Opposing effects of the purinergic P2X7 receptor on seizures in neurons and microglia in male mice.

BACKGROUND: The purinergic ATP-gated P2X7 receptor (P2X7R) is increasingly recognized to contribute to pathological neuroinflammation and brain hyperexcitability. P2X7R expression has been shown to be increased in the brain, including both microglia and neurons, in experimental models of epilepsy and patients. To date, the cell type-specific downstream effects of P2X7Rs during seizures remain, however, incompletely understood. METHODS: Effects of P2X7R signaling on seizures and epilepsy were analyzed in induced seizure models using male mice including the kainic acid model of status epilepticus and pentylenetetrazole model and in male and female mice in a genetic model of Dravet syndrome. RNA sequencing was used to analyze P2X7R downstream signaling during seizures. To investigate the cell type-specific role of the P2X7R during seizures and epilepsy, we generated mice lacking exon 2 of the P2rx7 gene in either microglia (P2rx7:Cx3cr1-Cre) or neurons (P2rx7:Thy-1-Cre). To investigate the protective potential of overexpressing P2X7R in GABAergic interneurons, P2X7Rs were overexpressed using adeno-associated virus transduction under the mDlx promoter. RESULTS: RNA sequencing of hippocampal tissue from wild-type and P2X7R knock-out mice identified both glial and neuronal genes, in particular genes involved in GABAergic signaling, under the control of the P2X7R following seizures. Mice with deleted P2rx7 in microglia displayed less severe acute seizures and developed a milder form of epilepsy, and microglia displayed an anti-inflammatory molecular profile. In contrast, mice lacking P2rx7 in neurons showed a more severe seizure phenotype when compared to epileptic wild-type mice. Analysis of single-cell expression data revealed that human P2RX7 expression is elevated in the hippocampus of patients with temporal lobe epilepsy in excitatory and inhibitory neurons. Functional studies determined that GABAergic interneurons display increased responses to P2X7R activation in experimental epilepsy. Finally, we show that viral transduction of P2X7R in GABAergic interneurons protects against evoked and spontaneous seizures in experimental temporal lobe epilepsy and in mice lacking Scn1a, a model of Dravet syndrome. CONCLUSIONS: Our results suggest a dual and opposing action of P2X7R in epilepsy and suggest P2X7R overexpression in GABAergic interneurons as a novel therapeutic strategy for acquired and, possibly, genetic forms of epilepsy.

Regulation of the voltage-dependent sodium channel NaV1.1 by AKT1.

The voltage-sensitive sodium channel NaV1.1 plays a critical role in regulating excitability of GABAergic neurons and mutations in the corresponding gene are associated to Dravet syndrome and other forms of epilepsy. The activity of this channel is regulated by several protein kinases. To identify novel regulatory kinases we screened a library of activated kinases and we found that AKT1 was able to directly phosphorylate NaV1.1. In vitro kinase assays revealed that the phosphorylation site was located in the C-terminal part of the large intracellular loop connecting domains I and II of NaV1.1, a region that is known to be targeted by other kinases like PKA and PKC. Electrophysiological recordings revealed that activated AKT1 strongly reduced peak Na+ currents and displaced the inactivation curve to more negative potentials in HEK-293 cell stably expressing NaV1.1. These alterations in current amplitude and steady-state inactivation were mimicked by SC79, a specific activator of AKT1, and largely reverted by triciribine, a selective inhibitor. Neurons expressing endogenous NaV1.1 in primary cultures were identified by expressing a fluorescent protein under the NaV1.1 promoter. There, we also observed a strong decrease in the current amplitude after addition of SC79, but small effects on the inactivation parameters. Altogether, we propose a novel mechanism that might regulate the excitability of neural networks in response to AKT1, a kinase that plays a pivotal role under physiological and pathological conditions, including epileptogenesis.

Salient brain entities labelled in P2rx7-EGFP reporter mouse embryos include the septum, roof plate glial specializations and circumventricular ependymal organs.

