Hypoxia attenuates purinergic P2X receptor-induced inflammatory gene expression in brainstem microglia (original) (raw)
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Brain Research Reviews, 2005
Microglial cells are the major cellular elements with immune function inside the CNS and play important roles in orchestrating inflammatory brain response to hypoxia and trauma. Although a complete knowledge of the endogenous factors leading to a prompt activation of microglia is not yet available, activation of P2 purinoreceptors by extracellular ATP has been indicated as a primary factor in microglial response. A still unresolved question, however, is which subtype(s) of P2 receptors mediate(s) the response to ATP. By a combination of RT-PCR, Western blotting, and single-cell calcium imaging, we assessed the presence and the activity of P2 receptor subtypes in the mouse microglial cell line N9. All members of the P2 receptor family, including the recently reported receptor for sugar nucleotides (P2Y 14 ), were found to be present in these cells at mRNA and/or protein level. The functionality of the receptors was assessed by analysis of the calcium responses evoked by specific agonists both in N9 cells and in primary microglia from rat brain. Interestingly, a different functional profile of P2 receptors was observed in resting or in LPS-activated N9 cells. Overnight exposure to LPS increased P2Y 6 and P2Y 14 , decreased P2X 7 , and left unchanged P2Y 1 and P2Y 2,4 receptor activity. The change in the P2 receptor profile in activated cells suggests selective roles for specific P2 receptor subtypes in microglial activation triggered by LPS. We speculate that modulation of microglial cell function via subtype-selective P2 receptor ligands may open up new strategies in the therapeutic management of inflammatory neurological diseases characterized by abnormal microglia response. D 2004 Elsevier B.V. All rights reserved.
P2X7 receptor activation regulates microglial cell death during oxygen-glucose deprivation
Brain-resident microglia may promote tissue repair following stroke but, like other cells, they are vulnerable to ischemia. Here we identify mechanisms involved in microglial ischemic vulnerability. Using time-lapse imaging of cultured BV2 microglia, we show that simulated ischemia (oxygen-glucose deprivation; OGD) induces BV2 microglial cell death. Removal of extracellular Ca 2þ or application of Brilliant Blue G (BBG), a potent P2X7 receptor (P2X7R) antagonist, protected BV2 microglia from death. To validate and extend these in vitro findings, we assessed parenchymal microglia in freshly isolated hippocampal tissue slices from GFP-reporter mice (CX3CR1 GFP/þ ). We confirmed that calcium removal or application of apyrase, an ATP-degrading enzyme, abolished OGD-induced microglial cell death in situ, consistent with involvement of ionotropic purinergic receptors. Indeed, whole cell recordings identified P2X7R-like currents in tissue microglia, and OGD-induced microglial cell death was inhibited by BBG. These pharmacological results were complemented by studies in tissue slices from P2X7R null mice, in which OGD-induced microglia cell death was reduced by nearly half. Together, these results indicate that stroke-like conditions induce calcium-dependent microglial cell death that is mediated in part by P2X7R. This is the first identification of a purinergic receptor regulating microglial survival in living brain tissues. From a therapeutic standpoint, these findings could help direct novel approaches to enhance microglial survival and function following stroke and other neuropathological conditions.
Journal of Neurochemistry, 2003
Under normal and pathological conditions, brain cells release nucleotides that regulate a wide range of cellular responses due to activation of P2 nucleotide receptors. In this study, the effect of extracellular nucleotides on IFNc-induced NO release in murine BV-2 microglial cells was investigated. BV-2 cells expressed mRNA for metabotropic P2Y and ionotropic P2X receptors. Among the P2 receptor agonists tested, ATP, ADP, 2¢,3¢-O-(4-benzoylbenzoyl)-ATP (BzATP), and 2-methylthio-ATP (2-MeSATP), but not UTP, enhanced IFNc-induced iNOS expression and NO production, suggesting that the uridine nucleotide receptors P2Y 2 and P2Y 6 are not involved in this response. U0126, an antagonist for MEK1/2, a kinase that phosphorylates the extracellular signal-regulated kinases ERK1/2, decreased IFNc-induced NO production. BzATP, a potent P2X 7 receptor agonist, was more effective than ATP, ADP, or 2-MeSATP at enhancing IFNc-induced ERK1/2 phosphorylation. Consistent with activation of the P2X 7 receptor, periodate-oxidized ATP, a P2X 7 receptor antagonist, and suramin, a non-specific P2 receptor antagonist, inhibited the effect of ATP or BzATP on IFNc-induced NO production, whereas pyridoxal-phosphate-6-azophenyl-2¢,4¢-disulfonic acid (PPADS), an antagonist of several P2X receptor subtypes, was ineffective. These results suggest that activation of P2X 7 receptors may contribute to inflammatory responses in microglial cells seen in neurodegenerative diseases.
