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.

Glycine-induced NMDA receptor internalization provides neuroprotection and preserves vasculature following ischemic stroke

“…Glycine is also thought to exert its neuroprotective effects via mediation of non-ionotropic NMDAR function ( Chen et al., 2015 , 2017 ; Hu et al., 2016 ), or by promoting microglial polarization ( Liu et al., 2019 ). Partial agonists at the GBS on NMDARs also afford neuroprotection following an OGD challenge ( Stanton et al., 2009 ) and during MCAO paradigm ( Zheng et al., 2017 ). Pharmacological elevation of brain glycine following NFPS administration potentiates ischemic preconditioning ( Pinto et al., 2015 ) and confers neuroprotection via global activation of ionotropic GlyRs during transient MCAO ( Huang et al., 2016 ).…”

“…A recent review suggests that glycine and d -serine may be therapeutically beneficial by down regulating NMDARs, such as rodent models of traumatic brain injury and lipopolysaccharide-induced neuroinflammation ( Biegon et al., 2018 ). There is also a growing body of evidence to suggest that extracellular glycine is neuroprotective in several rodent ischemic stroke models ( Huang et al., 2016 ; Zheng et al., 2017 ; Zhao et al., 2018 ; Liu et al., 2019 ; Chen et al., 2020 ; Yamamoto et al., 2016 ). Moreover, the level of extracellular glycine appears to be important in stroke outcome.…”

Glycine-induced NMDA receptor internalization provides neuroprotection and preserves vasculature following ischemic stroke

“…Recent work suggests that it is via modulation of intracellular pathways, including the Phosphatase and tensin homolog (PTEN)/protein kinase B (AKT) signaling pathway 34,35 , or vascular endothelial growth factor receptor 2 46 . Glycine is also thought to exert its neuroprotective effects via mediation of non-ionotropic NMDAR function 31-neuroprotection following an OGD challenge 48 and during MCAO paradigm 49 . Pharmacological 372 elevation of brain glycine following NFPS administration potentiates ischemic preconditioning 25 373 and confers neuroprotection via activation global activation of ionotropic glycine receptors during 374 transient MCAO 36 .…”

“…A recent review suggests that glycine and d-serine may be therapeutically beneficial by down 378 regulating NMDARs, such as rodent models of traumatic brain injury and lipopolysaccharide-379 induced neuroinflammation 50 . There is also a growing body of evidence to suggest that extracellular glycine is neuroprotective in several rodent ischemic stroke models 34,36,46,47,49,51 .…”

Ginkgo Biloba Extracts Inhibit Ischemic LTP Through Attenuating EPSCs In Rat Hippocampus

“…A pathological form of plasticity, named post-ischemic long-term potentiation (i-LTP), was observed in glutamate receptor-mediated neurotransmission after stroke and thought to exert a detrimental effect via facilitation of excitotoxic damage (Calabresi et al 2003;Costa et al 2011;Zheng et al 2017). This longterm enhancement in glutamate receptor-mediated excitatory responses was mainly attributed to excessively released synaptic glutamate and the followed increase of intracellular Ca 2+ through N-methyl-D-aspartate (NMDA) type glutamate receptors (Wu et al 2017;Zhou et al 2015).…”