Subcellular Localization of mRNAs in Mammalian Neurons

描述（申请人提供）：神经元树突是突触后信息的初始解释者和突触前信息的整合者。在过去的几十年里，从体外和体内实验中产生的数据表明，树突的数量和大小随着各种行为和药物操作的增加而增加。最近，mrna被定位在树突中。进一步的数据表明，mrna可以在树突中响应各种类型的调节剂进行局部翻译。我们最近通过监测从转染到分离树突的GFP合成的GFP的荧光，表明树突翻译发生在沿树突长度的固定热点上。此外，这些热点在响应mGluR1激动剂DHPG的翻译速率上存在异质性。大部分热点的响应表现为指数转换速率（EXP），其余热点的响应表现为线性转换速率（LIN）。这些翻译热点的存在表明亚树突对翻译后反应的特异性。我们建议进一步表征这些翻译热点，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例；3)每种类型的热点有哪些翻译机械成分共定位；4)树突脊柱相关蛋白是否优先与任何一种类型的热点共定位；5) mRNA结构的改变是否会改变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（IDAT）免疫检测扩增法，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文库，这些文库与IDAT一起富集（筛选）用于树突蛋白检测，以确定树突腔室中蛋白质的身份，并使用差异筛选程序，在树突功能药理学调节剂治疗后，这些蛋白质的丰度发生变化。这些数据将与已生成的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响应性调节的主要蛋白质谱的潜力。在我们的分析中包括了观察树突定位蛋白翻译后修饰的可能性。这些数据可能会对我们如何思考突触后参与Hebbian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