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Exosomes secreted by cortical neurons upon glutamatergic synapse activation specifically interact with neurons.

Chivet M, Javalet C, Laulagnier K, Blot B, Hemming FJ, Sadoul R - J Extracell Vesicles (2014)

Bottom Line: We found that exosomes from neuroblastoma cells bind indiscriminately to neurons and glial cells and could be endocytosed preferentially by glial cells.In contrast, exosomes secreted from stimulated cortical neurons bound to and were endocytosed only by neurons.Thus, our results demonstrate for the first time that exosomes released upon synaptic activation do not bind to glial cells but selectively to other neurons suggesting that they can underlie a novel aspect of interneuronal communication.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM), U836, Grenoble, France; Grenoble Institute of Neuroscience, University of Grenoble Alpes, Grenoble, France.

ABSTRACT
Exosomes are nano-sized vesicles of endocytic origin released into the extracellular space upon fusion of multivesicular bodies with the plasma membrane. Exosomes represent a novel mechanism of cell-cell communication allowing direct transfer of proteins, lipids and RNAs. In the nervous system, both glial and neuronal cells secrete exosomes in a way regulated by glutamate. It has been hypothesized that exosomes can be used for interneuronal communication implying that neuronal exosomes should bind to other neurons with some kind of specificity. Here, dissociated hippocampal cells were used to compare the specificity of binding of exosomes secreted by neuroblastoma cells to that of exosomes secreted by cortical neurons. We found that exosomes from neuroblastoma cells bind indiscriminately to neurons and glial cells and could be endocytosed preferentially by glial cells. In contrast, exosomes secreted from stimulated cortical neurons bound to and were endocytosed only by neurons. Thus, our results demonstrate for the first time that exosomes released upon synaptic activation do not bind to glial cells but selectively to other neurons suggesting that they can underlie a novel aspect of interneuronal communication.

No MeSH data available.


Related in: MedlinePlus

Soluble GFP–TTC is endocytosed before being secreted via neuronal exosomes. Cortical neurons were incubated at 37°C with GFP–TTC for 2 h (36 nM). (A) Confocal microscopy of neurons stained with anti-EEA1 (red), shows that GFP–TTC proteins (green) bind to and are endocytosed by neurons. (B) Density separation of extracellular vesicles secreted during a 15 min treatment with bicuculline and 4-AP; Western blot analysis using anti-GFP, anti-flotillin-1 and anti-Alix, shows GFP immunoreactivity in fractions containing exosomes. TCL: total cell lysates, Inp: input. (C) Immunogold labelling with anti-GFP of vesicles pelleted at 100,000×g demonstrates the presence of GFP–TTC on the exosomal surface. Scale bars: (A) 10 µm (C) 100 nm.
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Figure 0002: Soluble GFP–TTC is endocytosed before being secreted via neuronal exosomes. Cortical neurons were incubated at 37°C with GFP–TTC for 2 h (36 nM). (A) Confocal microscopy of neurons stained with anti-EEA1 (red), shows that GFP–TTC proteins (green) bind to and are endocytosed by neurons. (B) Density separation of extracellular vesicles secreted during a 15 min treatment with bicuculline and 4-AP; Western blot analysis using anti-GFP, anti-flotillin-1 and anti-Alix, shows GFP immunoreactivity in fractions containing exosomes. TCL: total cell lysates, Inp: input. (C) Immunogold labelling with anti-GFP of vesicles pelleted at 100,000×g demonstrates the presence of GFP–TTC on the exosomal surface. Scale bars: (A) 10 µm (C) 100 nm.

Mentions: Based on our previous work, we also used GFP–TTC to label neuronal exosomes. After 2 h incubation on cortical cells, the soluble bacterial recombinant GFP–TTC labelled neurons exclusively. Labelling was seen both on the surface and inside endosomes, some of which were labelled with the early endosomal marker EEA1 (Fig. 2A). GFP–TTC pre-labelled neurons were then extensively washed and incubated for 15 min in a medium containing bicuculline and 4-aminopyridine to trigger exosome release. Vesicles secreted during the 15 min incubation were harvested by ultracentrifugation and separated on a sucrose gradient. As seen in Fig. 2B, GFP–TTC protein could be detected in the exosome fractions containing flotillin-1 and Alix demonstrating that the soluble protein is bound to vesicles floating in sucrose at the density of exosomes. Immunoelectron microscopy using anti-GFP demonstrated that GFP–TTC was indeed bound to the surface of exosomes (Fig. 2C).


