Subcellular Localization of mRNAs in Mammalian Neurons

描述(申请人提供)：神经元树突是最初的突触后解释和整合突触前信息。在过去的几十年里，来自体外和体内实验的数据表明，树突的数量和大小随着各种行为和药物操作的反应而增加。最近，mRNAs被定位在树突中。进一步的数据表明，mRNAs可以在树突中局部翻译，对各种类型的调节剂做出反应。最近，我们通过监测从GFP合成的GFP的荧光发现，树突的平移发生在沿着树突长度的不动热点上。GFP是从GFP转染到分离的树突中的。此外，这些热点对mGluR1激动剂DHPG的翻译速率是不同的。大多数热点呈现指数翻译率(EXP)，而其余热点的响应是线性的(LIN)。这些翻译热点的存在表明了翻译后反应的树突下特异性。我们建议进一步确定这些翻译热点的特征，以确定1)EXP热点是否总是以EXP方式响应，或者它们是否也表现出LIN翻译速率，反之亦然；2)已知的树突翻译调节器是否改变了线性指数翻译热点的比例3)什么翻译机械成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何一种热点共定位，最后，5)mRNA结构的变化是否改变了EXP和LIN热点的分布或数量。此外，我们还将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来确定树突的蛋白质图谱，该方法名为免疫检测T7 RNA聚合酶扩增(IDAT)。我们建议利用两个不同的噬菌体展示文库，结合IDAT来检测树突状蛋白，以确定树突室中蛋白质的同一性，并使用差异筛选程序，在树突状功能的药物调节剂处理后，这些蛋白质的丰度的变化。这些数据将与已产生的mRNA表达谱进行比较，以区分体细胞或树突合成的蛋白质对树突调节反应的主要蛋白质谱做出贡献的可能性。我们的分析包括寻找树突定位蛋白的翻译后修饰的可能性。这些数据可能会对我们如何看待突触后参与调节Hebbian突触的方式产生重大影响

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