Cell-Produced α-Synuclein Is Secreted in a Calcium-Dependent Manner by Exosomes and Impacts Neuronal Survival

Abstract

␣-Synuclein is central in Parkinson's disease pathogenesis. Although initially ␣-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 ␣-synuclein in neuronal homeostasis, we have generated wild-type ␣-synuclein and ␤-galactosidase inducible SH-SY5Y cells. Soluble oligomeric and monomeric species of ␣-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, ␣-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 ␣-synuclein causes cell death of recipient neuronal cells, which can be reversed after ␣-synuclein immunodepletion from the CM. High-and low-molecular-weight ␣-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 ␣-synuclein is secreted via an exosomal, calcium-dependent mechanism and suggest that ␣-synuclein secretion serves to amplify and propagate Parkinson's diseaserelated pathology.

Prion-Like Propagation of Protein Aggregation and Related Therapeutic Strategies

“…α-synuclein secretion appears to be mediated by an unconventional mechanism, independent of the ER/Golgi [61,62]. Evidence suggests that this secretion is an active process as it is temperature sensitive, independent of cell death, and the amount of α-synuclein released from cells does not correlate with the release of other cytosolic proteins into the media [62].…”

“…Release of α-synuclein may occur through multiple mechanisms, however. Free α-synuclein can be immunoprecipitated from media and human CSF [60], and at least a subset of the toxic species in conditioned media is removed via α-synuclein-specific immunoprecipitation [61]. Calcium-mediated exosome release may also contribute to secretion, as α-synuclein can be found in the exosomal fraction of conditioned media [61,63].…”

“…Free α-synuclein can be immunoprecipitated from media and human CSF [60], and at least a subset of the toxic species in conditioned media is removed via α-synuclein-specific immunoprecipitation [61]. Calcium-mediated exosome release may also contribute to secretion, as α-synuclein can be found in the exosomal fraction of conditioned media [61,63]. Importantly, a subset of α-synuclein remains intact in the exosome fraction after trypsin digestion, but is absent after saponin-mediated membrane permeableization, confirming it is protected from protease digestion within exosomes [63].…”

Proteolytic clearance of extracellular α-synuclein as a new therapeutic approach against Parkinson disease

“…[8][9][10] In addition, α-syn and its aggregates have been shown to be secreted from neuronal cells by a non-classical exocytic pathway 11 and its release is increased under various protein misfolding stress conditions. 12 α-Syn has also been reported to be secreted in a calcium-dependent manner by exosomes, 13 and lysosomal dysfunction increases exosome-mediated α-syn release. 14 These studies imply that extracellular α-syn is not only due to cellular leakage by cell death, but also its release from cells.…”

Glucocerebrosidase, a new player changing the old rules in Lewy body diseases

“…Exocytosis of these vesicles would generate vesicle-free extracellular α -synuclein. Previous studies have shown that α -synuclein is secreted via unconventional exocytosis (Lee et al , 2005 ;Jang et al , 2010 ), and exosome-associated release has also been suggested (Emmanouilidou et al , 2010 ;Alvarez -Erviti et al, 2011 ;Danzer et al , 2012 ). In recipient cells, internalization of extracellular α -synuclein aggregates may occur through endocytosis of the free protein or through direct fusion of exosomes to the plasma membrane.…”