Antioxidants & Redox Signaling, 2006
Hypoxia/ischemic brain injury accompanies an inflammatory response involving an activation of glial cells. This study, using an in vitro model, investigated the signaling mechanisms mediating hypoxic responses of the two glial cell types (astrocytes and microglia) in relation to the expression of inducible nitric oxide synthase (iNOS). In cultures of rat brain microglia and astrocytes, hypoxia (8 h) followed by reoxygenation (24 h) (H/O) had little (microglia) or no (astrocytes) effect on the expression of iNOS. However, H/O elicited opposite effects on lipopolysaccharide (LPS) induction of iNOS in the two cell types: it potentiated LPS induction of iNOS in microglia but inhibited this response in astrocytes. Similar differential effects of hypoxia were observed on the production of tumor necrosis factor-␣ (TNF␣). In contrast, there was an upregulation of hemoxygenase-1 (HO-1), a counter-regulatory pathway, with astrocytes showing a bigger induction than microglia. While hypoxic activation of mitogen-activated protein kinases (MAPKs) was similar in the two glial types, the activation pattern of NFB was clearly different: hypoxia stimulated the activation of NFB pathway and NFB-dependent transcription in microglia but not in astrocytes. Lastly, the two cell types displayed differential vulnerabilities to hypoxia-induced cell death, the astrocytes being relatively more resistant than microglia.
Bioenergetic regulation of microglia
Glia, 2017
Microglia have diverse actions, ranging from synapse pruning in development to cytotoxic effects in disease. Brain energy metabolism and substrate availability vary under normal and disease states, but how these variations influence microglial function is relatively unknown. Microglia, like most other cell types, express the full complement of gene products required for both glycolytic and oxidative metabolism. Evidence suggests that microglia increase aerobic glycolysis and decrease respiration when activated by various stimuli. Mitochondrial function, glucose availability, and glycolytic rate influence pro-inflammatory gene expression at both transcriptional and post-translational levels. These effects are mediated through CtBP, an NADH-sensitive transcriptional co-repressor; through effects on NLRP3 inflammasome assembly and caspase-1 activation; through formation of advanced glycation end-products; and by less well-defined mechanisms. In addition to these transcriptional effects...
PLoS ONE, 2011
Cyclooxygenases (COX) are prostanoid synthesizing enzymes constitutively expressed in the brain that contribute to excitotoxic neuronal cell death. While the neurotoxic role of COX-2 is well established and has been linked to prostaglandin E 2 synthesis, the role of COX-1 is not clearly understood. In a model of N-Methyl-D-aspartic acid (NMDA) induced excitotoxicity in the mouse cerebral cortex we found a distinctive temporal profile of COX-1 and COX-2 activation where COX-1, located in microglia, is responsible for the early phase of prostaglandin E 2 synthesis (10 minutes after NMDA), while both COX-1 and COX-2 contribute to the second phase (3-24 hours after NMDA). Microglial COX-1 is strongly activated by ATP but not excitatory neurotransmitters or the Toll-like receptor 4 ligand bacterial lipopolysaccharide. ATP induced microglial COX-1 dependent prostaglandin E 2 synthesis is dependent on P2X7 receptors, extracellular Ca 2+ and cytoplasmic phospholipase A2. NMDA receptor activation induces ATP release from cultured neurons leading to microglial P2X7 receptor activation and COX-1 dependent prostaglandin E 2 synthesis in mixed microglial-neuronal cultures. Pharmacological inhibition of COX-1 has no effect on the cortical lesion produced by NMDA, but counteracts the neuroprotection exerted by inhibition of COX-2 or observed in mice lacking the prostaglandin E 2 receptor type 1. Similarly, the neuroprotection exerted by the prostaglandin E 2 receptor type 2 agonist butaprost is not observed after COX-1 inhibition. P2X7 receptors contribute to NMDA induced prostaglandin E 2 production in vivo and blockage of P2X7 receptors reverses the neuroprotection offered by COX-2 inhibition. These findings suggest that purinergic signaling in microglia triggered by neuronal ATP modulates excitotoxic cortical lesion by regulating COX-1 dependent prostanoid production and unveil a previously unrecognized protective role of microglial COX-1 in excitotoxic brain injury.
