DUSP1/MKP-1 represents another piece in the P2X7R intracellular signaling puzzle in cerebellar cells: our last journey with Mª Teresa along the purinergic pathways of Eden.

The P2X7 receptor (P2X7R) stands out within the purinergic family as it has exclusive pharmacological and regulatory features, and it fulfills distinct roles depending on the type of stimulation and cellular environment. Tonic activation of P2X7R promotes cell proliferation, whereas sustained activation is associated with cell death. Yet strikingly, prolonged P2X7R activation in rat cerebellar granule neurons and astrocytes does not affect cell survival. The intracellular pathways activated by P2X7Rs involve proteins like MAPKs, ERK1/2 and p38, and interactions with growth factor receptors could explain their behavior in populations of rat cerebellar cells. In this study, we set out to characterize the intracellular mechanisms through which P2X7Rs and Trk receptors, EGFR (epidermal growth factor receptor) and BDNFR (brain-derived neurotrophic factor receptor), regulate the dual-specificity phosphatase DUSP1. In cerebellar astrocytes, the regulation of DUSP1 expression by P2X7R depends on ERK and p38 activation. EGFR stimulation can also induce DUSP1 expression, albeit less strongly than P2X7R. Conversely, EGF was virtually ineffective in regulating DUSP1 in granule neurons, a cell type in which BDNF is the main regulator of DUSP1 expression and P2X7R only induces a mild response. Indeed, the regulation of DUSP1 elicited by BDNF reflects the balance between both transcriptional and post-transcriptional mechanisms. Importantly, when the regulation of DUSP1 expression is compromised, the viability of both astrocytes and neurons is impaired, suggesting this phosphatase is essential to maintain proper cell cytoarchitecture and functioning.

Changes in melatonin receptor expression in a murine model of glaucoma.

Purpose: The objective of this study was to evaluate the changes in the melatoninergic receptors of DBA/2J and C57BL/6J mice with the development of glaucoma. DBA/2J mice are widely used to study the physiopathology of glaucoma due to the similarities of their eyes to human eyes and the resulting similarity in the development of their pathology. In addition, melatoninergic receptors are known for their control of intraocular pressure (IOP), reducing the production of aqueous humor; however, little is known about their relationship with the development of this pathology. Methods: mRNA expression of MT1, MT2, and GPR50 melatoninergic receptors was performed with quantitative real-time PCR. In addition, receptor expression was performed with immunohistochemical techniques on the ciliary processes. To further investigate the effect of melatonin and its analog 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) on IOP, animals were instilled with these compounds and the corresponding melatoninergic antagonists to assess their effect on IOP. Results: All melatoninergic receptor expression decayed with the development of the glaucomatous pathology in the DBA/2J mice, and was especially visible for the MT2 receptor. However, receptor expression was consistent in the C57BL/6J control mice across all ages investigated. Furthermore, IOP blockage was stronger with 4PPDOT (MT2 antagonist) only in the DBA/2J mice which suggests a correlation of this receptor with the development of the glaucomatous pathology in DBA/2J animals. Conclusions: Melatonin receptor expression decays with the development of the glaucomatous pathology. This implies that the physiologic hypotensive effect of endogenous melatonin reducing IOP is not possible. A solution for such changes in receptor expression is the exogenous application of melatonin or any of its analogs that permit the activation of the remaining melatonin receptors.

Dual-Specificity Phosphatase Regulation in Neurons and Glial Cells.

Dual-specificity protein phosphatases comprise a protein phosphatase subfamily with selectivity towards mitogen-activated protein (MAP) kinases, also named MKPs, or mitogen-activated protein kinase (MAPK) phosphatases. As powerful regulators of the intensity and duration of MAPK signaling, a relevant role is envisioned for dual-specificity protein phosphatases (DUSPs) in the regulation of biological processes in the nervous system, such as differentiation, synaptic plasticity, and survival. Important neural mediators include nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) that contribute to DUSP transcriptional induction and post-translational mechanisms of DUSP protein stabilization to maintain neuronal survival and differentiation. Potent DUSP gene inducers also include cannabinoids, which preserve DUSP activity in inflammatory conditions. Additionally, nucleotides activating P2X7 and P2Y13 nucleotide receptors behave as novel players in the regulation of DUSP function. They increase cell survival in stressful conditions, regulating DUSP protein turnover and inducing DUSP gene expression. In general terms, in the context of neural cells exposed to damaging conditions, the recovery of DUSP activity is neuroprotective and counteracts pro-apoptotic over-activation of p38 and JNK. In addition, remarkable changes in DUSP function take place during the onset of neuropathologies. The restoration of proper DUSP levels and recovery of MAPK homeostasis underlie the therapeutic effect, indicating that DUSPs can be relevant targets for brain diseases.

