The Mechanisms Underlying NMDA Receptor Activity Change And Trafficking

NMDA receptor (NMDAR) is cation channel gated by glutamate, the main excitatory neurotransmitter in the mammalian CNS. NMDAR is permeable to Na+, which contribute to postsynaptic depolarization, and Ca2+ , which generate intracellular Ca2+ transient. NMDAR plays a vital role in physiological and pathological conditions. In physiological condition, NMDAR function as'molecular switch'to trigger synaptic efficacy change (LTP, long-term potentiation and LTD, long-term depression), which constitute the base of brain learning and study function. In pathological condition, NMDAR can cause cell death when intensely or chronically activated. After ,in fact, neu rons fail to generate sufficient ATP, ionic gradients are lost and glutamate is released, promoting overactivation of NMDAR and excessive neuronal calcium entry. Intracellular calcium overload activates calcium-depen dent kinases and proteases and finally"executioner"molecules such as caspases that promote cell death by suicidal mechanisms resembling apoptosis. So the molecular mechanisms underlying NMDAR activity change and trafficking is worth of intense study.1. Glycine exerts dual roles in ischaemic injury through distinct mechanismsLong-term changes in synaptic transmission, such as long-term potentiation, are thought to be the substrate of learning and memory. Many of the molecular processes involved in the induction or maintenance of LTP are the same as those activated during excitotoxicity. For example, NMDAR is activated and calciums enter into endocytoplasm in the induction phase of HFS stimuli-induced and post-ischemia LTP. Hence, an association between LTP and ischemia has been suggested. Study of mechanisms underlying ischemia-induced LTP is needed to elucidate ischemia and can supply experimental base for clinical treatment of ischemia.Ischemia elicits rapid release of various amino acids including glycine, which is the co-agonist of NMDA receptor (NMDAR) and one of main inhibitory neurotransmitters in central nervous system. Although accumulating studies has aimed to illustrate the exact effect of glycine in development of post-ischemic brain injury, the results remain quite controversial. These controversial mainly involves whether glycine exerts deleterious or neuroprotective effect in ischemia and by which mechanism glycine displays its effect.Here, we report that glycine exerts dose-dependent bidirectional effects on pathological plasticity in both in vitro and in vivo stroke models. In in vitro stroke model induced by oxygen and glucose deprivation (OGD) or in in vivo stroke model produced by middle cerebral artery occlusion (MCAO), exogenously applied glycine at relatively low level potentiated post-ischemic long-term potentiation (i-LTP). By contrast, glycine at high level totally abolished i-LTP. Similar bifurcated observation was obtained by endogenous glycine at different levels achieved by blockade of glycine transporter with antagonists at either subsaturating or saturating concentration. These data suggest that the level of glycine is a critical factor in determining the direction of its role in regulating ischemic injury. Further evidence demonstrated that the deleterious effect exerted by low level glycine was mediated by its modulation on NMDAR co-agonist site (site B), whereas neuroprotective effect of high level glycine was mediated by glycine receptor (site A) activation and subsequent differential regulation of NMDAR NR2 subunit components in in vitro and in vivo stroke models.Thus, our present results provide a molecular basis for the dual roles of glycine in facilitation or correction of post-ischemic pathological plasticity through distinct mechanisms and suggest that glycine receptor could be a potential target for clinical treatment of stroke.2. Myosin IIB regulate NMDAR trafficking to postsynaptic membrane during LTPLong-term potentiation (LTP) and Long-term depression (LTD) of excitatory synaptic transmission in CNS are indicators of long-term changes in synaptic efficacy and are considered to be the main model for investigating mechanisms underlying learning and memory. Furthermore, excitatory glutamate receptors play a critical role in the induction and maintenance of LTP and LTD. The channel conductance enhancement, the number increase and the subunits switch of postsynaptic AMPA receptors are the major causes of LTP. Recent studies have found that motor protein Myosin Vb or Myosin Va mobilize AMPA receptors-containing endosomes into dendritic spines for LTP. NMDA receptor, one type of the most important excitatory glutamate receptors, plays a critical role in excitatory synaptic transmission as'molecular switch'. Many forms of neural plasticity induction needs NMDA receptor activation and calcium influx into cytoplasm. The postsynaptic NMDA receptor number is not static, both PMA and TBS stimuli can induce NMDAR-LTP. NMDAR-LTP needs NMDAR insertion into cytomembrane, postsynaptic NMDA receptor number increase and NMDAR subunit switch. Then, how does NMDAR traffick to postsynaptic membrane, and who regulate the process of NMDAR trafficking?For illustrating these questions, we used PMA to induce chemical LTP in hippocampus CA1 pyramidal neurons. To record NMDAR-mediated excitatory postsynaptic currents (EPSCs), whole-cell recording technique was used and the cell membrane voltage was clamped to +40 mV with NBQX added in ACSF. Immunostaining, immunoblot and co-immunoprecipitation were also used with tenderly designed interference peptides to study the mechanisms of NMDAR trafficking in spines and clustering in postsynaptic membrane. We found that, PMA could induce postsynaptic NMDAR number increase, and the PSD Myosin IIB number also increased in the same while. PMA could also promote the interaction between NMDAR and Myosin IIB in PSD and cytoplasm. When interference peptide was used to disturb the binding between NMDAR and Myosin IIB, the PMA-induced interaction increase as well as the postsynaptic NMDAR number and the PSD Myosin IIB number increase were reversed. Our results illustrate that PKC induced NMDAR trafficking to PSD needs activated Myosin IIB and Myosin IIB interaction with NMDAR. Myosin IIB maybe an important signal molecule which participate in regulating NMDAR trafficking.