Abstract
Jimenez-Pacheco A, Diaz-Hernandez M, Arribas-Blázquez M, Sanz-Rodriguez A, Olivos-Oré LA, Artalejo AR, Alves M, Letavic M, Miras-Portugal MT, Conroy RM, Delanty N, Farrell MA, O'Brien DF, Bhattacharya A, Engel T, Henshall DC. J Neurosci 2016;36:5920–5932.
Neuroinflammation is thought to contribute to the pathogenesis and maintenance of temporal lobe epilepsy, but the underlying cell and molecular mechanisms are not fully understood. The P2X7 receptor is an ionotropic receptor predominantly expressed on the surface of microglia, although neuronal expression has also been reported. The receptor is activated by the release of ATP from intracellular sources that occurs during neurodegeneration, leading to microglial activation and inflammasome-mediated interleukin 1β release that contributes to neuroinflammation. Using a reporter mouse in which green fluorescent protein is induced in response to the transcription of P2rx7, we show that expression of the receptor is selectively increased in CA1 pyramidal and dentate granule neurons, as well as in microglia in mice that developed epilepsy after intra-amygdala kainic acid-induced status epilepticus. P2X7 receptor levels were increased in hippocampal subfields in the mice and in resected hippocampus from patients with pharmacoresistant temporal lobe epilepsy. Cells transcribing P2rx7 in hippocampal slices from epileptic mice displayed enhanced agonist-evoked P2X7 receptor currents, and synaptosomes from these animals showed increased P2X7 receptor levels and altered calcium responses. A 5 d treatment of epileptic mice with systemic injections of the centrally available, potent, and specific P2X7 receptor antagonist JNJ-47965567 (30 mg/kg) significantly reduced spontaneous seizures during continuous video-EEG monitoring that persisted beyond the time of drug presence in the brain. Hippocampal sections from JNJ-47965567-treated animals obtained >5 d after treatment ceased displayed strongly reduced microgliosis and astrogliosis. The present study suggests that targeting the P2X7 receptor has anticonvulsant and possibly disease-modifying effects in experimental epilepsy.
Commentary
Temporal lobe epilepsy (TLE) is the most common form of partial epilepsy in adults (1). Regrettably, TLE is also the most common form of refractory epilepsy; approximately 30 to 40 percent of patients do not respond to current drug options (2). Recently, greater attention has been placed on the potential role of inflammation in the pathogenesis of TLE and in promoting pharmacoresistance (3,4). A major player in inflammation is ATP, which can be released upon seizure activity in epileptogenic brain areas (5). This study focused on the role of a specific receptor for ATP, the P2X7 receptor (P2X7R), as previous work by the same group had shown that P2X7R expression is increased in the cortex of pharmacoresistant TLE patients (6). There is good agreement that microglia express P2X7R, but neuronal expression of P2X7R has been a matter of some debate. Here, the authors sought to determine whether both neurons and microglia in the hippocampus express P2X7R, whether this expression is altered in epilepsy, and to investigate the role of P2X7R activation in spontaneous recurrent seizures in a mouse model of TLE.
To establish a model of chronic TLE in adult male mice, the authors used unilateral injections of a small amount of the convulsant kainic acid into the basolateral amygdala. In this model, mice develop spontaneous recurrent seizures within 5 days, and eventually exhibit cellular damage in the ipsilateral hippocampus, primarily in the CA3 region. Here, the authors used a transgenic mouse in which transcription of the P2rx7 gene drives expression of enhanced green fluorescent protein (GFP) (P2rx7-GFP); therefore, cells expressing P2X7R should also express GFP. The primary advantage of this model is that it enables identification of P2X7R-expressing cells without the use of antibody-based immunohistochemistry, which can lack specificity and/or sensitivity. In these mice, hippocampal GFP expression co-localized with markers for both neurons and microglia. Two weeks after the intra-amygdalar kainic acid injection, GFP expression was increased in the ipsilateral CA1 and dentate gyrus areas of hippocampus, indicating that expression of P2X7R is increased with epilepsy in these hippocampal subregions.
