Endothelial prostaglandin E₂ regulates neuronal injury after seizure via activation of astrocytes

Takako Takemiya

^{Medical Research Institute, Tokyo Women’s Medical University, Shinjuku, Tokyo 162-8666, Japan}

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Abstract

Astrocytes interact closely with neurons via glutamate; this astrocyte-neuron circuit may play a pivotal role in synaptic transmission. In addition, astrocytes contact vascular endothelial cells (ECs) with their end-feet; therefore, ECs may have some role in regulating neuronal activity via astrocytes in the brain. In our studies, we found that kainic acid (KA) microinjection induced the expression of microsomal prostaglandin E synthase-1 (mPGES-1) in venous ECs and the expression of the prostaglandin E₂ (PGE₂) receptor EP3 on astrocytes. Moreover, endothelial mPGES-1 exacerbated KA-induced neuronal injury in the mouse brain. In in vitro experiments, mPGES-1 produced PGE₂, which increased astrocytic Ca²⁺ levels and Ca²⁺-dependent glutamate release, thus aggravating neuronal injury. We found ECs had a role under pathological conditions and brain ECs are not merely a physiological barrier between the blood and brain; instead, they may also act as a signal transducer or amplifier. Moreover, the endothelium-astrocyte-neuron signaling pathway may be crucial for driving neuronal injury elicited by repetitive seizures and may be a new therapeutic target for epilepsy.

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Introduction

Prostaglandin E₂ (PGE₂) is an important modulator in inflammation. In the brain, PGE₂ is associated with neuroinflammation because PGE₂ is involved in pathological processes such as seizure and cerebral ischemia^1,2. PGE₂ is sequentially synthesized from arachidonic acid by cyclooxygenase (COX) and PGE₂ synthase (PGES). Inducible COX-2 expression is known to be associated with acute neurotoxicity^1-4 and is also involved in delayed proinflammatory activities, which aggravate the neuronal damage found in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), multiple sclerosis (MS) and Alzheimer’s disease (AD)^4,5. We find that COX-2 is induced in non-neuronal cells late after seizure and facilitates neuronal injury in the hippocampus⁶.

In this review, we show the induction of microsomal prostaglandin E synthase-1 (mPGES-1) in brain endothelial cells (ECs) as well as the role of endothelial mPGES-1 in neuronal loss in the hippocampus after seizures. Furthermore, we present a novel mechanism for exacerbation of neuronal injury by PGE₂ derived from endothelial mPGES-1 and discuss the intercellular signaling pathway among endothelia, astrocytes and neurons in the process.

Induction of COX-2 and mPGES-1 in the brain

PGE₂ is synthesized by mPGES-1 coupling with COX-2 in brain ECs in lipopolysaccharide (LPS)-induced fever⁷, and mPGES-1 is also co-induced with COX-2 during fever or inflammation^8-11. Moreover, we find that mPGES-1 is induced in the hippocampus after epileptic seizures caused by kainic acid (KA) microinjection¹². KA is an analogue of the excitatory amino acid glutamate, and it is used in research to investigate the mechanisms of hippocampal neuronal loss after seizures because it induces generalized convulsion and causes neuronal loss in the hippocampus after seizures¹³. Unilateral KA microinjection induces COX-2 in bilateral neurons in the hippocampi, but in ipsilateral blood vessels both at 8 h and 24 h after KA injection⁶. Moreover, mPGES-1 is also localized in the blood vessels at 8 h, lasting until 24 h after KA microinjection¹². Double immunostaining for both mPGES-1 and von Willebrand factor (an endothelial cell marker) shows that mPGES-1 is induced with COX-2 in the ECs for 48 h after the microinjection¹². Finally, neuronal loss is caused in the KA microinjection side⁶, therefore we judge that PGE₂ synthesized by endothelial COX-2 and mPGES-1 facilitates neuronal injury in the hippocampus. Meanwhile, general injection of KA or pilocarpine causes general convulsion and induces COX-2 protein 18 h or 24 h after injection in hippocampal neurons. In addition COX-2 inhibitor blocks the neuronal injury^14,15, suggesting that neuronal COX-2 has an effect to facilitate neuronal injury after strong seizure.

