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Exosomes neutralize synaptic-plasticity-disrupting activity of Aβ assemblies in vivo.

An K, Klyubin I, Kim Y, Jung JH, Mably AJ, O'Dowd ST, Lynch T, Kanmert D, Lemere CA, Finan GM, Park JW, Kim TW, Walsh DM, Rowan MJ, Kim JH - Mol Brain (2013)

Bottom Line: We here provide in vivo evidence that exosomes derived from N2a cells or human cerebrospinal fluid can abrogate the synaptic-plasticity-disrupting activity of both synthetic and AD brain-derived Aβ.Mechanistically, this effect involves sequestration of synaptotoxic Aβ assemblies by exosomal surface proteins such as PrPC rather than Aβ proteolysis.These data suggest that exosomes can counteract the inhibitory action of Aβ, which contributes to perpetual capability for synaptic plasticity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Gyungbuk 790-784, Korea. joungkim@postech.ac.kr.

ABSTRACT

Background: Exosomes, small extracellular vesicles of endosomal origin, have been suggested to be involved in both the metabolism and aggregation of Alzheimer's disease (AD)-associated amyloid β-protein (Aβ). Despite their ubiquitous presence and the inclusion of components which can potentially interact with Aβ, the role of exosomes in regulating synaptic dysfunction induced by Aβ has not been explored.

Results: We here provide in vivo evidence that exosomes derived from N2a cells or human cerebrospinal fluid can abrogate the synaptic-plasticity-disrupting activity of both synthetic and AD brain-derived Aβ. Mechanistically, this effect involves sequestration of synaptotoxic Aβ assemblies by exosomal surface proteins such as PrPC rather than Aβ proteolysis.

Conclusions: These data suggest that exosomes can counteract the inhibitory action of Aβ, which contributes to perpetual capability for synaptic plasticity.

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Related in: MedlinePlus

Characterization of ADDLs and exosomes used for biochemical and physiological experiments. (A) ADDLs were analyzed by Western blotting using 6E10. SM, size markers (kDa). (B) DLS particle distribution analysis of ADDLs (red line) (14.3 ± 1.1 nm) and Aβ1-42 freshly dissolved in 10 mM NaOH (black line) (6.4 ± 0.3 nm) was expressed as hydrodynamic radii (RH). (C) A tapping AFM mode image of ADDLs (X-Y, 5 x 5 μm with an inset displaying a z-range in color from 0 to 15 nm). (D) By AFM, only small (3 - 6 nm) globular structures were detected. (E) Exosomes isolated from the conditioned medium of N2a cells had their density 1.13 g/ml to 1.19 g/ml, and contained the exosomal marker proteins Alix, Flotilin-1 and PrPC. Multiple (non-, mono- or di-) glycosylated PrPC proteins were detected between 20 ~ 35 kDa on SDS-PAGE. (F) By EM, exosomes appeared as closed vesicles of 30-120 nm in diameter (Scale bar: 100 nm), (G) a size range that agreed with that measured by DLS.
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Figure 1: Characterization of ADDLs and exosomes used for biochemical and physiological experiments. (A) ADDLs were analyzed by Western blotting using 6E10. SM, size markers (kDa). (B) DLS particle distribution analysis of ADDLs (red line) (14.3 ± 1.1 nm) and Aβ1-42 freshly dissolved in 10 mM NaOH (black line) (6.4 ± 0.3 nm) was expressed as hydrodynamic radii (RH). (C) A tapping AFM mode image of ADDLs (X-Y, 5 x 5 μm with an inset displaying a z-range in color from 0 to 15 nm). (D) By AFM, only small (3 - 6 nm) globular structures were detected. (E) Exosomes isolated from the conditioned medium of N2a cells had their density 1.13 g/ml to 1.19 g/ml, and contained the exosomal marker proteins Alix, Flotilin-1 and PrPC. Multiple (non-, mono- or di-) glycosylated PrPC proteins were detected between 20 ~ 35 kDa on SDS-PAGE. (F) By EM, exosomes appeared as closed vesicles of 30-120 nm in diameter (Scale bar: 100 nm), (G) a size range that agreed with that measured by DLS.

