【作者】 颜景芝；

【导师】 陆巍；

【作者基本信息】 南京医科大学 ， 药理学， 2010， 博士

【摘要】 谷氨酸受体是海马内主要的兴奋性神经递质受体,离子型谷氨酸受体包括NMDA受体、AMPA受体和KA受体。它们在神经发育、学习记忆、感知等方面起着重要作用。其中一个重要的功能就是参与突触传递的调节,产生突触可塑性。长时程增强（long-term potentiation, LTP）或长时程抑制（long-term depression, LTD）被认为是突触可塑性的重要细胞模型,突触后AMPA受体数量的增加或减少是LTP或LTD产生的重要机制之一。NMDA受体作为“分子开关”触发突触可塑性,主要决定LTP的诱导。而AMPA受体则主要参与LTP的表达和维持。当发生LTP时,通过增加突触后AMPA受体的数量和效率而增加兴奋性突触后电流。因此,NMDA和AMPA受体的膜转运被认为是突触可塑性调节的重要环节。AMPA受体和NMDA受体都可以被一系列的蛋白激酶磷酸化,而磷酸化的水平则直接影响了这些受体的功能特性,包括通道电导和受体膜定位等。近几年研究发现,PKC能促进NMDA受体和AMPA受体的转运上膜,但其具体的分子机制及两种转运的可能联系还有待于进一步探讨。本文主要目的旨在探讨PKC调控NMDA和AMPA受体转运上膜的可能分子机制。一、PKC通过间接激活CaMKII促进NMDA受体的转运NMDA受体在兴奋性突触功能中起着至关重要的作用。多种形式的突触可塑性都依赖于NMDARs的激活以及随后胞内Ca²⁺浓度的增加。因此,调节NMDARs功能对突触可塑性具有非常重要的意义。我们采用免疫印迹、免疫沉淀、免疫荧光染色,结合电生理学方法及小分子肽干扰技术,研究PKC激活后NMDA受体转运的可能分子机制。我们的研究结果发现,PKC激活不仅能够促进突触后NMDA受体表达增加,从而直接上调突触后NMDA受体功能,而且能通过间接激活Src酪氨酸激酶来上调NMDA受体功能。除此以外, PKC还可以通过与NMDA受体结合蛋白（NAPs）相互作用而促进NMDA受体转运到细胞膜。本文中我们发现CaMKII可能是一种介导PKC激活诱导的NMDA受体转运增加的NAP蛋白。PKC激活引起CaMKII自身磷酸化水平增加,以及随后CaMKII与NMDA受体结合增加,同时伴随功能性NMDA受体插入到突触后位点。PKC诱导的增强可以被CaMKII抑制剂AIP削弱,也可以被选择性的打断CaMKII与NR2A或NR2B结合的小肽Tat-NR2A和Tat-NR2B所抑制。而且,Tat-NR2A和Tat-NR2B各自选择性的阻断PKC诱导的LTP的诱导和表达阶段。进一步相互排除实验证明PKC和CaMKII共享某一共同信号通路增强NMDA受体的转运和LTP产生。总之,我们的结果支持这样一个解释或假设,即PKC通过间接激活CaMKII自身磷酸化使CaMKII与NMDA受体结合增加,从而促进NMDA受体的转运。二、PI3K通过激活aPKCλ调控AMPA受体的转运AMPA受体主要介导快速的突触传递,参与LTP的表达和维持。突触后AMPA受体数量变化和功能特性的调节可能是突触可塑性发生的一个主要机制。因此,了解AMPA受体转运的分子机制将具有非常重要的意义。我们采用免疫印迹、免疫沉淀、免疫荧光染色,结合电生理学方法及小分子肽干扰技术,研究PKC对AMPA受体转运的可能分子机制。我们的研究结果表明,一种特殊类型的非典型PKC——aPKCλ（atypical PKCλ）是PI3K诱导LTP的必要条件,可能是PI3K通过间接激活aPKCλ,促进功能性GluR1在突触后的表达,从而引起LTP;进一步研究发现,P62蛋白作为衔接蛋白（adaptor protein）辅助形成GluR1- P62-aPKCλ复合体,从空间上拉近aPKCλ与GluR1距离,促进aPKCλ对GluR1的磷酸化作用,进而调节其转运上膜。更多还原

