MECHANISMS OF MRNA TRANSPORT INTO NEURONAL DENDRITES

描述（来自申请人的摘要）：神经元接收大量的各种各样的神经元信号。 这些输入通常在其细胞外的离散结构域中分离， 面这种分布的不均匀性反映在数量上， 这些蛋白质的目标是细胞周围的不同突触。 突触的异质性也反映在不同的分布， 选择的mRNA被运输到树突中。有人认为， 位于树突内的mRNA在突触可塑性中起作用。的 本提案中所述实验的目的是为以下方面提供支持： 以下一般假设：局部电信号影响 通过顺式作用mRNA在神经元树突中的分布和利用 序列，这种控制水平需要提供一种适应 改变电刺激的模式和水平。这 该提案将利用浦肯野的独特属性， 细胞特异性mRNA，Pcp-2（L7），和小脑系统的研究， 树突状路由的mRNAs的分子和细胞机制。一个结合在 体内（转基因小鼠）和体外（将标记的mRNA直接注射到 小脑切片）方法将用于绘制RNA路由信号（Aim 1）。在 此外，动态方面的mRNA运输和影响，由于电 将检查mRNA路由、定位和利用的活性， 这些研究将通过一些新的分析手段来促进 合成的Pcp-2（L7）mRNA（Aim 2）。测试的功能相关性 树突状mRNA是编码Pcp-2（L7）蛋白但不编码Pcp-2（L7）蛋白的mRNA的变体形式。 将被路由到树突的mRNA与行为神经元中正常路由的mRNA进行比较。 在Pcp-2（L7）无效突变小鼠中的拯救实验（Aim 3）。因此，分子 目标1中的分析将提供工具来研究 Aim 3中的mRNA路线以及用于研究的测定（直接mRNA注射） 影响Aim 2中树突内定位的细胞外信号。 同样，在L7细胞中建立L7 mRNA和蛋白的诱导表达， 目标3中的零突变背景将使一个额外的工具能够用于 研究对Aim 2新合成的mRNA和蛋白质的刺激作用。

DESCRIPTION (from applicant's abstract): Neurons receive an enormous variety of inputs that are often segregated in discrete domains on their extracellular surface. This heterogeneity of distribution is reflected in the quantities and kinds of proteins that are targeted to different synapses around the cell. Synaptic heterogeneity is also reflected in the differential distributions of selected mRNAs that are transported into dendrites. It is thought that some mRNAs localized within dendrites play a role in synaptic plasticity. The purpose of the experiments described in this proposal is to provide support for the following general hypothesis: Local electrical signals affect the distribution and utilization of mRNAs in neuronal dendrites via cis-acting mRNA sequences, and this level of control is required to provide a means of adapting the cell to changing patterns and levels of electrical stimulation. This proposal will take advantage of the unique properties of a Purkinje cell-specific mRNA, Pcp-2(L7), and of the cerebellar system to investigate the molecular and cellular mechanisms of dendritic routing of mRNAs. A combined in vivo (transgenic mice) and in vitro (direct injection of labeled mRNAs in cerebellar slices) approach will be used to map RNA routing signals (Aim 1). In addition, dynamic aspects of mRNA trafficking and effects due to electrical activity on mRNA routing, localization and utilization will be examined, and these studies will be facilitated by some novel means of analyzing newly synthesized Pcp-2(L7) mRNA (Aim 2). To test the functional relevance of dendritic mRNA a variant form of mRNA that encodes Pcp-2(L7) protein but is not routed into dendrites will be compared to a normally routed mRNA in behavioral rescue experiments in Pcp-2(L7) null mutant mice (Aim 3). Thus, the molecular analysis in Aim 1 will provide tools to study the functional significance of mRNA routing in Aim 3 as well as an assay (direct mRNA injection) for studying extracellular signals that influence intradendritic localization in Aim 2. Likewise, establishment of inducible expression of L7 mRNA and protein in an L7 null mutant background in Aim 3 will enable an additional tool to be used to study stimulatory effects on newly synthesized mRNA and protein in Aim 2.