Mentions: We investigated whether exosomes affect Aβ-induced LTP impairment in the CA1 region of the dorsal hippocampus in vivo. We used ADDLs [5] prepared from synthetic Aβ1-42 and exosomes isolated from the conditioned media of cultured N2a neuroblastoma cells (Figure 1). On SDS-PAGE, ADDLs yielded 3 bands which migrated with molecular weights of ~4 (monomer), ~12 (trimer) and 16 (tetramer) kDa (Figure 1A). By dynamic light scattering (DLS), ADDLs contained a mixture of species with hydrodynamic radii (RH) ranging from ~10 to 30 nm (Figure 1B), but by atomic force microscopy (AFM) only small (3 - 6 nm) globular structures were detected (Figure 1C, D). The apparent size discrepancy for the Aβ species present in our ADDL preparation is likely to result from technical limits of the used methods. Specifically, since SDS-PAGE is highly denaturing, it might not be suitable for determination of native sizes of Aβ assembly, but could be used to distinguish the SDS-stable forms from labile Aβ species. While AFM could be used to detect oligomeric forms of Aβ, certain assemblies would not adhere to mica well enough and as a result, were not detected. Nonetheless, our characterization of ADDLs revealed the presence of a heterogeneous mixture of different Aβ species, some of which were at least partially stable in SDS and which existed as small globular structures of 3 - 6 nm [5,19]. To prepare exosome fractions, we had excluded plasma membrane-derived fragments and other non-exosomal vesicles through the optimized procedures [20]. Contrasting to the vesicles originated from Golgi body that float at 1.05 to 1.12 g/ml and endoplasmic reticulum-derived vesicles at 1.18 to 1.25 g/ml, exosomes are the only vesicles sizing 30 ~ 100 nm and gradient density ranging 1.13 ~ 1.19 g/ml (Figure 1E) [12,20]. Exosomes are further defined by their expression of marker proteins such as Flotillin-1, Alix or PrPC that are highly enriched in the exosomal fractions (Figure 1E), their ultrastructure and size (Figure 1F, G) [12]. Altogether, we verified that our procedures were able to yield relatively pure exosomes [12,20].


Exosomes neutralize synaptic-plasticity-disrupting activity of Aβ assemblies in vivo.

An K, Klyubin I, Kim Y, Jung JH, Mably AJ, O'Dowd ST, Lynch T, Kanmert D, Lemere CA, Finan GM, Park JW, Kim TW, Walsh DM, Rowan MJ, Kim JH - Mol Brain (2013)

Characterization of ADDLs and exosomes used for biochemical and physiological experiments. (A) ADDLs were analyzed by Western blotting using 6E10. SM, size markers (kDa). (B) DLS particle distribution analysis of ADDLs (red line) (14.3 ± 1.1 nm) and Aβ1-42 freshly dissolved in 10 mM NaOH (black line) (6.4 ± 0.3 nm) was expressed as hydrodynamic radii (RH). (C) A tapping AFM mode image of ADDLs (X-Y, 5 x 5 μm with an inset displaying a z-range in color from 0 to 15 nm). (D) By AFM, only small (3 - 6 nm) globular structures were detected. (E) Exosomes isolated from the conditioned medium of N2a cells had their density 1.13 g/ml to 1.19 g/ml, and contained the exosomal marker proteins Alix, Flotilin-1 and PrPC. Multiple (non-, mono- or di-) glycosylated PrPC proteins were detected between 20 ~ 35 kDa on SDS-PAGE. (F) By EM, exosomes appeared as closed vesicles of 30-120 nm in diameter (Scale bar: 100 nm), (G) a size range that agreed with that measured by DLS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4222117&req=5

