Endothelial prostaglandin E2 regulates neuronal injury after seizure via activation of astrocytes
Takako TakemiyaMedical Research Institute, Tokyo Women’s Medical University, Shinjuku, Tokyo 162-8666, Japan
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.
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 ischemia1,2. 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 neurotoxicity1-4 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 inflammation8-11. 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 injury14,15, 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 glutamate18-20, 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 brain30-32. Conversely, there is also mounting evidence for dynamic astrocyte-to-neuron interactions; for example, astrocytes modulate synaptic transmission18-20. The interactions are also involved in neuronal synchrony33 and epileptic discharges14,34, 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 loss21,36,37. 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.
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.
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.
- Takemiya T, Suzuki K, Sugiura H, et al. Inducible brain COX-2 facilitates the recurrence of hippocampal seizures in mouse rapid kindling. Prostaglandins Other Lipid Mediat. 2003; 71(3-4): 205-16.
- Sasaki T, Kitagawa K, Yamagata K, et al. Amelioration of hippocampal neuronal damage after transient forebrain ischemia in cyclooxygenase-2-deficient mice. J Cereb Blood Flow Metab. 2004; 24(1): 107-13.
- Takemiya T, Matsumura K, Yamagata K. Roles of prostaglandin synthesis in excitotoxic brain diseases. Neurochem Int. 2007; 51(2-4): 112-20.
- Takemiya T, Yamagata K. Chapter 4. Effects of COX-2 Inhibitors on Brain Diseases. in Trends in COX-2 Inhibitor Research. New York, NY, USA: Nova Science Publishers, 2007.
- Takemiya T, Yamagata K. Chapter 3. The Modulatory Role of COX-2 and Prostaglandins in Brain Diseases. in Prostaglandins: New Research. New York, NY, USA: Nova Science Publishers, 2006.
- Takemiya T, Maehara M, Matsumura K, et al. Prostaglandin E2 produced by late induced COX-2 stimulates hippocampal neuron loss after seizure in the CA3 region. Neurosci Res. 2006; 56(1): 103-10.
- Yamagata K, Matsumura K, Inoue W, et al. Coexpression of microsomal-type prostaglandin E synthase with cyclooxygenase-2 in brain endothelial cells of rats during endotoxin-induced fever. J Neurosci. 2001; 21(8): 2669-77.
- Engblom D, Saha S, Engstrom L, et al. Microsomal prostaglandin E synthase-1 is the central switch during immune-induced pyresis. Nat Neurosci. 2003; 6(11): 1137-8.
- Trebino CE, Stock JL, Gibbons CP, et al. Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase. Proc Natl Acad Sci U S A. 2003; 100(15): 9044-9.
- Inoue W, Matsumura K, Yamagata K, et al. Brain-specific endothelial induction of prostaglandin E(2) synthesis enzymes and its temporal relation to fever. Neurosci Res. 2002; 44(1): 51-61.
- Ek M, Engblom D, Saha S, et al. Inflammatory response: pathway across the blood-brain barrier. Nature. 2001; 410(6827): 430-1.
- Takemiya T, Matsumura K, Sugiura H, et al. Endothelial microsomal prostaglandin E synthase-1 exacerbates neuronal loss induced by kainate. J Neurosci Res. 2010; 88(2): 381-90.
- Cavalheiro EA, Riche DA, Le Gal La Salle G. Long-term effects of intrahippocampal kainic acid injection in rats: a method for inducing spontaneous recurrent seizures. Electroencephalogr Clin Neurophysiol. 1982; 53(6): 581-9.
- Kang N, Xu J, Xu Q, et al. Astrocytic glutamate release-induced transient depolarization and epileptiform discharges in hippocampal CA1 pyramidal neurons. J Neurophysiol. 2005; 94(6): 4121-30.
- Trandafir CC, Pouliot WA, Dudek FE, et al. Co-administration of subtherapeutic diazepam enhances neuroprotective effect of COX-2 inhibitor, NS-398, after lithium pilocarpine-induced status epilepticus. Neuroscience. 2015; 284: 601-10.
