quantified by Image j and normalized in relation to β-actin. (B–D) Statistical analysis of densitometric data from indicated experimental groups. The values are expressed as mean ± SEM, n = 3 independent experiments. One-way ANOVA and Newman-Keuls post test, *p < 0.05, vs. Sham; ^#p < 0.05, vs. Vehicle.

Figure 8

GLYX-13 has no effect on the loss of NR2B after 24 h reperfusion. (A) Representative images of western blot. The intensity of each band was quantified by image j and normalized in relation to β-actin. (B–D) Statistical analysis of densitometric data from indicated experimental groups. The values are expressed as mean ± SEM, n = 3 independent experiments. One-way ANOVA and Newman-Keuls post test *p < 0.05, vs. Sham.

Figure 9

GLYX-13 ameliorated pathological potentiation of excitatory postsynaptic current (EPSC). (A) Sample traces were presented; bar chart of the data showed the effects of GLYX-13 on ischemic long-term potentiation (i-LTP; n = 5, one-way ANOVA and Newman-Keuls post test, **p < 0.01, compared with control; ^##p < 0.01, compared with oxygen-glucose deprivation (OGD) group). (B) The changes in pathological i-LTP were under various conditions. GLYX-13 at 10 μmol/L completely abolished i-LTP. As a control, no obvious change in EPSCs was observed when hippocampal slices were not suffered from OGD treatment. (C) Suppression of i-LTP induced by GLYX-13 was reversed by full glycine site agonist D-Serine (100 μmol/L).

Figure 10

GLYX-13 suppressed the elevation of NR2B-containing NMDA receptors (NMDARs) component induced by OGD treatment. (A) Schematic paradigms showed experimental protocols adopted to detect the NR2 subunit components under control, OGD, OGD with 10 μmol/L GLYX-13, OGD with co-application of GLYX-13 and D-serine (100 μmol/L). Selective NR2B subunit antagonist Ro256981 (20 nmol/L) was used to exhibit the amplitude NR2A and NR2B (total amplitude subtract NR2A) containing NMDARs mediated EPSCs in CA1 neurons. The arrows in the paradigm represent the time points to record EPSCs. (B) Sample traces were presented and normalized NMDA EPSCs of total NMDA EPSCs, NR2A and NR2B-containing NMDAR subunit component under various conditions. (n = 5, one-way ANOVA and Newman-Keuls post test, **p < 0.01, compared with control; ^##p < 0.01, compared with OGD group).

Figure 11

OGD-induced i-LTP was completely abolished by selective NMDAR antagonist AP-5 (50 μmol/L, n = 5, student’s t-test, p < 0.01).

Figure 12

GLYX-13 reduced cerebral infarct volume through enhancing NR2A-containing components and decreasing NR2B-containing components in vivo MCAO ischemia model. Representative brain sections were stained with TTC under various treatments. Unstained area is infarct. GLYX-13 treatment along with selective NR2B antagonist (Ro256981 5 mg/kg) 2 h after 1 h MCAO challenge failed to display any additional neuroprotective effects; by contrast GLYX-13 treatment along with selective NR2A antagonist (NVP-AAM077 2.4 mg/kg) reversed the amelioration of infarct volume produced by GLYX-13 (n = 6, ***p < 0.001, compared with Vehicle).