Astrocytes shed large membrane vesicles that contain mitochondria, lipid droplets and ATP

Abstract

Various cells types, including stem and progenitor cells, can exchange complex information via plasma membrane-derived vesicles, which can carry signals both in their limiting membrane and lumen. Astrocytes, traditionally regarded as mere supportive cells, play previously unrecognized functions in neuronal modulation and are capable of releasing signalling molecules of different functional significance. In the present study, we provide direct evidence that human fetal astrocytes in culture, expressing the same feature as immature and reactive astrocytes, release membrane vesicles larger than the microvesicles described up to now. We found that these large vesicles, ranging from 1–5 to 8 μm in diameter and expressing on their surface β1-integrin proteins, contain mitochondria and lipid droplets together with ATP. We documented vesicle content with fluorescent-specific dyes and with the immunocytochemistry technique we confirmed that mitochondria and lipid droplets were co-localized in the same vesicle. Scanning electron microscopy and transmission electron microscopy confirmed that astrocytes shed from surface membrane vesicles of the same size as the ones detected by fluorescence microscopy. Our results report for the first time that cultured astrocytes, activated by repetitive stimulation of ATP released from neighboring cells, shed from their surface large membrane vesicles containing mitochondria and lipid droplets.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Agnati LF, Guidolin D, Guescini M, Genedani S, Fuxe K (2010) Understanding wiring and volume transmission. Brain Res Rev 64:137–159

    PubMed  Article  Google Scholar 

  2. Bergersen LH, Gundersen V (2009) Morphological evidence for vesicular glutamate release from astrocytes. Neuroscience 158:260–265

    PubMed  Article  CAS  Google Scholar 

  3. Bianco F, Pravettoni E, Colombo A, Schenk U, Moller T, Matteoli M, Verderio C (2005) Astrocyte-derived ATP induces vesicle shedding and IL-1β release from microglia. J Immunol 174:7268–7277

    PubMed  CAS  Google Scholar 

  4. Bianco F, Perrotta C, Novellino L, Francolini M, Riganti L, Menna E, Saglietti L, Schuman EH, Furlan R, Clementi E, Matteoli M, Verderio C (2009) Acid spingomyelinase activity triggers microparticle release from glial cells. EMBO J 28:1043–1054

    PubMed  Article  CAS  Google Scholar 

  5. Bodin P, Burnstock G (2001) Evidence that release of adenosine triphosphate from endothelial cells during increased shear stress is vesicular. J Cardiovasc Pharmacol 38:900–908

    PubMed  Article  CAS  Google Scholar 

  6. Cabras S, Saba F, Reali C, Scorciapino ML, Sirigu A, Talani G, Biggio G, Sogos V (2010) Antidepressant imipramine induces human astrocytes to differentiate into cells with neuronal phenotype. Int J Neuropsychopharmacol 31:1–13

    Google Scholar 

  7. Coco S, Calegari F, Pravettoni E, Pozzi D, Taverna E, Rosa P, Matteoli M, Verderio C (2003) Storage and release of ATP from astrocytes in culture. J Biol Chem 278:1354–1362

    PubMed  Article  CAS  Google Scholar 

  8. Cocucci E, Racchetti G, Podini P, Meldolesi J (2007) Enlargeosome traffic: exocytosis triggered by various signals is followed by endocytosis, membrane shedding or both. Traffic 8:742–757

    PubMed  Article  CAS  Google Scholar 

  9. Cocucci E, Racchetti G, Meldolesi J (2009) Shedding microvesicles: artefacts no more. Trends Cell Biol 19:43–51

    PubMed  Article  CAS  Google Scholar 

  10. Ducharme NA, Bickel PE (2008) Lipid droplets in lipogenesis and lipolysis. Endocrinology 149:942–949

    PubMed  Article  CAS  Google Scholar 

  11. Falchi AM, Battetta B, Sanna F, Piludu M, Sogos V, Serra M, Melis M, Putzolu M, Diaz G (2007) Intracellular cholesterol changes induced by translocator protein (18 kDa) TSPO/PBR ligands. Neuropharmacology 53:318–329

