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.