In vivo investigation of microRNA expression at the mammalian Post synapse by AGO-APP

In the brain, neurons communicate through synapses. In response to various stimuli, synapses undergo diverse forms of plasticity, leading to the remodelling of synapses and persistent alterations in synaptic strength. Synaptic plasticity serves as the fundamental substrate for processes associated with learning and memory. It is now established that local translation, facilitating rapid and spatially confined changes in the expression of numerous synaptic proteins, actively contributes to various facets of synaptic plasticity. MicroRNAs (miRNAs) are small non-coding regulatory elements. miRNAs can engage in base-pairing interactions with complementary mRNA sequences, mostly in the 3′ untranslated region (UTR), thus inducing silencing of the targeted mRNA molecules. Owing to their mode of action, miRNAs have been postulated to play a pivotal role in regulating synaptic plasticity by exerting control over mRNA localization, translation, and stability at post synapses. However, in vivo, evidence supporting such roles for miRNAs in the mammalian brain remains limited. The primary focus of my Ph.D. thesis was largely dedicated to the development of novel experimental approaches aimed at elucidating the localization and activity of miRNAs at the post synapse in the mouse brain. To accomplish this, a novel in vivo technique called Ago-APP (Argonaute Protein Affinity Purification by Peptides) was developed. This technique involves the targeted expression of a small peptide, T6B, derived from the GW182 protein TNRC6B, which allows for the selective purification of miRNAs bound to Argonaute proteins. Based on this peptide, we have defined the experimental conditions to purify AGO-bound, and therefore active, microRNAs from mouse brain tissue with high specificity. Transgenic mouse lines were generated to identify miRNAs enriched at excitatory post synapses in two distinct brain regions: the olfactory bulb and cortex. This involved creating transgenic mice conditionally expressing a tagged T6B peptide alone or fused to PSD95, a protein enriched at excitatory synapses. These transgenic mice were bred with two CreERT2 expressing mouse lines, one where the expression of the recombinase is under the control of Nestin promoter to target at the post-natal stage, the progenitors of the olfactory bulb interneurons, the other where the expression of cre is under the control of the NeuroD6 promoter to target cortical excitatory glutamatergic neurons. In addition, the fusion of the ERT2 sequence with cre restricts the expression of cre to the injection of tamoxifen. Following confirmation of transgene expression patterns using confocal imaging and electron microscopy, miRNA populations were purified from postnatal olfactory bulb interneurons and cortical glutamatergic neurons after transgene induction. This purification was achieved either in whole cells (with T6B alone) or specifically at the postsynaptic level (with PSD95-T6B). we subsequently sequenced these samples using an Illumina sequencing machine (miniSeq) enabling the identification of miRNAs specifically enriched at the post-synapses. This forced us to set 6 a new and original library preparation protocol, as the existing ones were either biased or incompatible with very low-concentration RNA samples. Comparison of miRNA expression patterns between samples expressing T6B alone and PSD95-T6B allowed for the identification of synapse-enriched miRNAs. The functional significance of these enrichments will be further investigated in future studies. KEYWORDS Brain, neurons, neuronal progenitor, synaptic plasticity, post-synapses, synaptic strength, excitatory synapses, local translation, microRNAs, mRNA, Argonaute, Ago-APP, T6B peptide, GW182 protein, TNRC6B, transgenic mouse, olfactory bulb, cortex, PSD95, Cre recombinase, nestin, NeuroD6, FACS