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Acceleration of α-synuclein aggregation by exosomes.

Grey M, Dunning CJ, Gaspar R, Grey C, Brundin P, Sparr E, Linse S - J. Biol. Chem. (2014)

Bottom Line: The exosomes reduce the lag time indicating that they provide catalytic environments for nucleation.The catalytic effects of exosomes derived from naive cells and cells that overexpress α-synuclein do not differ.Using mass spectrometry, we found several phospholipid classes in the exosomes, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and the gangliosides GM2 and GM3.

View Article: PubMed Central - PubMed

Affiliation: From the Departments of Physical Chemistry.

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cryo-TEM analysis of fibrils. 5 μl of α-syn fibrils formed in 10 mm MES, pH 5.5, with 140 mm NaCl was added to the glow-discharged grid for cryo-TEM imaging. A, exosomes isolated from N2a cells overexpressing wild-type α-syn reveal spherical unilamellar vesicles with the presence of darker gray areas inside and on top of the vesicles (exemplified by arrow). B, when recombinant α-syn is mixed with exosomes from cells overexpressing α-syn, an immediate increase in “gray shadows” appears in the same area of the grid as where exosomes are found. C, after 18 h of co-incubation of recombinant α-syn with exosomes from cells overexpressing wild-type α-syn fibrils associated with the exosomes are present. D, incubation of recombinant α-syn with the exosomes leads to formation of patches in the exosome membrane that are darker than the surrounding membrane (marked by arrows). E, incubation of recombinant α-syn with exosomes overexpressing α-syn at neutral pH 7.5. Here a surface decoration of the exosome membrane is observed (inset shows magnification).
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Figure 10: cryo-TEM analysis of fibrils. 5 μl of α-syn fibrils formed in 10 mm MES, pH 5.5, with 140 mm NaCl was added to the glow-discharged grid for cryo-TEM imaging. A, exosomes isolated from N2a cells overexpressing wild-type α-syn reveal spherical unilamellar vesicles with the presence of darker gray areas inside and on top of the vesicles (exemplified by arrow). B, when recombinant α-syn is mixed with exosomes from cells overexpressing α-syn, an immediate increase in “gray shadows” appears in the same area of the grid as where exosomes are found. C, after 18 h of co-incubation of recombinant α-syn with exosomes from cells overexpressing wild-type α-syn fibrils associated with the exosomes are present. D, incubation of recombinant α-syn with the exosomes leads to formation of patches in the exosome membrane that are darker than the surrounding membrane (marked by arrows). E, incubation of recombinant α-syn with exosomes overexpressing α-syn at neutral pH 7.5. Here a surface decoration of the exosome membrane is observed (inset shows magnification).

Mentions: The size distribution in the exosome samples was determined using DLS and NTA. The main species in the DLS measurements, representing around 99% of the vesicles by number, was found to have a diameter around 100 nm, which is typical for exosomes (42), and the minor portion (<1%) has a considerably larger diameter (Fig. 1D). NTA showed similar results (Fig. 1E). The measured ζ potential for exosomes was similar to that obtained for small unilamellar vesicles (SUVs) with anionic lipids (Table 1). We can thus conclude that the exosomes are negatively charged. The cryo-TEM images of the exosomes show unilamellar, spherical vesicles with a diameter of 96 ± 8 (±S.E.), in agreement with the DLS and NTA measurements. Also, a fraction of larger exosomes was visible, with a diameter of 185 ± 35 nm (±S.E.). The membrane thickness was found to be 6 nm, which is typical for cell membranes (normally 4–10 nm) (43, 44). Exosomes from cells overexpressing human α-syn contain dark gray fields (Fig. 10A), which may represent protein or some other components of the exosomes. Additional exposure leads to blistering of these fields, further supporting a protein component. However, from these images alone, it is not possible to tell whether this is α-syn, another protein, or several proteins. The cryo-TEM images also show that the vesicles deform when they approach each other, consistent with electrostatic repulsion between the charged membranes.


