Proteomic characterisation of leech microglia extracellular vesicles (EVs): comparison between differential ultracentrifugation and Optiprep™ density gradient isolation

Abstract

In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.

Keywords: Hirudo medicinalis; Optiprep™; extracellular vesicles; microglia; neurite outgrowth; protein content; ultracentrifugation.

Figure 1.

Extracellular Vesicles (EVs) collection and isolation strategies. (a) Leech CNS were dissected and mechanically dissociated. Microglia were separated from neurons by filtration and primary cultured. (b) Microglial cell and apoptotic bodies were removed from the conditioned medium by successive centrifugation steps. EVs were isolated from conditioned medium by differential ultracentrifugation (UC samples) or submitted to further density gradient ultracentrifugation step. (c) EVs were collected in different fractions after Optiprep^TM density gradient separation (ODG fractions).

Figure 2.

Nanoparticle Tracking Analysis (NTA) on microglial EVs. (a) Particles from UC samples (red dots) were quantified. Vehicle PBS (black triangles) was used as negative control. (b) Particles counting in ODG fractions F1 to F8 for each replicates (blue, red, and yellow dots) were compared to Optiprep^TM gradient fractions processed in the same way as control (black triangles). Each circle or triangle represents an individual count. Green and grey bars indicate the average for EVs or control fractions, respectively.

Figure 3.

Transmission Electron Microscopy of EVs. (a) Morphology of Optiprep^TM density gradient isolated EVs. Some vesicles are more electron-dense (arrow). (b) EVs purified by simple ultracentrifugation appear flattened. A layer of globular lipids was noticed in the background (arrow heads). (c) EVs aggregates were observed independently of the isolation protocols used.

Figure 4.

Venn’s diagrams of unique and shared proteins identified in UC and ODG samples. (a) Comparison between UC, ODG EV-rich (F4, F5, F6) and ODG EV-poor (F1, F2, F3, F7, F8) fractions. (b) Number of proteins identified in ODG EV-rich fraction F4, F5 and F6. (c) Unique and shared proteins identified in ODG EV-poor fractions F1, F2, F3, F7 and F8. (d) Proteins overlap between fractions (F3, F7) physically close to the EV-rich ones (F4 and F6).

Figure 5.

Gene Ontology (GO) analysis of EVs. Comparison between UC and ODG samples in term of number of genes identified for different GO terms.

Figure 6.

In vitro assays for EVs neurotrophic activity assessment of ODG purified EVs. (a) Outgrowth measurements of leech neurons exposed for 14 days to complete medium (Control, n = 38) or to vesicles from ODG EV-rich fractions (n = 37). Each open circle represents the percentage of neurite outgrowth from individual neurons. Bars indicate the average values for each series of measurements. A significant difference was observed between the two conditions with a p-value of 0.0029. (b) and (c) pictures display an example of neurite outrgrowth recorded for the same neurone at 6 and 20 days of culture with L15 medium alone (Control) or containing 10⁶ EVs/well (ODG EV-rich fractions). Scale bars correspond to 20 µm.