Exosomes secreted by cortical neurons upon glutamatergic synapse activation specifically interact with neurons.

Chivet M, Javalet C, Laulagnier K, Blot B, Hemming FJ, Sadoul R - J Extracell Vesicles (2014)

Soluble GFP–TTC is endocytosed before being secreted via neuronal exosomes. Cortical neurons were incubated at 37°C with GFP–TTC for 2 h (36 nM). (A) Confocal microscopy of neurons stained with anti-EEA1 (red), shows that GFP–TTC proteins (green) bind to and are endocytosed by neurons. (B) Density separation of extracellular vesicles secreted during a 15 min treatment with bicuculline and 4-AP; Western blot analysis using anti-GFP, anti-flotillin-1 and anti-Alix, shows GFP immunoreactivity in fractions containing exosomes. TCL: total cell lysates, Inp: input. (C) Immunogold labelling with anti-GFP of vesicles pelleted at 100,000×g demonstrates the presence of GFP–TTC on the exosomal surface. Scale bars: (A) 10 µm (C) 100 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4232649&req=5

Figure 0002: Soluble GFP–TTC is endocytosed before being secreted via neuronal exosomes. Cortical neurons were incubated at 37°C with GFP–TTC for 2 h (36 nM). (A) Confocal microscopy of neurons stained with anti-EEA1 (red), shows that GFP–TTC proteins (green) bind to and are endocytosed by neurons. (B) Density separation of extracellular vesicles secreted during a 15 min treatment with bicuculline and 4-AP; Western blot analysis using anti-GFP, anti-flotillin-1 and anti-Alix, shows GFP immunoreactivity in fractions containing exosomes. TCL: total cell lysates, Inp: input. (C) Immunogold labelling with anti-GFP of vesicles pelleted at 100,000×g demonstrates the presence of GFP–TTC on the exosomal surface. Scale bars: (A) 10 µm (C) 100 nm.
Mentions: Based on our previous work, we also used GFP–TTC to label neuronal exosomes. After 2 h incubation on cortical cells, the soluble bacterial recombinant GFP–TTC labelled neurons exclusively. Labelling was seen both on the surface and inside endosomes, some of which were labelled with the early endosomal marker EEA1 (Fig. 2A). GFP–TTC pre-labelled neurons were then extensively washed and incubated for 15 min in a medium containing bicuculline and 4-aminopyridine to trigger exosome release. Vesicles secreted during the 15 min incubation were harvested by ultracentrifugation and separated on a sucrose gradient. As seen in Fig. 2B, GFP–TTC protein could be detected in the exosome fractions containing flotillin-1 and Alix demonstrating that the soluble protein is bound to vesicles floating in sucrose at the density of exosomes. Immunoelectron microscopy using anti-GFP demonstrated that GFP–TTC was indeed bound to the surface of exosomes (Fig. 2C).

Bottom Line: We found that exosomes from neuroblastoma cells bind indiscriminately to neurons and glial cells and could be endocytosed preferentially by glial cells.In contrast, exosomes secreted from stimulated cortical neurons bound to and were endocytosed only by neurons.Thus, our results demonstrate for the first time that exosomes released upon synaptic activation do not bind to glial cells but selectively to other neurons suggesting that they can underlie a novel aspect of interneuronal communication.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM), U836, Grenoble, France; Grenoble Institute of Neuroscience, University of Grenoble Alpes, Grenoble, France.

ABSTRACT
Exosomes are nano-sized vesicles of endocytic origin released into the extracellular space upon fusion of multivesicular bodies with the plasma membrane. Exosomes represent a novel mechanism of cell-cell communication allowing direct transfer of proteins, lipids and RNAs. In the nervous system, both glial and neuronal cells secrete exosomes in a way regulated by glutamate. It has been hypothesized that exosomes can be used for interneuronal communication implying that neuronal exosomes should bind to other neurons with some kind of specificity. Here, dissociated hippocampal cells were used to compare the specificity of binding of exosomes secreted by neuroblastoma cells to that of exosomes secreted by cortical neurons. We found that exosomes from neuroblastoma cells bind indiscriminately to neurons and glial cells and could be endocytosed preferentially by glial cells. In contrast, exosomes secreted from stimulated cortical neurons bound to and were endocytosed only by neurons. Thus, our results demonstrate for the first time that exosomes released upon synaptic activation do not bind to glial cells but selectively to other neurons suggesting that they can underlie a novel aspect of interneuronal communication.

No MeSH data available.


Related in: MedlinePlus