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
• microglia
• extracellular vesicles
• protein content
• ultracentrifugation
• Optiprep™
• neurite outgrowth

Author’s contributions

Conceived and designed the experiments: TA, CL, JV and PES. Leech dissection: TA, ARR, QL and CVC. Proteomic sampling and NTA measurement: TA. Leech databases annotation: IG, HT. Proteomic data analyses TA and SA. Electron microscopy: ASL and CS. Funding and mass spectrometry facilities: MS and IF. Manuscript redaction: TA, JV and PES. Manuscript editing: TA, FLM, CL, FD, JV and PES. All authors have given approval to the final version of the manuscript.

Acknowledgments

TA received a PhD grant from the French Research Minister delivered by Lille University for three years. Authors would like to thank specially Dr Morvane Colin, Dr Etienne Audinat and Dr Guillaume van Niel for their precious advices, Loic Brunet for TEM observation. The TEM observations were performed on a device of the BICeL-Campus CS core facility acquired through a funding from the Agence Nationale de la Recherche, ANR (10-EQPX-04-01). The Bilille platform is affiliated to the French Institute for Bioinformatics and France Genomique. BlastP computations were performed on the HPC cloud of Bilille, supported by University of Lille.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

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Additional information

Funding

This work was supported by the Ministère de l’Enseignement Supérieur, de la Recherche Scientifique et des Technologies de l’Information et de la Communication.