Microglia respond to HIV-1 protein Nef expression by releasing distinct extracellular vesicle population

Background: Microglia not only protect the central nervous system against injury or infection but also promote neurodegeneration when activated improperly or serve as HIV-1 cellular reservoirs. We here examined the effect of HIV-1 protein Nef expression on intracellular biogenesis and extracellular...

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Bibliographic Details
Published in:Journal of extracellular vesicles 2018-01, Vol.7, p.197-197
Main Authors: Dominkuš, Pia Pužar, Stenovec, Matjaž, Ferdin, Jana, Sitar, Simona, Bobnar, Saša Trkov, Lasič, Eva, Plemenitaš, Ana, Peterlin, Boris Matija, Žagar, Ema, Kreft, Marko, Lenassi, Metka
Format: Article
Language:English
Subjects:
Biosynthesis
Calcium (intracellular)
CD81 antigen
CD9 antigen
Central nervous system
Dextran
Electron microscopy
Endosomes
Extracellular vesicles
Flow cytometry
HIV
Human immunodeficiency virus
Immunoblotting
Intracellular
Lysosomes
Microglia
Nanoparticles
Nef protein
Neurodegeneration
Organelles
Proteins
Sucrose
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Summary:Background: Microglia not only protect the central nervous system against injury or infection but also promote neurodegeneration when activated improperly or serve as HIV-1 cellular reservoirs. We here examined the effect of HIV-1 protein Nef expression on intracellular biogenesis and extracellular release of vesicles (extracellular vesicles, EVs) from human microglia. Methods: We have studied intracellular and extracellular vesicles in Nefexpressing (transfected or HIV-1 infected) immortalized human microglia by live confocal and electron microscopy, asymmetric-flow fieldflow fractionation connected to detectors, flow cytometry, nanoparticle tracking analysis and immunoblotting of subcellular fractions and EVs. Results: Nef-particles in Nef-expressing microglia comprise large, intracellular Ca2+ concentration-independent, non-directional mobile population, which differs in mobility to dextran-laden or Lysotracker-laden endo-/lysosomes. Nef-particles differ from late endosomes/lysosomes also in terms of abundance, size (area) and protein markers. Importantly, Nef-particles significantly co-localize with organelles immunopositive for tetraspanins CD9 and CD81, likely representing the plasma membrane-derived compartments previously connected to HIV-1 assembly. After release, EVs are in higher concentrations (up to 30x), smaller in size (average root mean square roughness (R-ms) 172 nm), float on sucrose gradient in exosome fractions (positive for flotillin, Tsg101, annexin) and some contain Nef (>2%), when compared to constitutively released EVs (around 5 x 10E7 EVs/10E6 cells; average Rrms 365 nm). Nef is released with flotillin-positive EVs also from HIV-1 infected microglia. Summary/Conclusion: Microglia respond to Nef expression by releasing distinct EV population, likely promoting HIV-1 pathogenesis. This is also the first report to propose that microglial CD9- and CD81-positive plasma membrane-derived compartments are associated with EV biogenesis and Nef release.
ISSN:2001-3078