Dendritic Protein Synthesis in Hippocampal Neurons

描述(申请人提供)：现在很清楚，动物建立长期记忆需要蛋白质合成。突触传递和蛋白质合成的失调在许多疾病中起着重要作用。直到最近，人们一直认为所有神经元功能所需的所有蛋白质都是在细胞体中制造的。在神经元突触底部发现的多核糖体表明，蛋白质可能是在树突中合成的，以响应突触的活动。在之前的资助期间，我们专注于开发成像技术来可视化树突中的蛋白质合成，并发现了几种通过局部蛋白质合成实现的可塑性形式。在这项提案中，我们将研究将微型突触传递(MINI)耦合到蛋白质翻译机制的信号机制。我们之前发现，Minis能够抑制树突状蛋白质的合成机制。迷你基因的缺失会导致翻译上调和快速的内环境平衡反应。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联系起来。我们还将确定树突中是否存在依赖刺激的核糖体组装和运输。以前对海马神经元树突中核糖体的观察表明，突触在脊椎中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局部组装成脊椎或被贩运到脊椎。一个悬而未决的大问题是关于躯体合成的蛋白质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，我们开发了一种技术，可以用来鉴定树突状蛋白质组的成分。在这项技术的基础上，我们将修改我们在裂解物中标记蛋白质的程序，以包括对完整细胞和组织切片中的蛋白质进行荧光标记。我们将开发多个荧光标记来分别跟踪在细胞体和树突中合成的蛋白质。将随着时间的推移分析在这两个隔室中合成的蛋白质的命运，以解决体细胞蛋白质合成与树突蛋白质合成对突触蛋白质群体的贡献，以及这些贡献如何随着突触活性和可塑性的变化而变化。

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.