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).