RESUMEN
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.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo , Receptores Purinérgicos P2X7 , Animales , Ratas , Receptores ErbB/metabolismo , Neuronas/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Transducción de SeñalRESUMEN
As members of the family of nucleotide receptors, P2X7 receptors are of particular interest due to their unique structural and pharmacological characteristics. As ATP-gated ionic channels, P2X7 receptors in their activation elicit membrane depolarization; extracellular calcium influx; and activation of several downstream intracellular signaling pathways, some of them independent of the ionic channel activity. Further interactions of P2X7 receptors and cytoskeleton-related proteins have also been confirmed, and we previously described the effects of P2X7 receptor stimulation on the morphology of rat cerebellar astrocytes. In the present work, we used time-lapse video microscopy and atomic force microscopy (AFM) to elucidate the effects of P2X7 receptor stimulation on the morphology, migratory capabilities, and mechanical properties of rat cerebellar astrocytes in vitro. Stimulation of P2X7 receptors with the selective agonist BzATP specifically caused an increase in cell size, motility, and number of membrane protrusions of the astrocytes in culture. These effects were reverted when cells were previously treated with the competitive antagonist of P2X7R, A 438079. AFM analysis also showed an increase in cell stiffness and viscosity after P2X7 receptor stimulation. Surprisingly, these effects on the mechanical properties of the cell were not blocked by the treatment with the antagonist. Fluorescence microscopy analysis of the actin cytoskeleton showed an increase in actin stress fibers after BzATP treatment, an effect that again was not blocked by previous treatment with the antagonist, further confirming that the effects of P2X7 receptors on the cytoskeleton of astrocytes are, at least in part, independent of the ionic channel activity.
Asunto(s)
Astrocitos , Nucleótidos , Actinas/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Astrocitos/metabolismo , Calcio/metabolismo , Señalización del Calcio , Células Cultivadas , Nucleótidos/metabolismo , Ratas , Receptores Purinérgicos P2X7/metabolismoRESUMEN
Purinergic signaling regulates a plethora of physiological processes and is an expanding research field [...].
Asunto(s)
Adenosina Trifosfato/metabolismo , Uniones Comunicantes/fisiología , Receptores Purinérgicos/metabolismo , Animales , Humanos , Transducción de SeñalRESUMEN
Endothelial cells and astrocytes preferentially express metabotropic P2Y nucleotide receptors, which are involved in the maintenance of vascular and neural function. Among these, P2Y1 and P2Y2 receptors appear as main actors, since their stimulation induces intracellular calcium mobilization and activates signaling cascades linked to cytoskeletal reorganization. In the present work, we have analyzed, by means of atomic force microscopy (AFM) in force spectroscopy mode, the mechanical response of human umbilical vein endothelial cells (HUVEC) and astrocytes upon 2MeSADP and UTP stimulation. This approach allows for simultaneous measurement of variations in factors such as Young's modulus, maximum adhesion force and rupture event formation, which reflect the potential changes in both the stiffness and adhesiveness of the plasma membrane. The largest effect was observed in both endothelial cells and astrocytes after P2Y2 receptor stimulation with UTP. Such exposure to UTP doubled the Young's modulus and reduced both the adhesion force and the number of rupture events. In astrocytes, 2MeSADP stimulation also had a remarkable effect on AFM parameters. Additional studies performed with the selective P2Y1 and P2Y13 receptor antagonists revealed that the 2MeSADP-induced mechanical changes were mediated by the P2Y13 receptor, although they were negatively modulated by P2Y1 receptor stimulation. Hence, our results demonstrate that AFM can be a very useful tool to evaluate functional native nucleotide receptors in living cells.
Asunto(s)
Adenosina Difosfato/análogos & derivados , Astrocitos/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2/metabolismo , Tionucleótidos/metabolismo , Uridina Trifosfato/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Astrocitos/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Microscopía de Fuerza Atómica , Transducción de Señal , Tionucleótidos/farmacología , Uridina Trifosfato/farmacologíaRESUMEN
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.
Asunto(s)
Fosfatasas de Especificidad Dual/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Animales , Encefalopatías/metabolismo , Encefalopatías/patología , Humanos , Inflamación/metabolismo , Inflamación/patología , Factores de Crecimiento Nervioso/metabolismo , Neurogénesis , Neuroglía/citología , Neuroglía/patología , Neuronas/citología , Neuronas/patología , Estrés Oxidativo , Dolor/metabolismo , Dolor/patologíaRESUMEN
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.
