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.
Eight G protein-coupled P2Y receptor subtypes respond to extracellular adenine and uracil mononucleotides and dinucleotides. P2Y receptors belong to the δ group of rhodopsin-like GPCRs and contain two structurally distinct subfamilies: P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 (principally G q protein-coupled P2Y 1 -like) and P2Y 12-14 (principally G i protein-coupled P2Y 12 -like) receptors. Brain P2Y receptors occur in neurons, glial cells, and vasculature. Endothelial P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 6 receptors induce vasodilation, while smooth muscle P2Y 2 , P2Y 4 , and P2Y 6 receptor activation leads to vasoconstriction. Pancreatic P2Y 1 and P2Y 6 receptors stimulate while P2Y 13 receptors inhibits insulin secretion. Antagonists of P2Y 12 receptors, and potentially P2Y 1 receptors, are anti-thrombotic agents, and a P2Y 2 /P2Y 4 receptor agonist treats dry eye syndrome in Asia. P2Y receptor agonists are generally pro-inflammatory, and antagonists may eventually treat inflammatory conditions. This article reviews recent developments in P2Y receptor pharmacology (using synthetic agonists and antagonists), structure and biophysical properties (using X-ray crystallography, mutagenesis and modelling), physiological and pathophysiological roles, and present and potentially future therapeutic targeting.Abbreviations: BMD, bone mineral density; DUSP, dual specificity protein phosphatase; ECL, extracellular loop; EPAC, exchange protein activated by cAMP; KO, knockout; MSD, musculoskeletal disorder; SNP, single nucleotide polymorphism; SS, Sjögren's syndrome; TM, transmembrane helix.
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 1 release that contributes to neuroinflammation.UsingareportermouseinwhichgreenfluorescentproteinisinducedinresponsetothetranscriptionofP2rx7,weshowthat 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 Ͼ5 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.
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.
There is solid evidence indicating that hyperphosphorylated tau protein, the main component of intracellular neurofibrillary tangles present in the brain of Alzheimer disease patients, plays a key role in progression of this disease. However, it has been recently reported that extracellular unmodified tau protein may also induce a neurotoxic effect on hippocampal neurons by activation of M1 and M3 muscarinic receptors. In the present work we show an essential component that links both effects, which is tissue-nonspecific alkaline phosphatase (TNAP). This enzyme is abundant in the central nervous system and is mainly required to keep control of extracellular levels of phosphorylated compounds. TNAP dephosphorylates the hyperphosphorylated tau protein once it is released upon neuronal death. Only the dephosphorylated tau protein behaves as an agonist of muscarinic M1 and M3 receptors, provoking a robust and sustained intracellular calcium increase finally triggering neuronal death. Interestingly, activation of muscarinic receptors by dephosphorylated tau increases the expression of TNAP in SH-SY5Y neuroblastoma cells. An increase in TNAP activity together with increases in protein and transcript levels were detected in Alzheimer disease patients when they were compared with healthy controls. Alzheimer disease (AD)3 is characterized by the loss of neurons and the presence of amyloid plaques and neurofibrillary tangles. The plaques are dense deposits of amyloid- peptide and cellular material outside and around neurons, whereas the tangles are aggregates of the microtubule-associated protein tau, which has become hyperphosphorylated and accumulates inside the cells (1). In AD, tau pathology follows a reproducible pattern, in which hyperphosphorylated and aggregated tau first appears in the entorhinal cortex and hippocampus, and from there the disease spreads to the surrounding areas (2). During this process, neuronal loss occurs and tau protein may be found in the extracellular space in monomeric form or in aggregated form, assembled in extracellular ghost tangles. Indeed, an inverse correlation can be found between the number of extracellular tangles and the number of living neurons in the hippocampus (3-5). It has been also suggested that extracellular aggregated tau can promote the aggregation of intracellular tau (6). Moreover, it has been reported that extracellular monomeric tau is toxic for neurons, playing a role in the spreading of AD pathology (7-9). Monomeric tau-dependent toxicity occurs when extracellular tau binds and activates cell membrane receptors, identified as M1 and M3 muscarinic receptors (7).Sluggish disassembly of aggregated tau and slow degradation of its monomeric form in extracellular media provide this protein with a long stay outside the cell. In this location, hyperphosphorylated monomeric tau can be recognized as a substrate of several extracellular enzymes, some of which can remove the phosphates from the protein (10, 11). One of these enzymes is tissue-nonspecific alkaline phosphatas...
The proposed presence of P2X7 receptor (P2X7R) in neurons has been the source of some contention. Initial studies suggested an absence of P2X7R mRNA in neurons, and the apparent nonspecificity of the antibodies used to identify P2X7R raised further doubts. However, subsequent studies using new pharmacological and biomolecular tools provided conclusive evidence supporting the existence of functional P2X7Rs in neurons. The P2X7 receptor has since been shown to play a leading role in multiple aspects of neuronal physiology, including axonal elongation and branching and neurotransmitter release. P2X7R has also been implicated in neuronal pathologies, in which it may influence neuronal survival. Together, this body of research suggests that P2X7R may constitute an important therapeutic target for a variety of neurological disorders.
A reduction in extracellular ATP levels by TNAP is essential for the development of neuritic processes by cultured hippocampal neurons. Results demonstrate that TNAP-mediated effects regulate both ligand availability and protein expression of P2X7 receptor in the axonal growth cone.
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