Study of exosomal microRNAs from microglia involved in neuroprotection in Hirudo medicinalis

Background: Unlike vertebrates, the medicinal leech (Hirudo medicinalis) can be lesioned only on axons without any contact on neuronal cell bodies due to the tubular structure of its nerve cord. At this time, the microglial cells migrate to the site of lesion in close contact with damaged axons. Those cells are able to release extracellular vesicles (EVs) to dialogue with neurons. We showed that microglial EVs are massively present in lesioned connectives and in ganglion around the neuronal cell bodies following injury. Taking into account that EVs contain proteins, lipids and nucleic acids (mRNAs and microRNAs) we focused on microRNA populations mediating the microglia-neurons crosstalk for a better understanding of neuroprotection. Methods: The methodology is based on (1) the collection of activated microglia from injured leech nerve cord, (2) the isolation of microglial EVs by a differential centrifugation with a density gradient, (3) the characterization of vesicular microRNAs by NGS (RNAseq) and (4) quantification of microRNAs representation in microglial EVs. Results: The first results show that EVs from microglia contain many identified microRNAs. We start the quantitative study to explore their differential representation in EVs from a primary culture of microglia under early vs. late activated state. The preliminary results show that some microRNAs are more represented in EVs in an early activated state compare to a late activated state. Taking into account these results, we study target mRNAs which could be under the influence of these microRNAs using bio-informatics and we show that numerous mRNAs involved in neuroinflammation pathways (wnt or TGF-) could be regulated by this microRNAs. Summary/Conclusion: The further studies (1) will use fluorescent molecular beacons specific for each miRNA to determine the percentage of positive EV subpopulations and (2) will measure the impact of miRNAs on neuronal survival (neurite outgrowth and neuronal protein signatures) by using synthetics miRNAs (mimics or inhibitors).