Isolation of microglia-derived extracellular vesicles: towards miRNA signatures and neuroprotection

Abstract

The functional preservation of the central nervous system (CNS) is based on the neuronal plasticity and survival. In this context, the neuroinflammatory state plays a key role and involves the microglial cells, the CNS-resident macrophages. In order to better understand the microglial contribution to the neuroprotection, microglia-derived extracellular vesicles (EVs) were isolated and molecularly characterized to be then studied in neurite outgrowth assays. The EVs, mainly composed of exosomes and microparticles, are an important cell-to-cell communication process as they exhibit different types of mediators (proteins, lipids, nucleic acids) to recipient cells. The medicinal leech CNS was initially used as an interesting model of microglia/neuron crosstalk due to their easy collection for primary cultures. After the microglia-derived EV isolation following successive methods, we developed their large-scale and non-targeted proteomic analysis to (i) detect as many EV protein markers as possible, (ii) better understand the biologically active proteins in EVs and (iii) evaluate the resulting protein signatures in EV-activated neurons. The EV functional properties were also evaluated in neurite outgrowth assays on rat primary neurons and the RNAseq analysis of the microglia-derived EVs was performed to propose the most representative miRNAs in microglia-derived EVs. This strategy allowed validating the EV isolation, identify major biological pathways in EVs and corroborate the regenerative process in EV-activated neurons. In parallel, six different miRNAs were originally identified in microglia-derived EVs including 3 which were only known in plants until now. The analysis of the neuronal proteins under the microglial EV activation suggested possible miRNA-dependent regulation mechanisms. Taken together, this combination of methodologies showed the leech microglial EVs as neuroprotective cargos across species and contributed to propose original EV-associated miRNAs whose functions will have to be evaluated in the EV-dependent dialog between microglia and neurons.

Emerging Roles of Extracellular Vesicles in the Central Nervous System: Physiology, Pathology, and Therapeutic Perspectives

“…Hence, Prada et al (2018) found miR-146a, a miRNA associated to several inflammation-related diseases, to be enriched in microglia-derived EVs. This finding was later confirmed by a study of Lemaire et al (2019) . As CNS sentinels, microglia are also responsible for the overall homeostasis of the brain: they are able to sense extracellular ATP, which may be the result of damaged cells, and to react to these high ATP levels by shedding EVs containing pro-inflammatory factors, like IL-1β, thus enhancing neuroinflammation ( Bianco et al, 2005 ).…”

Glia-Derived Extracellular Vesicles: Role in Central Nervous System Communication in Health and Disease

“…Interestingly, leech microglia EVs have been described to trigger a significant increase of rat PC12 cell differentiation, suggesting the presence of common molecular mediators and the evolutionary conservation of EV-mediated communication systems (Raffo-Romero et al, 2018). In addition, a proteomic analysis and studies on miRNA signatures of EVs released from leech microglia further support the presence of conserved neuroprotective cargo throughout the evolution (Arab et al, 2019;Lemaire et al, 2019).…”

Microglial Extracellular Vesicles as Vehicles for Neurodegeneration Spreading

“…Exosomes derived from microglia upon toxic exposure to alcohol promote C1q mediated neuronal cell death [ 154 ]. Interestingly, a recent report demonstrated that the glia–neuron communication mediated by EVs is maintained even between species [ 155 ]. The authors isolated exosomes from leach microglial cells and observed an increase in neurite outgrowth in rat primary neurons [ 155 ].…”