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Acid sphingomyelinase activity triggers microparticle release from glial cells.

Bianco F, Perrotta C, Novellino L, Francolini M, Riganti L, Menna E, Saglietti L, Schuchman EH, Furlan R, Clementi E, Matteoli M, Verderio C - EMBO J. (2009)

Bottom Line: ATP-induced shedding and IL-1beta release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice.We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1beta release.Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1beta release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

View Article: PubMed Central - PubMed

Affiliation: CNR Institute of Neuroscience and Department of Medical Pharmacology, University of Milano, Milano, Italy.

ABSTRACT
We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL-1beta, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X7, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP-induced shedding and IL-1beta release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1beta release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1beta release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

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A-SMase is activated downstream of P2X7R stimulation. (A) Time course of A-SMase activation by 100 μM BzATP versus not stimulated cells as control, determined in cell lysates by measuring hydrolysis of sphingomyelin to phosphorylcholine at pH 5.5. Values are expressed as fold increases over basal A-SMase activity (1.12±0.3 nmol/mg h−1) conventionally indicated as 1 (n=3). (B) A-SMase translocation onto the PM on P2X7R stimulation as determined by FACS analysis of intact N9 microglial cells. The relative fluorescence intensity (RFI) was calculated as ratio of sample mean fluorescence over negative control mean fluorescence. The RFI values reported in the panels are the mean±s.e.m. measured in the three experiments. The results shown are from one out of three experiments. (C) Surface exposure of A-SMase on P2X7R stimulation, as detected by western blotting of biotinylated PM proteins with the A-SMase antibody. (D) Western blot analysis for A-SMase of P2, P3 and P4 vesicles accumulated in the supernatant of microglia cells under basal conditions or on stimulation with 100 μM BzATP for 30 min. (E) FACS analysis of intact P2, P3 and P4 vesicles for surface A-SMase, showing most of association of the enzyme to the outer leaflet of P2 and P3 MPs. (F) Spectrophotometric analysis of fluorescent MPs present in total supernatants collected from either FM1-43-labelled N9 microglial cells (black bars) or FM1-43-labelled N9 microglial clone, not expressing the P2X7R (white bars), 20 min after A-SMase (2 U/ml) or BzATP (100 μM) addition. (G) Quantitative analysis of FM1-43-labelled vesicles in the total supernatants of microglial cells exposed to exogenous A-SMase in the presence/absence of the P2X7R antagonist KN-62 or the ATP degrading enzyme apyrase.
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f3: A-SMase is activated downstream of P2X7R stimulation. (A) Time course of A-SMase activation by 100 μM BzATP versus not stimulated cells as control, determined in cell lysates by measuring hydrolysis of sphingomyelin to phosphorylcholine at pH 5.5. Values are expressed as fold increases over basal A-SMase activity (1.12±0.3 nmol/mg h−1) conventionally indicated as 1 (n=3). (B) A-SMase translocation onto the PM on P2X7R stimulation as determined by FACS analysis of intact N9 microglial cells. The relative fluorescence intensity (RFI) was calculated as ratio of sample mean fluorescence over negative control mean fluorescence. The RFI values reported in the panels are the mean±s.e.m. measured in the three experiments. The results shown are from one out of three experiments. (C) Surface exposure of A-SMase on P2X7R stimulation, as detected by western blotting of biotinylated PM proteins with the A-SMase antibody. (D) Western blot analysis for A-SMase of P2, P3 and P4 vesicles accumulated in the supernatant of microglia cells under basal conditions or on stimulation with 100 μM BzATP for 30 min. (E) FACS analysis of intact P2, P3 and P4 vesicles for surface A-SMase, showing most of association of the enzyme to the outer leaflet of P2 and P3 MPs. (F) Spectrophotometric analysis of fluorescent MPs present in total supernatants collected from either FM1-43-labelled N9 microglial cells (black bars) or FM1-43-labelled N9 microglial clone, not expressing the P2X7R (white bars), 20 min after A-SMase (2 U/ml) or BzATP (100 μM) addition. (G) Quantitative analysis of FM1-43-labelled vesicles in the total supernatants of microglial cells exposed to exogenous A-SMase in the presence/absence of the P2X7R antagonist KN-62 or the ATP degrading enzyme apyrase.

