Subcellular Localization of mRNAs in Mammalian Neurons

描述（由申请人提供）：神经元树突是突触前信息的初始突触后解释者和整合者。在过去的几十年中，已经从体外和体内实验中产生的数据表明，树突的数量和大小增加，以响应各种行为和药理学操作。最近，mRNA被定位在树突中。进一步的数据显示，mRNA可以在树突中响应于各种类型的调节剂而局部翻译。我们最近已经表明，树突翻译发生在固定热点沿着树突的长度，通过监测荧光从绿色荧光蛋白合成，从绿色荧光蛋白已转染到分离的树突。此外，这些热点在响应于DHPG（mGluR1激动剂）的翻译速率中是异质的。大多数热点在其响应中表现出指数翻译速率（EXP），而其余热点是线性的（LIN）。这些翻译热点的存在表明，亚树突特异性翻译后反应。我们建议进一步表征这些翻译热点以确定1）EXP热点总是以EXP方式响应还是它们也可以表现出LIN翻译速率，反之亦然，2）已知的树突翻译调节剂是否改变线性指数翻译热点的比率，3）什么翻译机制组分与每种类型的热点共定位，4）树突棘相关蛋白是否优先与任一类型的热点共定位，以及5）mRNA结构的变化是否改变EXP和LIN热点的分布或数量。此外，我们将更进一步，使用我们实验室最近开发的一种新的蛋白质组学方法，称为T7 RNA聚合酶扩增的免疫检测（IDAT），确定树突的蛋白质谱。我们建议利用两种不同的噬菌体展示库，富集（淘选）树突状蛋白检测结合IDAT确定的身份的蛋白质在树突状隔室，并使用差异筛选程序，这些蛋白质的丰度与树突状功能的药理学调节剂治疗后的变化。将这些数据与已经生成的mRNA表达谱进行比较，以区分体细胞或树突合成蛋白质对树突对调节的响应性的主要蛋白质谱的贡献的潜力。包括在我们的分析是在翻译后修饰的树突状定位蛋白质的可能性。这些数据可能会对我们如何看待突触后参与赫布突触的调节产生重大影响

DESCRIPTION (provided by applicant): Neuronal dendrites are the initial post-synaptic interpreters and integrators of presynapfic information. Over the last several decades, data has been generated from both in vitro and in vivo experiments that show that dendrites increase in number and size in response to various behavioral and pharmacological manipulations. More recently mRNAs have been localized in dendrites. Further data shows that mRNAs can be translated locally, in the dendrite, in response to various types of modulators. We have recently shown that dendritic translation occurs in immobile hotspots along the length of the dendrite through the monitoring of fluorescence from GFP that was synthesized from GFP that had been transfected into isolated dendrites. Further these hotspots were heterogeneous in their translational rate in response to DHPG, a mGluR1 agonist. Most of the hotspots exhibited exponential translation rates (EXP) while the remaining hotspots were linear (LIN) in their response. The existence of these translational hotspots suggests a subdendritic specificity to post-translational responsiveness. We propose to further characterize these translational hotspots to determine 1) do the EXP hotspots always respond in an EXP manner or can they also exhibit LIN translation rates and vice-versa, 2) whether known modulators of dendritic translation alter the ratio of linear exponential translation hotspots 3) what translational machinery component co-localize with each type of hotspot, 4) do dendritic spine associated proteins colocalize with either type of hotspot preferentially and finally, 5) do changes in mRNA structure alter the distribution or number of EXP and LIN hotspots. In addition, we will go one step further and determine the protein profile of dendrites using a novel proteomics method recently developed in our lab called Immuno-Detectlon Amplified by T7 RNA Polymerase (IDAT). We propose to utilize two different phage display libraries that are enriched (panned) for dendritic protein detection in conjunction with IDAT to determine the identity of proteins in the dendritic compartment and, using a differential screening procedure, changes in the abundance of these proteins after treatment with pharmacological modulators of dendritic function. These data will be compared with the mRNA expression profiles that have been generated to distinguish between the potential for somatic or dendritically synthesized proteins to contribute to the predominant protein profile of dendritic responsiveness to modulation. Included in our analysis is the possibility of looking at post-translational modification of dendritically localized proteins. These data will likely have significant impact upon how we think about post-synaptic involvement in regulation of the Hebbian synapse