Acceleration of α-synuclein aggregation by exosomes.

Grey M, Dunning CJ, Gaspar R, Grey C, Brundin P, Sparr E, Linse S - J. Biol. Chem. (2014)

cryo-TEM analysis of fibrils. 5 μl of α-syn fibrils formed in 10 mm MES, pH 5.5, with 140 mm NaCl was added to the glow-discharged grid for cryo-TEM imaging. A, exosomes isolated from N2a cells overexpressing wild-type α-syn reveal spherical unilamellar vesicles with the presence of darker gray areas inside and on top of the vesicles (exemplified by arrow). B, when recombinant α-syn is mixed with exosomes from cells overexpressing α-syn, an immediate increase in “gray shadows” appears in the same area of the grid as where exosomes are found. C, after 18 h of co-incubation of recombinant α-syn with exosomes from cells overexpressing wild-type α-syn fibrils associated with the exosomes are present. D, incubation of recombinant α-syn with the exosomes leads to formation of patches in the exosome membrane that are darker than the surrounding membrane (marked by arrows). E, incubation of recombinant α-syn with exosomes overexpressing α-syn at neutral pH 7.5. Here a surface decoration of the exosome membrane is observed (inset shows magnification).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 10: cryo-TEM analysis of fibrils. 5 μl of α-syn fibrils formed in 10 mm MES, pH 5.5, with 140 mm NaCl was added to the glow-discharged grid for cryo-TEM imaging. A, exosomes isolated from N2a cells overexpressing wild-type α-syn reveal spherical unilamellar vesicles with the presence of darker gray areas inside and on top of the vesicles (exemplified by arrow). B, when recombinant α-syn is mixed with exosomes from cells overexpressing α-syn, an immediate increase in “gray shadows” appears in the same area of the grid as where exosomes are found. C, after 18 h of co-incubation of recombinant α-syn with exosomes from cells overexpressing wild-type α-syn fibrils associated with the exosomes are present. D, incubation of recombinant α-syn with the exosomes leads to formation of patches in the exosome membrane that are darker than the surrounding membrane (marked by arrows). E, incubation of recombinant α-syn with exosomes overexpressing α-syn at neutral pH 7.5. Here a surface decoration of the exosome membrane is observed (inset shows magnification).
Mentions: The size distribution in the exosome samples was determined using DLS and NTA. The main species in the DLS measurements, representing around 99% of the vesicles by number, was found to have a diameter around 100 nm, which is typical for exosomes (42), and the minor portion (<1%) has a considerably larger diameter (Fig. 1D). NTA showed similar results (Fig. 1E). The measured ζ potential for exosomes was similar to that obtained for small unilamellar vesicles (SUVs) with anionic lipids (Table 1). We can thus conclude that the exosomes are negatively charged. The cryo-TEM images of the exosomes show unilamellar, spherical vesicles with a diameter of 96 ± 8 (±S.E.), in agreement with the DLS and NTA measurements. Also, a fraction of larger exosomes was visible, with a diameter of 185 ± 35 nm (±S.E.). The membrane thickness was found to be 6 nm, which is typical for cell membranes (normally 4–10 nm) (43, 44). Exosomes from cells overexpressing human α-syn contain dark gray fields (Fig. 10A), which may represent protein or some other components of the exosomes. Additional exposure leads to blistering of these fields, further supporting a protein component. However, from these images alone, it is not possible to tell whether this is α-syn, another protein, or several proteins. The cryo-TEM images also show that the vesicles deform when they approach each other, consistent with electrostatic repulsion between the charged membranes.

Bottom Line: The exosomes reduce the lag time indicating that they provide catalytic environments for nucleation.The catalytic effects of exosomes derived from naive cells and cells that overexpress α-synuclein do not differ.Using mass spectrometry, we found several phospholipid classes in the exosomes, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and the gangliosides GM2 and GM3.

View Article: PubMed Central - PubMed

Affiliation: From the Departments of Physical Chemistry.

Show MeSH
Related in: MedlinePlus