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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Analysis of Variance
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Animals
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Brefeldin A / pharmacology
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Calcium / metabolism*
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Calcium / pharmacology
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Cell Death / drug effects
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Cell Differentiation / drug effects
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Cell Line, Tumor
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Cell Proliferation / drug effects
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Cell Survival / drug effects
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Cell Survival / genetics
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Cells, Cultured
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Cerebral Cortex / cytology
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Chromatography, High Pressure Liquid / methods
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Culture Media, Conditioned / chemistry
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Culture Media, Conditioned / pharmacology
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Cytotoxicity Tests, Immunologic / methods
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Dose-Response Relationship, Drug
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Endocytosis / drug effects
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Exosomes / physiology*
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Exosomes / ultrastructure
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Gene Expression Regulation, Neoplastic / drug effects
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Humans
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Hydrogen Peroxide / pharmacology
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Immunoprecipitation / methods
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Mass Spectrometry / methods
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Microscopy, Confocal / methods
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Microscopy, Electron, Transmission / methods
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Molecular Weight
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Multivesicular Bodies / drug effects
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Multivesicular Bodies / pathology*
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Multivesicular Bodies / ultrastructure
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Nerve Tissue Proteins / metabolism
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Neuroblastoma / pathology
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Neuroblastoma / ultrastructure
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Neurons / drug effects
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Neurons / metabolism
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Neurons / ultrastructure
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Peptides / pharmacology
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Piperidines / pharmacology
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Presenilin-1 / pharmacology
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Protein Synthesis Inhibitors / pharmacology
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Pyrazoles / pharmacology
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Rats
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Receptors, Transferrin / metabolism
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Serum / metabolism
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Subcellular Fractions / drug effects
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Subcellular Fractions / metabolism
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Subcellular Fractions / ultrastructure
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Temperature
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Transfection
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alpha-Synuclein / genetics*
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alpha-Synuclein / metabolism*
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beta-Galactosidase / genetics
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beta-Galactosidase / metabolism
Substances
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1,3-bis(4-hydroxyphenyl)-4-methyl-5-(4-(2-piperidinylethoxy)phenol)-1H-pyrazole
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Culture Media, Conditioned
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Nerve Tissue Proteins
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Peptides
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Piperidines
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Presenilin-1
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Protein Synthesis Inhibitors
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Pyrazoles
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Receptors, Transferrin
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alpha-Synuclein
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Brefeldin A
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Hydrogen Peroxide
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beta-Galactosidase
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Calcium