MicroRNA networks in synaptic plasticity

描述(申请人提供)：本申请涉及挑战领域06，使能技术；特定挑战主题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、抽动症、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，很大程度上是因为每个microRNA可以有数百个信使核糖核酸靶标，每个靶标可能受到数十个微RNA的调节。这一领域的研究遇到了障碍，因为对microRNA功能的理解存在两个主要差距。首先，是什么决定了哪些microRNAs与特定的mRNA靶标结合在一起，人们对此知之甚少。这个问题一直是无法解决的，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microRNAs调控的信使核糖核酸靶标的群体定义不明确。将microRNA途径的失调与特定的信使核糖核酸靶标联系起来需要这种信息，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，并将应用它来识别与编码p250GAP和LimKI的mRNAs的3‘UTR序列相互作用的microRNAs，这两种蛋白质与树突形成和突触可塑性有关。我们还优化了一种互补的方法来识别两个microRNAs的额外靶点，miR132和miR134，这两个microRNAs被认为调节p250GAP和LimKI在突触的表达。该提案中概述的新方法将对理解microRNAs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，该基因含有一个连接到序列MS2的3‘非编码区探针，该序列可被噬菌体MS2结合蛋白识别。MS2标记的3‘非编码区探针将被引入原代大脑皮层细胞和转基因小鼠中，含有相关microRNAs的复合体将通过亲和层析纯化并通过多重PCR进行鉴定。我们已经确定了这种方法的有效性，现在将利用细胞培养和转基因小鼠模型来表征与p250GAP和LimKI转录本相互作用的microRNAs群体，并确定这些关联是否受到生长因子和神经元活动的影响。然后，我们将通过免疫纯化包含抗原表位标记版本的Ago2的RNA诱导沉默复合体(RISC)来鉴定miR132和miR134的新的mRNA靶点。已经使用各种预测算法识别了这些microRNAs的一些靶标，但大多数仍有待发现。这些研究将使我们能够表征miR132和miR134靶标的全部组成，并确定miR132和miR134是否调控不同的、重叠的或功能相关的一组mRNA靶标。我们预测，大多数miR132和miR134靶点将是不同的，但这些靶点将聚集在参与介导突触可塑性的途径上。与公共卫生相关：该项目描述了一种新的方法来确定哪些microRNAs与p250GAP和LimKI的3‘UTR序列结合，这两种蛋白质被认为可以调节突触脊椎的形成，以响应生长因子和神经元的活动。这种方法对于理解microRNA通路对神经和精神疾病的贡献至关重要。

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.