Increased Ap4A levels and ecto-nucleotidase activity in glaucomatous mice retina.

The pathogenesis of glaucoma involves numerous intracellular mechanisms including the purinergic system contribution. Furthermore, the presence and release of nucleotides and dinucleotides during the glaucomatous damage and the maintenance of degradation machinery through ecto-nucleotidase activity are participating in the modulation of the suitable extracellular complex balance. The aim of this study was to investigate the levels of diadenosine tetraphosphate (Ap4A) and the pattern of ecto-nucleotidase activity expression in glaucomatous retinas during the progress the pathology. Ap4A levels were analyzed by HPLC in glaucomatous retinas from the DBA/2J mice at 3, 9, 15, and 23 months of age. For that, retinas were dissected as flattened whole-mounts and stimulated in Ringer buffer with or without 59 mM KCl. NPP1 expression was analyzed by RT-PCR and western blot and its distribution was assessed by immunohistochemistry studies examined under confocal microscopy. Glaucomatous mice exhibited Ap4A values, which changed in stimulated retinas as long as the pathology progressed varying from 0.73 ± 0.04 (3 months) to 0.170 ± 0.05 pmol/mg retina (23 months). Concomitantly, NPP1 expression was significantly increased (82.15%) in the DBA/2J mice at 15 months. Furthermore, immunohistochemical studies showed that NPP1 labeling was stronger in OPL and IPL labeling tangentially in the vitreal part of the retina and was upregulated at 15 months of age. Our findings demonstrate that Ap4A decreased levels may be related with exacerbated activity of NPP1 protein in glaucomatous degeneration and in this way contributing to elucidate different mechanisms involved in retinal impairment in glaucomatous degeneration.

Expression and function of the metabotropic purinergic P2Y receptor family in experimental seizure models and patients with drug-refractory epilepsy.

OBJECTIVE: ATP is released into the extracellular space during pathologic processes including increased neuronal firing. Once released, ATP acts on P2 receptors including ionotropic P2X and metabotropic P2Y receptors, resulting in changes to glial function and neuronal network excitability. Evidence suggests an involvement of P2Y receptors in the pathogenesis of epilepsy, but there has been no systematic effort to characterize the expression and function of the P2Y receptor family during seizures and in experimental and human epilepsy. METHODS: Status epilepticus was induced using either intra-amygdala kainic acid or pilocarpine to characterize the acute- and long-term changes in hippocampal P2Y expression. P2Y expression was also investigated in brain tissue from patients with temporal lobe epilepsy. Finally, we analyzed the effects of two specific P2Y agonists, ADP and UTP, on seizure severity and seizure-induced cell death. RESULTS: Both intra-amygdala kainic acid and pilocarpine-induced status epilepticus increased the transcription of the uracil-sensitive P2Y receptors P2ry2 , P2ry4 , and P2ry6 and decreased the transcription of the adenine-sensitive P2Y receptors P2ry1 , P2ry12 , P2ry13 . Protein levels of P2Y1 , P2Y2 , P2Y4 , and P2Y6 were increased after status epilepticus, whereas P2Y12 expression was decreased. In the chronic phase, P2ry1 , P2ry2 , and P2ry6 transcription and P2Y1 , P2Y2 , and P2Y12 protein levels were increased with no changes for the other P2Y receptors. In hippocampal samples from patients with temporal lobe epilepsy, P2Y1 and P2Y2 protein expression was increased, whereas P2Y13 levels were lower. Demonstrating a functional contribution of P2Y receptors to seizures, central injection of ADP exacerbated seizure severity, whereas treatment with UTP decreased seizure severity during status epilepticus in mice. SIGNIFICANCE: The present study is the first to establish the specific hippocampal expression profile and function of the P2Y receptor family after experimental status epilepticus and in human temporal lobe epilepsy and offers potential new targets for seizure control and disease modification.

