title: Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival.
abstract: alpha-Synuclein is central in Parkinson's disease pathogenesis. Although initially alpha-synuclein was considered a purely intracellular protein, recent data suggest that it can be detected in the plasma and CSF of humans and in the culture media of neuronal cells. To address a role of secreted alpha-synuclein in neuronal homeostasis, we have generated wild-type alpha-synuclein and beta-galactosidase inducible SH-SY5Y cells. Soluble oligomeric and monomeric species of alpha-synuclein are readily detected in the conditioned media (CM) of these cells at concentrations similar to those observed in human CSF. We have found that, in this model, alpha-synuclein is secreted by externalized vesicles in a calcium-dependent manner. Electron microscopy and liquid chromatography-mass spectrometry proteomic analysis demonstrate that these vesicles have the characteristic hallmarks of exosomes, secreted intraluminar vesicles of multivesicular bodies. Application of CM containing secreted alpha-synuclein causes cell death of recipient neuronal cells, which can be reversed after alpha-synuclein immunodepletion from the CM. High- and low-molecular-weight alpha-synuclein species, isolated from this CM, significantly decrease cell viability. Importantly, treatment of the CM with oligomer-interfering compounds before application rescues the recipient neuronal cells from the observed toxicity. Our results show for the first time that cell-produced alpha-synuclein is secreted via an exosomal, calcium-dependent mechanism and suggest that alpha-synuclein secretion serves to amplify and propagate Parkinson's disease-related pathology.
authors.affiliation: Division of Basic Neurosciences, Biomedical Research Foundation of the Academy of Athens, Athens 11527, Greece.
authors.forename: Evangelia
authors.initials: E
authors.lastname: Emmanouilidou
authors.forename: Katerina
authors.initials: K
authors.lastname: Melachroinou
authors.forename: Theodoros
authors.initials: T
authors.lastname: Roumeliotis
authors.forename: Spiros D
authors.initials: SD
authors.lastname: Garbis
authors.forename: Maria
authors.initials: M
authors.lastname: Ntzouni
authors.forename: Lukas H
authors.initials: LH
authors.lastname: Margaritis
authors.forename: Leonidas
authors.initials: L
authors.lastname: Stefanis
authors.forename: Kostas
authors.initials: K
authors.lastname: Vekrellis
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects; D055354:Exosomes / Q000502:physiology* / Q000648:ultrastructure; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D047468:Immunoprecipitation / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008970:Molecular Weight; D057146:Multivesicular Bodies / Q000187:drug effects / Q000473:pathology* / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009447:Neuroblastoma / Q000473:pathology / Q000648:ultrastructure; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D010455:Peptides / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D053764:Presenilin-1 / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D051381:Rats; D011990:Receptors, Transferrin / Q000378:metabolism; D044967:Serum / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D013696:Temperature; D014162:Transfection; D051844:alpha-Synuclein / Q000235:genetics* / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: C524478:1,3-bis(4-hydroxyphenyl)-4-methyl-5-(4-(2-piperidinylethoxy)phenol)-1H-pyrazole; D017077:Culture Media, Conditioned; D009419:Nerve Tissue Proteins; D010455:Peptides; D010880:Piperidines; D053764:Presenilin-1; D011500:Protein Synthesis Inhibitors; D011720:Pyrazoles; D011990:Receptors, Transferrin; D051844:alpha-Synuclein; D020126:Brefeldin A; D006861:Hydrogen Peroxide; D001616:beta-Galactosidase; D002118:Calcium