Role of MicroRNAs in Activity Regulated Synthesis and Insertion of Membrane Proteins

 描述（由申请人提供）：长期以来一直假设树突棘内的局部蛋白质合成为控制棘发育和突触可塑性提供突触特异性机制。检验这一假设的一个固有困难是， 缺乏合适的方法来直接可视化和量化具有足够的空间和时间分辨率的局部平移。虽然许多mRNA已被定位到树突的原位杂交方法，只有少数已被直接显示为局部翻译的树突使用荧光报告，和技术的限制往往妨碍可视化的精确位点发生翻译和翻译反应是如何调节的生理信号在形态学背景下。因此，一个关键的差距是缺乏合适的方法来解决有关在脊柱中翻译的mRNA类型的基本问题，并询问有关蛋白质分选和脊柱发育中的潜在调控机制及其功能的问题。与这一重大知识差距相关的一个基本但尚未回答的问题是，膜蛋白是否可以在棘中或棘附近翻译，以及是否存在机制将合成与响应于可塑性诱导刺激的细胞表面运输耦合。我们提出的研究目标是开发和应用一种单分子成像方法，在微流体装置中使用荧光报告分子来可视化和量化树突和棘内的翻译事件。目的1将探讨这一假设，即受体和通道的局部翻译发生在本地的脊柱和合成和表面表达的机制是协调调节可塑性诱导刺激。目的2：研究树突状细胞microRNA在可塑性刺激下的活性调节、膜蛋白的局部翻译和表面表达中的作用。我们预计，这种技术方法的开发和应用将揭示受体介导的调节局部mRNA翻译的树突棘，在蛋白质运输和神经元发育中发挥重要作用的新机制。我们预计，拟议中的研究将导致一个新的视角，如何本地翻译是时空控制下的突触机制，可能受损的神经系统疾病，包括癫痫，脆性X综合征和精神分裂症。

 DESCRIPTION (provided by applicant): Local protein synthesis within dendritic spines has long been hypothesized to provide a synapse specific mechanism for the control of spine development and synaptic plasticity. An inherent difficulty in testing this hypothesis has been the lack of suitable methods to directly visualize and quantify local translation with sufficient spatil and temporal resolution. While numerous mRNAs have been localized to dendrites by in situ hybridization methods, only a few have been directly shown to be locally translated in dendrites using fluorescent reporters, and technical limitations often preclude visualization of the precise loci where translation occurs and how translational responses are regulated by physiological signals within a morphologic context. Thus, a critical gap is lack of suitable methods to address fundamental questions about the types of mRNAs that are translated in spines, and to ask questions about the underlying regulatory mechanisms and their function in protein sorting and spine development. Related to this major gap in knowledge, a fundamental but as yet unanswered question is whether membrane proteins can be translated in or near spines, and whether mechanisms exist to couple synthesis with trafficking to the cell surface in response to plasticity inducing stimuli. Our goal for the proposed research is to develop and apply a single molecule imaging approach using fluorescent reporters in microfluidic devices to visualize and quantify translational events within dendrites and spines. Aim 1 will investigate the hypothesis that local translation of receptors and channels occurs locally in spines and that mechanisms of synthesis and surface expression are coordinately regulated by plasticity inducing stimuli. Aim 2 will investigate the hypothesis that dendritic microRNAs are necessary for both the activity regulated local translation and surface expression of membrane proteins in response to plasticity inducing stimuli. We anticipate that development and application of this technological approach will uncover novel mechanisms for receptor-mediated regulation of local mRNA translation in dendritic spines that play important roles in protein trafficking and neuronal development. We anticipate that the proposed research will lead to a new perspective on how local translation is under spatiotemporal control by synaptic mechanisms that are likely impaired in neurologic disorders, including epilepsy, fragile x syndrome and schizophrenia.