- Janzer RC, Raff MC. Astrocytes induce blood-brain barrier properties in endothelial cells. Nature. 1987; 325(6101): 253-7.
- Bezzi P, Carmignoto G, Pasti L, et al. Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature. 1998; 391(6664): 281-5.
- Volterra A, Steinhauser C. Glial modulation of synaptic transmission in the hippocampus. Glia. 2004; 47(3): 249-57.
- Haydon PG, Carmignoto G. Astrocyte control of synaptic transmission and neurovascular coupling. Physiol Rev. 2006; 86(3): 1009-31.
- Perea G, Araque A. Astrocytes potentiate transmitter release at single hippocampal synapses. Science. 2007; 317(5841): 1083-6.
- Ding S, Fellin T, Zhu Y, et al. Enhanced astrocytic Ca2+ signals contribute to neuronal excitotoxicity after status epilepticus. J Neurosci. 2007; 27(40): 10674-84.
- Takemiya T, Matsumura K, Sugiura H, et al. Endothelial microsomal prostaglandin E synthase-1 facilitates neurotoxicity by elevating astrocytic Ca2+ levels. Neurochem Int. 2011; 58(4): 489-96.
- Amano H, Hayashi I, Endo H, et al. Host prostaglandin E(2)-EP3 signaling regulates tumor-associated angiogenesis and tumor growth. J Exp Med. 2003; 197(2): 221-32.
- Kitanaka J, Hashimoto H, Gotoh M, et al. Expression pattern of messenger RNAs for prostanoid receptors in glial cell cultures. Brain Res. 1996; 707(2): 282-7.
- Waschbisch A, Fiebich BL, Akundi RS, et al. Interleukin-1 beta-induced expression of the prostaglandin E-receptor subtype EP3 in U373 astrocytoma cells depends on protein kinase C and nuclear factor-kappaB. J Neurochem. 2006; 96(3): 680-93.
- Dey A, Kang X, Qiu J, et al. Anti-Inflammatory Small Molecules To Treat Seizures and Epilepsy: From Bench to Bedside. Trends Pharmacol Sci. 2016; 37(6): 463-84.
- Rojas A, Gueorguieva P, Lelutiu N, et al. The prostaglandin EP1 receptor potentiates kainate receptor activation via a protein kinase C pathway and exacerbates status epilepticus. Neurobiol Dis. 2014; 70: 74-89.
- Jiang J, Ganesh T, Du Y, et al. Small molecule antagonist reveals seizure-induced mediation of neuronal injury by prostaglandin E2 receptor subtype EP2. Proc Natl Acad Sci U S A. 2012; 109(8): 3149-54.
- Fujikawa R, Higuchi S, Nakatsuji M, et al. EP4 Receptor-Associated Protein in Microglia Promotes Inflammation in the Brain. Am J Pathol. 2016; 186(8): 1982-8.
- Zonta M, Angulo MC, Gobbo S, et al. Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation. Nat Neurosci. 2003; 6(1): 43-50.
- Takano T, Tian GF, Peng W, et al. Astrocyte-mediated control of cerebral blood flow. Nat Neurosci. 2006; 9(2): 260-7.
- Iadecola C, Nedergaard M. Glial regulation of the cerebral microvasculature. Nat Neurosci. 2007; 10(11): 1369-76.
- Fellin T, Pascual O, Gobbo S, et al. Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors. Neuron. 2004; 43(5): 729-43.
- Tian GF, Azmi H, Takano T, et al. An astrocytic basis of epilepsy. Nat Med. 2005; 11(9): 973-81.
- Kambe Y, Nakamichi N, Georgiev DD, et al. Insensitivity to glutamate neurotoxicity mediated by NMDA receptors in association with delayed mitochondrial membrane potential disruption in cultured rat cortical neurons. J Neurochem. 2008; 105(5): 1886-900.
- Hardingham GE, Fukunaga Y, Bading H. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat Neurosci. 2002; 5(5): 405-14.
- Vizi ES, Fekete A, Karoly R, et al. Non-synaptic receptors and transporters involved in brain functions and targets of drug treatment. Br J Pharmacol. 2010; 160(4): 785-809.