1. Introduction
Epilepsy is a common brain disease characterized by spontaneous recurrent seizures that affects up to 70 million people worldwide (Thijs et al., 2019). Epilepsy can be caused by a variety of factors including inherited and de novo gene mutations or can be acquired as a result of a brain injury (Pitkanen et al., 2015; Steinlein, 2008). While there are over 30 approved anti-seizure medications (ASMs) in clinical use, pharmacoresistance remains as high as 30%, a rate that has not changed in decades (Klein et al., 2024). Moreover, ASMs have shown no apparent impact on disease progression and can cause serious side effects (e.g ., dizziness, headaches) (Perucca et al., 2023), highlighting the need for novel therapies.
The cell and molecular hallmarks of neuroinflammation are a common finding in brain samples from patients who underwent neurosurgical resection of seizure-generating tissue. Accordingly, there is interest in therapeutic targeting of neuroinflammatory pathways such as interleukin-1β (IL-1β) for seizure control and possibly disease modification (Aronica et al., 2017; Devinsky et al., 2013; Vezzani et al., 2011). Extracellularly released adenosine triphosphate (eATP) functions as a damage-activated molecular pattern (DAMP), mediating pro- and anti-inflammatory effects via ionotropic P2X and metabotropic P2Y receptors (Andrejew et al., 2020; Beamer et al., 2021). Among these, the P2X7 receptor (P2X7R) has attracted particular attention (Beamer et al., 2017; Engel et al., 2021). Activation of the P2X7R requires high amounts of eATP, limiting its function mainly to pathophysiologic circumstances, and results in opening of a non-selective cationic channel permeable to Na+, K+ and Ca2+, leading to the later release of IL-1β. The receptor is normally expressed at low levels in the brain, in particular on microglia and oligodendrocytes (Alves et al., 2024; Illes et al., 2017; Kaczmarek-Hajek et al., 2018). P2X7R expression increases in the brain in experimental models of epilepsy and is elevated within the hippocampus from patients with treatment-resistant epilepsy (Alves et al., 2024; Dona et al., 2009; Engel et al., 2012; Jimenez-Pacheco et al., 2016; Morgan et al., 2020). P2X7Rs have been implicated in a variety of pathological processes relevant to epilepsy including increased permeability of the blood brain-barrier (BBB), altered neurotransmitter release, cell death and neuroinflammation (Andrejew et al., 2020; Klein et al., 2018; Sperlagh et al., 2014).
Functional studies indicate that targeting the P2X7R can attenuate seizures. For example, P2X7R antagonists, such as AFC-5128 and JNJ-47965567, have been shown to reduce the severity of evoked and spontaneous seizures in mice (Amhaoul et al., 2016; Amorim et al., 2017; Engel et al., 2012; Jimenez-Mateos et al., 2015; Jimenez-Pacheco et al., 2016; Jimenez-Pacheco et al., 2013; Mamad et al., 2023; Rozmer et al., 2017). It is unclear, however, whether these therapeutic effects will translate to humans as there are sequence and functional differences between the rodent and human P2X7R and controversy over whether neurons express functional P2X7Rs (Illes et al., 2017).
Human induced pluripotent stem cells (hiPSCs) represent invaluable human model systems to test the effects of new drugs on human cells, thereby advancing treatments further towards a clinical application (Autar et al., 2022; Jones et al., 2016; Rivetti di Val Cervo et al., 2021). While P2X7Rs have been shown to be present on several hiPSCs-induced cell lines including microglia, astrocytes and neurons (Francistiova et al., 2021; Kesavan et al., 2023), whether P2X7R signaling contributes to hyperexcitable networks using hiPSCs has not been investigated. Here, we show, using an in vitro model of epileptiform-like events in hiPSCs-derived neurons, that P2X7R antagonism can reduce epileptiform activity but only in the context of a primed inflammatory state. We further show that P2X7R antagonism has a synergistic effect when co-applied with a conventional ASM, providing support for the use of P2X7R-based drugs as stand-alone or adjunctive treatments for pharmacoresistant epilepsy.