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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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Morphological and biochemical characterisation of MPs released by cortical astrocytes. (A) Negative staining electron microscopy of P2 (bar, 300 nm), P3 (bar, 300 nm) and P4 (100 nm) vesicles pelleted from supernatant of astrocyte exposed to 100 μM BzATP for 20 min was carried out as described in Supplementary Figure 2. Number of analysed vesicles from three different preparations: n=50, P2; n=161, P3; n=184, P4. (B) Fluorescence images of P2, P3 and P4 vesicles stained by NBD, annexin V, the exosome marker CD63 and Na+/K+ ATPase or GLAST, as PM markers. Bar, 5 μm. (C) FACS of astrocyte-derived P2, P3 and P4 vesicles labelled by annexin V-PE and NBD. (D) Western blotting of P2, P3 and P4 lysates obtained from medium conditioned for 30 min by 100 μM BzATP-treated astrocytes for IL-1β, A-SMase, membrane (GLAST) and exosome (CD63, HSP70) markers. Vesicle were loaded as described in Supplementary data. HSP70 staining is below ECL detectability in P4 vesicles 30 min after BzATP stimulation, but become clearly detectable in P4 vesicles on longer conditioning (24 h). For IL-1β blotting, vesicle fractions were obtained from supernatant of astrocytes exposed to BzATP for 15 min. (E) Immunoblot analysis of A-SMase and Na+/K+ ATPase in glial lysates and PM-derived MPs isolated by annexin V-coated beads (Supplementary data).
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f2: Morphological and biochemical characterisation of MPs released by cortical astrocytes. (A) Negative staining electron microscopy of P2 (bar, 300 nm), P3 (bar, 300 nm) and P4 (100 nm) vesicles pelleted from supernatant of astrocyte exposed to 100 μM BzATP for 20 min was carried out as described in Supplementary Figure 2. Number of analysed vesicles from three different preparations: n=50, P2; n=161, P3; n=184, P4. (B) Fluorescence images of P2, P3 and P4 vesicles stained by NBD, annexin V, the exosome marker CD63 and Na+/K+ ATPase or GLAST, as PM markers. Bar, 5 μm. (C) FACS of astrocyte-derived P2, P3 and P4 vesicles labelled by annexin V-PE and NBD. (D) Western blotting of P2, P3 and P4 lysates obtained from medium conditioned for 30 min by 100 μM BzATP-treated astrocytes for IL-1β, A-SMase, membrane (GLAST) and exosome (CD63, HSP70) markers. Vesicle were loaded as described in Supplementary data. HSP70 staining is below ECL detectability in P4 vesicles 30 min after BzATP stimulation, but become clearly detectable in P4 vesicles on longer conditioning (24 h). For IL-1β blotting, vesicle fractions were obtained from supernatant of astrocytes exposed to BzATP for 15 min. (E) Immunoblot analysis of A-SMase and Na+/K+ ATPase in glial lysates and PM-derived MPs isolated by annexin V-coated beads (Supplementary data).

Mentions: We have earlier shown that stimulation of P2X7R by ATP or the selective agonist Benzoyl-ATP (BzATP) induces MP shedding from microglia cell surface (Bianco et al, 2005). In this study, we show that MP shedding occurs also in astrocytes. To study the dynamics of MP formation, we briefly labelled the lipid bilayers of primary cortical astrocytes or glial cell lines with the fluorescent styryl dye FM1–43 or with the fluorophore-conjugated phosphocholine compound NBD C6-HPC and observed the cells by time-lapse fluorescence video microscopy. By this approach, we observed not only the formation (Figure 1A) but also the shedding from the PM of variably sized vesicles on 100 μM BzATP exposure (Figure 1B and C; Supplementary Movie). To quantify the shedding process, supernatant of glial cells exposed to BzATP for 20 min were pelleted at 10 000 g, to separate MPs as pellet (Heijnen et al, 1999). Fluorescence of pelleted MPs was then quantified by spectrophotometric analysis (Supplementary Figure 1; see also Figure 2). Several perturbants were tested for their effects on MP shedding, including the P2X7 antagonists Brilliant Blue G (100 nM) and KN-62 (10 μM), the P2X2,4 antagonist TNP-ATP (3 nM), the Rho-effector kinase inhibitor Y-27632 dihydrochloride (100 μM) and the actin polymerisation inhibitor cytochalasin D (2 μg/ml), as well as perturbants of Ca2+ homeostasis, such as the Ca2+ chelators EDTA (500 μM) and BAPTA (30 μM) and the Ca2+ ionophore ionomycin (10 μM). Phorbol ester PMA (1 μM) was also used as positive control for the shedding process. The results of this analysis indicated that BzATP-induced MP shedding in astrocytes requires both P2X7R activation and cytoskeleton reorganisation, and it is dependent on both extracellular and cytoplasmic Ca2+ (Figure 1D and E).