    PubMed  Article  CAS  Google Scholar 

  12. Faurè J, Lachenal G, Court M et al (2006) Exosomes are resealed by cultured cortival neurones. Mol Cell Neurosci 31:642–648

    PubMed  Article  Google Scholar 

  13. Fellin T (2009) Communication between neurons and astrocytes: relevance to the modulation of synaptic and network activity. J Neurochem 108:533–544

    PubMed  Article  CAS  Google Scholar 

  14. Greenspan P, Mayer EP, Fowler SD (1985) Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100:965–973

    PubMed  Article  CAS  Google Scholar 

  15. Guescini M, Genedani S, Stocchi V, Agnati LF (2010) Astrocytes and Glioblastoma cells release exosomes carrying mtDNA. J Neural Transm 117:1–4

    PubMed  Article  CAS  Google Scholar 

  16. György B, Szabó TG, Pásztói M, Pál Z, Misják P, Aradi B, László V, Pállinger É, Pap E, Kittel Á, Nagy G, Falus A, Buzá EI (2011) Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci 68:2667–2688

    PubMed  Article  Google Scholar 

  17. Hamilton NB, Attwell D (2010) Do astrocytes really exocytose neurotransmitters? Nat Rev Neurosci 11:227–238

    PubMed  Article  CAS  Google Scholar 

  18. Herrera MB, Bruno S, Buttiglieri S, Tetta C, Gatti S, Deregibus MC, Bussolati B, Camussi G (2006) Isolation and characterization of a stem cell population from adult human liver. Stem Cells 24:2840–2850

    PubMed  Article  CAS  Google Scholar 

  19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin-Phenol reagents. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  20. Martin S, Parton RG (2006) Lipid droplets: a unified view of a dynamic organelle. Nat Rev Mol Cell Biol 7:373–378

    PubMed  Article  CAS  Google Scholar 

  21. Mauch DH, Nagler K, Schumacher S, Goritz C, Muller EC, Otto A, Pfrieger FW (2001) CNS synaptogenesis promoted by glia-derived cholesterol. Science 294:1354–1357

    PubMed  Article  CAS  Google Scholar 

  22. Medina JM, Tabernero A (2002) Astrocyte-synthesized oleic acid behaves as a neurotrophic factor for neurons. J Physiol Paris 96:265–271

    PubMed  Article  CAS  Google Scholar 

  23. Mulder M (2009) Sterols in the central nervous system. Curr Opin Clin Nutr Metab Care 12:152–158

    PubMed  Article  CAS  Google Scholar 

  24. Nieweg K, Schaller H, Pfrieger FW (2009) Marked differences in cholesterol synthesis between neurons and glial cells from postnatal rats. J Neurochem 109:125–134

    PubMed  Article  CAS  Google Scholar 

  25. Olofsson SO, Bostrom P, Andersson L, Rutberg M, Perman J, Borén J (2009) Lipid droplets as dynamic organelles connecting storage and efflux of lipids. Biochim Biophys Acta 1791:448–458

    PubMed  Article  CAS  Google Scholar 

  26. Pangrsic T, Potokar M, Stenovec M, Kreft M, Fabbretti E, Nistri A, Pryazhnikov E, Khiroug L, Giniatullin R, Zorec R (2007) Exocytotic release of ATP from cultured astrocytes. J Biol Chem 282:28749–28758

    PubMed  Article  CAS  Google Scholar 

  27. Pfrieger FW (2002) Outsourcing in the brain: do neurons depend on cholesterol delivery by astrocytes? BioEssays 25:72–78

    Article  CAS  Google Scholar 

  28. Potokar M, Kreft M, Lee S-Y, Takano H, Haydon PG, Zorec R (2009) Trafficking of astrocytic vesicles in hippocampal slices. Biochem Biophys Res Commun 25:1192–1196

