Abstract
Smith J, Menéndez Méndez A, Alves M, Parras A, Conte G, Bhattacharya A, Ceusters M, Nicke A, Henshall DC, Jimenez-Mateos EM, Engel T. Br J Pharmacol. 2023. Epub ahead of print. PMID: 36637008. doi:10.1111/bph.16033
Neonatal seizures represent a clinical emergency. However, current anti-seizure medications fail to resolve seizures in ∼50% of infants. The P2X7 receptor (P2X7 R) is an important driver of inflammation, and evidence suggests that P2X7 R contributes to seizures and epilepsy in adults. However, no genetic proof has yet been provided to determine what contribution P2X7 R makes to neonatal seizures, its effects on inflammatory signalling during neonatal seizures, and the therapeutic potential of P2X7R-based treatments on long-lasting brain excitability. Neonatal seizures were induced by global hypoxia in 7-day-old mouse pups (P7). The role of P2X7Rs during seizures was analysed in P2X7R-overexpressing and knockout mice. Treatment of wild-type mice after hypoxia with the P2X7 R antagonist JNJ-47965567 was used to determine the effects of the P2X7 R on long-lasting brain hyperexcitability. Cell type-specific P2X7 R expression was analysed in P2X7R-EGFP reporter mice. RNA sequencing was used to monitor P2X7R-dependent hippocampal downstream signalling. P2X7 R deletion reduced seizure severity, whereas P2X7 R overexpression exacerbated seizure severity and reduced responsiveness to anti-seizure medication. P2X7 R deficiency led to an anti-inflammatory phenotype in microglia, and treatment of mice with a P2X7 R antagonist reduced long-lasting brain hyperexcitability. RNA sequencing identified several pathways altered in P2X7 R knockout mice after neonatal hypoxia, including a down-regulation of genes implicated in inflammation and glutamatergic signalling. Treatments based on targeting the P2X7 R may represent a novel therapeutic strategy for neonatal seizures with P2X7Rs contributing to the generation of neonatal seizures, driving inflammatory processes and long-term hyperexcitability states.Background and Purpose:
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Commentary
Some 60 years ago Geoff Burnstock, a young, goateed junior faculty, was working with a smooth muscle prep of the guinea pig colon and noticed something odd. When he stimulated the nerve innervating the muscle in the presence of both adrenergic and muscarinic receptor antagonists, he saw a small tetrodotoxin-sensitive muscle hyperpolarization when he expected no response at all based on the current autonomic nervous system dichotomy at the time. With a decade’s further work and recalling an observation made 35 years prior by Szent-Györgyi, he pitched a controversial new class of neurotransmitters that was exemplified by ATP, which he called a “purinergic signaling” system.
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He was laughed out of many rooms for this preposterous proposal. The idea slowly took root, however, and he was eventually thoroughly vindicated by the cloning of an extensive family of 15 purinergic receptors. This family is similar to other neurotransmitter receptor families in that members include both G-protein coupled receptors and ligand-gated ion channels. The P2X7 receptor is an ATP-gated cation channel. In contrast to other P2X receptors, high concentrations of ATP are required to activate P2X7 receptors. This receptor might be stimulated by vesicular release of AT
Knowing all this, Smith and colleagues exploited the connection between hypoxia and ATP release to explore a role for P2X7 receptor activation in seizures produced in neonatal mice by an episode of mild hypoxia. 3 Global hypoxia was induced in 7-day-old pups by switching from room air to 95% N2/5% O2 for 15 minutes, which elicited a peculiar behavior featuring automatisms with occasional spasms that was interpreted as a seizure and confirmed by cortical surface EEG. Following the return to normoxia, seizures continued for at least 75 minutes. A major finding of this paper is that seizure intensity in the post-hypoxic period, scored both behaviorally and by EEG, was reduced in P2X7 receptor knockout mice and conversely enhanced in mice overexpressing P2X7 receptors under control of a BAC-derived P2RX7 promoter. These experiments are thorough and supplement the groups’ previous finding that 2 different P2X7 receptor antagonists, when administered before the hypoxic episode, reduce seizure severity. 4 In their previous study, 4 both behavioral and electrographic seizures were attenuated by P2X7 receptor block during the period of hypoxia, whereas in the current study, 3 seizure severity in the hypoxic period itself was reported to be unaffected in the P2X7 KO. However, inspection of Fig 1 k in this study reveals visibly smaller electrographic seizure power during the hypoxic period in the P2X7 receptor knockout mice that did not reach statistical significance. This potential discrepancy between the 2 studies likely reflects technical challenges with this P7 mouse seizure model and possibly suboptimal sample sizes, but also demonstrates the value of performing both genetic and pharmacologic targeting of a protein—each approach addresses limitations of the other. As expected from this lab, procedures that increase confidence in the findings were built into the experimental design (e.g., prospective determination of group sizes by power analysis, breeding schemes that allow comparison among littermates of different genotypes, blinded evaluation of seizure phenotypes, requirement for homogeneity of variance in ANOVA before carrying out post-hoc tests). The overall conclusion from this 3 and the previous 4 study is that P2X7 receptor activation during and after a period of severe hypoxia exacerbates seizures in neonatal mice.
