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. 2011 Jun;42(3):360-7.
doi: 10.1016/j.nbd.2011.01.029. Epub 2011 Feb 18.

Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission

Lydia Alvarez-Erviti  1 , Yiqi Seow, Anthony H Schapira, Chris Gardiner, Ian L Sargent, Matthew J A Wood, J Mark Cooper
Affiliations

Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission

Lydia Alvarez-Erviti et al. Neurobiol Dis. 2011 Jun.

Abstract

Alpha-synuclein aggregation plays a central role in Parkinson's disease pathology. Direct transmission of alpha-synuclein from pathologically affected to healthy unaffected neurons may be important in the anatomical spread of the disease through the nervous system. We have demonstrated that exosomes released from alpha-synuclein over-expressing SH-SY5Y cells contained alpha-synuclein and these exosomes were capable of efficiently transferring alpha-synuclein protein to normal SH-SY5Y cells. Moreover, the incubation of cells with ammonium chloride or bafilomycin A1 to produce the lysosomal dysfunction recently reported in Parkinson's disease led to an increase in the release of alpha-synuclein in exosomes and a concomitant increase in alpha-synuclein transmission to recipient cells. This study clearly demonstrates the importance of exosomes in both the release of alpha synuclein and its transmission between cells and suggests that factors associated with PD pathology accelerate this process. These mechanisms may play an important role in PD pathology and provide a suitable _target for therapeutic intervention.