    Article  Google Scholar 

  29. Proia P, Schiera G, Mineo M, Ingrassia AM, Santoro G, Savettieri G, Di Liegro I (2008) Astrocytes shed extracellular vesicles that contain fibroblast growth factor-2 and vascular endothelial growth factor. Int J Mol Med 21:63–67

    PubMed  CAS  Google Scholar 

  30. Ratajczak J, Wysoczynski M, Hayek F, Janowska-Wieczorek A, Ratajczak MZ (2006) Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication. Leukemia 20:1487–1495

    PubMed  Article  CAS  Google Scholar 

  31. Robenek H, Robenek MJ, Troyer D (2005) PAT family proteins pervade lipid droplet cores. J Lipid Res 46:1331–1338

    PubMed  Article  CAS  Google Scholar 

  32. Sbai O, Ould-Yahoui A, Ferhat L, Gueye Y, Bernard A, Charrat E, Mehanna A, Risso JJ, Chauvin JP, Fenouillet E, Rivera S, Khrestchatisky M (2010) Differential vesicular distribution and trafficking of MMP-2, MMP-9, and their inhibitors in astrocytes. Glia 58:344–366

    PubMed  Google Scholar 

  33. Sergent-Tanguy S, Michel DC, Neveu I, Naveilhan P (2006) Long-lasting coexpression of nestin and glial fibrillary acidic protein in primary cultures of astroglial cells with a major participation of nestin+/GFAP cells in cell proliferation. J Neurosci Res 83:1515–1524

    PubMed  Article  CAS  Google Scholar 

  34. Spees JL, Olson SD, Whitney MJ, Prockop DJ (2006) Mitochondrial transfer between cells can rescue aerobic respiration. Proc Natl Acad Sci USA 103:1283–1288

    PubMed  Article  CAS  Google Scholar 

  35. Stevens B (2008) Neuron-astrocyte signaling in the development and plasticity of neural circuits. Neurosignals 16:278–288

    PubMed  Article  CAS  Google Scholar 

  36. Striedinger K, Meda P, Scemes E (2007) Exocytosis of ATP from astrocyte progenitors modulates spontaneous Ca2+ oscillations and cell migration. Glia 55:652–662

    PubMed  Article  Google Scholar 

  37. Tabernero A, Lavado EM, Granda B, Velasco A, Medina JM (2001) Neuronal differentiation is triggered by oleic acid synthesized and released by astrocytes. J Neurochem 79:606–616

    PubMed  Article  CAS  Google Scholar 

  38. Taylor AR, Robinson MB, Gifondorwa DJ, Tytell M, Milligan CE (2007) Regulation of heat shock protein 70 release in astrocytes: role of signaling kinases. Dev Neurobiol 67:1815–1829

    PubMed  Article  CAS  Google Scholar 

  39. Wang S, Cesca F, Loers G, Schweizer M, Buck F, Benfenati F, Schachner M, Kleene R (2011) Synapsin I is an oligomannose-carrying glycoprotein, acts as an oligomannose-binding lectin, and promotes neurite outgrowth and neuronal survival when released via glia-derived exosomes. J Neurosci 18:7275–7290

    Article  Google Scholar 

  40. Xu J, Peng H, Kang N, Zhao Z, Lin J, Stanton PK, Kang J (2007) Glutamate-induced exocytosis of glutamate from astrocytes. J Biol Chem 282:24185–24197

    PubMed  Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors wish to thank David Nilson for improving the English text. This work was supported by the Fondazione Banco di Sardegna (Sassari, Italy).

Author information

Affiliations

Authors

Corresponding author

Correspondence to Angela Maria Falchi.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Falchi, A.M., Sogos, V., Saba, F. et al. Astrocytes shed large membrane vesicles that contain mitochondria, lipid droplets and ATP. Histochem Cell Biol 139, 221–231 (2013). https://doi.org/10.1007/s00418-012-1045-x

Download citation

Keywords

  • Membrane vesicles
  • Astrocytes
  • Lipid droplets
  • Mitochondria
  • ATP