Expression, Function and Targeting of the ATP-gated P2X7 receptor in epilepsy
Epilepsy is a common and disabling neurologic disease characterized by the occurrence of recurrent, unprovoked seizures, which affects about 1 % of the population. Seizures are the result of abnormal electrical activity in the brain and can be profoundly disabling, affecting work, social activity and increasing risk of harm. In at least 30 - 40 % of patients, treatment is inadequate, with patients continuing to experience seizures; therefore, there is an important need to develop new anti-epileptogenic drugs. To this end, the application of animal models of evoked seizure, such as focal-onset status epilepticus models, allows for researchers to characterize the early molecular events associated with seizure associated-pathology and the onset of epilepsy.
ATP is an essential transmitter/co-transmitter in neuron function and pathophysiology and has recently emerged as a potential contributor to prolonged seizures (status epilepticus) through the activation of the purinergic ionotropic P2X7 receptor (P2X7R). Prolonged and excessive temporal lobe seizures following experimental focal-onset status epilepticus are associated with seizure-induced neuronal cell death and sclerosis of the hippocampus, and herald the emergence of spontaneous seizures. Increased P2X7 receptor expression has been reported in the hippocampus and cortex after prolonged seizures in animal models and P2X7 receptor antagonists reduced seizure-induced damage to these brain regions. The body of work presented here in this thesis arose from the presumption that P2X7R is expressed and functional in epilepsy and its activation contributes to the development of seizures in status epilepticus and spontaneous seizures during epilepsy. The P2X7R inhibition was explored for its possible contribution in the development of spontaneous seizures and its potential disease-modifying effects in epilepsy.
First, increased P2X7R expression has been reported in the hippocampus after status epilepticus, and P2X7R antagonists reduced seizure-induced damage to this brain region (Engel et al., 2012b). However, status epilepticus also produces damage to the neocortex. The present study was designed to characterize P2X7R in the neocortex and assess effects of P2X7R antagonists on cortical injury after status epilepticus. Status epilepticus resulted in increased P2X7R protein levels in the neocortex of mice and also neocortical P2X7R levels were observed elevated in epilepsy. Immunohistochemistry determined that neurons were the major cell population transcribing the P2X7R in the neocortex within the first 8 h after status epilepticus. In epileptic mice, P2X7R up-regulation occurred in microglia as well as in neurons. Pretreatment of mice with the specific P2X7R inhibitor A-438079 reduced electrographic and clinical seizure severity during status epilepticus and reduced seizure-induced neuronal death in the neocortex.
Second, P2X7R expression and transcriptional control was assessed in the hippocampus in epilepsy, including functional evidence of the receptor. Using GFPexpressing P2X7R reporter mice we localized the increased expression mainly to neuron and microglia in epileptic animals. Isolated synaptosomes were also analysed and an increased presence of the P2X7R was also observed in the hippocampus in epilepsy. Moreover, functional studies including patch clamp technique and microfluorometric Ca2+ measurements showed increased functional activation of P2X7R in epileptic mice. These findings support a role for P2X7R in the pathophysiology of chronic epilepsy and suggest P2X7 receptor antagonists may have therapeutic effects against recurrent seizures or progression of disease pathology.
Third, the contribution of brain inflammation in the development of spontaneous seizures has gained support over the years, with P2X7R being a potential target due to its contribution to the processing and release of IL-1β (Vezzani et al., 2011). This study assessed the effect of a novel P2X7R antagonist, JNJ-47965567, on the number of spontaneous seizures in epilepsy, which was unexplored until now, in the kainic acid-induced status epilepticus model. The P2X7R antagonist succeeded 12 to reduce the number of spontaneous seizures in epileptic mice. Additionally, microglia and astrocytes reactivity was observed to be reduced in the hippocampus of mice treated with the P2X7R antagonist. These results suggest possible diseasemodifying effects through the P2X7R inhibition. Continuous administration of P2X7 receptor antagonists to epileptic mice will validate the effectiveness of P2X7 receptor inhibitors in stopping seizure occurrence and the progression of disease pathology.
In summary, this thesis shows evidence of the involvement of the P2X7R activation in the recurrence of spontaneous seizures in epilepsy and supports the development of new P2X7R inhibitors as a novel therapeutic target with diseasemodifying effects to treat and prevent epilepsy.