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Proteomic characterisation of leech microglia extracellular vesicles (EVs): comparison between differential

T Arab1, A Raffo-Romero1, C Van Camp1

  • 1U-1192 - Laboratoire Protéomique, Réponse Inflammatoire et Spectrométrie de Masse - PRISM, Univ. Lille, Inserm, Lille, France.

Journal of Extracellular Vesicles
|May 10, 2019
PubMed

Insights

Leech microglia release extracellular vesicles (EVs) upon central nervous system (CNS) injury. Optimized isolation methods revealed EV-specific proteins and neurotrophic activity, promoting neurite outgrowth in neurons.

Area of Science:

  • Neuroimmunology
  • Cellular Biology
  • Extracellular Vesicles Research

Background:

  • Microglial cells are the primary immune cells of the central nervous system (CNS) in mammals.
  • Microglial activation and migration to lesion sites are observed following CNS injury.
  • Activated microglia release extracellular vesicles (EVs), but their characterization in non-mammalian models is limited.

Purpose of the Study:

  • To investigate microglial activation and extracellular vesicle (EV) release in an annelid (leech) model.
  • To compare the efficacy of two isolation protocols for microglia-derived EVs.
  • To characterize the proteomic content and functional neurotrophic activity of leech microglial EVs.

Main Methods:

  • Primary microglia cultures were established from leeches.
  • Extracellular vesicles (EVs) were isolated using differential ultracentrifugation (UC) and Opti-prep™ Density Gradient (ODG) ultracentrifugation.
  • Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and mass spectrometry-based proteomics were employed for characterization.

Main Results:

  • Both isolation protocols yielded EVs containing EV-specific proteins.
  • The ODG protocol demonstrated superior efficiency in reducing co-isolated protein aggregates and membranous particles.
  • Proteomic analysis identified known EV proteins, and purified EVs promoted neurite outgrowth in primary neurons, indicating neurotrophic activity.

Conclusions:

  • This study provides the first characterization of microglial EV protein content in an annelid model.
  • The Opti-prep™ Density Gradient ultracentrifugation protocol enhances the purity of isolated microglial EVs.
  • Leech microglial EVs possess neurotrophic properties and can stimulate neurite outgrowth, suggesting conserved functions across species.
Keywords:
Hirudo medicinalisOptiprep™extracellular vesiclesmicroglianeurite outgrowthprotein contentultracentrifugation

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