The P2X7 receptor contributes to seizures and inflammation‐driven long‐lasting brain hyperexcitability following hypoxia in neonatal mice

Abstract

Background and Purpose Neonatal seizures represent a clinical emergency. However, current anti‐seizure medications fail to resolve seizures in ~50% of infants. The P2X7 receptor (P2X7R) is an important driver of inflammation, and evidence suggests that P2X7R contributes to seizures and epilepsy in adults. However, no genetic proof has yet been provided to determine what contribution P2X7R 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. Experimental Approach 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 P2X7R antagonist JNJ‐47965567 was used to determine the effects of the P2X7R on long‐lasting brain hyperexcitability. Cell type‐specific P2X7R expression was analysed in P2X7R‐EGFP reporter mice. RNA sequencing was used to monitor P2X7R‐dependent hippocampal downstream signalling. Key Results P2X7R deletion reduced seizure severity, whereas P2X7R overexpression exacerbated seizure severity and reduced responsiveness to anti‐seizure medication. P2X7R deficiency led to an anti‐inflammatory phenotype in microglia, and treatment of mice with a P2X7R antagonist reduced long‐lasting brain hyperexcitability. RNA sequencing identified several pathways altered in P2X7R knockout mice after neonatal hypoxia, including a down‐regulation of genes implicated in inflammation and glutamatergic signalling. Conclusion and Implications Treatments based on targeting the P2X7R 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.

The P2X7 Receptor as a Mechanistic Biomarker for Epilepsy

“…In contrast to seizures, however, P2X7R antagonism has been shown to protect against seizure-induced cell death and reduce neuroinflammation in both KA-induced and pilocarpine-induced status epilepticus models [ 83 , 90 , 112 ]. Notably, P2X7R antagonism also reduced seizure severity in a mouse model of hypoxia-induced neonatal seizures [ 98 , 106 ] and a model of early life seizures in rat pups [ 89 ], suggesting that the observed anticonvulsive effects are not age-dependent. No, or only weak, effects of P2X7R antagonism were observed on non-damaging seizures.…”

“…pilocarpine rat model [ 103 ]. In addition, P2X7R antagonisms applied after hypoxia-induced seizures in mouse pups reduced long-lasting brain hyperexcitability [ 98 ], further suggesting anti-epileptogenic potential. In the same line, P2X7R antagonism during epilepsy reduced seizure severity without altering the frequency of seizures in a model where epilepsy was induced via i.p.…”

“…While P2X7R driving inflammatory processes is the most likely explanation [ 116 ], it is important to keep in mind that P2X7R signalling has been involved in numerous pathways in the brain, with several of these having a known role during epileptogenesis (e.g., maintenance of the BBB, synaptic plasticity, neurogenesis, control of neurotransmitter release, et cetera) [ 71 , 72 , 73 , 74 , 75 , 117 ]. Evidence supporting P2X7R contributing to brain hyperexcitability via driving inflammation include that (i) P2X7Rs are highly expressed on microglia, which increases following status epilepticus and increases during epilepsy [ 87 , 100 ], (ii) blocking of P2X7Rs during status epilepticus reduced hippocampal IL-1β levels [ 83 ] (iii) and the fact that while microglia from P2X7R-overexpressing mice showed a pro-inflammatory phenotype during status epilepticus [ 100 ], microglia from P2X7R KO mice presented an anti-inflammatory phenotype following hypoxia-induced seizures in neonatal mouse pups [ 98 ]. In addition, treatment of mice with an anti-inflammatory agent (i.e., minocycline) abolishes P2X7R-mediated effects during seizures [ 98 , 100 ].…”

“…Evidence supporting P2X7R contributing to brain hyperexcitability via driving inflammation include that (i) P2X7Rs are highly expressed on microglia, which increases following status epilepticus and increases during epilepsy [ 87 , 100 ], (ii) blocking of P2X7Rs during status epilepticus reduced hippocampal IL-1β levels [ 83 ] (iii) and the fact that while microglia from P2X7R-overexpressing mice showed a pro-inflammatory phenotype during status epilepticus [ 100 ], microglia from P2X7R KO mice presented an anti-inflammatory phenotype following hypoxia-induced seizures in neonatal mouse pups [ 98 ]. In addition, treatment of mice with an anti-inflammatory agent (i.e., minocycline) abolishes P2X7R-mediated effects during seizures [ 98 , 100 ]. If pro-convulsant effects of P2X7Rs are mediated via driving inflammation, this may also partly explain differences in P2X7R-based treatment responses observed between different models and disease stages.…”

P2X7 Forges Ahead in Neonatal Hypoxia

“…2 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% N 2 /5% O 2 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.…”