P2X7 receptor antagonism suppresses epileptiform‐like activity in an inflammation‐primed human iPSC‐derived neuron model of drug‐resistant epilepsy

Neuroinflammation is increasingly recognised to contribute to drug-resistant epilepsy. Activation of ATP-gated P2X7 receptors has emerged as an important upstream mechanism, and increased P2X7 receptor expression is present in the seizure focus in rodent models and patients. Pharmacological antagonists of P2X7 receptors attenuate seizures in rodents, but this has not been explored in human neural networks. Human neurons were differentiated from two human induced pluripotent stem cell (hiPSC) lines. P2X7 receptor function on neurons was assessed via the calcium transients evoked by the agonist BzATP. Acute or chronic models of epileptiform-like events were generated by exposing hiPSC cultures to the GABAA receptor antagonist picrotoxin or a cocktail of picrotoxin and neuroinflammatory agents, with or without P2X7 receptor antagonists. Epileptiform-like activity was measured via single cell patch-clamp recordings. BzATP application (300 μM) resulted in increased calcium influx in hiPSC-derived neurons which was blocked by the P2X7 receptor antagonists JNJ-47965567 (100 nM) and AFC-5128 (30 nM). Single-cell patch-clamp recordings showed that, while treatment with AFC-5128 did not reduce epileptiform-like activity triggered by picrotoxin alone, AFC-5128 reduced the severity of epileptiform-like activity under inflammatory conditions. Notably, epileptiform-like events in the inflammation-primed picrotoxin model were refractory to the anti-seizure medication carbamazepine alone but were reduced by the co-application of carbamazepine with AFC-5128. Our findings demonstrate anti-seizure effects of targeting P2X7 receptors in a human neuronal network model of epilepsy and suggest P2X7 receptor-based treatments may be effective add-on treatments for controlling drug-resistant seizures.