Bianco, F. et al. Astrocyte-derived ATP induces vesicle shedding and IL-1 release from microglia. J. Immunol. 174, 7268-7277

Consiglio Nazionale delle Ricerche-Institute of Neuroscience, Cellular and Molecular Pharmacology and Department of Medical Pharmacology, University of Milan, Italy.
The Journal of Immunology (Impact Factor: 4.92). 07/2005; 174(11):7268-77.
Source: PubMed


ATP has been indicated as a primary factor in microglial response to brain injury and inflammation. By acting on different purinergic receptors 2, ATP is known to induce chemotaxis and stimulate the release of several cytokines from these cells. The activation of purinergic receptors 2 in microglia can be triggered either by ATP deriving from dying cells, at sites of brain injury or by ATP released from astrocytes, in the absence of cell damage. By the use of a biochemical approach integrated with video microscopy experiments, we investigated the functional consequences triggered in microglia by ATP released from mechanically stimulated astrocytes, in mixed glial cocultures. Astrocyte-derived ATP induced in nearby microglia the formation and the shedding of membrane vesicles. Vesicle formation was inhibited by the ATP-degrading enzyme apyrase or by P2X(7)R antagonists. Isolation of shed vesicles, followed by IL-1beta evaluation by a specific ELISA revealed the presence of the cytokine inside the vesicular organelles and its subsequent efflux into the extracellular medium. IL-1beta efflux from shed vesicles was enhanced by ATP stimulation and inhibited by pretreatment with the P2X(7) antagonist oxidized ATP, thus indicating a crucial involvement of the pore-forming P2X(7)R in the release of the cytokine. Our data identify astrocyte-derived ATP as the endogenous factor responsible for microvesicle shedding in microglia and reveal the mechanisms by which astrocyte-derived ATP triggers IL-1beta release from these cells.