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Sistema de Señalización de MAP Quinasas , Polimorfismo Genético , Receptores Purinérgicos P2 , Animales , Cromosomas Humanos Par 3/genética , Cromosomas Humanos Par 3/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Ratones , Ratones Noqueados , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Relación Estructura-ActividadRESUMEN
Nucleotides activating P2Y13 receptors display neuroprotective actions against different apoptotic stimuli in cerebellar granule neurons. In the present study, P2Y13 neuroprotection was analyzed in conditions of genotoxic stress. Exposure to cisplatin and UV radiation induced caspase-3-dependent apoptotic cell death, and p38 MAPK signaling de-regulation. Pre-treatment with P2Y13 nucleotide agonist, 2methyl-thio-ADP (2MeSADP), restored granule neuron survival and prevented p38 long-lasting activation induced by cytotoxic treatments. Microarray gene expression analysis in 2MeSADP-stimulated cells revealed over-representation of genes related to protein phosphatase activity. Among them, dual-specificity phosphatase-2, DUSP2, was validated as a transcriptional target for P2Y13 receptors by QPCR. This effect could explain 2MeSADP ability to dephosphorylate a DUSP2 substrate, p38, reestablishing the inactive form. In addition, cisplatin-induced p38 sustained activation correlated perfectly with progressive reduction in DUSP2 expression. In conclusion, P2Y13 receptors regulate DUSP2 expression and contribute to p38 signaling homeostasis and survival in granule neurons.
Asunto(s)
Daño del ADN , Fosfatasa 2 de Especificidad Dual/metabolismo , Sistema de Señalización de MAP Quinasas , Fármacos Neuroprotectores/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Animales , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Núcleo Celular/efectos de la radiación , Cerebelo/patología , Cisplatino/farmacología , Citoprotección/efectos de los fármacos , Citoprotección/efectos de la radiación , Activación Enzimática/efectos de los fármacos , Activación Enzimática/efectos de la radiación , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de la radiación , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/patología , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Ratas , Ratas Wistar , Tionucleótidos/farmacología , Rayos Ultravioleta , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Extracellular nucleotides have been recognized as important modulators of inflammation via their action on specific pyrimidine receptors (P2). This regulation coexists with the temporal framework of proinflammatory and proresolution mediators released by the cells involved in the inflammatory response, including macrophages. Under proinflammatory conditions, the expression of cyclooxygenase-2 leads to the release of large amounts of PGs, such as PGE2, that exert their effects through EP receptors and other intracellular targets. The effect of these PGs on P2 receptors expressed in murine and human macrophages was investigated. In thioglycollate-elicited and alternatively activated macrophages, PGE2 selectively impairs P2Y but not P2X7 Ca(2+) mobilization. This effect is absent in LPS-activated cells and is specific for PGE2 because it cannot be reproduced by other PGs with cyclopentenone structure. The inhibition of P2Y responses by PGE2 involves the activation of nPKCs (PKCε) and PKD that can be abrogated by selective inhibitors or by expression of dominant-negative forms of PKD. The inhibition of P2Y signaling by PGE2 has an impact on the cell migration elicited by P2Y agonists in thioglycollate-elicited and alternatively activated macrophages, which provide new clues to understand the resolution phase of inflammation, when accumulation of PGE2, anti-inflammatory and proresolving mediators occurs.
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Calcio/fisiología , Dinoprostona/fisiología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Receptores Purinérgicos P2Y/fisiología , Transducción de Señal/inmunología , Animales , Señalización del Calcio/inmunología , Células Cultivadas , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Dinoprostona/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Macrófagos Peritoneales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Purinérgicos P2Y/deficiencia , Receptores Purinérgicos P2Y/metabolismoRESUMEN
The nucleotide uridine trisphosphate (UTP) released to the extracellular milieu acts as a signaling molecule via activation of specific pyrimidine receptors (P2Y). P2Y receptors are G protein-coupled receptors expressed in many cell types. These receptors mediate several cell responses and they are involved in intracellular calcium mobilization. We investigated the role of the prostanoid PGE2 in P2Y signaling in mouse embryonic fibroblasts (MEFs), since these cells are involved in different ontogenic and physiopathological processes, among them is tissue repair following proinflammatory activation. Interestingly, Ca(2+)-mobilization induced by UTP-dependent P2Y activation was reduced by PGE2 when this prostanoid was produced by MEFs transfected with COX-2 or when PGE2 was added exogenously to the culture medium. This Ca(2+)-mobilization was important for the activation of different metabolic pathways in fibroblasts. Moreover, inhibition of COX-2 with selective coxibs prevented UTP-dependent P2Y activation in these cells. The inhibition of P2Y responses by PGE2 involves the activation of PKCs and PKD, a response that can be suppressed after pharmacological inhibition of these protein kinases. In addition to this, PGE2 reduces the fibroblast migration induced by P2Y-agonists such as UTP. Taken together, these data demonstrate that PGE2 is involved in the regulation of P2Y signaling in these cells.