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Figures

Fig. 1
Fig. 1
Analysis of exosomes released by SH-SY5Y. Characterisation of exosomes isolated from conditioned medium (CM) from normal and alpha-synuclein over-expressing SH-SY5Y cells: a, Electron micrograph of phosphotungstic acid-stained exosomes; scale bar = 200 nm. b, NTA analysis of particle diameters in exosome preparations from CM from normal (green line) or alpha synuclein expressing (red line) SH-SY5Y cells. c, Western blot analysis of exosome markers (LAMP-1, Alix and flotillin-1) and alpha-synuclein (alpha-synuclein and HA). d, Conditioned medium from alpha synuclein expressing cells was immunoprecipitated (IP) with anti-tubulin or anti-flotillin-1 antibodies. All the eluted IP protein and 40% (15 μg) of the flow through (FT) were loaded and probed with anti-LAMP-1 and anti-alpha-synuclein antibodies. e, ELISA quantitation of alpha-synuclein levels in the flow through after IP with flotillin-1 presented as a percentage of the level in the CM.
Fig. 2
Fig. 2
Analysis of the inter-cellular transfer of alpha synuclein by exosomes, a. Western blot analysis of alpha-synuclein levels (using anti-alpha-synuclein and HA antibodies) relative to actin in normal SH-SY5Y cells following overnight treatment with exosomes isolated from CM from normal and alpha-synuclein over-expressing SH-SY5Y cells. b, Immunofluoresence staining for alpha-synuclein (i and ii, anti-alpha-synuclein antibody; iii and iv, anti-HA antibody, arrows) in normal SH-SY5Y cells (i and iii) and after 16 h exposure to exosomes isolated from CM from alpha-synuclein over-expressing cells (ii and iv). c, Serial confocal Z-projection images (4 μm) of normal SH-SY5Y cells 16 h after incubation with exosomes isolated from WT alpha-synuclein over-expressing cells, anti-alpha-synuclein antibody (green) and DAPI (blue). d. Western blot analysis of alpha-synuclein levels (alpha-synuclein antibody) relative to actin in normal SH-SY5Y cells following overnight treatment with intact and sonicated exosomes isolated from CM from alpha-synuclein over-expressing SH-SY5Y cells. e. Immunofluoresence staining of alpha-synuclein (alpha-synuclein antibody, green) relative to DAPI (blue) in differentiated normal SH-SY5Y cells grown under control conditions (i) or 16 h after treatment with exosomes isolated from CM from alpha-synuclein over-expressing cells (ii).
Fig. 3
Fig. 3
Influence of lysosomal inhibition on alpha-synuclein levels. Analysis of alpha-synuclein overexpressing cells under normal conditions (control) or after treatment with ammonium chloride or bafilomycin A1. a, Influence of ammonium chloride (20 mM) treatment for 7 days and bafilomycin A1 (200 nM) treatment for 24 and 72 h upon cell proliferation as determined by the Celltiter Blue kit in alpha synuclein over-expressing SH-SY5Y cells. Statistical analysis compared to controls *p < 0.05. b and c, Western Blot analysis of alpha-synuclein (alpha-synuclein antibody) and LC3-I and II levels relative to actin after 7 days ammonium chloride or 24 and 72 h bafilomycin A1 treatment. Statistical analysis compared to controls *p < 0.05, **p < 0.01. d, Immunofluoresence staining for alpha-synuclein (anti-HA antibody, green) after 7 days of treatment with ammonium chloride or 24 h of treatment with bafilomycin A1. e, Western Blot analysis of alpha-synuclein levels in SH-SY5Y cells treated with ammonium chloride or bafilomycin A1. Cells were sequential extracted with high salt (HS), HS/Triton (Triton) and SDS/urea (urea), and 3%, 3% and 20% of each extract respectively were separated and detected with an anti-alpha-synuclein antibody.
Fig. 4
Fig. 4
Influence of lysosomal inhibition upon exosome-mediated alpha-synuclein transmission to normal cells. a, ELISA quantitation of total alpha-synuclein release under control conditions or after ammonium chloride or bafilomycin A1 treatment. Data compared to controls, *p < 0.05, **p < 0.01. b and c, Western Blot analysis of exosomes isolated from CM from normal and alpha-synuclein over expressing cells grown under control conditions or after ammonium chloride (20 mM for 7 days) or bafilomycin A1 (200 nM) treatment. Alpha-synuclein levels (alpha-synuclein antibody) were evaluated relative to an exosome marker, Flotillin-1. Statistical analysis compared to controls **p < 0.01. d and e, Western Blot analysis of alpha-synuclein levels (alpha-synuclein antibody) relative to actin in normal SH-SY5Y cells following 16 h incubation with exosomes isolated from CM from normal and alpha-synuclein over-expressing SH-SY5Y cells grown in the absence or presence of ammonium chloride (7 days). Statistical analysis compared to controls **p < 0.01. f, Immunofluoresence staining of alpha-synuclein (alpha-synuclein antibody) in (i) normal untreated SH-SY5Y cells, and (ii) normal SH-SY5Y cells after 16 h incubation with exosomes isolated from alpha-synuclein over-expressing cells, (iii–v) alpha-synuclein over-expressing cells after ammonium chloride treatment, (iv and v) demonstrating peri-nuclear alpha-synuclein accumulation (arrows). All studies were with dividing SH-SY5Y cells except for panel v where the recipient cells had been differentiated for 7 days. g, Serial confocal Z-projections of normal SH-SY5Y cells 16 h after incubation with exosomes isolated from WT alpha-synuclein over-expressing cells treated with ammonium chloride, anti-alpha-synuclein antibody (green) and DAPI (blue).
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References

    1. Alvarez-Erviti L., Rodriguez-Oroz M.C., Cooper J.M., Ferrer I., Obeso J.A., Schapira A.H. Chaperone-mediated autophagy markers in Parkinson's disease brains. Arch. Neurol. 2010;67:1464–1472. - PubMed
    1. Alais S., Simoes S., Baas D., Lehmann S., Raposo G., Darlix J.L., Leblanc P. Mouse neuroblastoma cells release prion infectivity associated with exosomal vesicles. Biol. Cell. 2008;100:603–615. - PubMed
    1. Borghi R., Marchese R., Negro A., Marinelli L., Forloni G., Zaccheo D., Abbruzzese G., Tabaton M. Full length alpha-synuclein is present in cerebrospinal fluid from Parkinson's disease and normal subjects. Neurosci. Lett. 2000;287:65–67. - PubMed
    1. Braak H., Del Tredici K., Rüb U., de Vos R.A., Jansen Steur E.N., Braak E. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol. Aging. 2003;24:197–211. - PubMed
    1. Chau K.Y., Ching H.L., Schapira A.H., Cooper J.M. Relationship between alpha synuclein phosphorylation, proteasomal inhibition and cell death: relevance to Parkinson's disease pathogenesis. J. Neurochem. 2009;110:1005–1013. - PubMed

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