Purinergic Signaling Induces Cyclooxygenase-1-Dependent Prostanoid Synthesis in Microglia: Roles in the Outcome of Excitotoxic Brain Injury

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 E2 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 E2 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 E2 synthesis is dependent on P2X7 receptors, extracellular Ca2+ and cytoplasmic phospholipase A2. NMDA receptor activation induces ATP release from cultured neurons leading to microglial P2X7 receptor activation and COX-1 dependent prostaglandin E2 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 E2 receptor type 1. Similarly, the neuroprotection exerted by the prostaglandin E2 receptor type 2 agonist butaprost is not observed after COX-1 inhibition. P2X7 receptors contribute to NMDA induced prostaglandin E2 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

COX-1 inhibition reverses protection obtained by COX-2 inhibition, EP1 receptor deletion, and EP2 receptor activation.

<p>(<b>A</b>) Cortical lesion volumes were determined from thionine stained serial brain sections 24 hours after NMDA injection. Where indicated, animals received NS398 and/or SC560 as described in <i>Methods</i>. Vehicle consisted of 100 µl DMSO∶Saline (1∶9) administered i.p. (n = 6–9/group, * p<0.05). (<b>B</b>) NMDA lesion volumes in EP1^−/− mice systemically treated with vehicle (as in A) or SC560 (n = 5/group, * p<0.05 from vehicle). (<b>C</b>) Mice received the EP2 receptor agonist Butaprost (170 fmol) together with NMDA resulting in reduced lesion volumes. Lesions are enlarged in mice receiving SC560 (vehicle as in A; n = 9/group; * p<0.05 from vehicle). (<b>D</b>) Reduced lesion in nNOS^−/− mice is not reversed by SC560 (n = 6/group; n.s. = non significant).</p

NMDA triggers neuronal ATP release and induces PGE₂ production in neuron-microglia co-cultures.

<p>(<b>A</b>) Neurons were exposed to NMDA (1 mM) and extracellular ATP (ecATP) levels were determined in the supernatant (n = 8/group derived from 4 independent experiments, * p<0.05 from 0). (<b>B</b>) Co-cultured microglia/neurons or separately cultured microglial and neuronal sister cultures were exposed to ATP (1 mM) or NMDA (1 mM) for 30 minutes. Vehicle consisted of 5 µl PBS. PGE₂ was determined in supernatants (n = 12/group derived from 6 independent experiments, * p<0.05 from vehicle of the respective group). (<b>C</b>) wt neurons were co-cultured with wt or P2X7^−/− microglia and stimulated as described in B (n = 6/group derived from 3 independent experiments, * p<0.05 from vehicle of the respective group).</p

<i>In vivo</i> inhibition of P2X7 receptors blocks NMDA induced PGE₂ production and reverts the protective effect of NS398.

<p>(<b>A</b>) Animals were injected i.c.v. with saline (ve; 3 µl) or the P2X7 receptor blocker oxATP (300 nmol in 3 µl). Thirty minutes later NMDA was delivered stereotaxically to the parietal cortex. Brain tissue was collected 10 minutes after end of NMDA injection and PGE₂ tissue levels were determined (n = 5/group; * p<0.05 from NMDA+ve). (<b>B</b>) Animals were treated as in A and both groups received NS398 (20 mg/kg i.p.). Lesion volumes were determined 24 hours after NMDA injections (n = 5/group; * p<0.05 from NMDA+ve). Note the slightly increased lesion volumes in NS398+ve treated as compared to animals presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025916#pone-0025916-g002" target="_blank">Fig. 2A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025916#pone-0025916-g007" target="_blank">7C</a>, a fact that could be related to surgical procedures associated with i.c.v. injections. (<b>C</b>) Animals received NS398 (20 mg/kg i.p.) together with A438079 (200 µmol/kg in physiol. saline; i.p.) or vehicle (100 µl physiol. saline; i.p.). Lesion volumes were determined 24 hours after NMDA injections (n = 4/group; * p<0.05 from NMDA+ve).</p

ATP is an immediate inducer of microglial PGE₂ production.

<p>Cultured cortical microglia (<b>A</b>) or neurons (<b>B</b>) were exposed to NMDA (1 mM), glutamate (Glu; 300 µM), LPS (1 µg/ml) or ATP (1 mM) for 30 minutes. PGE₂ levels in supernatants were determined by ELISA (n = 6/group derived from 3 independent experiments, * p<0.05). (<b>C</b>) Microglia were exposed to LPS (1 µg/ml) for indicated time points and PGE₂ was assayed in the supernatant by ELISA (n = 6/group derived from 3 independent experiments). (<b>D</b>) Microglial COX-1 and COX-2 expression levels after LPS (1 µg/ml) treatment were determined by western blot analysis. Blots are representative for four independent experiments.</p

P2X7 receptors mediate ATP but not LPS induced PGE₂ release in microglial cultures.

<p>(<b>A</b>) Microglia were treated with ATP (1 mM) or the P2X7 receptor agonist BzATP (100 µM) alone or together with the P2X7 receptor inhibitor oxATP (1 mM), the PLA2 inhibitor AACOCF3 (20 µM), the COX-1 inhibitor SC560 (1 µM), or the Ca²⁺-independent PLA2 inhibitor BEL (10 µM). Some experiments were conducted in Ca²⁺-free extracellular solution (Ca-free). PGE₂ levels in supernatants were determined by ELISA (n = 8–12/group derived from 4–6 independent experiments, * p<0.05 from all others except ATP, BzATP, and BEL+ATP groups). (<b>B</b>) Microglia derived from wild type, COX1^−/−, or P2X7^−/− animals were stimulated with ATP (1 mM) for 30 minutes or LPS (1 µg/ml) for 16 hours. Vehicle consisted of 5 µl PBS. PGE₂ levels in supernatants were determined by ELISA (n = 6/group derived from 3 independent experiments, * p<0.05 from other groups with same treatment).</p