Endothelial mPGES-1 exacerbates neuronal injury

PGE₂ produced in ECs could have a direct effect on the adjacent astrocytes because brain ECs are surrounded by astrocytic end-feet¹⁶. In addition, several lines of evidence indicate that prostaglandin E receptors (EPs) are present on astrocytes, and exogenous PGE₂ immediately evokes Ca²⁺-dependent glutamate release from astrocytes¹⁷; therefore, astrocytes may be directly activated by endogenous PGE₂ to elevate the intracellular Ca²⁺ levels ([Ca²⁺]_i) through the EP receptors. Furthermore, astrocytes can modulate synaptic transmission through the release of glutamate^18-20, which may stimulate delayed neuronal injury after seizures²¹. Therefore, we hypothesized that PGE2 produced by endothelial mPGES-1 directly activated EP receptors on astrocytes, elevating the astrocytic [Ca²⁺]_i, subsequently evoking sustained glutamate release and ultimately facilitating neuronal injury.

We found that the PGE₂ concentration was significantly elevated by KA treatment in cultured hippocampal slices from wild type (wt) mice, but that increase was not observed in slices from mPGES-1 knockout mice (mPGES-1^−/−)²². The astrocytic [Ca²⁺]_i was significantly higher in the hippocampal CA3 region in the wt slice cultures than in the mPGES-1^−/− slice cultures²². These results suggest that the PGE₂ derived from mPGES-1 upregulates the astrocytic [Ca²⁺]_i in the hippocampal CA3 region.

Next, we examined the effects of each EP receptor antagonist and agonist on the [Ca²⁺]_i in astrocytes in the KA-treated wt and mPGES-1^−/− slice cultures. An EP3 receptor antagonist²³ decreased the [Ca²⁺]_i in astrocytes in the KA-treated wt slices²², conversely, an EP3 receptor agonist²³ increased the [Ca²⁺]_i in astrocytes in the KA-treated mPGES-1^−/− slices, suggesting that the EP3 receptor has a crucial role in astrocytic Ca²⁺ elevation²². EP3 immunoreactivity was rarely detected in the astrocytes in naive control mice; in contrast, it was enhanced in the astrocytic end-feet with swelling after KA microinjection²². In the mPGES-1^−/− mice, the end-feet also showed swelling, but the EP3 immunoreactivity was not increased as much as in the wt mice²², indicating that the EP3 receptor was locally induced by KA in hippocampal astrocytes, which might receive PGE₂ from ECs. Previous publications have reported that EP3 mRNA is expressed in cultured astrocytes²⁴ and that EP3 protein expression is induced in astrocytomas by interleukin-1β²⁵. These findings indicate that astrocytic EP3 receptors may be upregulated under pathological conditions, and endothelial PGE₂ may directly activate EP3 receptors on astrocytic end-feet in neurotoxic brain diseases, such as epileptic seizures. PGE₂ also acts on other three receptors, namely EP1, EP2 and EP4, and activation of their receptors has been found to contribute to PGE₂-mediated neurotoxicity²⁶. Block of EP1 receptor reduces proinflammatory responses and neuronal damage in the hippocampus after KA injection in mice²⁷. Moreover, selective EP2 antagonism by small molecules prevents up-regulation of COX-2 in microglia, leading to reduce neuronal injury induced by pilocarpine²⁸. In addition, EP4 receptor-associated protein promotes proinflammatory activation of microglia which modulates neuronal damage²⁹. Activation of these receptors is concerned with regulation of neuronal injury by PGE₂.

Furthermore, we observed that treatment with KA for 17 h dramatically increased the level of glutamate release in the wt slices but not in the mPGES-1^−/− slices²². To verify whether mPGES-1 regulates hippocampal neuronal death, we stained the cells with propidium iodide (PI). The results showed greater PI uptake in the CA3 region of the wt slices than in that of the mPGES-1^−/− slices²². This significant increase in PI uptake in the wt slices suggests that neuronal injury may be enhanced by mPGES-1, which regulates the Ca²⁺-dependent glutamate release from astrocytes.

