Nr2a And Nr2b Reverse Regulation Of Neuronal Death And Its Molecular Mechanism

Stroke is one of the most common causes of death and contributes to serious and long-term disability. The only current pharmacologic treatment option for stroke is thrombolytic therapy. Clinical results demonstrated that patients were 32% more likely to recover with little or no disability following treatment with rt-PA given within 3 h of the onset of ischemic stroke. However, in clinical practice the use of rt-PA has been limited to fewer than 5% of patients with ischemic stroke owing to the narrow therapeutic window and contraindications that increase the risk of hemorrhagic transformation.Glutamate is the major excitatory neurotransmitter in the mammalian brain and key mediator of intracellular communication, plasticity, growth and differentiation. NMDA receptors is one kind of Glutamate receptors. The N-methyl-D-aspartate (NMDA) receptor is a heteromeric ligand-gated ion channel that interacts with multiple intracellular proteins by way of different subunits. NMDA receptors is primarily expressed in forebrain structures, such as the cortex, hippocampus, striatum, thalamus, and olfactory bulb. NMDA receptor play a central role in hypoxia or ischemia. Major events in the cascade triggered by hypoxia or ischemia include overstimulation of NMDA-type glutamate receptors. Modulating glutamate activation of NMDA receptors could potentially have a neuroprotective effect in ischemic stroke. Glutamate-receptor antagonists such as etomidate and MK-801 have demonstrated efficacy in decreasing neuronal damage in animal models. However, NMDA-receptor antagonists have not been shown to improve outcome in clinical trials for treatment of ischemic stroke. This is understandable given the important role of physiological NMDA receptor signalling in the brain, as well as in crucial adaptive processes such as synaptic plasticity and neuronal survival and resistance to trauma. It indicated that too little or too much NMDA receptor activity is harmful to neurons. It might prove possible to compensate for them in cases whereNMDAreceptor activity is suppressed. Likewise, if NMDA-receptor-dependent prodeath signalling is via the specific coupling of NMDA receptors to a molecule involved in the neurotoxic process, then small compounds designed to break this interaction might prove a useful therapeutic tool. It requires discrimination of the pro-death and pro-survival signal of NMDA receptor. The comparative studys on synaptic and extrasynaptic NMDA receptor or NR2A and NR2B become the most interesting aspects for researchers.The ultimate goal is to be able to interfere selectively with the pro-death effects of pathological NMDA receptor activation without affecting pro-survival or other physiological signalling events. For example, if extrasynaptic NMDA receptors do indeed prove to be particularly strongly coupled to cell death, then their lack of apparent physiological role and differences in subunit composition from synaptic receptors will make them an attractive therapeutic target.Part I The opposite role of NR2A and NR2B subunit on neuron deathAIM: To investigate the role of NR2A and NR2B on glu-induced neuron death by pharmacological inhibition of NR2A or NR2B and explore the possible mechanisms. METHODS: Cultured cortical neurons at DIV10 were bathed with glu alone or in in combination with NVP-AAM007 or Ro25-6981 for 24h and measured the LDH release ratio and NO_x content. Cultured cortical neurons at DIV11 were bathed with glu alone or in in combination with NVP-AAM007 or Ro25-6981 for 1h and measured the NOS activity. Cultured cortical neurons at DIV10 were bathed with NVP-AAM007 or Ro25-6981 for 24h and measures the expression of nNOS by Western-blot analyses. Cultured cortical neurons at DIV10 were bathed with glu or in combination wit 7-NI for 24h and measured the LDH release ratio. RESULTS: NVP-AAM007 could further increase the LDH release ratio, nNOS activity and NO_x content enhanced by glu bath. While, Ro25-6981 has an opposite effect compared with NVP-AAM007. They have no effect on nNOS expression. 7-NI could reduced LDH release ratio enhanced by glu bath. CONCLUSION: By regulating nNOS activity, NR2A and NR2B exercise their different role in neuron death induced by glu.Part II The role of NR2B on CA1 neuron death induced by global brain ischemia in gerbilAIM: To study the role of NR2B subtype in CAl neuron death induce by global brain ischemia in gerbil. METHODS: By administration of vehicle, Ro25-6981 or 7-NI in the global brain ischemia model gerbil and using three immunohistochemical methods(NeuN, crystal violet and Fluoro-Jade) to observe CAl neuron death. RESULTS: Less neuron death was observed in sham, Ro25-6981 or 7-NI group compared with vehicle group. CONCLUSION: During brain ischemia, NR2B can up-regulate nNOS activity and aggravate brain damage.Part III Study on the target of NR2B/PSD-95 couplingAIM: By pharmacological and molecular biological methods, to study if expression of NR2B C- terminal polypeptide can antagonize glutamate excitotoxicity. METHODS: According to PCR method, gain genic sequence of NR2B C-terminal polypeptide. Insert these genic sequence into AAV-IRES-GFP vector. Using the AAV-helper free system of Stratagene company to construct recombinant AAV(AAV-GFP, AAV-33C-IRES-GFP and AAV-200C-IRES-GFP). The AAV-100-IRES- GFP and AAV-600-IRES-GFP can expression 33 or 200 amino acid of NR2B C- terminal respectively. After infected by rAAV , the neuron were incubated with glu for 24h and observed the morphology of neurons. RESULTS: After incubated with glu for 24h, neuron infected with rAAV (AAV-33C-IRES-GFP and AAV-200C-IRES-GFP) has similar morphological feature compared with normal group. While infected with AAV-GFP, most of neurons were death. CONCLUSION: Expression of NR2B C- terminal polypeptide can antagonize glutamate excitotoxicity.