Mentions: To investigate the specific involvement of A-SMase in MP production, we directly measured A-SMase activity in microglia exposed for different time points to BzATP. Kinetic analysis of A-SMase activity revealed a peak of enzyme activity at 2 min after agonist addition (Figure 3A), indicating that the enzyme acts as a downstream effector of the receptor. Although A-SMase activation period is shorter than that of MP release (Figure 1F), it is plausible that increased generated ceramide has long-lasting effects on membrane fluidity required for P2X7-dependent blebbing. Activation of A-SMase by P2X7R was accompanied by enzyme translocation to the PM outer leaflet, as shown by the increased A-SMase staining detected by FACS in intact cells (Figure 3B). A-SMase translocation to the PM outer leaflet on P2X7R activation was confirmed by biotynilation and western blotting experiments that revealed surface expression of the enzyme in cultured astrocytes during BzATP exposure (Figure 3C). Finally, A-SMase translocation was also suggested by movement of the enzyme from the exosomal fraction (P4) to PM-derived fractions, P2 and P3, on BzATP stimulation of microglial cells (Figure 3D). Interestingly, FACS analysis of intact P2, P3 and P4 vesicles for A-SMase revealed immunoreactivity for the enzyme in P2 and P3 but not in P4 pellet (Figure 3E). P4 vesicles were negative for outer A-SMase also under non-stimulating conditions (not shown), when these vesicles have highest luminal concentration of the enzyme (Figure 3D). These results, together with the presence of A-SMase in P4 fraction (Figures 2D and 3D), indicated both association of the enzyme to the outer leaflet of MPs and luminal localisation of A-SMase in exosomes. Furthermore, FACS experiments on intact vesicles doubled labelled with A-SMase and annexin V revealed that A-SMase immunoreactivity represented 87±5% of total MPs exposing PS (not shown), indicating that the presence of the enzyme on MPs is instrumental for vesicle budding from PM.


Acid sphingomyelinase activity triggers microparticle release from glial cells.

Bianco F, Perrotta C, Novellino L, Francolini M, Riganti L, Menna E, Saglietti L, Schuchman EH, Furlan R, Clementi E, Matteoli M, Verderio C - EMBO J. (2009)

A-SMase is activated downstream of P2X7R stimulation. (A) Time course of A-SMase activation by 100 μM BzATP versus not stimulated cells as control, determined in cell lysates by measuring hydrolysis of sphingomyelin to phosphorylcholine at pH 5.5. Values are expressed as fold increases over basal A-SMase activity (1.12±0.3 nmol/mg h−1) conventionally indicated as 1 (n=3). (B) A-SMase translocation onto the PM on P2X7R stimulation as determined by FACS analysis of intact N9 microglial cells. The relative fluorescence intensity (RFI) was calculated as ratio of sample mean fluorescence over negative control mean fluorescence. The RFI values reported in the panels are the mean±s.e.m. measured in the three experiments. The results shown are from one out of three experiments. (C) Surface exposure of A-SMase on P2X7R stimulation, as detected by western blotting of biotinylated PM proteins with the A-SMase antibody. (D) Western blot analysis for A-SMase of P2, P3 and P4 vesicles accumulated in the supernatant of microglia cells under basal conditions or on stimulation with 100 μM BzATP for 30 min. (E) FACS analysis of intact P2, P3 and P4 vesicles for surface A-SMase, showing most of association of the enzyme to the outer leaflet of P2 and P3 MPs. (F) Spectrophotometric analysis of fluorescent MPs present in total supernatants collected from either FM1-43-labelled N9 microglial cells (black bars) or FM1-43-labelled N9 microglial clone, not expressing the P2X7R (white bars), 20 min after A-SMase (2 U/ml) or BzATP (100 μM) addition. (G) Quantitative analysis of FM1-43-labelled vesicles in the total supernatants of microglial cells exposed to exogenous A-SMase in the presence/absence of the P2X7R antagonist KN-62 or the ATP degrading enzyme apyrase.
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Related In: Results  -  Collection

