Signaling via the prostaglandin E2 receptor EP4 exerts neuronal and vascular protection in a mouse model of cerebral ischemia
Xibin Liang, Lu Lin, Nathaniel S. Woodling, Qian Wang, Christoph Anacker, Tingting Pan, Milton Merchant, Katrin Andreasson
Xibin Liang, Lu Lin, Nathaniel S. Woodling, Qian Wang, Christoph Anacker, Tingting Pan, Milton Merchant, Katrin Andreasson
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Research Article Neuroscience

Signaling via the prostaglandin E2 receptor EP4 exerts neuronal and vascular protection in a mouse model of cerebral ischemia

Abstract

Stroke is the third leading cause of death in the United States. Fewer than 5% of patients benefit from the only intervention approved to treat stroke. Thus, there is an enormous need to identify new therapeutic targets. The role of inducible cyclooxygenase (COX-2) activity in stroke and other neurologic diseases is complex, as both activation and sustained inhibition can engender cerebral injury. Whether COX-2 induces cerebroprotective or injurious effects is probably dependent on which downstream prostaglandin receptors are activated. Here, we investigated the function of the PGE2 receptor EP4 in a mouse model of cerebral ischemia. Systemic administration of a selective EP4 agonist after ischemia reduced infarct volume and ameliorated long-term behavioral deficits. Expression of EP4 was robust in neurons and markedly induced in endothelial cells after ischemia-reperfusion, suggesting that neuronal and/or endothelial EP4 signaling imparts cerebroprotection. Conditional genetic inactivation of neuronal EP4 worsened stroke outcome, consistent with an endogenous protective role of neuronal EP4 signaling in vivo. However, endothelial deletion of EP4 also worsened stroke injury and decreased cerebral reperfusion. Systemic administration of an EP4 agonist increased levels of activated eNOS in cerebral microvessels, an effect that was abolished with conditional deletion of endothelial EP4. Thus, our data support the concept of targeting protective prostaglandin receptors therapeutically after stroke.

Authors

Xibin Liang, Lu Lin, Nathaniel S. Woodling, Qian Wang, Christoph Anacker, Tingting Pan, Milton Merchant, Katrin Andreasson

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Figure 2

EP4 signaling protects neurons in ischemic and excitotoxic paradigms in vitro.

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EP4 signaling protects neurons in ischemic and excitotoxic paradigms in ...
See Results for detailed statistical reporting. (A) EP4 expression in mouse cortical neurons stained for MAP2 (see Supplemental Figure 1). Scale bar: 10 μm. (B) Rapid induction of phospho-CREB after AE1-329 (100 nM) stimulation of cortical neurons from day 10 in vitro C57B6 mice. A representative Western blot is shown; lanes were run on the same gel but were noncontiguous (white lines). (C) Effect of AE1-329 on CREB phosphorylation. Densitometry values were calculated relative to 0 minutes, assigned as 100% (gray line). (D) Dose response demonstrated a protective effect of AE1-329 (0–1,000 nM) in cortical neurons stimulated with 250 μM glutamate for 24 hours, assayed by LDH release. n = 6–8 per group. (E) AE1-329 rescued cortical neurons subjected to 3 hours of OGD and assayed for PI fluorescence relative to total Hoechst nuclei. Cotreatment with 1 μM H89 reversed EP4 neuroprotection at all doses. n = 6 per group. *P < 0.05; **P < 0.01. (F) In cortical neurons subjected to OGD for 3 hours, AE1-329 reduced caspase-3 activation; this was reversed with 1 μM H89 cotreatment. *P < 0.01 vs. AE1-329 plus vehicle. (G and H) Dose response of AE1-329 rescue of CA1 neurons in organotypic hippocampal slices subjected to 75 minutes OGD (G) or 1 hour NMDA (H; 10 μM) and assayed for PI fluorescence of the CA1 subregion (Supplemental Figure 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. 0 nM AE1-329.