Identification of functional microRNA/mRNA complexes in the mouse brain: From neurogenesis to behavior and pathology

microRNAs （miRNAs）是大脑发育和功能的关键调节因子，控制着神经干细胞的决定、增殖、迁移和整合等过程。此外，突触的稳定性和可塑性受mirna和靶mrna之间的局部相互作用的调节。miRNA的表达和功能分析在技术上要求很高，因为它们体积小，序列同源性高，而且它们可以被限制在细胞间室（如突触）中。此外，并非所有的mirna都可能存在于功能复合物中。因此，区分活性和非活性mirna是功能分析的关键问题。因此，考虑到mirna在大脑中的重要作用以及研究其表达和功能的困难，显然需要创新的实验方法。为了开发富有成效的新方法，miRNA生物学的机械/生化方面的专家和神经生物学家之间的跨学科互动是必要的。在这个联盟中，由H. Cremer和E. Gascon领导的法国两个神经生物学研究小组与德国G. Meister的生物化学实验室联合，开发并应用新的工具和技术来研究大脑中miRNA-mRNA的相互作用。我们鉴定并表征了一种来自risc复合物蛋白TNRC6B的小肽，称为T6B，它以高亲和力结合所有已知的Argonaute （Ago）蛋白，并允许其有效的免疫沉淀（Ago - app）。在这个项目中，我们将在两个方向上使用这个独特的新工具。首先，分离和分析从神经干细胞生物学到动物行为等各种神经生物学背景下的活性mirna。其次，我们将使用T6B来研究miRNA通路的机制方面，并对Ago蛋白复合物的性质和运输获得新的见解。为了评估mirna在小鼠脑中的生物学作用，我们将使用活体脑电穿孔和以cre依赖方式表达T6B的新型转基因小鼠模型。我们将使用特定策略将T6B靶向gaba能和谷氨酸能突触，并将突触mirna与细胞质mirna进行比较。
此外，我们将使用T6B系统来研究复杂行为中受影响的特定神经元群体中的miRNA变化。最后，我们将研究miRNA复合物是如何运输的，以及它们的组成和修饰如何在不同的细胞区室中发生变化。

microRNAs (miRNAs) are key regulators of brain development and function, controlling processes like neural stem cell determination, proliferation, migration and integration. Moreover, synapse stability and plasticity are regulated by local interactions between miRNAs and target mRNAs.Analysis of miRNA expression and function is technically demanding due to their small size, their high degree of sequence homology and the fact that they can be confined to cellular compartments like synapses. Moreover, not all miRNAs might reside in functional complexes. Thus, discriminating active from inactive miRNAs is a key problem for functional analyses.Thus, given the important role of miRNAs in the brain and the difficulties to study their expression and function, it is evident that innovative experimental approaches are needed. To develop fruitful novel approaches, interdisciplinary interactions between specialists in mechanistic/biochemical aspects of miRNA biology and neurobiologists are necessary. In this consortium, two neurobiological research groups in France, led by H. Cremer and E. Gascon, united with the biochemistry laboratory of G. Meister in Germany to generate and apply new tools and technologies to investigate miRNA-mRNA interactions in the brain.We identified and characterized a small peptide derived from the RISC-complex protein TNRC6B, called T6B, which binds all known Argonaute (Ago) proteins with high affinity and allows for their efficient immunoprecipitation (AGO-APP). In this project we will use this unique new tool in two directions. First, to isolate and analyze active miRNAs in a variety of neurobiological contexts ranging from neural stem cell biology to animal behavior. Second, we will use T6B to approach mechanistic aspects of the miRNA pathway and gain novel insight into the properties and transport of Ago protein complexes.To assess the biological role of miRNAs in the mouse brain, we will use in vivo brain electroporation and new transgenic mouse models expressing T6B in a CRE-dependent manner. We will use strategies to target T6B specifically to GABAergic and glutamatergic synapses and compare the synaptic miRNome to cytosolic miRNAs.
 In addition, we will use the T6B system to study miRNA changes in specific neuronal populations affected during complex behaviors. Finally, we will investigate how miRNA complexes are transported and how their composition and modification changes in different cellular compartments.