The purinergic system is one of the oldest cell-to-cell communication mechanisms and exhibits relevant functions in the regulation of the central nervous system (CNS) development. Amongst the components of the purinergic system, the ionotropic P2X7 receptor (P2X7R) stands out as a potential regulator of brain pathology and physiology. Thus, P2X7R is known to regulate crucial aspects of neuronal cell biology, including axonal elongation, path-finding, synapse formation and neuroprotection. Moreover, P2X7R modulates neuroinflammation and is posed as a therapeutic target in inflammatory, oncogenic and degenerative disorders. However, the lack of reliable technical and pharmacological approaches to detect this receptor represents a major hurdle in its study. Here, we took advantage of the P2rx7-EGFP reporter mouse, which expresses enhanced green fluorescence protein (EGFP) immediately downstream of the P2rx7 proximal promoter, to conduct a detailed study of its distribution. We performed a comprehensive analysis of the pattern of P2X7R expression in the brain of E18.5 mouse embryos revealing interesting areas within the CNS. Particularly, strong labelling was found in the septum, as well as along the entire neural roof plate zone of the brain, except chorioidal roof areas, but including specialized circumventricular roof formations, such as the subfornical and subcommissural organs (SFO; SCO). Moreover, our results reveal what seems a novel circumventricular organ, named by us postarcuate organ (PArcO). Furthermore, this study sheds light on the ongoing debate regarding the specific presence of P2X7R in neurons and may be of interest for the elucidation of additional roles of P2X7R in the idiosyncratic histologic development of the CNS and related systemic functions.

Neurodevelopmental alterations and seizures developed by mouse model of infantile hypophosphatasia are associated with purinergic signalling deregulation.

Hypomorphic mutations in the gene encoding the tissue-nonspecific alkaline phosphatase (TNAP) enzyme, ALPL in human or Akp2 in mice, cause hypophosphatasia (HPP), an inherited metabolic bone disease also characterized by spontaneous seizures. Initially, these seizures were attributed to the impairment of GABAergic neurotransmission caused by altered vitamin B6 (vit-B6) metabolism. However, clinical cases in human newborns and adults whose convulsions are refractory to pro-GABAergic drugs but controlled by the vit-B6 administration, suggest that other factors are involved. Here, to evaluate whether neurodevelopmental alterations are underlying the seizures associated to HPP, we performed morphological and functional characterization of postnatal homozygous TNAP null mice, a model of HPP. These analyses revealed that TNAP deficient mice present an increased proliferation of neural precursors, an altered neuronal morphology, and an augmented neuronal activity. We found that these alterations were associated with a partial downregulation of the purinergic P2X7 receptor (P2X7R). Even though deficient P2X7R mice present similar neurodevelopmental alterations, they do not develop neonatal seizures. Accordingly, we found that the additional blockage of P2X7R prevent convulsions and extend the lifespan of mice lacking TNAP. In agreement with these findings, we also found that exogenous administration of ATP or TNAP antagonists induced seizures in adult wild-type mice by activating P2X7R. Finally, our results also indicate that the anticonvulsive effects attributed to vit-B6 may be due to its capacity to block P2X7R. Altogether, these findings suggest that the purinergic signalling regulates the neurodevelopmental alteration and the neonatal seizures associated to HPP.

Transient P2X7 Receptor Antagonism Produces Lasting Reductions in Spontaneous Seizures and Gliosis in Experimental Temporal Lobe Epilepsy.

UNLABELLED: 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 1ß 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. SIGNIFICANCE STATEMENT: Temporal lobe epilepsy is the most common and drug-resistant form of epilepsy in adults. Neuroinflammation is implicated as a pathomechanism, but the upstream mechanisms driving gliosis and how important this is for seizures remain unclear. In our study, we show that the ATP-gated P2X7 receptor is upregulated in experimental epilepsy and resected hippocampus from epilepsy patients. Targeting the receptor with a new centrally available antagonist, JNJ-47965567, suppressed epileptic seizures well beyond the time of treatment and reduced underlying gliosis in the hippocampus. The findings suggest a potential disease-modifying treatment for epilepsy based on targeting the P2X7 receptor.