Glia-to-neuron shuttling of miR-146a via extracellular microvesicles modulates synaptotagminI translation in neurons

It is widely accepted that glial cells secrete soluble molecules that mediate homeostatic synaptic plasticity and influence neurotransmission upon injury and inflammation. An additional secretory mechanism has been recently described in glial cells, which may play a role in glia-to-neuron signalling: upon activation, astrocytes and microglia, release circular membrane fragments, known as extracellular vesicles (EVs), which contain several components of donor glia (RNAs, proteins, lipids) and may function as efficient intercellular delivery mechanism. Using miRNA real-time PCR panels, we identified a set of miRNAs differentially expressed in EVs produced by pro-inflammatory vs. pro-regenerative microglia. Among them we found miR-146a, a glial-enriched miRNA, which is altered in brain disorders and targets neuron specific genes. To investigate whether glial EVs may transfer their miR-146a cargo to neurons, cultured hippocampal neurons were transfected with a Renilla Luciferase-based miR-146a sensor and exposed to EVs for 24-72h. By this approach we showed that miR-146a-storing EVs but not EVs produced by astrocytes treated with the anti-miR-146a inhibitor induce a significant increase in neuronal miR-146a levels. This increase was strongly inhibited when neuron-EV interaction was prevented by clocking phosphatidyl serine residues on EVs, a determinant for EV-recognition. Finally, we found that exposure to miR-146a-storing EVs results in decreased immunoreactivity of a validated miR-146a target, i.e. the synaptic vesicle protein synaptotagmin I. Taken together, our data indicate that EVs deliver to neurons biologically active miR-146a, highlighting the capability of glial cells to modulate neuronal gene expression. In order to investigate how glial EVs transfer their miRNA cargo to neurons we took advantage of optical manipulation combined with live imaging. This approach revealed that EVs positioned on the cell body make a stable interaction with neurons, staying attached to the neuronal surface up to 1h. Together with confocal analysis of fixed neurons exposed to EVs for different time points, this observation ruled out the possibility that EVs undergo rapid internalization or full fusion with cell membrane. Further investigation is ongoing to identify by proteomic analysis the surface proteins mediating EVs-neuron interaction and to clarify whether EVs can open a transient pore to transfer their cargo to neurons.