Figure 1: Characterization of ADDLs and exosomes used for biochemical and physiological experiments. (A) ADDLs were analyzed by Western blotting using 6E10. SM, size markers (kDa). (B) DLS particle distribution analysis of ADDLs (red line) (14.3 ± 1.1 nm) and Aβ1-42 freshly dissolved in 10 mM NaOH (black line) (6.4 ± 0.3 nm) was expressed as hydrodynamic radii (RH). (C) A tapping AFM mode image of ADDLs (X-Y, 5 x 5 μm with an inset displaying a z-range in color from 0 to 15 nm). (D) By AFM, only small (3 - 6 nm) globular structures were detected. (E) Exosomes isolated from the conditioned medium of N2a cells had their density 1.13 g/ml to 1.19 g/ml, and contained the exosomal marker proteins Alix, Flotilin-1 and PrPC. Multiple (non-, mono- or di-) glycosylated PrPC proteins were detected between 20 ~ 35 kDa on SDS-PAGE. (F) By EM, exosomes appeared as closed vesicles of 30-120 nm in diameter (Scale bar: 100 nm), (G) a size range that agreed with that measured by DLS.
Mentions: We investigated whether exosomes affect Aβ-induced LTP impairment in the CA1 region of the dorsal hippocampus in vivo. We used ADDLs [5] prepared from synthetic Aβ1-42 and exosomes isolated from the conditioned media of cultured N2a neuroblastoma cells (Figure 1). On SDS-PAGE, ADDLs yielded 3 bands which migrated with molecular weights of ~4 (monomer), ~12 (trimer) and 16 (tetramer) kDa (Figure 1A). By dynamic light scattering (DLS), ADDLs contained a mixture of species with hydrodynamic radii (RH) ranging from ~10 to 30 nm (Figure 1B), but by atomic force microscopy (AFM) only small (3 - 6 nm) globular structures were detected (Figure 1C, D). The apparent size discrepancy for the Aβ species present in our ADDL preparation is likely to result from technical limits of the used methods. Specifically, since SDS-PAGE is highly denaturing, it might not be suitable for determination of native sizes of Aβ assembly, but could be used to distinguish the SDS-stable forms from labile Aβ species. While AFM could be used to detect oligomeric forms of Aβ, certain assemblies would not adhere to mica well enough and as a result, were not detected. Nonetheless, our characterization of ADDLs revealed the presence of a heterogeneous mixture of different Aβ species, some of which were at least partially stable in SDS and which existed as small globular structures of 3 - 6 nm [5,19]. To prepare exosome fractions, we had excluded plasma membrane-derived fragments and other non-exosomal vesicles through the optimized procedures [20]. Contrasting to the vesicles originated from Golgi body that float at 1.05 to 1.12 g/ml and endoplasmic reticulum-derived vesicles at 1.18 to 1.25 g/ml, exosomes are the only vesicles sizing 30 ~ 100 nm and gradient density ranging 1.13 ~ 1.19 g/ml (Figure 1E) [12,20]. Exosomes are further defined by their expression of marker proteins such as Flotillin-1, Alix or PrPC that are highly enriched in the exosomal fractions (Figure 1E), their ultrastructure and size (Figure 1F, G) [12]. Altogether, we verified that our procedures were able to yield relatively pure exosomes [12,20].

Bottom Line: We here provide in vivo evidence that exosomes derived from N2a cells or human cerebrospinal fluid can abrogate the synaptic-plasticity-disrupting activity of both synthetic and AD brain-derived Aβ.Mechanistically, this effect involves sequestration of synaptotoxic Aβ assemblies by exosomal surface proteins such as PrPC rather than Aβ proteolysis.These data suggest that exosomes can counteract the inhibitory action of Aβ, which contributes to perpetual capability for synaptic plasticity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Gyungbuk 790-784, Korea. joungkim@postech.ac.kr.

ABSTRACT

Background: Exosomes, small extracellular vesicles of endosomal origin, have been suggested to be involved in both the metabolism and aggregation of Alzheimer's disease (AD)-associated amyloid β-protein (Aβ). Despite their ubiquitous presence and the inclusion of components which can potentially interact with Aβ, the role of exosomes in regulating synaptic dysfunction induced by Aβ has not been explored.

Results: We here provide in vivo evidence that exosomes derived from N2a cells or human cerebrospinal fluid can abrogate the synaptic-plasticity-disrupting activity of both synthetic and AD brain-derived Aβ. Mechanistically, this effect involves sequestration of synaptotoxic Aβ assemblies by exosomal surface proteins such as PrPC rather than Aβ proteolysis.

Conclusions: These data suggest that exosomes can counteract the inhibitory action of Aβ, which contributes to perpetual capability for synaptic plasticity.

Show MeSH
Related in: MedlinePlus