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.

Unraveling the Molecular Signature of Extracellular Vesicles From Endometrial-Derived Mesenchymal Stem Cells: Potential Modulatory Effects and Therapeutic Applications

“…proteins in ExoCarta database. Since ExoCarta is one of the most reliable database about EVs biomolecules (Rosa-Fernandes et al, 2017) and it has been commonly used by several authors to characterize the proteomic profiles of EVs (Jeannin et al, 2018;Arab et al, 2019;Göran Ronquist, 2019), our results further confirmed the vesicular origin of identified proteins. Surprisingly, the first identified protein was serotransferrin (see Supplementary Table 1).…”

Streptococcus mutans Membrane Vesicles Harboring Glucosyltransferases Augment Candida albicans Biofilm Development

“…A 10 μL suspension of MVs was adhered to formvar/carbon-coated nickel TEM grids, negatively stained for 1 min with 3% uranyl acetate, washed with ddH 2 O and observed by TEM at an acceleration voltage of 80 kV. The size distribution and diameter of MVs were measured by NTA (NanoSight NS300, Malvern, United Kingdom), as previously described ( Arab et al, 2019 ).…”

Molecular Mapping of Hydrogen Sulfide Targets in Normal Human Keratinocytes

“…Epidermal cell sheets were then treated or not for 24 h with 0.25 mM NaHS then lysed using a RLT lysis buffer (Qiagen, Hilden, Germany) for RNA-seq analyses or mechanically detached using a cell scraper for proteomics analyses. RNA-seq and proteomics analyses were performed by the Lyon-1 university genomics platform ProfileXpert and the Lille university proteomics platform PRISM (Proteomics Inflammatory Response Mass Spectrometry, INSERM U1192), respectively, as previously described [ 50 , 51 , 52 , 53 ] and detailed in the Supplementary Data.…”