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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-1b, 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 P2X 7 , microparticle shedding is associated with rapid activation of acid sphingomyelinase, which moves to plasma membrane outer leaflet. ATPinduced shedding and IL-1b 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-1b 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-1b release, thus, opening new strategies for the treatment of neuroinflammatory diseases.

The authors report that the sequence of an RNA interference oligonucleotide for Mcm21R/CENP-O was incorrectly annotated in the Supplementary Materials and Methods to the article cited above. We had reported the oligonucleotide (ATATGAGTCTGGTCTCCTA) to comprise positions 959-977 of the coding sequence of Mcm21R (as measured from the start codon) whereas it actually lies between positions 884-902. This error arose because several sequences for Mcm21R cDNA versions existed in the NCBI database in 2006. Now that the sequence has been curated, we noticed our error. We apologize for any inconvenience caused.

Microvesicles (MVs) are released from almost all cell brain types into the microenvironment and are emerging as a novel way of cell-to-cell communication. This review focuses on MVs discharged by microglial cells, the brain resident myeloid cells, which comprise ∼10–12% of brain population. We summarize first evidence indicating that MV shedding is a process activated by the ATP receptor P2X7 and that shed MVs represent a secretory pathway for the inflammatory cytokine IL-β. We then discuss subsequent findings which clarify how IL-1 β can be locally processed and released from MVs into the extracellular environment. In addition, we describe the current understanding about the mechanism of P2X7-dependent MV formation and membrane abscission, which, by involving sphingomyelinase activity and ceramide formation, may share similarities with exosome biogenesis. Finally we report our recent results which show that microglia-derived MVs can stimulate neuronal activity and participate to the propagation of inflammatory signals, and suggest new areas for future investigation.

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The triggering receptor expressed in myeloid (TREM) cells 2, referred to with the acronym TREM2, is a single-spanning membrane receptor of an immunoglobulin/lectin-like family. The members of this family operate through the interaction with a common coupling protein, DNAX-activation protein of 12 kDa (DAP12) which upon phosphorylation induces the activation of various intracellular signalling pathways (tyrosine kinases, phospholipase Cc). Activation of other Received December 20, 2008; revised manuscript received February 11, 2009; accepted April 21, 2009. Abbreviations used: BSA, bovine serum albumin; DAP12, DNAXactivation protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hHsp60, human heat shock protein 60; HRP, horseradish peroxidase; Hsp60, heat shock protein 60; PBS, phosphate-buffered saline; PLOSL, polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy; rHsp60, rat heat shock protein 60; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; siRNA, short interfering RNA; TREM, triggering receptor expressed in myeloid cells. AbstractTriggering receptor expressed in myeloid (TREM) cells 2, a receptor expressed by myeloid cells, osteoclasts and microglia, is known to play a protective role in bones and brain. Mutations of the receptor (or of its coupling protein, DAP12) sustain in fact a genetic disease affecting the two organs, the polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy (PLOSL or Nasu-Hakola disease). So far, specific agonist(s) of TREM2 have not been identified and its (their) transduction mechanisms are largely unknown. Heat shock protein 60 (Hsp60) is a mitochondrial chaperone that can also be harboured at the cell surface. By using constructs including the extracellular domain of TREM2 and the Fc domain of IgGs we have identified Hsp60 as the only TREM2-binding protein exposed at the surface of neuroblastoma N2A cells and astrocytes, and lacking in U373 astrocytoma. Treatment with Hsp60 was found to stimulate the best known TREM2-dependent process, phagocytosis, however, only in the microglial N9 cells rich in the receptor. Upon TREM2 down-regulation, the Hsp60-induced stimulation of N9 phagocytosis was greatly attenuated. Hsp60 is also released by many cell types, segregated within exosomes or shedding vesicles which might then undergo dissolution. However, the affinity of its binding (K d = 3.8 lM) might be too low for the soluble chaperone released from the vesicles to the extracellular space to induce a significant activation of TREM2. It might in contrast be appropriate for the binding of TREM2 to Hsp60 exposed at the surface of cells closely interacting with microglia. The ensuing stimulation of phagocytosis could play protective effects on the brain. Keywords: receptor binding, surface-harboured agonist, western and far-western blotting, phagocytosis, polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy.