Microglia trigger astrocyte-mediated neuroprotection via purinergic gliotransmission
Scientific Reports, 2014
Microglia are highly sensitive to even small changes in the brain environment, such as invasion of non-hazardous toxicants or the presymptomatic state of diseases. However, the physiological or pathophysiological consequences of their responses remain unknown. Here, we report that cultured microglia sense low concentrations of the neurotoxicant methylmercury (MeHg low ) and provide neuroprotection against MeHg, for which astrocytes are also required. When exposed to MeHg low , microglia exocytosed ATP via p38 MAPK-and vesicular nucleotide transporter (VNUT)-dependent mechanisms. Astrocytes responded to the microglia-derived ATP via P2Y 1 receptors and released interleukin-6 (IL-6), thereby protecting neurons against MeHg low . These neuroprotective actions were also observed in organotypic hippocampal slices from wild-type mice, but not in slices prepared from VNUT knockout or P2Y 1 receptor knockout mice. These findings suggest that microglia sense and respond to even non-hazardous toxicants such as MeHg low and change their phenotype into a neuroprotective one, for which astrocytic support is required. M icroglia, the resident immune cells in the brain, continuously monitor the brain microenvironment 1,2 and immediately respond to CNS disorders. Microglia are characterized by a very low threshold of activation, and their activation occurs within a few tens of minutes 3 . Microglial activation is a common feature of the early stage of CNS disorders 4,5 , during which they migrate toward the lesion, phagocytose debris and mediate inflammatory reactions 6 . The P2 receptor, a receptor for extracellular nucleotides, is highly expressed in microglia and participates in these reactions 7,8 . Extracellular ATP and other nucleotides are released or leak from injured cells under pathological conditions. For example, ATP released from apoptotic neurons contributes a 'find-me' signal 9 that activates microglial P2Y 12 receptors and induces process extension 8 . In contrast, UDP functions as an 'eat-me' signal and triggers microglial phagocytosis via P2Y 6 receptors 7 . In addition to the nucleotide-activated microglial function, microglia can release ATP 10-12 . Furthermore, microglia-derived ATP has recently been shown to activate the astrocytic P2Y 1 receptor and trigger ATP release, which in turn regulates synaptic transmission 13 . P2Y 1 receptor activation in astrocytes has been reported to induce neuroprotective effects against oxidative stress 14 , photothrombosis 15 , trauma 16 and neurotoxicants 17 . Although the physiological consequences of purinergic signal-mediated gliotransmission between microglia and astrocytes have begun to be identified, their neuroprotective roles are mostly unknown.
Redox Control of Microglial Function: Molecular Mechanisms and Functional Significance
Antioxidants & Redox Signaling, 2014
Neurodegenerative diseases are characterized by chronic microglial over-activation and oxidative stress. It is now beginning to be recognized that reactive oxygen species (ROS) produced by either microglia or the surrounding environment not only impact neurons but also modulate microglial activity. In this review, we first analyze the hallmarks of pro-inflammatory and anti-inflammatory phenotypes of microglia and their regulation by ROS. Then, we consider the production of reactive oxygen and nitrogen species by NADPH oxidases and nitric oxide synthases and the new findings that also indicate an essential role of glutathione (c-glutamyl-lcysteinylglycine) in redox homeostasis of microglia. The effect of oxidant modification of macromolecules on signaling is analyzed at the level of oxidized lipid by-products and sulfhydryl modification of microglial proteins. Redox signaling has a profound impact on two transcription factors that modulate microglial fate, nuclear factor kappa-light-chain-enhancer of activated B cells, and nuclear factor (erythroid-derived 2)-like 2, master regulators of the pro-inflammatory and antioxidant responses of microglia, respectively. The relevance of these proteins in the modulation of microglial activity and the interplay between them will be evaluated. Finally, the relevance of ROS in altering blood brain barrier permeability is discussed. Recent examples of the importance of these findings in the onset or progression of neurodegenerative diseases are also discussed. This review should provide a profound insight into the role of redox homeostasis in microglial activity and help in the identification of new promising targets to control neuroinflammation through redox control of the brain.
Journal of Molecular Neuroscience, 2014
Microglia cells are the primary mediators of the CNS immune defense system and crucial for the outcome of shaping inflammatory responses. They are highly dynamic, moving constantly, and become activated by neuronal signaling under pathological conditions. They fulfill a dual role by not only regulating local neuroinflammation but also conferring neuronal protection. Gonadal steroids are known to exert anti-inflammatory effects in the CNS. Recently, we have shown that the microglial-like cell line BV-2 is hypoxia-sensitive and regulated by gonadal steroids. The present study used primary rat cerebral cortex-derived microglia to analyze whether this cell type directly perceive and respond to acute hypoxia. Second, we investigated whether 17βestradiol (E2) and progesterone (P) interfere with hypoxiainduced changes. Short-term hypoxia increased the expression of a subset of pro-inflammatory (TNFa, IL1b) and oxidative stress-related (Hif1a) genes. The induction of TNFa and IL1b was counteracted by P. Hypoxia shifted the primary microglia to the pro-inflammatory M1 phenotype. The administration of E2 and P favored the neuroprotective M2 phenotype. Our findings extend previous data obtained with BV-2 cells and show that the primary microglia directly perceive hypoxia which increase their inflammatory activity. Both steroid hormones directly and indirectly interact with the microglia cells by reducing the inflammatory scenario and stimulating neuroprotection.
Glia, 2013
Microglia are sensitive to environmental changes and are immediately transformed into several phenotypes. For such dynamic ''modal shifts'', purinergic receptors have central roles. When microglia sense ATP/ADP leaked from injured cells by P2Y 12 receptors, they are transformed into a moving phenotype, showing process extension and migration toward the injured sites. Microglia upregulate adenosine A 2A receptors, by which they retract the processes showing an amoeboid-shaped, activated phenotype. Microglia also upregulate P2Y 6 receptors, and if they meet UDP leaked from dead cells, microglia start to engulf and eat the dead cells as a phagocytic phenotype. The P2Y 12 receptor-mediated responses are modulated by other P2 or P1 receptors. In contrast, the P2Y 6 receptor-mediated responses were not influenced by P2Y 12 receptors and vice versa. Microglia appear to use purinergic signals either cooperatively or distinctively to cause their modal shifts.