Here, we show for the first time the anti-epileptic potential of blocking the P2X7R using a human in vitro seizure model that included the key contribution of inflammatory signaling. Notably, P2X7R antagonism restored the anti-seizure effects of the ASM carbamazepine in the chronic hiPSC model, suggesting P2X7R-based treatments as add-on therapy for drug-refractory epilepsy.
A key obstacle hampering the translation of results from pre-clinical disease models into the clinic is the lack of their validation in human model systems. In the epilepsy field, new drug development remains largely reliant on pre-clinical drug screening in animal models (Loscher et al., 2013). Human cell models (e.g., hiPSC) offer a promising tool to bridge the translational gap between animal models and human clinical trials. Here, the P2X7R represents no exception. While several P2X7R antagonists have passed onto the clinical trial stage (e.g., depression (Recourt et al., 2023)), the decision to move P2X7R antagonist towards these trials was most likely based on results from animal studies. A growing body of evidence supports the P2X7R as a drug target for epilepsy but this remains in the pre-clinical domain (Engel, 2023). h iPSCs represent a human disease-relevant cellular model to assess phenotypic alterations and drug responses in neurological diseases including epilepsy (Hirose et al., 2020; Lu et al., 2022).
One of the major findings of our study was that P2X7R antagonism reduces epileptiform activity in hiPSCs. This was, however, only evident when an inflammatory tone was present, evoked by co-culturing with a cocktail of cytokines. These results are in line with previous data suggesting the pro-excitability effects of the P2X7R emerge only in the context of neuroinflammation (Beamer et al., 2022; Smith et al., 2023). The mechanism of how P2X7Rs promote hyperexcitability in our hiPSC model and whether this is mediated via P2X7Rs expressed in neurons remains to be established. For our epileptiform-like activity induction studies, we used co-cultures of iPSC-derived neurons and human primary astrocytes. While our data confirmed functional P2X7R expression on neurons, we have previously shown P2X7Rs to be functional also on iPSC-derived astrocytes (Kesavan et al., 2023). It is well established that astrocytes play an important role during synaptic communication via their interaction with pre- and post-synaptic compartments (i.e ., tri-partite synapse) (Halassa et al., 2010). Astrocytes contribute to neurotransmitter release, such as glutamate and GABA (Cuellar-Santoyo et al., 2022), which may contribute to network hyperexcitability (Khan et al., 2019). In this scenario, P2X7Rs on astrocytes and activated during inflammatory conditions, contribute to the activation of astrocytes and the subsequent increase in neurotransmitter release contributing thereby to the observed epileptiform discharges. Thus, blocking P2X7R activity on astrocytes would reduce astrocyte activation, neurotransmitter release and network hyperexcitability. P2X7Rs are, however also expressed on neurons where they have been shown to regulate, upon activation, the release of neurotransmitters such as glutamate and GABA (Alves et al., 2024; Barros-Barbosa et al., 2018; Sperlagh et al., 2002), which potentially also contributes to the observed changes in network hyperexcitability. The most likely scenario is, however, a combination of both astrocyte and neuronal P2X7R-mediate effects and the exact contribution from each cell type should be further investigated in future studies. Why the observed effects are restricted to neuroinflammatory conditions and do not impact on network hyperexcitability in an acute setting is likely due to the need for the P2X7R to be primed, for example via increased inflammation, which is in line with the observed absence on acute seizures in experimental animal models (Dogan et al., 2020; Fischer et al., 2016).
A second major finding of our study is the fact that P2X7R antagonism restores the anticonvulsive effects of the ASM carbamazepine. We have previously shown increased P2X7R expression to contribute to drug-unresponsiveness during status epilepticus, including the ASM carbamazepine (Beamer et al., 2022). The same study also showed that increased inflammation increases status epilepticus-induced drug-refractoriness, which can be overcome by genetic deletion of the P2X7R or via treatment with P2X7R antagonists (Beamer et al., 2022). Future studies should reveal how P2X7R antagonism under neuroinflammatory conditions restores the anti-convulsant actions of ASMs.
While our study provides the evidence of the anti-seizure potential of P2X7R antagonism in human model systems, there are several shortcomings which should be considered. While our study clearly demonstrates the functional expression of P2X7Rs in neurons, whether there are differences according to neuronal subpopulations has not been addressed and should be carried out in future experiments. As mentioned before, for our hiPSC in vitro model of epileptiform-like events we have used co-cultures of neurons and astrocytes with both cell types expressing P2X7Rs. Moreover, apart from neurons and astrocytes, P2X7Rs are highly expressed in microglia and previous studies have shown P2X7Rs to be expressed also in hiPSC-derived microglia (Francistiova et al., 2021). Future studies should clarify what cell type(s) contribute to P2X7R-mediated hyperexcitability. Our data suggest P2X7R antagonism to restore/potentiate the effects of the ASM carbamazepine. Future studies should establish whether these effects are specific for carbamazepine. Previous studies in animal models of status epilepticus have, however, shown that increased P2X7R expression reduces the responsiveness to several ASMs (i.e ., lorazepam, midazolam, carbamazepine and phenytoin) (Beamer et al., 2022).