An Update on P2Y13 Receptor Signalling and Function.

The distribution of nucleotide P2Y receptors across different tissues suggests that they fulfil key roles in a number of physiological and pathological conditions. P2Y13 is one of the latest P2Y receptors identified, a novel member of the Gi-coupled P2Y receptor subfamily that responds to ADP, together with P2Y12 and P2Y14. Pharmacological studies drew attention to this new ADP receptor, with a pharmacology that overlaps that of P2Y12 receptors but with unique features and roles. The P2RY12-14 genes all reside on human chromosome 3 at 3q25.1 and their strong sequence homology supports their evolutionary origin through gene duplication. Polymorphisms of P2Y13 receptors have been reported in different human populations, yet their consequences remain unknown. The P2Y13 receptor is versatile in its signalling, extending beyond the canonical signalling of a Gi-coupled receptor. Not only can it couple to different G proteins (Gs/Gq) but the P2Y13 receptor can also trigger several intracellular pathways related to the activation of MAPKs (mitogen-activated protein kinases) and the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3 axis. Moreover, the availability of P2Y13 receptor knockout mice has highlighted the specific functions in which it is involved, mainly in the regulation of cholesterol and glucose metabolism, bone homeostasis and aspects of central nervous system function like pain transmission and neuroprotection. This review summarizes our current understanding of this elusive receptor, not only at the pharmacological and molecular level but also, in terms of its signalling properties and specific functions, helping to clarify the involvement of P2Y13 receptors in pathological situations.

Ectonucleotide pyrophosphatase/phosphodiesterase activity in Neuro-2a neuroblastoma cells: changes in expression associated with neuronal differentiation.

Neuro-2a (N2a) neuroblastoma cells display an ectoenzymatic hydrolytic activity capable of degrading diadenosine polyphosphates. The Apn A-cleaving activity has been analysed with the use of the fluorogenic compound BODIPY FL guanosine 5'-O-(3-thiotriphosphate) thioester. Hydrolysis of this dinucleotide analogue showed a hyperbolic kinetic with a Km value of 4.9 ± 1.3 µM. Diadenosine pentaphosphate, diadenosine tetraphosphate, diadenosine triphosphate, and the nucleoside monophosphate AMP behaved as an inhibitor of BODIPY FL guanosine 5'-O-(3-thiotriphosphate) thioester extracellular degradation. Ectoenzymatic activity shared the typical characteristics of the ectonucleotide pyrophosphatase/phosphodiesterase family, as hydrolysis reached maximal activity at alkaline pH and was dependent on the presence of divalent cations, being strongly inhibited by EDTA and activated by Zn(2+) ions. Both NPP1 and NPP3 isozymes are expressed in N2a cells, their expression levels substantially changing when cells differentiate into a neuronal-like phenotype. In this sense, it is relevant to point the expression pattern of the NPP3 protein, whose levels were drastically reduced in the differentiated cells, being almost completely absent after 24 h of differentiation. Enzymatic activity assays carried out with differentiated N2a cells showed that NPP1 is the main isozyme involved in the extracellular degradation of dinucleotides in these cells, this enzyme reducing its activity and changing its subcellular location following neuronal differentiation. We described the presence of an ectoenzymatic activity able to hydrolyse diadenosine polyphosphates (ApnA) in N2a cells. This activity displays biochemical features that are typical of the ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) family members, as demonstrated by the use of the fluorogenic compound BODIPY-FL-GTPγS. Both NPP1 and NPP3 ectoenzymes are expressed in N2a cells, their levels dramatically changing when cells differentiate into a neuronal-like phenotype. Activity assays in differentiated cells showed that the ApnA-hydrolytic activity largely depends on the NPP1 isozyme.

Adenylate cyclase 5 coordinates the action of ADP, P2Y1, P2Y13 and ATP-gated P2X7 receptors on axonal elongation.

