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

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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.