Prolonged seizures [status epilepticus (SE)] constitute a neurological emergency that can permanently damage the brain. SE results from a failure of the normal mechanisms to terminate seizures; in particular, γ-amino butyric acid-mediated inhibition, and benzodiazepine anticonvulsants are often incompletely effective. ATP acts as a fast neurotransmitter via ionotropic ligand-gated P2X receptors. Here we report that SE induced by intra-amygdala kainic acid in mice selectively increased hippocampal levels of P2X7 receptors relative to other P2X receptors. Using transgenic P2X7 reporter mice expressing enhanced green fluorescent protein, we identify dentate granule neurons as the major cell population transcribing the P2X7 receptor after SE. Pretreatment of mice with an intracerebroventricular microinjection of 1.75 nmol A438079, a P2X7 receptor antagonist, reduced seizure duration by 58% and reduced seizure-induced neuronal death by 61%. Injection of brilliant blue G (1 pmol), another selective antagonist, reduced seizure duration by 48% and was also neuroprotective. A438079 was seizure-suppressive when injected shortly after induction of SE, and coinjection of A438079 with lorazepam 60 min after triggering SE, when electrographic seizure-responsiveness to lorazepam had decreased, also terminated SE. Our results suggest that P2X7 receptor antagonists may be a promising class of drug for seizure abrogation and neuroprotection in SE.

Summary Purpose ATP is an essential transmitter/cotransmitter 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). Increased P2X7R expression has been reported in the hippocampus, and P2X7R antagonists reduced seizure‐induced damage to this brain region. 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. Methods Status epilepticus was induced in mice by intraamygdala microinjection of kainic acid. Specific P2X7R inhibitors were administered into the ventricle before seizure induction, and cortical electroencephalography and behavior was recorded to assess seizure severity. P2X7R expression was examined in neocortex up to 24 h after status epilepticus, in epileptic mice, and in resected neocortex from patients with pharmacoresistent temporal lobe epilepsy (TLE). In addition, the induction of P2X7R after status epilepticus was investigated using transgenic P2X7R reporter mice, which express enhanced green fluorescent protein under the control of the p2x7r promoter. Key Findings Status epilepticus resulted in increased P2X7R protein levels in the neocortex of mice. Neocortical P2X7 receptor levels were also elevated in mice that developed epilepsy after status epilepticus and in resected neocortex from patients with pharmacoresistent TLE. Immunohistochemistry determined that neurons were the major cell population transcribing the P2X7R in the neocortex within the first 8 h after status epilepticus, whereas 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. Significance Our findings identify neurons in the neocortex as an important site of P2X7R up‐regulation after status epilepticus and in epilepsy, and provide support for the possible use of P2X7R antagonists for the treatment of status epilepticus and prevention of seizure‐induced brain damage.

The ATP-gated ionotropic P2X7 receptor (P2X7R) modulates glial activation, cytokine production and neurotransmitter release following brain injury. Levels of the P2X7R are increased in experimental and human epilepsy but the mechanisms controlling P2X7R expression remain poorly understood. Here we investigated P2X7R responses after focal-onset status epilepticus in mice, comparing changes in the damaged, ipsilateral hippocampus to the spared, contralateral hippocampus. P2X7R-gated inward currents were suppressed in the contralateral hippocampus and P2rx7 mRNA was selectively uploaded into the RNA-induced silencing complex (RISC), suggesting microRNA targeting. Analysis of RISC-loaded microRNAs using a high-throughput platform, as well as functional assays, suggested the P2X7R is a target of microRNA-22. Inhibition of microRNA-22 increased P2X7R expression and cytokine levels in the contralateral hippocampus after status epilepticus and resulted in more frequent spontaneous seizures in mice. The major pro-inflammatory and hyperexcitability effects of microRNA-22 silencing were prevented in P2rx7−/− mice or by treatment with a specific P2X7R antagonist. Finally, in vivo injection of microRNA-22 mimics transiently suppressed spontaneous seizures in mice. The present study supports a role for post-transcriptional regulation of the P2X7R and suggests therapeutic targeting of microRNA-22 may prevent inflammation and development of a secondary epileptogenic focus in the brain.

Hippocampal sclerosis is a frequent pathological finding in patients with temporal lobe epilepsy and can be caused by prolonged single or repeated brief seizures. Both DNA damage and endoplasmic reticulum stress have been implicated as underlying molecular mechanisms in seizure-induced brain injury. The CCAAT/enhancer-binding protein homologous protein (CHOP) is a transcriptional regulator induced downstream of DNA damage and endoplasmic reticulum stress, which can promote or inhibit apoptosis according to context. Recent work has proposed inhibition of CHOP as a suitable neuroprotective strategy. Here, we show that transcript and protein levels of CHOP increase in surviving subfields of the hippocampus after prolonged seizures (status epilepticus) in mouse models. CHOP was also elevated in the hippocampus from epileptic mice and patients with pharmacoresistant epilepsy. The hippocampus of CHOP-deficient mice was much more vulnerable to damage in mouse models of status epilepticus. Moreover, compared with wild-type animals, CHOP-deficient mice subject to status epilepticus developed more spontaneous seizures, displayed protracted hippocampal neurodegeneration and a deficit in a hippocampus-dependent object-place recognition task. The absence of CHOP was associated with a supra-maximal induction of p53 after status epilepticus, and inhibition of p53 abolished the cell death-promoting consequences of CHOP deficiency. The protective effect of CHOP could be partly explained by activating transcription of murine double minute 2 that targets p53 for degradation. These data demonstrate that CHOP is required for neuronal survival after seizures and caution against inhibition of CHOP as a neuroprotective strategy where excitotoxicity is an underlying pathomechanism.

These results suggest P2X7 receptor antagonists may be suitable as frontline or adjunctive treatments of pediatric status epilepticus or other early-life seizures, particularly when associated with brain damage.