Abstract
Exosomes are vesicles released by most cells into their environment upon fusion of multivesicular endosomes with the plasma membrane. Exosomes are vesicles of 60–100 nm in diameter, floating in sucrose at a density of ~1.15 g/mL and carrying a number of marker proteins such as Alix, Tsg101, and Flotillin-1. We use dissociated cortical neurons cultured for around two weeks as exosome-releasing cells. In these conditions, neurons make mature synapses and form networks that can be activated by physiological stimuli. Here, we describe methods to culture differentiated cortical neurons, induce exosome release by increasing glutamatergic synapse activity, and purify exosomes by differential centrifugations followed by density separation using sucrose gradients. These protocols allow purification of neuronal exosomes released within minutes of activation of glutamatergic synapses.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Thery C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9:581–593
Keller S, Sanderson MP, Stoeck A, Altevogt P (2006) Exosomes: from biogenesis and secretion to biological function. Immunol Lett 107:102–108
Kalra H, Simpson RJ, Ji H et al (2012) Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation. Plos Biol 10(12):e1001450
Lasser C, Alikhani VS, Ekstrom K et al (2011) Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J Transl Med 9:9
Vella LJ, Greenwood DL, Cappai R et al (2008) Enrichment of prion protein in exosomes derived from ovine cerebral spinal fluid. Vet Immunol Immunopathol 124:385–393
Raposo G, Nijman HW, Stoorvogel W et al (1996) B lymphocytes secrete antigen-presenting vesicles. J Exp Med 183:1161–1172
Thery C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2:569–579
Wang G, Dinkins M, He Q et al (2012) Astrocytes secrete exosomes enriched with proapoptotic ceramide and prostate apoptosis response 4 (PAR-4) potential mechanism of apoptosis induction in Alzheimer disease (AD). J Biol Chem 287:21384–21395
Potolicchio I, Carven GJ, Xu X et al (2005) Proteomic analysis of microglia-derived exosomes: metabolic role of the aminopeptidase CD13 in neuropeptide catabolism. J Immunol 175:2237–2243
Faure J, Lachenal G, Court M et al (2006) Exosomes are released by cultured cortical neurones. Mol Cell Neurosci 31:642–648
Fevrier B, Vilette D, Laude H, Raposo G (2005) Exosomes: a buble ride of prions? Traffic 6:10–17
Smalheiser NR (2007) Exosomal transfer of proteins and RNAs at synapses in the nervous system. Biol Direct 2:35
Kramer-Albers EM, Bretz N, Tenzer S et al (2007) Oligodendrocytes secrete exosomes containing major myelin and stress-protective proteins: trophic support for axons? Proteomics Clin Appl 1:1446–1461
Lopez-Verrilli MA, Picou F, Court FA (2013) Schwann cell-derived exosomes enhance axonal regeneration in the peripheral nervous system. Glia 61:1795–1806
Laulagnier K, Motta C, Hamdi S et al (2004) Mast cell- and dendritic cell-derived exosomes display a specific lipid composition and an unsual membrane organization. Biochem J 380:161–171
Savina A, Vidal M, Colombo MI (2002) The exosome pathway in K562 cells is regulated by Rab11. J Cell Sci 115:2505–2515
Vincent-Schneider H, Stumptner-Cuvelette P, Lankar D et al (2002) Exosomes bearing HLA-DR1 molecules need dendritic cells to efficiently stimulate specific T cells. Int Immunol 14:713–722
Bading H, Greenberg ME (1991) Stimulation of protein tyrosine phosphorylation by NMDA receptor activation. Science 253:912–914
Ichikawa M, Muramoto K, Kobayashi K et al (1993) Formation and maturation of synapses in primary cultures or rat cerebral cortical cells: an electron microscopic study. Neurosci Res 16:95–103
Lachenal G, Pernet-Gallay K, Chivet M et al (2011) Release of exosomes from differentiated neurons and its regulation by synaptic glutamatergic activity. Mol Cell Neurosci 46:409–418
Beaudoin GM 3rd, Lee SH, Singh D et al (2012) Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat Protoc 7:1741–1754
Marks MS (2001) Determination of molecular size by zonal sedimentation analysis on sucrose density gradients. Curr Protoc Cell Biol Chapter 5:Unit 3
Acknowledgment
K.L. was supported by “Fondation Plan Alzheimer.” C.J. and M.C. were supported by the “Ministère de l’Enseignement Supérieur et de la Recherche.” This work was funded by INSERM, Université Grenoble Alpes, and ANR (08-Blanc-0271 to R.S.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Laulagnier, K., Javalet, C., Hemming, F.J., Sadoul, R. (2017). Purification and Analysis of Exosomes Released by Mature Cortical Neurons Following Synaptic Activation. In: Hill, A. (eds) Exosomes and Microvesicles. Methods in Molecular Biology, vol 1545. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6728-5_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6728-5_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6726-1
Online ISBN: 978-1-4939-6728-5
eBook Packages: Springer Protocols