P2X receptors as targets for the treatment of status epilepticus.

Prolonged seizures are amongst the most common neurological emergencies. Status epilepticus is a state of continuous seizures that is life-threatening and prompt termination of status epilepticus is critical to protect the brain from permanent damage. Frontline treatment comprises parenteral administration of anticonvulsants such as lorazepam that facilitate γ-amino butyric acid (GABA) transmission. Because status epilepticus can become refractory to anticonvulsants in a significant proportion of patients, drugs which act on different neurotransmitter systems may represent potential adjunctive treatments. P2X receptors are a class of ligand-gated ion channel activated by ATP that contributes to neuro- and glio-transmission. P2X receptors are expressed by both neurons and glia in various brain regions, including the hippocampus. Electrophysiology, pharmacology and genetic studies suggest certain P2X receptors are activated during pathologic brain activity. Expression of several members of the family including P2X2, P2X4, and P2X7 receptors has been reported to be altered in the hippocampus following status epilepticus. Recent studies have shown that ligands of the P2X7 receptor can have potent effects on seizure severity during status epilepticus and mice lacking this receptor display altered seizures in response to chemoconvulsants. Antagonists of the P2X7 receptor also modulate neuronal death, microglial responses and neuroinflammatory signaling. Recent work also found altered neuronal injury and inflammation after status epilepticus in mice lacking the P2X4 receptor. In summary, members of the P2X receptor family may serve important roles in the pathophysiology of status epilepticus and represent novel targets for seizure control and neuroprotection

Functional characterization of P2Y1 and P2X4 receptors in human neuroblastoma SK-N-MC cells

Nucleotides are important signalling molecules in both the peripheral and central nervous system. However, the in vitro study of their receptors can be hampered by the heterogeneity of primary neuronal cultures. The use of clonal neuroblastoma cell lines allows to circumvent this difficulty, so these lines are often used as a model to analyze the properties, regulation and physiological role of nucleotide receptors in neural tissues. Expression studies indicated the presence of P2Y1, P2Y6, P2Y11, P2Y13, P2X1, P2X4, P2X5, P2X6 and P2X7 proteins in SK-N-MC cells. Functional analyses showed transient [Ca2+]i increases upon application of ADP, 2-MeSADP or ADPβS. Responses to these agonists seem to be mediated by a P2Y1 receptor, as demonstrated by the almost complete blockade exerted by the P2Y1-selective antagonist MRS2179. ATP was also able to induce [Ca2+]i increases in SK-N-MC cells. Responses to ATP were partially blocked by MRS2179 and the P2X antagonist TNP-ATP, thus suggesting that ATP can interact with two different P2 receptors: a P2Y1 receptor, inhibited by MRS2179, and a TNP-ATP sensitive P2X receptor. To characterize the P2X receptor responsible for the MRS2179-resistant component of the ATP response, we analyze the effect of several P2X agonists on [Ca2+]i. Cells did not show responses to either α,β-meATP or BzATP, although [Ca2+]i increases could be observed when cells were challenged with CTP. Both the response to CTP and the MRS2179-resistant component of ATP response were potentiated by ivermectin. Such pharmacological profile is consistent with the presence of a functional P2X4 receptor in SK-N-MC cell lineLos nucleótidos son importantes moléculas señalizadoras en el sistema nervioso. El estudio in vitro de sus receptores puede verse obstaculizado por la heterogeneidad de los cultivos neuronales. El uso de líneas celulares de neuroblastoma permite eludir esta dificultad y dichas líneas se utilizan frecuentemente como un modelo con el que analizar las propiedades, regulación y función de los receptores de nucleótidos en tejidos neurales. Estudios de expresión indicaron la presencia de proteínas P2Y1, P2Y6, P2Y11, P2Y13, P2X1, P2X4, P2X5, P2X6 y P2X7 en las células SK-N-MC. Análisis funcionales mostraron incrementos transitorios de [Ca2+]i tras la aplicación de ADP, 2- MeSADP o ADPβS, respuestas que parecen estar mediadas a través un receptor P2Y1, como se pone de manifiesto por el bloqueo casi total ejercido por el antagonista selectivo P2Y1, MRS2179. El ATP también indujo incrementos de [Ca2+]i en las células SK-N-MC, siendo su respuesta parcialmente bloqueada por MRS2179 y por el antagonista P2X TNP-ATP, lo que sugiere que el ATP puede interactuar con dos receptores P2 diferentes: un receptor P2Y1, inhibido por MRS2179, y un receptor P2X sensible a TNP-ATP. Se caracterizó el receptor P2X analizando el efecto de varios agonistas en la [Ca2+]i. Ninguna célula mostró respuestas a αβ- meATP o BzATP, aunque se observaron incrementos de [Ca2+]i cuando las células fueron estimuladas con CTP. Tanto la respuesta a CTP como el componente de la respuesta a ATP resistente a MRS2179, se potenciaron en presencia de ivermectina. Todos estos datos sugieren la presencia de un receptor P2X4 funcional en las células SK-N-MCThis work was supported by research grants from Ministerio de Ciencia e Innovación (BFU2014-53654-P) and Comunidad de Madrid (S2013/ICE-2958 BRADECM

