Dendritic Protein Synthesis in Hippocampal Neurons

描述（由申请人提供）：现在很清楚，蛋白质合成是动物建立长期记忆所必需的。突触传递和蛋白质合成的失调在许多疾病中起重要作用。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。在神经元突触基部发现多聚核糖体，提示了树突中合成蛋白质以响应突触活动的可能性。在上一个资助期，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。在这个建议中，我们将研究耦合微型突触传递（minis）的蛋白质翻译机制的信号机制。我们以前发现minis紧张性抑制树突状蛋白合成机制。minis的丢失导致翻译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偶联。我们还将确定是否有刺激依赖的装配和运输的树突中的核糖体。先前对海马神经元树突中核糖体的观察表明，棘中突触的翻译能力受到树突池中核糖体数量的限制。利用生物化学方法和动态延时成像，我们将研究多聚核糖体是否可能在脊椎中局部组装或贩运到脊椎。其中一个尚未回答的大问题是关于体细胞与树突合成的蛋白质对突触功能和可塑性的相对贡献。最近，我们已经开发出一种技术，可用于确定的成分的树突蛋白质组。在此技术的基础上，我们将修改我们的程序标记蛋白质裂解物，包括荧光标记的蛋白质在完整的细胞和组织切片。我们将开发多个荧光标记来分别追踪在细胞体和树突中合成的蛋白质。随着时间的推移，将分析在这两个隔室中合成的蛋白质的命运，以解决体细胞与树突蛋白质合成对突触蛋白质群体的贡献比例，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.