Dataset Information

Secretion and Uptake of ?-Synuclein Via Extracellular Vesicles in Cultured Cells.

ABSTRACT: In Parkinson's disease and other Lewy body disorders, the propagation of pathology has been accredited to the spreading of extracellular ?-synuclein (?-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of ?-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered molecular properties of ?-syn can influence the distribution and secretion of ?-syn in human neuroblastoma cells. Different ?-syn variants, including ?-syn:hemi-Venus and disease-causing mutants, were overexpressed and EVs were isolated from the conditioned medium. Of the secreted ?-syn, 0.1-2% was associated with vesicles. The major part of EV ?-syn was attached to the outer membrane of vesicles, whereas a smaller fraction was found in their lumen. For ?-syn expressed with N-terminal hemi-Venus, the relative levels associated with EVs were higher than for WT ?-syn. Moreover, such EV-associated ?-syn:hemi-Venus species were internalized in recipient cells to a higher degree than the corresponding free-floating forms. Among the disease-causing mutants, A53T ?-syn displayed an increased association with EVs. Taken together, our data suggest that ?-syn species with presumably lost physiological functions or altered aggregation properties may shift the cellular processing towards vesicular secretion. Our findings thus lend further support to the tenet that EVs can mediate spreading of harmful ?-syn species and thereby contribute to the pathology in ?-synucleinopathies.

SUBMITTER: Gustafsson G

PROVIDER: S-EPMC6223723 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Secretion and Uptake of α-Synuclein Via Extracellular Vesicles in Cultured Cells.

Gustafsson Gabriel G Lööv Camilla C Persson Emma E Lázaro Diana F DF Takeda Shuko S Bergström Joakim J Erlandsson Anna A Sehlin Dag D Balaj Leonora L György Bence B Hallbeck Martin M Outeiro Tiago F TF Breakefield Xandra O XO Hyman Bradley T BT Ingelsson Martin M

Cellular and molecular neurobiology 20181004 8

In Parkinson's disease and other Lewy body disorders, the propagation of pathology has been accredited to the spreading of extracellular α-synuclein (α-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of α-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered molecular properties of α-syn can influence the distribution and secretion of α-syn in human neuroblastoma cells. Different α-syn variants, i ...[more]

PMID: 30288631

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Project description:Exosomes, a type of extracellular vesicles, can be collected from the conditioned medium of cultured cells, and are expected to be used in disease therapy and drug delivery systems. However, since the yield of exosomes from conditioned medium is generally low, investigations to develop new methods to increase exosome secretion and to elucidate the secretion mechanism have been performed. Our previous studies demonstrated that activation of intracellular signaling including Rho GTPase and subsequent endocytosis of extraneous molecules in cells could be induced by low level electricity (0.3-0.5 mA/cm2). Since exosomes are produced in the process of endocytosis and secreted by exocytosis via certain signaling pathways, we hypothesized that low level electric treatment (ET) would increase exosome secretion from cultured cells via intracellular signaling activation. In the present study, the influence of ET (0.34 mA/cm2) on extracellular vesicle (EV) secretion from cultured cells was examined by using murine melanoma and murine fibroblast cells. The results showed that the number of EV particles collected by ultracentrifugation was remarkably increased by ET in both cell lines without cellular toxicity or changes in the particle distribution. Also, protein amounts of the collected EVs were significantly increased in both cells by ET without alteration of expression of representative exosome marker proteins. Moreover, in both cells, the ratio of particle numbers to protein amount was not significantly changed by ET. Rho GTPase inhibition significantly suppressed ET-mediated increase of EV secretion in murine melanoma, indicating that Rho GTPase activation could be involved in ET-mediated EV secretion in the cell. Additionally, there were almost no differences in uptake of each EV into each donor cell regardless of whether the cells had been exposed to ET for EV collection. Taken together, these results suggest that ET could increase EV secretion from both cancer and normal cells without apparent changes in EV quality.

Project description:BackgroundThe accumulation of α-synuclein (α-syn), an essential step in PD development and progression, is observed not only in neurons but also in glia, including astrocytes. The mechanisms regulating astrocytic α-syn level and aggregation remain unclear. More recently, it has been demonstrated that a part of α-syn spreading occurs through extracellular vesicles (EVs), although it is unknown whether this process is involved in astrocytes of PD. It is known, however, that EVs derived from the central nervous system exist in the blood and are extensively explored as biomarkers for PD and other neurodegenerative disorders.MethodsPrimary astrocytes were transfected with A53T α-syn plasmid or exposed to α-syn aggregates. The level of astrocyte-derived EVs (AEVs) was assessed by nanoparticle tracking analysis and immunofluorescence. The lysosomal function was evaluated by Cathepsin assays, immunofluorescence for levels of Lamp1 and Lamp2, and LysoTracker Red staining. The Apogee assays were optimized to measure the GLT-1+ AEVs in clinical cohorts of 106 PD, 47 multiple system atrophy (MSA), and 103 healthy control (HC) to test the potential of plasma AEVs as a biomarker to differentiate PD from other forms of parkinsonism.ResultsThe number of AEVs significantly increased in primary astrocytes with α-syn deposition. The mechanism of increased AEVs was partially attributed to lysosomal dysfunction. The number of α-syn-carrying AEVs was significantly higher in patients with PD than in HC and MSA. The integrative model combining AEVs with total and aggregated α-syn exhibited efficient diagnostic power in differentiating PD from HC with an AUC of 0.915, and from MSA with an AUC of 0.877.ConclusionsPathological α-syn deposition could increase the astrocytic secretion of EVs, possibly through α-syn-induced lysosomal dysfunction. The α-syn-containing AEVs in the peripheral blood may be an effective biomarker for clinical diagnosis or differential diagnosis of PD.

Dataset Information

Secretion and Uptake of ?-Synuclein Via Extracellular Vesicles in Cultured Cells.

Publications

Secretion and Uptake of α-Synuclein Via Extracellular Vesicles in Cultured Cells.

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