SPECIFIC PROTEOME OF MAMMALIAN CORTEX INHIBITORY & EXCITATORY SYNAPSES

哺乳动物皮层抑制的特定蛋白质组

• 批准号: 8361533
• 负责人: NATHANIEL HEINTZ
• 金额: $ 7.82万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361533
• 关键词: Bacterial Artificial Chromosomes; Biochemical; Biology; Brain; Cell Adhesion Molecules; Cells; Cerebellum; Collaborations; Complement; Complex; Dissection; Electrons; Equilibrium; Excitatory Synapse; Fiber; Funding; GABA Receptor; Grant; Hares; Inhibitory Synapse; Manuscripts; Membrane; Microscopic; Modeling; Modification; Mus; National Center for Research Resources; Nervous system structure; Neurons; Neurotransmitter Receptor; Paper; Population; Principal Investigator; Proteins; Proteome; Proteomics; Publishing; Purkinje Cells; Reporting; Research; Research Infrastructure; Resources; Signal Transduction; Signaling Protein; Source; Synapses; Synaptic plasticity; United States National Institutes of Health; Universities; Work; abstracting; base; computerized data processing; cost; density; in vivo; macromolecule; neural circuit; novel; postsynaptic; presynaptic; scaffold

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Four years ago, in collaboration with Nat Heintz (Rockefeller University), we initiated the study of the protein complement present at excitatory synapses in Purkinje cells. We used the Bacterial Artificial Chromosome (BAC) modification strategy to target the specific in vivo expression of GFP-fused GRID 2 to Purkinje cell's excitatory synapses. We performed dissections of mouse cerebella, and purified synapses bearing GFP-GRID2. Although challenging, our approach proved successful, as we isolated synapses and analyzed low-femtomol levels of proteins. During this last year, we continued our mass spectrometric analyses and identified ~70 synaptic proteins, confirming known excitatory proteins, the absence of inhibitory proteins, and identifying novel signatures of excitatory synapses. We have published a manuscript describing this work (F. Selimi, I. Cristea, E. Heller, B.T. Chait, N. Heintz "Proteomic studies of a single CNS synapse type: the parallel fiber/Purkinje cell synapse" PLoS Biology, 2009 Apr 14;7(4):e83). Using a similar approach to the one described above, we hare currently studying the protein composition of inhibitory synapses by isolating GABA receptors from specific cell populations in the Cortex. A paper describing this work has been prepared for submission. The following is the abstract from this manuscript: Electron microscopic studies of the mammalian brain revealed that there are two major classes of synapses (1). Type 1, excitatory synapses were defined as "asymmetric" based on the electron dense material directly apposed to the post- but not the presynaptic membrane. Biochemical studies of this postsynaptic density (PSD) have established it as a complex signal-processing machine that controls synaptic plasticity (2-5). Type 2, inhibitory synapses were defined as "symmetric" because the PSD is greatly reduced or absent. We report here that symmetric synapses contain a variety of neurotransmitter receptors, neural cell-scaffolding and adhesion molecules, but that they are entirely lacking in cell signaling proteins. This fundamental distinction between the functions of excitatory and inhibitory synapses in the nervous system has far reaching implications for models of synaptic plasticity, rapid adaptations in neural circuits, and longer term homeostatic mechanisms controlling the balance of excitation and inhibition in the mature brain.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 四年前，我们与Nat Heintz(洛克菲勒大学)合作，开始了对浦肯野细胞兴奋性突触中存在的蛋白质补体的研究。我们使用细菌人工染色体(BAC)修饰策略来靶向GFP融合的网格2到浦肯野细胞的兴奋性突触的体内特异性表达。我们对小鼠小脑进行了解剖，并纯化了带有GFP-GRID2的突触。尽管具有挑战性，但我们的方法被证明是成功的，因为我们分离了突触并分析了低水平的Femtomol蛋白质。在过去的一年里，我们继续我们的质谱学分析，鉴定了约70个突触蛋白，证实了已知的兴奋性蛋白，不存在抑制性蛋白，并鉴定了兴奋性突触的新特征。我们已经发表了一份描述这项工作的手稿(F.Slimi，I.Cristea，E.Heller，B.T.Chait，N.Heintz：《单一中枢神经系统突触类型的蛋白质组学研究：平行纤维/浦肯野细胞突触》PLoS Biology，2009年4月14日；7(4)：E83)。 使用与上述方法类似的方法，我们目前正在通过从大脑皮层特定细胞群中分离GABA受体来研究抑制性突触的蛋白质组成。已经编写了一份介绍这项工作的文件，准备提交。 以下是这篇手稿的摘要： 对哺乳动物大脑的电子显微镜研究表明，存在两种主要类型的突触(1)。类型1，兴奋性突触被定义为基于直接与突触后膜相对的电子致密物质而不是突触前膜的“不对称”突触。对这种突触后密度(PSD)的生化研究证实，它是一台控制突触可塑性的复杂信号处理机器(2-5)。类型2，抑制性突触被定义为“对称”，因为PSD大大减少或消失。我们在这里报道，对称性突触包含各种神经递质受体、神经细胞支架和黏附分子，但它们完全缺乏细胞信号蛋白。神经系统中兴奋性和抑制性突触功能的这种根本区别对突触可塑性的模型、神经回路的快速适应以及控制成熟大脑中兴奋和抑制平衡的长期稳态机制具有深远的影响。