Dendritic Protein Synthesis in Hippocampal Neurons

描述（由申请人提供）：现在很清楚，动物需要蛋白质合成来建立长期记忆。突触传递和蛋白质合成的错误调控在许多疾病中起着重要作用。直到最近，人们还认为所有神经元功能所需的所有蛋白质都是在细胞体中产生的。在神经元突触底部发现多核糖体表明，蛋白质可能在树突中合成以响应突触活动。在之前的资助期间，我们将注意力集中在成像技术的发展上，以可视化树突中的蛋白质合成，并发现了几种由局部蛋白质合成实现的可塑性形式。在本研究中，我们将研究微型突触传递（minis）与蛋白质翻译机制之间的信号机制。我们以前发现，小分子抑制性抑制树突蛋白的合成机制。miniis的缺失导致翻译的上调和快速的稳态反应。我们希望研究哪些细胞内信号通路将神经递质受体激活与蛋白质合成机制相结合。我们还将确定树突中是否存在刺激依赖性核糖体的组装和贩运。先前对海马神经元树突核糖体的观察表明，棘突触的翻译能力受到树突池中可用核糖体数量的限制。使用生化方法和动态延时成像，我们将检查多核糖体是否可能在脊柱中局部组装或运输到脊柱。一个悬而未决的大问题是，体细胞合成蛋白和树突合成蛋白对突触功能和可塑性的相对贡献。最近我们开发了一种技术，可以用来鉴定树突蛋白质组的成分。基于这项技术，我们将修改裂解物中蛋白质标记的程序，以包括完整细胞和组织切片中蛋白质的荧光标记。我们将开发多种荧光标记来分别跟踪细胞体和树突中产生的蛋白质。在这两个区室中合成的蛋白质的命运将随着时间的推移进行分析，以解决体细胞和树突蛋白合成对突触蛋白种群的部分贡献，以及这些贡献如何随着突触活性和可塑性而变化。

DESCRIPTION (provided by applicant): It is now clear that protein synthesis is required for animals to establish long-term memories. Misregulation of synaptic tranmission and protein synthesis plays an important role in many diseases. Until recently, it was assumed that all of the proteins required for all neuronal function were made in the cell body. The discovery of polyribosomes at the base of neuronal synapses suggested the possibility that proteins might be synthesized in dendrites in response to synaptic activity. In the previous grant period, we focussed our attention on the development of imaging techniques to visualize protein synthesis in dendrites and discovered several forms of plasticity that are implemented by local protein synthesis. In this proposal we will examine the signaling mechanisms that couple miniature synaptic transmission (minis) to the protein translation machinery. We previously discovered that minis tonically inhibit the dendritic protein synthesis machinery. Loss of minis leads to an upregulation of translation and a rapid homeostatic response. We wish to examine which intracellular signaling pathways couple neurotransmitter receptor activation to the protein synthesis machinery. We will also determine whether there is stimulation-dependent assembly and trafficking of ribosomes in dendrites. Previous observations of ribosomes in dendrites of hippocampal neurons suggest that the translational capacity of synapses in spines is limited by the number of ribosomes available in the dendritic pool. Using biochemical approaches and dynamic time-lapse imaging, we will examine whether polyribosomes might be assembled locally in spines or trafficked to spines. One of big unanswered questions concerns the relative contributions of somatically vs. dendritic synthesized proteins to synaptic function and plasticity. Recently we have developed a technique that can be used to identify the constituents of the dendritically proteome. Building on this technology, we will modify our procedure for labelling proteins in lysates to include fluorescent labelling of proteins in intact cells and tissue slices. We will develop multiple fluorescent tags to separately track proteins made in the cell body and the dendrites.The fate of proteins synthesized in these two compartments will be analyzed over time to address the fractional contribution of somatic vs. dendritic protein synthesis to the synaptic protein population and how these contributions change with synaptic activity and plasticity.