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    • "In recent years there has been a growing awareness that adenosine triphosphate (ATP) is not only critical for intracellular phosphorylation-dependent processes but is also extensively involved in extracellular signalling. ATP is now recognised to be released by neurons (Fields, 2011), astrocytes (Coco et al., 2003; Stout et al., 2002; Zhang et al., 2007), and microglia (Imura et al., 2013), and play key roles in modulating pre-and post-synaptic function (Fields, 2011; Khakh, 2001), glia–glia interactions (Bianco et al., 2005; Pascual et al., 2012) and glia–neuron interactions (Fields and Burnstock, 2006). The physiological actions of ATP are principally mediated by what are referred to as P2 nucleotide receptors, which are divided into the ionotropic ligand gated P2X receptors (P2X1, 2, 3, 4, 5, 6, and 7) and the metabotropic P2Y receptors (P2Y1, 2, 4, 6, 11, 12, 13 and 14) (Khakh, 2001; Abbracchio et al., 2006; North, 2002; North and Surprenant, 2000; Ralevic and Burnstock, 1998). "
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    ABSTRACT: A number of studies have identified that mutations in the P2X7 receptor occur with a significantly higher incidence in individuals with major depression. Consistent with these findings, a number of preclinical studies have identified that mice in which the P2X7 receptor has been deleted exhibit a higher level of resilience-like behaviour to acutely aversive situations. At present, however, no studies have examined changes in P2X7 receptor expression in otherwise healthy animals exposed to persistently stressful situations. This is significant as several lines of evidence have demonstrated that it is exposure to persistently aversive, rather than acutely aversive, situations that is associated with the emergence of mood disturbance. Accordingly, the objective of the current study was to examine whether chronic exposure to restraint stress was associated with alterations in the expression of P2X7 within the hippocampal formation. The study involved three principal groups: acute stress (1 session), chronic stress (21 sessions, 1 per day) and a chronic stress with recovery group (21 sessions, 1 per day followed by 7 days of no stress) and appropriate control groups. The results of the analysis indicate that all forms of stress, regardless of the duration, provoked a reduction in P2X7 receptor expression. Comparative analysis on normalised data indicated that the magnitude of the P2X7 reduction was significantly greater in the chronic stress relative to the acute stress group. We additionally found that there was a gradual rebound in P2X7 expression, in two of nine regions examined, in animals that were allowed to recover for 7 days following the final stress session. Collectively, these findings provide the first evidence that exposure to chronic restraint stress produces a pronounced and relatively persistent suppression of the P2X7 receptor within the hippocampus.
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    • "Several mechanisms have now been identifi ed showing that microglia actively infl uence the synaptic properties in hippocampal slices. For instance, upon stimulation with the nucleotide ATP, cultured microglia can rapidly shed micro-vesicles, most probably by a mechanism depending on the P2X7 purinergic receptor (Bianco et al. 2005 ). When these vesicles were applied to cultured hippocampal neurons, they increased the frequency of mEPSCs (Antonucci et al. 2012 ), raising the hypothesis that neurotransmission could be regulated by physical contacts. "
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    ABSTRACT: Microglia are the resident immune cells of the brain. As such, they rapidly detect changes in normal brain homeostasis and accurately respond by fine- tuning in a tightly regulated manner their morphology, gene expression, and functional behaviour. Depending on the nature of these changes, microglia can thicken and retract their processes, proliferate and migrate, release numerous signalling factors and compounds influencing neuronal physiology (e.g., cytokines and trophic factors), in addition to secreting proteases able to transform the extracellular matrix, and phagocytosing various types of cellular debris, etc. Because microglia also transform rapidly (on a time scale of minutes) during experimental procedures, studying these very special cells requires methods that are specifically non-invasive. The development of such methods has provided unprecedented insights, these past few years, into the roles of microglia during normal physiological conditions. In particular, transcranial two-photon in vivo imaging revealed that presumably “resting” microglia continuously survey the brain parenchyma with their highly motile processes, in addition to modulating their structural and functional interactions with neuronal circuits along the changes in neuronal activity and behavioural experience occurring throughout the lifespan. In this chapter, we will describe how surveillant microglia interact with synaptic elements, and modulate the number, maturation, function, and plasticity of synapses in the healthy developing, mature, and aging brain, with consequences on neuronal activity, learning and memory, and the behavioural outcome.
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    • "Apyrase hydrolyses tri-and di-phosphate nucleotides (Kurpius et al., 2007; Warny et al., 2001) and should therefore abrogate both ATP-and UDP-signaling. Because ATP release is also known to be involved in early inflammatory responses to LPS stimulation (such as IL-1b release; Bianco et al., 2005; Inoue, 2002), and because we have reported previously that the majority of neurons are lost between 2 and 3 days after LPSor LTA-treatment (Fricker et al., 2012a; Neher et al., 2011b; Neniskyte et al., 2011), apyrase was added to mixed cultures 2 days after LPS-or LTA-treatment. Apyrase efficiently inhibited neuronal loss measured at 3 days after stimulation with either LTA or LPS, without affecting the number of necrotic or apoptotic neurons (Fig. 1). "
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    ABSTRACT: Microglia activated through Toll-like receptor (TLR)-2 or -4 can cause neuronal death by phagocytosing otherwise-viable neurons—a form of cell death called “phagoptosis.” UDP release from neurons has been shown to provoke microglial phagocytosis of neurons via microglial P2Y6 receptors, but whether inhibition of this process affects neuronal survival is unknown. We tested here whether inhibition of P2Y6 signaling could prevent neuronal death in inflammatory conditions, and whether UDP signaling can induce phagoptosis of stressed but viable neurons. We find that delayed neuronal loss and death in mixed neuronal/glial cultures induced by the TLR ligands lipopolysaccharide (LPS) or lipoteichoic acid was prevented by: apyrase (to degrade nucleotides), Reactive Blue 2 (to inhibit purinergic signaling), or MRS2578 (to specifically block P2Y6 receptors). In each case, inflammatory activation of microglia was not affected, and the rescued neurons remained viable for at least 7 days. Blocking P2Y6 receptors with MRS2578 also prevented phagoptosis of neurons induced by 250 nM amyloid beta 1–42, 5 μM peroxynitrite, or 50 μM 3-morpholinosydnonimine (which releases reactive oxygen and nitrogen species). Furthermore, the P2Y6 receptor agonist UDP by itself was sufficient to stimulate microglial phagocytosis and to induce rapid neuronal loss that was prevented by eliminating microglia or inhibiting phagocytosis. In vivo, injection of LPS into rat striatum induced microglial activation and delayed neuronal loss and blocking P2Y6 receptors with MRS2578 prevented this neuronal loss. Thus, blocking UDP/P2Y6 signaling is sufficient to prevent neuronal loss and death induced by a wide range of stimuli that activate microglial phagocytosis of neurons. GLIA 2014
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