Phenobarbital (20 mg/kg intravenous) is still the first line therapy for neonatal seizures 5 although concerns with efficacy and safety remain. The authors confirm previous reports that phenobarbital (25 mg/kg intraperitoneally) reduces hypoxic seizures and go on to show that the anti-seizure effect of phenobarbital is nearly eliminated in P2X7-overexpressing mice. Given that hypoxia upregulates P2X7 receptors, 6 this could provide an explanation for the development of phenobarbital-resistant seizures in neonates. For balance one would like to have seen whether phenobarbital is more effective in the P2X7 knockouts.
Interestingly, brief hypoxia increased the severity of kainate-induced seizures tested 5 weeks later, and administration of a P2X7 receptor competitive antagonist, JNJ-47965567, daily for 7 days beginning after the hypoxic period eliminated this excess seizure intensity. This suggests a disease-modifying effect of P2X7, and the next question tackled was the general mechanism by which P2X7 receptors influence seizure threshold and severity.
Pre-hypoxic treatment with minocycline, a broad-spectrum antibiotic structurally related to tetracycline, prevented the excess seizure severity observed in the P2X7-overexpressing mice. Minocycline, like other tetracyclines, possesses anti-inflammatory and anti-apoptotic effects that have led to its evaluation in multiple clinical indications that have an inflammatory component, 7 so far without a payday. In the current study, P2X7 immunoreactivity in the overexpressing mice was limited to microglia and oligodendrocytes, with no observable expression by neurons or astrocytes. Hypoxia caused an increase in CD16, a microglial marker of the pro-inflammatory phenotype, but this was not observed in the P2X7 KO. Conversely, microglia in P2X7 KO mice overexpressed the anti-inflammatory microglial protein, CD206, after hypoxia, and this was not seen in the WT mice. These observations were interpreted, reasonably, in a tentative conclusion that P2X7 activation after hypoxia contributes to an inflammatory milieu that exacerbates seizures.
In further pursuit of mechanism, they found numerous genes to be modestly up- or downregulated in hippocampus 24 hours after hypoxia, most with <1.6-fold change in messenger RNA level. When comparing WT and P2X7 KO mice, the authors reported a number of genes that were differentially up- or downregulated following hypoxia that may relate to seizure susceptibility or epileptogenesis, such as glutamatergic signaling and inflammatory pathways. However, their observations that genes with altered expression levels after hypoxia were almost entirely different in WT and P2X7 KO mice, and that the average difference in expression changes was <30% between the genotypes is, to this reader, suggestive of a plethora of false positives in this particular experiment. Nevertheless, this study does provide potential ideas for more targeted mechanistic studies in the future.
Altogether, this study builds momentum toward the introduction of P2X7 receptor antagonists as adjunctive therapy for neonatal seizures, working alongside phenobarbital and other anti-seizure drugs. The paper adds a genetic argument to this therapeutic use, and so provides valuable complement to previous studies of P2X7 receptor antagonists in the same preclinical model. 4 Future attention to the occupancy-efficacy relationship in this and other efficacy models, and a search for conditions in which pathophysiology is dominated by P2X7 activation, may help entice pharmaceutical interest and push P2X7 into a clinical niche.