We next added exogenous PGE₂ to the mPGES-1^−/− slices to validate the above findings on the endogenous PGE₂. PGE₂ enhanced the astrocytic [Ca²⁺]_i in the CA3 region²². Moreover, PGE₂ caused an increase in the glutamate concentration and exacerbated the PI uptake in the CA3 region²². These results indicate that the PGE₂ derived from mPGES-1 is an important mediator that regulates neuronal injury. Exogenous PGE₂ also increased neuronal [Ca²⁺]_i in co-cultures with astrocytes, but the increase was not found without astrocytes (unpublished data). In addition, the [Ca2+]i increase in neurons was observed to follow the [Ca²⁺]_i increase in astrocytes (unpublished data). These results suggest that PGE₂ indirectly increases the neuronal [Ca²⁺]_i via the astrocytic [Ca²⁺]_i increase and subsequent glutamate release. Finally, we investigated whether this PGE₂-evoked glutamate release occurred in a Ca²⁺-dependent manner. A membrane-permeable Ca²⁺ chelator, BAPTA-AM, diminished the increase in the [Ca²⁺]_i in the astrocytes in the wt slices and abolished the increase in glutamate concentration²². Moreover, BAPTA-AM partially ameliorated the neuronal injury in the CA3 region, suggesting that CA3 neuronal injury is locally regulated by Ca²⁺-dependent glutamate release from neighboring astrocytes²². Together, these results suggest that the PGE₂ produced by endothelial mPGES-1 activates the astrocytic EP3 receptors to elevate the [Ca²⁺]i in astrocytes, causing Ca²⁺-dependent glutamate release and facilitating neuronal injury²².

Intercellular signaling pathway among endothelia, astrocytes and neurons

Neuron-to-astrocyte signaling controls arterial blood flow in the brain^30-32. Conversely, there is also mounting evidence for dynamic astrocyte-to-neuron interactions; for example, astrocytes modulate synaptic transmission^18-20. The interactions are also involved in neuronal synchrony³³ and epileptic discharges^14,34, which contribute to a delayed neuronal injury after seizures²¹. Neurons are vulnerable to glutamate in the hippocampus, and it is thought to be mediated by N-methyl-D-aspartate (NMDA) receptors (NMDARs)³⁵. In particular, glutamate release from astrocytes activates the extrasynaptic NMDAR subunit NR2B, which induces neuronal currents21 or triggers neuronal loss^21,36,37. This suggests that extrasynaptic NR2B receptors play crucial roles in the neurotoxicity caused by the glutamate released from astrocytes. Conversely, neuronal glutamate activates astrocytic mGluR5 to cause an increase in [Ca²⁺]_i in astrocytes, which may in turn release glutamate and generate feedback to extrasynaptic NR2B²¹. Thus, the neuron-astrocyte circuit may amplify the glutamate signaling, which aggravates neuronal excitotoxicity following seizures.

In this review, we propose an advanced mechanism for excitotoxicity via ECs and astrocytes. We demonstrated that endothelial mPGES-1 regulated Ca²⁺ signaling in astrocytes and Ca²⁺-dependent glutamate release, consistent with the findings that application of exogenous PGE2 propagated astrocytic [Ca²⁺]_i and evoked Ca²⁺-dependent glutamate release¹⁷. However, PGE₂ alone did not increase astrocytic [Ca²⁺]_i (unpublished data); therefore, PGE₂ may require another factor, such as a concomitant activation of astrocytic EP3, to elevate [Ca²⁺]_i in astrocytes. Brain ECs are not merely a physiological barrier between the blood and brain; instead, they may also act as a signal transducer or amplifier. In particular, we found ECs had a role under pathological conditions, such as in epileptic seizure. The interaction among neurons, astrocytes and ECs may be key to understanding the processes of seizure-induced neuronal injury in epilepsy.

Conclusions

PGE₂ is synthesized by inducible mPGES-1 and COX-2 in vascular ECs in response to KA microinjection. In addition, endothelial PGE₂ activates astrocytic EP3 receptor to elevate [Ca²⁺]_i levels in astrocytes, causing Ca²⁺-dependent glutamate release which stimulates neuronal injury. This is a new mechanism underlying neuronal injury regulated by ECs; therefore, this review emphasizes that brain ECs act as a signal transducer or amplifier, especially, under pathological conditions, such as epileptic seizure. The analysis of the interactions among neurons, astrocytes and ECs provides a better understanding of the processes of seizure induced neuronal injury and will facilitate the development of new treatments.

Acknowledgments

This work was supported by KAKENHI (17K10064) from the Japan Society for the Promotion of Science.

Conflict of Interest

The authors declare no conflict of interest.

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