MicroRNA-132 regulation of dendritic growth

描述（由申请人提供）：MicroRNAs有助于关键的大脑功能，并参与多种神经和精神疾病。然而，由于几个原因，具体的microrna如何与这些过程相关尚不清楚。首先，大脑特异性microrna的基因敲除很难产生。其次，预测microRNA靶点仍然极具挑战性。第三，缺乏测量单个细胞中microRNA水平的方法，更重要的是，缺乏将这些水平与特定靶标的调节联系起来的方法。本应用程序的总体目标是确定creb调控的microRNA miR-132如何指导树突生长和可塑性。我们的具体目标是：1)利用条件敲除表征miR-132在脑中的作用。我们已经生成了一种含有带通量的miR-132等位基因的小鼠菌株，并使用表达cre的逆转录病毒载体显示，缺乏miR-132的新生海马神经元在树突生长和分支方面显着减少。这是神经特异性microRNA敲除的第一个例子，我们将使用这些小鼠来检查miR-132在成人神经发生，树突生长以及与活动诱导的形态变化相关的行为中的作用，如空间学习和恐惧条件反射。2)确定肝素结合(Hb)-EGF和CDK抑制剂p21这两个在ago2免疫纯化实验中发现的miR-132靶点是否有助于miR-132对神经元存活、形态和功能的影响。3)确定单个神经元中microRNA表达的变化如何与特定microRNA靶点的表达相关。microRNA功能的测定通常是对大量细胞的平均反应。使用我们实验室开发的一种新型比例microRNA传感器，我们观察到单个神经元在miR-132的表达上存在很大差异。我们现在将询问miR-132水平的这些差异是否与目标mRNA表达的差异有关，首先使用培养的神经元，然后使用体内模型。

DESCRIPTION (provided by applicant): MicroRNAs contribute to critical brain functions and participate in multiple neurological and psychiatric conditions. Exactly how specific microRNAs relate to these processes remains unclear, however, for several reasons. First, genetic knockouts of brain-specific microRNAs have been difficult to generate. Second, predicting microRNA targets remains extraordinarily challenging. Third, methods for measuring microRNA levels in individual cells and, more importantly, linking these levels to the regulation of particuar targets are lacking. The overall objective of this application is to determine how the CREB-regulated microRNA, miR-132, directs dendritic growth and plasticity. Our specific aims are to: 1) Characterize the role of miR-132 in brain using a conditional knockout. We have already generated a mouse strain containing a floxed miR-132 allele and have shown, using a Cre-expressing retroviral vector, that newborn hippocampal neurons lacking miR-132 display a dramatic decrease in dendritic growth and branching. This was the first example of a neural-specific microRNA knockout, and we will use these mice to examine the role of miR-132 in adult neurogenesis, dendritic growth, and behaviors associated with activity-induced morphological changes, such as spatial learning and fear conditioning. 2) Determine whether heparin-binding (Hb)-EGF and the CDK inhibitor, p21, two miR-132 targets identified in Ago2-immunopurification assays, contribute to miR-132 effects on neuronal survival, morphology, and function. 3) Determine how changes in microRNA expression in individual neurons correlates with expression of specific microRNA targets. Assays of microRNA function typically average responses of large populations of cells. Using a new ratiometric microRNA sensor developed in our laboratory, we observed that individual neurons display large differences in the expression of miR-132. We will now ask whether these differences in miR-132 levels are associated with differences in target mRNA expression using, initially, cultured neurons and, subsequently, in vivo models.