Identification of the P2X7 Receptor Signature in the Brain and in the Blood Following Seizures and During Epilepsy

Current pharmacological approaches to treat epilepsy are mainly focused on enhancing neuronal inhibition or decreasing neuronal excitation, however, these have no significant impact on disease progression and, critically, over 30% of patients are refractory to antiseizure drugs (ASDs) available in the clinic. This demonstrates the urgent need for drug targets with a different mechanism of action. Experimental models of induced status epilepticus are the main models for the identification of potential anti-epileptogenic drugs but whether these models mimic faithfully the human condition remains incomplete explored. The adenosine-triphosphate (ATP)-gated P2X7 receptor (P2X7R), described as a gatekeeper of inflammation, has been suggested as an important player in epilepsy, with P2X7R antagonists providing both anticonvulsant and disease-modifying effects in animal models of epilepsy. However, before P2X7R-based treatments can enter the clinic for epilepsy, we must establish P2X7R down-stream signalling in vivo, and, identify P2X7R-dependent biomarkers for the identification of patients possibly benefiting from P2X7R-targeting drugs.

During my PhD, I first compared the expression profiles in the hippocampus, main structure affected during temporal lobe epilepsy (TLE), between different animal models of epilepsy with data from patients, thereby testing whether these models reproduce the human condition also at the molecular level. The second part of my PhD studies aimed to determine the P2X7R signalling profile in the brain. Here we established the P2X7Rspecific microRNA profile in hippocampi from wild-type and P2X7R knock-out mice following intraamygdala kainic acid-induced status epilepticus. Finally, to identify a potential biomarker for the diagnosis of epilepsy, I took part in a study investigating changes in purines level in the blood in patients and animal model of status epilepticus, and I established a P2X7R-dependent blood signature during epilepsy. For this, I measured P2X7R protein levels in the plasma of patients with epilepsy, investigated P2X7R-dependent changes in cytokine release in plasma in P2X7R deficient mice and, tested the diagnostic potential of blood purine changes for seizures and epilepsy.

My results show that mice injected with kainic acid into the intra-amygdala display a higher dysregulation of gene expression after status epilepticus (2729 genes) when compared to epilepsy (412). Comparing the expression profile of our mouse model with the expression profile of patients with TLE showed a very strong overlap of dysregulated genes, confirming the suitability of our mouse model to study epilepsy. To investigate P2X7R-dependent downstream signalling in the brain, I compared P2X7R-deficient mice with wild-type (WT) mice under physiological conditions and 8 h following status epilepticus focusing on microRNAs. My main results showed numerous differentially regulated microRNAs in P2X7R-deficient mice suggesting an involvement of P2X7R in the regulation of miRNA expression under physiological and pathological conditions (i.e. status epilepticus). In a collaborative study with clinicians at Beaumont Hospital (Dublin), we demonstrated the diagnostic potential of newly-developed purine-measuring biosensors for seizures and epilepsy. Finally, I measured P2X7R protein levels in plasma from epileptic patients. Here my results showed increased P2X7R protein levels in plasma of patients with TLE when compared to healthy controls and patients with nonepileptic attack disorder (NEAD), suggesting that P2X7R itself might be a good biomarker for the diagnosis of epilepsy. Measuring cytokine level comparing WT with P2X7Rdeficient mice, I found differences in cytokines expression in the blood, mainly following status epilepticus, suggesting that P2X7R can modulate the release of cytokines and chemokines in the blood.

Taken together, our results demonstrate that, (i) the intra-amygdala kainic acid model of epilepsy is a reliable model to study human TLE, (ii) P2X7R activation modulates the expression profile of microRNAs in the brain and, (iii) that purine and P2X7R-based biomarkers have a strong potential to support the diagnosis of seizures and epilepsy.