【Abstract】 Glutamate receptors are the predominant excitatory neurotransmitter in hippocampus. Ionotropic glutamate receptors (iGluRs) are a major class of heteromeric ligand-gated ion channels and mediate the majority of the excitatory neurotransmission in the vertebrate central nervous system (CNS). iGluRs can be classified as N-methyl-D-aspartate (NMDA) receptor or the non-NMDA receptor, which can be further subdivided intoα-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, or kainate (KA) receptor. These receptors are important for neural development, learning and memory and cognition. One of the major functions of glutamate receptors appears to be the modulation of synaptic transmission, resulting in synaptic plasticity. An important cellular models of synaptic plasticity in the mammalian brain are the long-term depression (LTD) and long-term potentiation (LTP) that are generated at excitatory synapses on hippocampal CA1 pyramidal cells.An increase or decrease in the number of ionotropic glutamate receptors on a post-synaptic cell may lead to long-term potentiation or long-term depression of that cell, respectively. NMDA receptor, as a“molecular switch”, triggers synaptic plasticity and contributes to the induction of LTP. While AMPA receptor is important for memory storage and participates in the expression and maintainance of LTP. During LTP, excitatory postsynaptic current (EPSC) is enhanced by the increased numbers and efficiency of AMPA receptor. Therefore, the trafficking of NMDA or AMPA receptor is critical for the regulation of syanptic plasticity.NMDA and AMPA receptor can be phosphorylated by a series of protein kinase (such as PKC, PKA, CaMKII et al.), which directly influence their functional property, including channel localization, conductance, and open probability. Recent studies have shown that PKC promotes delivery of NMDA or AMPA receptor to surface membrane. However, the detailed molecular mechanisms remain unclear. Thus, The primary aim of the study was to investigate the possible molecular mechanism of NMDA or AMPA receptor trafficking regulated by PKC.1. Protein kinase C promotes NMDA receptor trafficking by indirectly triggering CaMKII autophosphorylationThe NMDA receptor (NMDAR) plays a central role in the function of excitatory synapses. Many forms of synaptic plasticity depend on NMDA receptor activation and subsequent increase of intracellular Ca2+. Therefore, it is very important for synaptic plasticity to regulate functional NMDAR. Using the methods of western blotting, immunoprecipitation, immunofluorescene and electrophysiology, we demonstrate the possible mechanism of NMDAR trafficking induced by protein kinase C (PKC). Our results showed that PKC promotes NMDA receptor trafficking to the cell surface or indirectly activates Src and upregulates NMDA receptor function. PKC promotes NMDA receptor trafficking to the cell surface via interaction with NMDAR-associated proteins (NAPs). Here we show that CaMKII is such a NAP that mediates potentiation of NMDAR trafficking by PKC. PKC activation elicits concurrent potentiation in both CaMKII autophosphorylation and association between CaMKII and NMDARs, accompanying by functional NMDAR insertion, at postsynaptic site. This PKC-induced potentiation was abolished by CaMKII antagonist AIP or by selectively disturbing the interaction between CaMKII and NR2A or NR2B with peptide Tat-NR2A and Tat-NR2B. The PKC-tyrosine kinase Src signaling pathway contributed to the potentiation of NMDA channel activity but was not required for PKC-induced NMDAR trafficking. Moreover, Tat-NR2A and Tat-NR2B completely abolished PKC-induced long-term potentiation (LTP) respectively. Further mutual occluding experiments demonstrate that PKC and CaMKII share common signaling pathway in potentiation of NMDAR trafficking and LTP production. Taken together, our present results support the interpretation that PKC promotes NMDA receptor trafficking by indirectly triggering CaMKII autophosphorylation and subsequent increased association with NMDARs, , which appears to be critical for synaptic palsticity..2. Activation of aPKCλis required for AMPA receptors insertion during PI3K-induced LTP.AMPA receptors mediate fast synaptic transmission at a majority of excitatory synapses, and participate in the expression and maintainance of LTP. The number and functional property of AMPA receptor (AMPAR) in postsynaptic sites is widely regarded as a major determinant of synaptic plasticity. Therefore, it is very important for us to understand an underlying mechanism of AMPA receptor trafficking. Using the methods of western blotting, immunoprecipitation, immunofluorescene and electrophysiology, we illustrate the role of PKC in modulating the trafficking of AMPA receptor in synapse. Our results demonstrated that aPKCλ, a specifically atypical PKC, is necessary and sufficient for PI3K-induced LTP. aPKCλmay be activated indirectly by PI3K and promotes GluR1 insertion to postsynaptic membrane. Further studies have shown that P62, as adaptor protein, mediateds the formation of GluR1-P62-aPKCλcomplex, and recruits GluR1 closer to aPKCλ. Then aPKCλphosphorylates GluR1 at ser818 and regulates AMPA receptor trafficking to postsynaptic membrane.更多还原