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f3: A-SMase is activated downstream of P2X7R stimulation. (A) Time course of A-SMase activation by 100 μM BzATP versus not stimulated cells as control, determined in cell lysates by measuring hydrolysis of sphingomyelin to phosphorylcholine at pH 5.5. Values are expressed as fold increases over basal A-SMase activity (1.12±0.3 nmol/mg h−1) conventionally indicated as 1 (n=3). (B) A-SMase translocation onto the PM on P2X7R stimulation as determined by FACS analysis of intact N9 microglial cells. The relative fluorescence intensity (RFI) was calculated as ratio of sample mean fluorescence over negative control mean fluorescence. The RFI values reported in the panels are the mean±s.e.m. measured in the three experiments. The results shown are from one out of three experiments. (C) Surface exposure of A-SMase on P2X7R stimulation, as detected by western blotting of biotinylated PM proteins with the A-SMase antibody. (D) Western blot analysis for A-SMase of P2, P3 and P4 vesicles accumulated in the supernatant of microglia cells under basal conditions or on stimulation with 100 μM BzATP for 30 min. (E) FACS analysis of intact P2, P3 and P4 vesicles for surface A-SMase, showing most of association of the enzyme to the outer leaflet of P2 and P3 MPs. (F) Spectrophotometric analysis of fluorescent MPs present in total supernatants collected from either FM1-43-labelled N9 microglial cells (black bars) or FM1-43-labelled N9 microglial clone, not expressing the P2X7R (white bars), 20 min after A-SMase (2 U/ml) or BzATP (100 μM) addition. (G) Quantitative analysis of FM1-43-labelled vesicles in the total supernatants of microglial cells exposed to exogenous A-SMase in the presence/absence of the P2X7R antagonist KN-62 or the ATP degrading enzyme apyrase.
Mentions: To investigate the specific involvement of A-SMase in MP production, we directly measured A-SMase activity in microglia exposed for different time points to BzATP. Kinetic analysis of A-SMase activity revealed a peak of enzyme activity at 2 min after agonist addition (Figure 3A), indicating that the enzyme acts as a downstream effector of the receptor. Although A-SMase activation period is shorter than that of MP release (Figure 1F), it is plausible that increased generated ceramide has long-lasting effects on membrane fluidity required for P2X7-dependent blebbing. Activation of A-SMase by P2X7R was accompanied by enzyme translocation to the PM outer leaflet, as shown by the increased A-SMase staining detected by FACS in intact cells (Figure 3B). A-SMase translocation to the PM outer leaflet on P2X7R activation was confirmed by biotynilation and western blotting experiments that revealed surface expression of the enzyme in cultured astrocytes during BzATP exposure (Figure 3C). Finally, A-SMase translocation was also suggested by movement of the enzyme from the exosomal fraction (P4) to PM-derived fractions, P2 and P3, on BzATP stimulation of microglial cells (Figure 3D). Interestingly, FACS analysis of intact P2, P3 and P4 vesicles for A-SMase revealed immunoreactivity for the enzyme in P2 and P3 but not in P4 pellet (Figure 3E). P4 vesicles were negative for outer A-SMase also under non-stimulating conditions (not shown), when these vesicles have highest luminal concentration of the enzyme (Figure 3D). These results, together with the presence of A-SMase in P4 fraction (Figures 2D and 3D), indicated both association of the enzyme to the outer leaflet of MPs and luminal localisation of A-SMase in exosomes. Furthermore, FACS experiments on intact vesicles doubled labelled with A-SMase and annexin V revealed that A-SMase immunoreactivity represented 87±5% of total MPs exposing PS (not shown), indicating that the presence of the enzyme on MPs is instrumental for vesicle budding from PM.

Bottom Line: ATP-induced shedding and IL-1beta release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice.We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1beta release.Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1beta release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

View Article: PubMed Central - PubMed

Affiliation: CNR Institute of Neuroscience and Department of Medical Pharmacology, University of Milano, Milano, Italy.

ABSTRACT
We have earlier shown that microglia, the immune cells of the CNS, release microparticles from cell plasma membrane after ATP stimulation. These vesicles contain and release IL-1beta, a crucial cytokine in CNS inflammatory events. In this study, we show that microparticles are also released by astrocytes and we get insights into the mechanism of their shedding. We show that, on activation of the ATP receptor P2X7, microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATP-induced shedding and IL-1beta release are markedly reduced by the inhibition of acid sphingomyelinase, and completely blocked in glial cultures from acid sphingomyelinase knockout mice. We also show that p38 MAPK cascade is relevant for the whole process, as specific kinase inhibitors strongly reduce acid sphingomyelinase activation, microparticle shedding and IL-1beta release. Our results represent the first demonstration that activation of acid sphingomyelinase is necessary and sufficient for microparticle release from glial cells and define key molecular effectors of microparticle formation and IL-1beta release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

Show MeSH
Related in: MedlinePlus