In adult brains, ionotropic or metabotropic purinergic receptors are widely expressed in neurons and glial cells. They play an essential role in inflammation and neurotransmission in response to purines secreted to the extracellular medium. Recent studies have demonstrated a role for purinergic receptors in proliferation and differentiation of neural stem cells although little is known about their role in regulating the initial neuronal development and axon elongation. The objective of our study was to investigate the role of some different types of purinergic receptors, P2Y1, P2Y13 and P2X7, which are activated by ADP or ATP. To study the role and crosstalk of P2Y1, P2Y13 and P2X7 purinergic receptors in axonal elongation, we treated neurons with specific agonists and antagonists, and we nucleofected neurons with expression or shRNA plasmids. ADP and P2Y1-GFP expression improved axonal elongation; conversely, P2Y13 and ATP-gated P2X7 receptors halted axonal elongation. Signaling through each of these receptor types was coordinated by adenylate cyclase 5. In neurons nucleofected with a cAMP FRET biosensor (ICUE3), addition of ADP or Blue Brilliant G, a P2X7 antagonist, increased cAMP levels in the distal region of the axon. Adenylate cyclase 5 inhibition or suppression impaired these cAMP increments. In conclusion, our results demonstrate a crosstalk between two metabotropic and one ionotropic purinergic receptor that regulates cAMP levels through adenylate cyclase 5 and modulates axonal elongation triggered by neurotropic factors and the PI3K-Akt-GSK3 pathway.

Presence of diadenosine polyphosphates in microdialysis samples from rat cerebellum in vivo: effect of mild hyperammonemia on their receptors.

Diadenosine triphosphate (Ap(3)A), diadenosine tetraphosphate (Ap(4)A), and diadenosine pentaphosphate (Ap(5)A) have been identified in microdialysis samples from the cerebellum of conscious freely moving rats, under basal conditions, by means of a high-performance liquid chromatography method. The occurrence of Ap(3)A in the cerebellar microdyalisates is noteworthy, as the presence of this compound in the interstitial medium in neural tissues has not been previously described. The concentrations measured for the diadenosine polyphosphates in the cerebellar dialysate were (in nanomolar) 10.5 ± 2.9, 5.4 ± 1.2, and 5.8 ± 1.3 for Ap(3)A, Ap(4)A, and Ap(5)A, respectively. These concentrations are in the range that allows the activation of the presynaptic dinucleotide receptor in nerve terminals. However, a possible interaction of these dinucleotides with other purinergic receptors cannot be ruled out, as rat cerebellum expresses a variety of P2X or P2Y receptors susceptible to be activated by diadenosine polyphosphates, such as the P2X1-4, P2Y(1), P2Y(2), P2Y(4), and P2Y(12) receptors, as demonstrated by quantitative real-time PCR. Also, the ecto-nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP3, able to hydrolyze the diadenosine polyphosphates and terminate their extracellular actions, are expressed in the rat cerebellum. All these evidences contribute to reinforce the role of diadenosine polyphosphates as signaling molecules in the central nervous system. Finally, we have analyzed the possible differences in the concentration of diadenosine polyphosphates in the cerebellar extracellular medium and changes in the expression levels of their receptors and hydrolyzing enzymes in an animal model of moderate hyperammonemia.

The specificity protein factor Sp1 mediates transcriptional regulation of P2X7 receptors in the nervous system.

P2X7 receptors are involved not only in physiological functions but also in pathological brain processes. Although an increasing number of findings indicate that altered receptor expression has a causative role in neurodegenerative diseases and cancer, little is known about how expression of P2rx7 gene is controlled. Here we reported the first molecular and functional evidence that Specificity protein 1 (Sp1) transcription factor plays a pivotal role in the transcriptional regulation of P2X7 receptor. We delimited a minimal region in the murine P2rx7 promoter containing four SP1 sites, two of them being highly conserved in mammals. The functionality of these SP1 sites was confirmed by site-directed mutagenesis and Sp1 overexpression/down-regulation in neuroblastoma cells. Inhibition of Sp1-mediated transcriptional activation by mithramycin A reduced endogenous P2X7 receptor levels in primary cultures of cortical neurons and astrocytes. Using P2rx7-EGFP transgenic mice that express enhanced green fluorescent protein under the control of P2rx7 promoter, we found a high correlation between reporter expression and Sp1 levels in the brain, demonstrating that Sp1 is a key element in the transcriptional regulation of P2X7 receptor in the nervous system. Finally, we found that Sp1 mediates P2X7 receptor up-regulation in neuroblastoma cells cultured in the absence of serum, a condition that enhances chromatin accessibility and facilitates the exposure of SP1 binding sites.

