吸入麻醉药通过激活CK2磷酸化NR2B导致发育期神经元兴奋性毒性损伤机制研究

The NMDA receptor (NMDAR) is a ligand-gated ion channel permeable to Na+, K+, and Ca2+, and is found at excitatory synapses throughout the brain. NMDARs are required for many forms of learning and memory, and are implicated in numerous neurological disorders。NMDARs are heterotetramers composed of two NR1 subunits and two NR2 subunits。In cerebral cortex there is a developmental switch from NR2B- to NR2A-containing NMDA receptors。NMDAR subunit composition varies between different brain regions and throughout development。synaptic NMDARs switch from those predominantly containing NR2B to those containing NR2A. The switch in NR2 subunit composition is dependent on neuronal activity and experience.The NR2B to NR2A switch causes important changes to NMDAR function, altering the amount of calcium influx through the pore and the types of proteins interacting with the intracellular domain of the receptor。NR2 subunit composition at developing hippocampal synapses requires CK2 activity.Casein kinase 2 (CK2) phosphorylates the NR2B subunit within its PDZ-binding domain，on serine 1480 (S1480).phosphorylation of NR2B on S1480 increas in response to NMDAR activity and is regulated by NR2A expression..Researches have revealed the toxcity of inhalation anesthectics such as isoflurane to developing hippocampal neurons.But the mechanism is still uncertain.Our study is based on the hypothesis that inhalation anesthectics can act on developing neuronal specific GABA A receptor to increase intracellular free calcium concentration and to activate CK2 to phosphorylate NR2B，then to promote endocytosis of NR2A that the NMDAR facilitate NR2A insertion by removing NR2B from the synaptic sites via S1480 phosphorylation，and further elevation of calcium concentration, so that the growth and differentiation of neurapophysis can be inhibited. We want to detect the mechanisms of inhalation anesthectics on the growth and differentiation of developing neurapophysis and synaptogenesis from ion channel, neuron, brain slice and whole animal levels.

NMDA受体亚单位在发育期海马神经元的生长分化、突触形成、可塑性及学习和记忆功能中起着十分重要的作用，研究发现，吸入麻醉药异氟烷等可致发育期神经元毒性，但其机制尚不十分明确。本项目依据吸入麻醉药作用于发育期神经元阶段特异性的GABA A受体，升高细胞内自由钙离子浓度，从而激活CK2磷酸化NR2B,促进NR2B胞吞NR2A插入突触后膜，进一步升高钙离子浓度，抑制神经元神经突生长分化的理论假设，拟从离子通道、神经元、脑片和整体动物水平，探讨吸入麻醉药对发育期神经元神经突生长分化和神经元之间突触形成的作用及其机制，并筛选出防治该毒性的可能干预靶点，为临床安全使用吸入麻醉药提供理论依据

大量证据显示，多次暴露于异氟烷等吸入麻醉药会导致发育期大鼠学习记忆功能受损。鉴于NMDA受体在发育期海马神经元生长分化、突触形成、可塑性及学习和记忆功能中所起的十分重要的作用， 其可能为在异氟烷所致上述神经毒性的机制之一。经研究本课题组证实异氟烷致发育期海马神经元及发育期大鼠海马的神经毒性时NMDA受体各亚单位中是NR2B而不是NR2A或者NR1表达及磷酸化增加。导致NR2B表达及磷酸化增加的原因，经研究证实可能与第10 号染色体缺失的磷酸酶及张力蛋白同源基因（hosphatase and tensin homologue deleted on chromosome 10，PTEN）有关。.异氟烷不仅能够使得非活性状态的p-PTEN表达减少、致使相应tau蛋白磷酸化增加、神经元突触标志蛋白PSD-95、SNAP-45表达减少、学习记忆功能受损，且PTEN特异性拮抗剂BPV能够逆转上述改变，且可以使得NR2B表达及磷酸化减少。证实了上述假设。.除了直接导致发育期大脑或者离体神经元凋亡及突触形成减少而导致学习记忆功能受损外，异氟烷对神经干细胞的影响也是其导致远期学习记忆功能受损的另外一个可能的机制。本研究显示随着暴露时间的延长，神经干细胞增殖减少且向胶质细胞分化增加这可能与激活了钙蛋白Calpain有关。特异性抑制Calpain会使得异氟烷所致上述改变得到改善。.通过对上述可能机制进行干预，异氟烷的神经毒性得到相应的逆转，可能为降低异氟烷神经毒性的提供相应靶点和手段。