P2X7 Mediates Superoxide Production in Primary Microglia and Is Up-regulated in a Transgenic Mouse Model of Alzheimer's Disease

Abstract

Primary rat microglia stimulated with either ATP or 2-and 3-O-(4-benzoylbenzoyl)-ATP (BzATPActivated microglia have been observed in patients suffering from both acute (stroke) and chronic (Alzheimer's disease) neurological disorders (1, 2). Microglia are believed to contribute to the progression of Alzheimer's disease (AD) 1 because these cells can release pro-inflammatory substances known to induce neurotoxicity (3). Reactive oxygen intermediates (ROIs), one of several pro-inflammatory substances released by microglia (4), are likely to play a very important role in AD because hallmark modifications of ROI damage such as lipid peroxidation and nitrotyrosine conjugates are characteristic of post-mortem AD brains (3). Hence, pro-inflammatory stimuli that promote microglial ROI production might contribute to the pathogenesis of AD.ATP is an important messenger in the brain and can be released from cells by both lytic and non-lytic mechanisms (5). ATP evokes a variety of biological responses in microglia (6 -9).The effects of ATP are mediated through interactions with the P2 purinoceptors, broadly classified into P2Y metabotropic and P2X ionotropic receptors (10). The P2Y receptors are G proteincoupled and P2X receptors are ligand-gated ion channels (10). Whereas the P2Y receptors are responsible for Ca 2ϩ release predominantly from intracellular stores, P2X receptors are responsible for Ca 2ϩ influx from extracellular sources. Microglia possess both P2Y and P2X receptors (11-13). The P2X 7 receptor is highly expressed by cells of the macrophage lineage, such as dendritic cells, alveolar macrophages, and microglia. Activation of the P2X 7 receptor is unique in triggering the formation of large nonselective membrane pores, permeable to molecules up to 900 Da which ultimately results in death of the cell (9, 14). ATP and ATP analogs have been used to characterize the role of P2 receptors in microglial activation. Micromolar concentrations of ATP are required to activate the P2Y receptors, whereas millimolar (1-5 mM) concentrations of ATP are required to activate the P2X receptors. The ATP analog BzATP is a selective agonist at the P2X receptor and does not bind P2Y receptors (15,16). Oxidized ATP (oATP) is a specific antagonist of P2X 7 that binds irreversibly to the receptor and prevents its activation by ATP (17). In this study, these pharmacological tools were used to determine the purinergic receptors involved in O 2. production in microglia.The P2X 7 receptor plays a role in the generation of superoxide in microglia. Our studies elucidate a putative signal transduction pathway that mediates this response. These studies also demonstrate that BzATP-and ATP-activated microglia can mediate neurotoxicity. Finally, a distinct alteration was detected in the staining pattern for P2X 7 receptor in a transgenic mouse model of AD, suggesting that P2X 7 receptor activation could play a contributing role in AD. MATERIALS AND METHODSReagents-Reagents not specified otherwise were obtained from Sigma. PD98059, SB203580, LY2940...

Physiological and pathological functions of P2X7 receptor in the spinal cord

“…Consistent with this hypothesis an increase in P2X7R immunoreactivity has been observed in activated microglia of two transgenic models of Alzheimer's disease [71,72] in microglia/ macrophages of spinal cord undergoing multiple or amyotrophic lateral sclerosis [73], in ischemic cortical tissue [74], and in a model of kainite-induced seizures [62] suggesting that microglial P2X7R, together with P2X4R, might be a general mediator of stress during pathological states. Besides uptake and release of inflammatory molecules, other pathways might contribute to microglial effects on neuronal excitability and disease outcome.…”

Microglial Activation in Stroke: Therapeutic Targets

“…62 The P2X 7 receptors are expressed by resting microglia, and this expression is upregulated after brain injury. 63 Activation of P2X 7 receptors triggers microglia proliferation, 64,65 superoxide production, 66 release of interleukin 1␤ (IL-1␤), 67,68 and release of TNF␣. 64,69 However, microglia also exhibit reduced phagocytosis during P2X 7 receptor stimulation.…”

Adenosine 5′-triphosphate stimulates the increase of TGF-β1 in rat mesangial cells under high-glucose conditions via reactive oxygen species and ERK1/2

“…Primary rat microglial cells stimulated with ATP rapidly generate hydrogen peroxide (H 2 O 2 ) through the activation of NADPH oxidase. H 2 O 2 production was sustained for at least 90 min after stimulation [15] . Additionally, ATP-induced apoptosis in murine macrophages is mediated in part by ROS produced by NADPH oxidase [14] .…”

“…High glucose levels induce the generation of reactive oxygen species (ROS) by mesangial cells; ROS, Adenosine 5′-triphosphate stimulates the increase of TGF-β1 in rat mesangial cells under high-glucose conditions via reactive oxygen species and ERK1/2 in turn, activate mitogen-activated protein kinase (MAPK) in mesangial cells, leading to their secretion of transforming growth factor-β1 (TGF-β1) and fibronectin [13] . In addition, extracellular ATP has been shown to stimulate ROS generation in macrophages and microglia [14,15] . As there is evidence showing that mesangial cells express almost all P2 receptor subtypes, it became interesting to us to determine whether, under high-glucose conditions, ATP and P2 receptors mediate the production of ROS by mesangial cells.…”