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

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, for example, their small size, their high degree of sequence homology and the fact that they can be confined to cellular sub-compartments like synapses. Moreover, only 40% of the expressed miRNA molecules in a cell are included in RNA Induced Silencing Complexes (RISC) and therefore involved in inhibitory interactions while the rest is silent. Thus, discriminating active from inactive miRNAs is a key problem for functional analyses.

Thus, given the important role of miRNAs in the brain on one hand, and the problems to study their expression and function on the other, it is evident that innovative experimental approaches based on deep understanding of the miRNA pathway are needed. It is also evident that to develop such approaches we need to establish close interactions between specialists in mechanistic and biochemical aspects of miRNA biology and neurobiologists. 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 their efficient immunoprecipitation (AGO-APP). In collaborative work we demonstrated that the AGO-APP approach allows the efficient isolation of active miRNAs directly from brain tissue

In this project we will use unique new tool in two contexts. First, to isolate and analyze active miRNAs in a variety of neurobiological contexts ranging from neural stem cell biology to animal behavior, to understand the impact of this regulatory pathway in brain development and function. Second, we will use T6B to approach mechanistic aspects of the miRNA pathway and gain new insight into the properties and transport of Ago proteins and the RISC complex.

At the level of the biological role of miRNAs we will ask:
1. How do miRNAs/mRNA interactions control neural stem cell determination and neuronal differentiation? Based on in vivo brain electroporation and new transgenic mouse models expressing T6B in a CRE-dependent manner we will study these aspects in both, postnatal olfactory bulb neurogenesis and the developing neocortex.
2. Which miRNAs are expressed at the neuronal synapse and what is their function?
We devised strategies to target T6B specifically to GABAergic and glutamatergic synapses. We will compare the synaptic miRNome to cytosolic miRNAs.
3. How do miRNAs influence complex behaviors?
We will use the T6B system to study miRNA changes in specific neuronal populations activated upon precise behavioral paradigms.

At the level of mechanistic aspects we will use T6B as a tool to investigate:
1. How and when is the RISC complex assembled in neurons? Are preassembled complexes or individual components transported along the axon to the synapse?
2. How do posttranslational modifications of Ago impact on its properties? We have evidence that phosphorylation regulates Ago's interactions with mRNA and will investigate the function of this regulation.