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)

Morphological and biochemical characterisation of MPs released by cortical astrocytes. (A) Negative staining electron microscopy of P2 (bar, 300 nm), P3 (bar, 300 nm) and P4 (100 nm) vesicles pelleted from supernatant of astrocyte exposed to 100 μM BzATP for 20 min was carried out as described in Supplementary Figure 2. Number of analysed vesicles from three different preparations: n=50, P2; n=161, P3; n=184, P4. (B) Fluorescence images of P2, P3 and P4 vesicles stained by NBD, annexin V, the exosome marker CD63 and Na+/K+ ATPase or GLAST, as PM markers. Bar, 5 μm. (C) FACS of astrocyte-derived P2, P3 and P4 vesicles labelled by annexin V-PE and NBD. (D) Western blotting of P2, P3 and P4 lysates obtained from medium conditioned for 30 min by 100 μM BzATP-treated astrocytes for IL-1β, A-SMase, membrane (GLAST) and exosome (CD63, HSP70) markers. Vesicle were loaded as described in Supplementary data. HSP70 staining is below ECL detectability in P4 vesicles 30 min after BzATP stimulation, but become clearly detectable in P4 vesicles on longer conditioning (24 h). For IL-1β blotting, vesicle fractions were obtained from supernatant of astrocytes exposed to BzATP for 15 min. (E) Immunoblot analysis of A-SMase and Na+/K+ ATPase in glial lysates and PM-derived MPs isolated by annexin V-coated beads (Supplementary data).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: Morphological and biochemical characterisation of MPs released by cortical astrocytes. (A) Negative staining electron microscopy of P2 (bar, 300 nm), P3 (bar, 300 nm) and P4 (100 nm) vesicles pelleted from supernatant of astrocyte exposed to 100 μM BzATP for 20 min was carried out as described in Supplementary Figure 2. Number of analysed vesicles from three different preparations: n=50, P2; n=161, P3; n=184, P4. (B) Fluorescence images of P2, P3 and P4 vesicles stained by NBD, annexin V, the exosome marker CD63 and Na+/K+ ATPase or GLAST, as PM markers. Bar, 5 μm. (C) FACS of astrocyte-derived P2, P3 and P4 vesicles labelled by annexin V-PE and NBD. (D) Western blotting of P2, P3 and P4 lysates obtained from medium conditioned for 30 min by 100 μM BzATP-treated astrocytes for IL-1β, A-SMase, membrane (GLAST) and exosome (CD63, HSP70) markers. Vesicle were loaded as described in Supplementary data. HSP70 staining is below ECL detectability in P4 vesicles 30 min after BzATP stimulation, but become clearly detectable in P4 vesicles on longer conditioning (24 h). For IL-1β blotting, vesicle fractions were obtained from supernatant of astrocytes exposed to BzATP for 15 min. (E) Immunoblot analysis of A-SMase and Na+/K+ ATPase in glial lysates and PM-derived MPs isolated by annexin V-coated beads (Supplementary data).
Mentions: We have earlier shown that stimulation of P2X7R by ATP or the selective agonist Benzoyl-ATP (BzATP) induces MP shedding from microglia cell surface (Bianco et al, 2005). In this study, we show that MP shedding occurs also in astrocytes. To study the dynamics of MP formation, we briefly labelled the lipid bilayers of primary cortical astrocytes or glial cell lines with the fluorescent styryl dye FM1–43 or with the fluorophore-conjugated phosphocholine compound NBD C6-HPC and observed the cells by time-lapse fluorescence video microscopy. By this approach, we observed not only the formation (Figure 1A) but also the shedding from the PM of variably sized vesicles on 100 μM BzATP exposure (Figure 1B and C; Supplementary Movie). To quantify the shedding process, supernatant of glial cells exposed to BzATP for 20 min were pelleted at 10 000 g, to separate MPs as pellet (Heijnen et al, 1999). Fluorescence of pelleted MPs was then quantified by spectrophotometric analysis (Supplementary Figure 1; see also Figure 2). Several perturbants were tested for their effects on MP shedding, including the P2X7 antagonists Brilliant Blue G (100 nM) and KN-62 (10 μM), the P2X2,4 antagonist TNP-ATP (3 nM), the Rho-effector kinase inhibitor Y-27632 dihydrochloride (100 μM) and the actin polymerisation inhibitor cytochalasin D (2 μg/ml), as well as perturbants of Ca2+ homeostasis, such as the Ca2+ chelators EDTA (500 μM) and BAPTA (30 μM) and the Ca2+ ionophore ionomycin (10 μM). Phorbol ester PMA (1 μM) was also used as positive control for the shedding process. The results of this analysis indicated that BzATP-induced MP shedding in astrocytes requires both P2X7R activation and cytoskeleton reorganisation, and it is dependent on both extracellular and cytoplasmic Ca2+ (Figure 1D and E).

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