MicroRNA networks in synaptic plasticity

描述（由申请人提供）：本申请涉及挑战领域06，使能技术；具体挑战主题06-MH-103，“神经科学研究的新技术”microrna被认为与精神分裂症、自闭症、图雷特综合征、情绪障碍和脆性X综合征有关。然而，大脑中的MicroRNA网络相当复杂，主要是因为每个MicroRNA可以有数百个mRNA靶标，每个靶标可能由数十个MicroRNA调节。这一领域的研究遇到了障碍，因为在对microRNA功能的理解上存在两个主要差距。首先，是什么决定了哪些microrna与特定的mRNA靶标结合，人们知之甚少。这个问题还无法解决，因为还没有一种经验方法来确定在特定条件下发生哪些特定的microRNA相互作用。其次，受单个microrna调控的mRNA靶点群体定义不清。将microRNA通路的失调与特定mRNA靶点联系起来需要这些信息，而这些信息不能仅通过生物信息学获得。我们已经开发了一种通用的方法来解决第一个问题，并将其应用于鉴定与编码p250GAP和LimKI的mrna的3'UTR序列相互作用的microrna，这两种蛋白质与树突棘的形成和突触可塑性有关。我们还优化了一种互补方法，用于鉴定两种microrna miR132和miR134的其他靶点，这两种microrna已被提出用于调节突触中的p250GAP和LimKI表达。本提案中概述的新方法将对理解microrna如何促进突触可塑性产生重大影响，从而可能导致新的治疗方法。我们开发的方法利用了一个融合基因，该基因包含一个3'UTR探针，连接到MS2序列，该序列被噬菌体MS2结合蛋白识别。将ms2标记的3'UTR探针引入原代皮质细胞和转基因小鼠，通过亲和层析纯化含有相关microrna的复合物，并通过多重PCR进行鉴定。我们已经确定了这种方法的有效性，现在将表征与p250GAP和LimKI转录物相互作用的microrna群体，并使用细胞培养和转基因小鼠模型确定这些关联是否受到生长因子和神经元活性的影响。然后，我们将通过免疫纯化含有抗原表位标记版本Ago2的rna诱导沉默复合物（RISC）来鉴定miR132和miR134的新mRNA靶点。这些microrna的一些靶标已经通过各种预测算法被确定，但大多数仍有待发现。这些研究将使我们能够表征miR132和miR134靶点的完整补体，并确定miR132和miR134是否调节不同的、重叠的或功能相关的mRNA靶点集。我们预测，大多数miR132和miR134靶点将是不同的，但这些靶点将会聚在介导突触可塑性的通路上。公共卫生相关性：该项目描述了一种新方法，用于确定哪些microrna与p250GAP和LimKI的3'UTR序列结合，这两种蛋白质已被提出在响应生长因子和神经元活动时调节突触棘的形成。这种方法对于理解microRNA通路在神经和精神疾病中的作用至关重要。

DESCRIPTION (provided by applicant): This application addresses Challenge Area 06, Enabling technologies; Specific challenge topic 06-MH-103, "New technologies for neuroscience research" MicroRNAs are believed to contribute to schizophrenia, autism, Tourette's syndrome, mood disorders, and fragile X syndrome. MicroRNA networks in brain are quite complex, however, largely because each microRNA can have hundreds of mRNA targets and each target can, potentially, be regulated by dozens of microRNAs. Research in this area has hit a roadblock because of two major gaps in the understanding of microRNA function. First, what determines which microRNAs bind to a specific mRNA target is very poorly understood. This problem has not been approachable because there has not been an empirical way to determine which particular microRNA interactions occur under specific conditions. Second, the populations of mRNA targets regulated by individual microRNAs are poorly defined. Linking the dysregulation of a microRNA pathway to a particular mRNA target requires this information, which cannot be obtained through bioinformatics alone. We have developed a general method to solve the first problem and will apply it to identify microRNAs that interact with the 3'UTR sequences of mRNAs encoding p250GAP and LimKI, two proteins that have been linked to dendritic spine formation and synaptic plasticity. We have also optimized a complementary method for identifying additional targets of the two microRNAs, miR132 and miR134, that have been proposed to regulate p250GAP and LimKI expression at the synapse. The novel methods outlined in this proposal will have a major impact on the understanding of how microRNAs contribute to synaptic plasticity that could lead to new therapeutic approaches. The approach that we have developed utilizes a fusion gene containing a 3'UTR probe linked to the sequence, MS2, which is recognized by the bacteriophage MS2 binding protein. The MS2-tagged 3'UTR probes will be introduced into primary cortical cells and transgenic mice, and complexes containing the associated microRNAs will be purified by affinity chromatography and identified by multiplex PCR. We have already established the efficacy of this approach and will now characterize the populations of microRNAs interacting with the p250GAP and LimKI transcripts and determine whether these associations are influenced by growth factors and neuronal activity using cell culture and transgenic mouse models. We will then identify new mRNA targets of miR132 and miR134 by immunopurifying RNA-induced silencing complexes (RISC) containing an epitope-tagged version of Ago2. A few targets of these microRNAs have been identified using various prediction algorithms, but most remain to be discovered. These studies will allow us to characterize the full complement of miR132 and miR134 targets and determine whether miR132 and miR134 regulate distinct, overlapping, or functionally related sets of mRNA targets. We predict that the majority of miR132 and miR134 targets will be distinct, but that these targets will converge on pathways involved in mediating synaptic plasticity. PUBLIC HEALTH RELEVANCE: This project describes a novel method for determining which microRNAs bind to the 3'UTR sequences of p250GAP and LimKI, two proteins that have been proposed to regulate the formation of synaptic spines in response to growth factors and neuronal activity. This method is critical for understanding the contribution of microRNA pathways to neurological and psychiatric disease.