Limits...
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

Neuronal exosomes bearing GFP–TTC bind specifically to neurons. (A) Exosomes released by cortical neurons pre-incubated with GFP–TTC were harvested, pelleted at 100,000×g and separated on a sucrose gradient. Three GFP–TTC containing fractions (sucrose density of 1.1–1.15 g/ml) were pooled, pelleted, resuspended in incubation medium and incubated for 1 h on hippocampal cell cultures (16 DIV) (A, B, C). A) After washing, cells were immunostained with anti-MAP2 antibody (red) to label neurons. Hoechst nuclear staining shows the presence of numerous MAP2 negative cells which are not stained with GFP–TTC exosomes (maxima intensity). (B) After washing, cells were immunostained with anti-MAP2 antibody (red) to label neurons and anti-GFAP to stain astrocytes (magenta). (C) Cells were washed and processed for immunogold labelling using anti-GFP and processed for EM observation. Single or aggregated exosomes carrying gold-labelled GFP–TTC can be seen on the surface of neurons. Scale bars: (A, B) 10 µm, (C) 500 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4232649&req=5

Figure 0004: Neuronal exosomes bearing GFP–TTC bind specifically to neurons. (A) Exosomes released by cortical neurons pre-incubated with GFP–TTC were harvested, pelleted at 100,000×g and separated on a sucrose gradient. Three GFP–TTC containing fractions (sucrose density of 1.1–1.15 g/ml) were pooled, pelleted, resuspended in incubation medium and incubated for 1 h on hippocampal cell cultures (16 DIV) (A, B, C). A) After washing, cells were immunostained with anti-MAP2 antibody (red) to label neurons. Hoechst nuclear staining shows the presence of numerous MAP2 negative cells which are not stained with GFP–TTC exosomes (maxima intensity). (B) After washing, cells were immunostained with anti-MAP2 antibody (red) to label neurons and anti-GFAP to stain astrocytes (magenta). (C) Cells were washed and processed for immunogold labelling using anti-GFP and processed for EM observation. Single or aggregated exosomes carrying gold-labelled GFP–TTC can be seen on the surface of neurons. Scale bars: (A, B) 10 µm, (C) 500 nm.

Mentions: We next tested the binding on mixed cultures of hippocampal cells (14 DIV) of GFP–TTC exosomes released by cortical neurons upon synaptic activation. Exosomes secreted from cortical neurons, which had endocytosed GFP–TTC, were separated on sucrose gradients (pooled fractions 4, 5, 6 in Fig. 2B). This purification step made sure that all GFP–TTC was bound to exosomes and precluded any contamination with soluble GFP–TTC. As seen in Fig. 4A, GFP–TTC-exosomes exclusively decorated MAP2-positive soma and dendrites and did not bind to MAP2 negative cells of the same field. Co-staining with anti-GFAP antibodies further demonstrated that GFP–TTC exosomes bind only to MAP2 positive neurons and not to GFAP positive astrocytes (Fig. 4B). EM observation using immunogold against GFP of hippocampal neurons incubated with exosomes, confirmed the presence of single (Fig. 4C, left panel) or of aggregated exosomes (Fig. 4C, right panel) bound to the neuronal surface.


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)

Neuronal exosomes bearing GFP–TTC bind specifically to neurons. (A) Exosomes released by cortical neurons pre-incubated with GFP–TTC were harvested, pelleted at 100,000×g and separated on a sucrose gradient. Three GFP–TTC containing fractions (sucrose density of 1.1–1.15 g/ml) were pooled, pelleted, resuspended in incubation medium and incubated for 1 h on hippocampal cell cultures (16 DIV) (A, B, C). A) After washing, cells were immunostained with anti-MAP2 antibody (red) to label neurons. Hoechst nuclear staining shows the presence of numerous MAP2 negative cells which are not stained with GFP–TTC exosomes (maxima intensity). (B) After washing, cells were immunostained with anti-MAP2 antibody (red) to label neurons and anti-GFAP to stain astrocytes (magenta). (C) Cells were washed and processed for immunogold labelling using anti-GFP and processed for EM observation. Single or aggregated exosomes carrying gold-labelled GFP–TTC can be seen on the surface of neurons. Scale bars: (A, B) 10 µm, (C) 500 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0004: Neuronal exosomes bearing GFP–TTC bind specifically to neurons. (A) Exosomes released by cortical neurons pre-incubated with GFP–TTC were harvested, pelleted at 100,000×g and separated on a sucrose gradient. Three GFP–TTC containing fractions (sucrose density of 1.1–1.15 g/ml) were pooled, pelleted, resuspended in incubation medium and incubated for 1 h on hippocampal cell cultures (16 DIV) (A, B, C). A) After washing, cells were immunostained with anti-MAP2 antibody (red) to label neurons. Hoechst nuclear staining shows the presence of numerous MAP2 negative cells which are not stained with GFP–TTC exosomes (maxima intensity). (B) After washing, cells were immunostained with anti-MAP2 antibody (red) to label neurons and anti-GFAP to stain astrocytes (magenta). (C) Cells were washed and processed for immunogold labelling using anti-GFP and processed for EM observation. Single or aggregated exosomes carrying gold-labelled GFP–TTC can be seen on the surface of neurons. Scale bars: (A, B) 10 µm, (C) 500 nm.
Mentions: We next tested the binding on mixed cultures of hippocampal cells (14 DIV) of GFP–TTC exosomes released by cortical neurons upon synaptic activation. Exosomes secreted from cortical neurons, which had endocytosed GFP–TTC, were separated on sucrose gradients (pooled fractions 4, 5, 6 in Fig. 2B). This purification step made sure that all GFP–TTC was bound to exosomes and precluded any contamination with soluble GFP–TTC. As seen in Fig. 4A, GFP–TTC-exosomes exclusively decorated MAP2-positive soma and dendrites and did not bind to MAP2 negative cells of the same field. Co-staining with anti-GFAP antibodies further demonstrated that GFP–TTC exosomes bind only to MAP2 positive neurons and not to GFAP positive astrocytes (Fig. 4B). EM observation using immunogold against GFP of hippocampal neurons incubated with exosomes, confirmed the presence of single (Fig. 4C, left panel) or of aggregated exosomes (Fig. 4C, right panel) bound to the neuronal surface.

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