title: Isolation of Exosomes and Microvesicles from Cell Culture Systems to Study Prion Transmission.
abstract: Extracellular vesicles (EVs) are composed of microvesicles and exosomes. Exosomes are small membrane vesicles (40-120 nm sized) of endosomal origin released in the extracellular medium from cells when multivesicular bodies fuse with the plasma membrane, whereas microvesicles (i.e., shedding vesicles, 100 nm to 1 μm sized) bud from the plasma membrane. Exosomes and microvesicles carry functional proteins and nucleic acids (especially mRNAs and microRNAs) that can be transferred to surrounding cells and tissues and can impact multiple dimensions of the cellular life. Most of the cells, if not all, from neuronal to immune cells, release exosomes and microvesicles in the extracellular medium, and all biological fluids including blood (serum/plasma), urine, cerebrospinal fluid, and saliva contain EVs.Prion-infected cultured cells are known to secrete infectivity into their environment. We characterized this cell-free form of prions and showed that infectivity was associated with exosomes. Since exosomes are produced by a variety of cells, including cells that actively accumulate prions, they could be a vehicle for infectivity in body fluids and could participate to the dissemination of prions in the organism. In addition, such infectious exosomes also represent a natural, simple, biological material to get key information on the abnormal PrP forms associated with infectivity.In this chapter, we describe first a method that allows exosomes and microvesicles isolation from prion-infected cell cultures and in a second time the strategies to characterize the prions containing exosomes and their ability to disseminate the prion agent.
authors.affiliation: CNRS UMR5239, LBMC, Ecole Normale Supérieure de Lyon, Lyon, 69007, France. Pascal.Leblanc@ens-lyon.fr.|Institut NeuroMyoGène (INMG), CNRS UMR5310 - INSERM U1217, Université de Lyon - Université Claude Bernard, Lyon, 69000, France. Pascal.Leblanc@ens-lyon.fr.
authors.forename: Pascal
authors.initials: P
authors.lastname: Leblanc
authors.affiliation: IHAP, Université de Toulouse, INRA, ENVT, Toulouse, France.
authors.forename: Zaira E
authors.initials: ZE
authors.lastname: Arellano-Anaya
authors.affiliation: Hôpital Neurologique Pierre Wertheimer, Bron-Lyon, France.
authors.forename: Emilien
authors.initials: E
authors.lastname: Bernard
authors.affiliation: CNRS UMR5239, LBMC, Ecole Normale Supérieure de Lyon, Lyon, 69007, France.|Institut NeuroMyoGène (INMG), CNRS UMR5310 - INSERM U1217, Université de Lyon - Université Claude Bernard, Lyon, 69000, France.
authors.forename: Laure
authors.initials: L
authors.lastname: Gallay
authors.affiliation: IRB, Hôpital St Eloi, Montpellier, 34295, France.
authors.forename: Monique
authors.initials: M
authors.lastname: Provansal
authors.affiliation: IRB, Hôpital St Eloi, Montpellier, 34295, France.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Lehmann
authors.affiliation: CNRS UMR5239, LBMC, Ecole Normale Supérieure de Lyon, Lyon, 69007, France.|Institut NeuroMyoGène (INMG), CNRS UMR5310 - INSERM U1217, Université de Lyon - Université Claude Bernard, Lyon, 69000, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Schaeffer
authors.affiliation: CNRS UMR144, Institut Curie, Paris, 75248, France.
authors.forename: Graça
authors.initials: G
authors.lastname: Raposo
authors.affiliation: IHAP, Université de Toulouse, INRA, ENVT, Toulouse, France. d.vilette@envt.fr.
authors.forename: Didier
authors.initials: D
authors.lastname: Vilette
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D002478:Cells, Cultured; D055354:Exosomes / Q000378:metabolism*; D051379:Mice; D018621:PrPC Proteins / Q000378:metabolism; D018620:PrPSc Proteins / Q000378:metabolism; D017096:Prion Diseases / Q000635:transmission*; D011328:Prions / Q000378:metabolism*
chemical_list: D018621:PrPC Proteins; D018620:PrPSc Proteins; D011328:Prions
keywords: Exosomes; Extracellular vesicles; Microvesicles; PrPC; PrPRes; PrPSc; Prions; Spreading