Homenaje a Ramón y Cajal en el centenario de la concesión del Premio Nobel

Scientific Meeting: Honoring to Ramon and Cajal in the centenary of the grant of the Nobel Prize.Mesa Redonda: Homenaje a Ramón y Cajal en el centenario de la concesión del Premio Nobe

Nucleotides in neuroregeneration and neuroprotection

AbstractBrain injury generates the release of a multitude of factors including extracellular nucleotides, which exhibit bi-functional properties and contribute to both detrimental actions in the acute phase and also protective and reparative actions in the later recovery phase to allow neuroregeneration. A promising strategy toward restoration of neuronal function is based on activation of endogenous adult neural stem/progenitor cells. The implication of purinergic signaling in stem cell biology, including regulation of proliferation, differentiation, and cell death has become evident in the last decade. In this regard, current strategies of acute transplantation of ependymal stem/progenitor cells after spinal cord injury restore altered expression of P2X4 and P2X7 receptors and improve functional locomotor recovery. The expression of both receptors is transcriptionally regulated by Sp1 factor, which plays a key role in the startup of the transcription machinery to induce regeneration-associated genes expression. Finally, general signaling pathways triggered by nucleotide receptors in neuronal populations converge on several intracellular kinases, such as PI3K/Akt, GSK3 and ERK1,2, as well as the Nrf-2/heme oxigenase-1 axis, which specifically link them to neuroprotection. In this regard, regulation of dual specificity protein phosphatases can become novel mechanism of actions for nucleotide receptors that associate them to cell homeostasis regulation.This article is part of the Special Issue entitled ‘Purines in Neurodegeneration and Neuroregeneration’

Cerebellar astrocytes co-express several ADP receptors. Presence of functional P2Y13-like receptors

Astrocytes exhibit a form of excitability based on variations of intracellular Ca2+ concentration in response to various stimuli, including ADP, ATP, UTP and dinucleotides. Here, we investigate the presence of the recently cloned ADP-sensitive receptors, P2Y12 and P2Y13 subtypes, which are negatively coupled to adenylate cyclase, in cerebellar astrocytes. We checked the effect of specific agonists, 2-methylthioadenosine diphosphate (2MeSADP) and ADP, on adenylate cyclase stimulation induced by isoproterenol. Both agonists significantly reduced the cAMP accumulation induced by isoproterenol. The inhibitory effect was concentration-dependent with IC50 values of 46 ± 13 and 23 ± 14 nM for 2MeSADP and ADP, respectively. The experiments were carried out in the presence of MRS-2179, a specific antagonist of P2Y1 receptor, to avoid any contribution of this receptor. Using fura-2 microfluorimetry we also proved that astrocytes responded to 2MeSADP stimulations with calcium responses in the absence and also in the presence of MRS-2179. Both effects, inhibition of adenylate cyclase and intracellular calcium mobilization, were not modified by 2MeSAMP, an antagonist of P2Y12 receptor, suggesting that were mediated by P2Y13-like receptors

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

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. 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.</p

MicroRNA-22 Controls Aberrant Neurogenesis and Changes in Neuronal Morphology After Status Epilepticus

Prolonged seizures (status epilepticus, SE) may drive hippocampal dysfunction and epileptogenesis, at least partly, through an elevation in neurogenesis, dysregulation of migration and aberrant dendritic arborization of newly-formed neurons. MicroRNA-22 was recently found to protect against the development of epileptic foci, but the mechanisms remain incompletely understood. Here, we investigated the contribution of microRNA-22 to SE-induced aberrant adult neurogenesis. SE was induced by intraamygdala microinjection of kainic acid (KA) to model unilateral hippocampal neuropathology in mice. MicroRNA-22 expression was suppressed using specific oligonucleotide inhibitors (antagomir-22) and newly-formed neurons were visualized using the thymidine analog iodo-deoxyuridine (IdU) and a green fluorescent protein (GFP)-expressing retrovirus to visualize the dendritic tree and synaptic spines. Using this approach, we quantified differences in the rate of neurogenesis and migration, the structure of the apical dendritic tree and density and morphology of dendritic spines in newly-formed neurons.SE resulted in an increased rate of hippocampal neurogenesis, including within the undamaged contralateral dentate gyrus (DG). Newly-formed neurons underwent aberrant migration, both within the granule cell layer and into ectopic sites. Inhibition of microRNA-22 exacerbated these changes. The dendritic diameter and the density and average volume of dendritic spines were unaffected by SE, but these parameters were all elevated in mice in which microRNA-22 was suppressed. MicroRNA-22 inhibition also reduced the length and complexity of the dendritic tree, independently of SE. These data indicate that microRNA-22 is an important regulator of morphogenesis of newly-formed neurons in adults and plays a role in supressing aberrant neurogenesis associated with SE