Ca2+-activated nucleotidase 1, a novel target gene for the transcriptional repressor DREAM (downstream regulatory element antagonist modulator), is involved in protein folding and degradation.

DREAM is a Ca(2+)-dependent transcriptional repressor highly expressed in neuronal cells. A number of genes have already been identified as the target of its regulation. Targeted analysis performed on cerebella from transgenic mice expressing a dominant active DREAM mutant (daDREAM) showed a drastic reduction of the amount of transcript of Ca(2+)-activated nucleotidase 1 (CANT1), an endoplasmic reticulum (ER)-Golgi resident Ca(2+)-dependent nucleoside diphosphatase that has been suggested to have a role in glucosylation reactions related to the quality control of proteins in the ER and the Golgi apparatus. CANT1 down-regulation was also found in neuroblastoma SH-SY5Y cells stably overexpressing wild type (wt) DREAM or daDREAM, thus providing a simple cell model to investigate the protein maturation pathway. Pulse-chase experiments demonstrated that the down-regulation of CANT1 is associated with reduced protein secretion and increased degradation rates. Importantly, overexpression of wtDREAM or daDREAM augmented the expression of the EDEM1 gene, which encodes a key component of the ER-associated degradation pathway, suggesting an alternative pathway to enhanced protein degradation. Restoring CANT1 levels in neuroblastoma clones recovered the phenotype, thus confirming a key role of CANT1, and of the regulation of its gene by DREAM, in the control of protein synthesis and degradation.

Seizure suppression and neuroprotection by targeting the purinergic P2X7 receptor during status epilepticus in mice.

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.

P2X7 receptors trigger ATP exocytosis and modify secretory vesicle dynamics in neuroblastoma cells.

Previously, we reported that purinergic ionotropic P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca(2+)/calmodulin-dependent kinase II-related mechanism. In the present study we used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca(2+)-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy, we have observed both the increase in near-membrane Ca(2+) concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus, involving this receptor type in the control of early steps (docking and priming) of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as vesicular nucleotide and monoamine (VMAT-1 and VMAT-2) transporters. Biochemical measurements indicated that ionomycin induced a significant release of ATP from N2a cells. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents, reminiscent of postsynaptic quantal events observed at synapses. Small transient inward currents were dependent on extracellular Ca(2+) and were abolished by Brilliant Blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation.

BCI, an inhibitor of the DUSP1 and DUSP6 dual specificity phosphatases, enhances P2X7 receptor expression in neuroblastoma cells.

P2X7 receptor (P2RX7) is expressed strongly by most human cancers, including neuroblastoma, where high levels of P2RX7 are correlated with a poor prognosis for patients. Tonic activation of P2X7 receptor favors cell metabolism and angiogenesis, thereby promoting cancer cell proliferation, immunosuppression, and metastasis. Although understanding the mechanisms that control P2X7 receptor levels in neuroblastoma cells could be biologically and clinically relevant, the intracellular signaling pathways involved in this regulation remain poorly understood. Here we show that (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), an allosteric inhibitor of dual specificity phosphatases (DUSP) 1 and 6, enhances the expression of P2X7 receptor in N2a neuroblastoma cells. We found that exposure to BCI induces the phosphorylation of mitogen-activated protein kinases p38 and JNK, while it prevents the phosphorylation of ERK1/2. BCI enhanced dual specificity phosphatase 1 expression, whereas it induced a decrease in the dual specificity phosphatase 6 transcripts, suggesting that BCI-dependent inhibition of dual specificity phosphatase 1 may be responsible for the increase in p38 and JNK phosphorylation. The weaker ERK phosphorylation induced by BCI was reversed by p38 inhibition, indicating that this MAPK is involved in the regulatory loop that dampens ERK activity. The PP2A phosphatase appears to be implicated in the p38-dependent dephosphorylation of ERK1/2. In addition, the PTEN phosphatase inhibition also prevented ERK1/2 dephosphorylation, probably through p38 downregulation. By contrast, inhibition of the p53 nuclear factor decreased ERK phosphorylation, probably enhancing the activity of p38. Finally, the inhibition of either p38 or Sp1-dependent transcription halved the increase in P2X7 receptor expression induced by BCI. Moreover, the combined inhibition of both p38 and Sp1 completely prevented the effect exerted by BCI. Together, our results indicate that dual specificity phosphatase 1 acts as a novel negative regulator of P2X7 receptor expression in neuroblastoma cells due to the downregulation of the p38 pathway.

