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

 描述(申请人提供)：长期以来，树突棘中的局部蛋白质合成一直被认为是为控制脊柱发育和突触可塑性提供突触特有的机制。检验这一假说的一个固有困难是 缺乏合适的方法来直接可视化和量化具有足够空间和时间分辨率的本地翻译。虽然许多mRNA已经通过原位杂交方法定位于树突，但使用荧光记者直接在树突中局部翻译的只有一小部分，而且技术限制往往阻碍了对翻译发生的准确位置以及翻译反应如何在形态背景下受到生理信号调控的可视化。因此，一个关键的差距是缺乏适当的方法来解决关于翻译成脊柱的mRNAs类型的基本问题，并询问关于潜在的调控机制及其在蛋白质分选和脊柱发育中的功能。与这一重大的知识差距有关，一个基本但尚未回答的问题是，膜蛋白是否可以在脊椎内或附近翻译，以及是否存在将合成与运输耦合到细胞表面的机制，以响应可塑性诱导刺激。我们提出的研究的目标是开发和应用一种在微流控设备中使用荧光报告器的单分子成像方法，以可视化和量化树突和棘内的翻译事件。目的1将探讨一种假说，即受体和通道的局部翻译发生在脊椎局部，合成和表面表达机制受可塑性诱导刺激的协调调节。目的2将探讨树突状microRNAs对于膜蛋白在可塑性诱导刺激下的活性调节的局部翻译和表面表达都是必需的假设。我们期待这一技术方法的发展和应用将揭示受体介导的树突棘局部mRNA翻译的新机制，树突棘在蛋白质运输和神经元发育中发挥重要作用。我们预计，这项拟议的研究将带来一个新的视角，即局部翻译如何受到突触机制的时空控制，这些突触机制可能在包括癫痫、脆性X综合征和精神分裂症在内的神经疾病中受损。

 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.