Microglia, glial cells with an immunocompetent role in the CNS, react to stimuli from the surrounding environment with alterations of their phenotypic response. Amongst other activating signals, the endotoxin lipopolysaccharide (LPS) is widely used as a tool to mimic bacterial infection in the CNS. LPS-activated microglia undergo dramatic changes in cell morphology/activity; in particular, they stop proliferating and differentiate from resting to effector cells. Activated microglia also show modifications of purinoreceptor signalling with a significant decrease in P2X 7 expression. In this study, we demonstrate that the down-regulation of the P2X 7 receptor in activated microglia may play an important role in the antiproliferative effect of LPS. Indeed, chronic blockade of the P2X 7 receptor by antagonists (oxidized ATP, KN62 and Brilliant Blue G), or treatment with the ATP-hydrolase apyrase, severely decreases microglial proliferation, down-regulation of P2X 7 receptor expression by small RNA interference (siRNA) decreases cell proliferation, and the proliferation of P2X 7 -deficient N9 clones and primary microglia, in which P2X 7 expression is down-regulated by siRNA, is unaffected by either LPS or P2X 7 antagonists. Furthermore, flow cytometric analysis indicates that exposure to oxidized ATP or treatment with LPS reversibly decreases cell cycle progression, without increasing the percentage of apoptotic cells. Overall, our data show that the P2X 7 receptor plays an important role in controlling microglial proliferation by supporting cell cycle progression. The P2X 7 receptor is an ATP-gated ion channel that has the unusual property of losing its ion selectivity and undergoing dilation to form a non-selective pore during prolonged ATP exposure. Because of this property, the P2X 7 receptor causes massive calcium entry, and this is often linked to an apoptotic fate of the cell (Di Virgilio 1995;Ferrari et al. 1997a). However, recent data have shown that tonic activation of this receptor mediates a proliferative rather than a death signal. The activation of transfected P2X 7 receptors by endogenously released ATP has been shown to enhance cell proliferation in human lymphoblastoid cells (Baricordi et al. 1999). The mechanism by which P2X 7 receptor stimulates cell growth has been recently linked to an increase in cellular energy stores (Adinolfi et al. 2005). The P2X 7 receptor is highly expressed in microglia, the resident cells of the brain which contribute to innate immunity. Microglial cells are normally in a resting state but, during infection or after traumatic stimuli, they differentiate into effector cells and acquire cytotoxic/phagocytic features to fight the pathogenic stimulus (Miyake 2004). Microglial activation is a complex process which proceeds through several steps and involves multiple factors, including cytokines and a wide variety of bacterial components (Norenberg et al. 1997 Abbreviations used: BAPTA-AM, acetoxymethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid; BzATP, 2...

It is now well-established that an active cross-talk occurs between neurons and glial cells, in the adult as well as in the developing and regenerating nervous systems. These functional interactions not only actively modulate synaptic transmission, but also support neuronal growth and differentiation. We have investigated the possible existence of a reciprocal interaction between inner ear vestibular neurons and Schwann cells maintained in primary cultures. We show that ATP released by the extending vestibular axons elevates intracellular calcium levels within Schwann cells. Purinergic activation of the Schwann P2X(7) receptor induces the release of neurotrophin BDNF, which occurs via a regulated, tetanus-toxin sensitive, vesicular pathway. BDNF, in turn, is required by the vestibular neuron to support its own survival and growth. Given the massive release of ATP during tissue damage, cross-talk between vestibular neurons and Schwann cells could play a primary role during regeneration.