The authors next sought to determine whether epilepsy alters the function of the P2X7R. Because the P2X7R can be expressed on presynaptic terminals, they prepared synaptosomes, or isolated nerve terminals, from the hippocampal tissue of control and epileptic mice. Using calcium imaging to measure calcium influx and activation of the synaptosomes, the authors found that the synaptosomes prepared from epileptic mice showed a stronger degree of activation by the P2X7R agonist BzATP than synaptosomes from control mice. Furthermore, whereas control synaptosomes responded to BzATP with a single, transient elevation in intracellular calcium levels, the synaptosomes from epileptic mice showed multiple, sustained elevations in calcium, indicating a stronger and more prolonged functional response.
The P2X7R has very low affinity for ATP binding. Therefore, it is typically activated only when ATP levels are very high due to pathologic ATP release, such as during seizures. The authors thus postulated that blocking P2X7R activation would reduce the pathologic consequences of robust seizure-induced ATP release. Here, they took advantage of a recently developed P2X7R antagonist, JNJ-47965567, which can cross the blood–brain barrier when injected systemically. Mice that were implanted with EEG electrodes received the intra-amygdalar injection of kainic acid to induce status epilepticus and subsequent epileptogenesis as in the previous experiments. After a baseline period of 10 days following kainic acid injection, the mice were treated twice daily with the antagonist or vehicle for 5 days, followed by 5 days of no treatment for a washout period. The frequency of seizures was reduced during the treatment, indicating suppression of seizure activity by blockade of P2X7R. Interestingly, however, this effect continued after treatment stopped, at least through the 5-day washout period of observation. Although an average rate of two seizures per day was still observed in the treated mice (compared with 6/day in control animals), and thus seizures were not completely abolished in all animals, this finding suggests that transient P2X7R antagonism produces long-lasting seizure-reducing effects. The same laboratory has since reported that P2X7R antagonist treatment in neonatal mice can reduce seizures elicited during hypoxia, but in contrast to the persistent effects observed in the model of TLE in adult mice, posthypoxia seizures are not suppressed (7). Therefore, although the therapeutic effects of P2X7R antagonism are not limited to adult animals or to epilepsies typically seen after neural development is complete, the time windows for therapeutic benefits may be distinct.
Proliferation of microglia and astrocytes (microgliosis and astrogliosis, respectively) are pathologic hallmarks of TLE (8–10). By staining hippocampal tissue from the treated and untreated epileptic mice, the authors found reduced expression of both a microglial marker, Iba1, and an astrocytic marker, glial fibrillary acidic protein (GFAP), in the JNJ-47965567–treated mice compared with vehicle-treated epileptic mice. Remarkably, this suppression persisted after the treatment ended, a cellular benefit typically not seen with treatment using antiepileptic drugs currently in clinical use.
Although this study provides intriguing evidence supporting P2X7R as a pharmacologic target in treating TLE, key questions remain. First, the timing of treatment in this study was soon after status epilepticus (starting 11 days after kainic acid treatment). It could be that there is a window during which P2X7R antagonism can exert beneficial effects on the ongoing epileptogenesis, particularly with respect to microgliosis and astrogliosis. Therefore, it is unclear whether the persistent effects of JNJ-47965567 treatment observed in these experiments would be recapitulated in mice that are treated later, in the state of chronic, fully developed epilepsy. This is important given the suggested potential for application of P2X7R targeting to patients with pharmacoresistant epilepsy; in the time needed to screen different antiepileptic drugs in these patients and determine the diagnosis of pharmacoresistance, the underlying epilepsy may have progressed too far to benefit from P2X7R antagonism. On the other hand, a P2X7R antagonist could be an appropriate adjunct in post-status epilepticus treatment as a prophylactic measure against the progression of epileptogenesis. It would also be interesting to determine how long the effects of P2X7R antagonism last, and whether the beneficial effects can be extended with multiple treatments. Second, this study focused exclusively on male animals. Although it is not clear whether inflammatory processes specific to TLE exhibit sex differences, microglial activation and inflammatory responses may differ between male and female animals in models of stroke and with aging (11,12). Therefore, it would be interesting to determine whether the therapeutic efficacy of JNJ-47965567 differs in females compared with males. It also remains unclear what the relative contributions of microglial vs. neuronal P2X7R activation are in these processes. Nevertheless, this work provides an intriguing basis for continued focus on the P2X7R in the bench-to-bedside development of novel therapies for TLE.