Tissue-nonspecific alkaline phosphatase promotes the neurotoxicity effect of extracellular tau.

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.

P2X7 receptors in the central nervous system.

For the past three decades, our laboratory has conducted pioneering research to elucidate the complexity of purinergic signaling in the CNS, alone and in collaboration with other groups, inspired by the ground-breaking efforts of Geoffrey Burnstock. This review summarizes our contribution to understand the nucleotide receptor signaling in the CNS with a special focus on the P2X7 receptor.

Combining low-density cell culture, single-cell tracking, and patch-clamp to monitor the behavior of postnatal murine cerebellar neural stem cells.

Low-density cell culture of the postnatal cerebellum, combined with live imaging and single-cell tracking, allows the behavior of postnatal cerebellar neural stem cells (NSCs) and their progeny to be monitored. Cultured cerebellar NSCs maintain their neurogenic nature giving rise, in the same relative proportions that exist in vivo, to the neuronal progeny generated by the three postnatal cerebellar neurogenic niches. This protocol describes the identification of the nature of the progeny through both post-imaging immunocytochemistry and patch-clamp recordings. For complete details on the use and execution of this protocol, please refer to Paniagua-Herranz et al. (2020b).

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.

Altered P2X7-receptor level and function in mouse models of Huntington's disease and therapeutic efficacy of antagonist administration.

The precise mechanism by which mutant huntingtin elicits its toxicity remains unknown. However, synaptic alterations and increased susceptibility to neuronal death are known contributors to Huntington's disease (HD) symptomatology. While decreased metabolism has long been associated with HD, recent findings have surprisingly demonstrated reduced neuronal apoptosis in Caenorhabditis elegans and Drosophila models of HD by drugs that diminish ATP production. Interestingly, extracellular ATP has been recently reported to elicit neuronal death through stimulation of P2X7 receptors. These are ATP-gated cation channels known to modulate neurotransmitter release from neuronal presynaptic terminals and to regulate cytokine production and release from microglia. We hypothesized that alteration in P2X7-mediated calcium permeability may contribute to HD synaptic dysfunction and increased neuronal apoptosis. Using mouse and cellular models of HD, we demonstrate increased P2X7-receptor level and altered P2X7-mediated calcium permeability in somata and terminals of HD neurons. Furthermore, cultured neurons expressing mutant huntingtin showed increased susceptibility to apoptosis triggered by P2X7-receptor stimulation. Finally, in vivo administration of the P2X7-antagonist Brilliant Blue-G (BBG) to HD mice prevented neuronal apoptosis and attenuated body weight loss and motor-coordination deficits. These in vivo data strongly suggest that altered P2X7-receptor level and function contribute to HD pathogenesis and highlight the therapeutic potential of P2X7 receptor antagonists.

Intracellular Calcium Recording After Purinoceptor Activation Using a Video-Microscopy Equipment.

Calcium is one of the most important intracellular messengers, triggering a wide range of cellular responses. Changes in intracellular free calcium concentration can be measured using calcium sensitive fluorescent dyes, which are either EGTA- or BAPTA-based organic molecules that change their spectral properties in response to Ca2+ binding. One of the most common calcium indicators is the ratiometric dye Fura-2. The main advantage of using ratiometric dyes is that the ratio signal is independent of the illumination intensity, dye concentration, photobleaching, and focus changes among others, allowing for the concentration of intracellular calcium to be determined independently of these artifacts. In this protocol, we describe the use of Fura-2 to measure intracellular calcium elevations in single cultured cells after purinoceptor activation using a video-microscopy equipment. This method, usually known as calcium imaging, allows for real-time quantification of intracellular calcium dynamics and can be adapted to measure agonist mediated intracellular calcium responses due to the activation of different purinergic receptors in several cellular models using the appropriate growth conditions.