Asunto(s)
Calcio/metabolismo , Ciclooxigenasa 2/metabolismo , Fibroblastos/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Animales , Núcleo Celular/metabolismo , Inmunoensayo , Ratones , Ratones Endogámicos C57BLRESUMEN
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.
Asunto(s)
Encéfalo/metabolismo , Regulación de la Expresión Génica , Receptores Purinérgicos P2X7/genética , Factor de Transcripción Sp1/metabolismo , Animales , Encéfalo/citología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Sistema Nervioso/metabolismo , Neuronas/metabolismo , Regiones Promotoras Genéticas , Receptores Purinérgicos P2X7/metabolismo , Factor de Transcripción Sp1/genética , Activación TranscripcionalRESUMEN
The brain distribution and functional role of glial P2X7 receptors are broader and more complex than initially anticipated. We characterized P2X7 receptors from cerebellar astrocytes at the molecular, immunocytochemical, biophysical, and cell physiologic levels. Mouse cerebellar astrocytes in culture express mRNA coding for P2X7 receptors, which is translated into P2X7 receptor protein as proven by Western blot analysis and immunocytochemistry. Fura-2 imaging showed cytosolic calcium responses to ATP and the synthetic analog 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) exhibited two components, namely an initial transient and metabotropic component followed by a sustained one that depended on extracellular calcium. This latter component, which was absent in astrocytes from P2X7 receptor knockout mice (P2X7 KO), was modulated by extracellular Mg(2+), and was sensitive to Brilliant Blue G (BBG) and 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A438079) antagonism. BzATP also elicited inwardly directed nondesensitizing whole-cell ionic currents that were reduced by extracellular Mg(2+) and P2X7 antagonists (BBG and calmidazolium). In contrast to that previously reported in rat cerebellar astrocytes, sustained BzATP application induced a gradual increase in membrane permeability to large cations, such as N-methyl-d-glucamine and 4-[3-methyl-2(3H)-benzoxazolylidene)-methyl]-1-[3-(triethylammonio)propyl]diiodide, which ultimately led to the death of mouse astrocytes. Cerebellar astrocyte cell death was prevented by BBG but not by calmidazolium, removal of extracellular calcium, or treatment with the caspase-3 inhibitor, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone, thus suggesting a necrotic-type mechanism of cell death. Since this cellular response was not observed in astrocytes from P2X7 KO mice, this study suggests that stimulation of P2X7 receptor may convey a cell death signal to cerebellar astrocytes in a species-specific manner.
Asunto(s)
Astrocitos/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Cerebelo/citología , Receptores Purinérgicos P2X7/fisiología , Animales , Animales Recién Nacidos , Astrocitos/ultraestructura , Benzoxazoles/metabolismo , Western Blotting , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/efectos de los fármacos , Cerebelo/ultraestructura , Citosol/metabolismo , Femenino , Colorantes Fluorescentes , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Nucleótidos/farmacología , Técnicas de Placa-Clamp , Cultivo Primario de Células , Compuestos de Quinolinio/metabolismo , Receptores Purinérgicos P2X7/efectos de los fármacosRESUMEN
The sodium- and chloride-coupled glycine neurotransmitter transporters (GLYTs) control the availability of glycine at glycine-mediated synapses. The mainly glial GLYT1 is the key regulator of the glycine levels in glycinergic and glutamatergic pathways, whereas the neuronal GLYT2 is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft. In this study, we report that stimulation of P2Y purinergic receptors with 2-methylthioadenosine 5'-diphosphate in rat brainstem/spinal cord primary neuronal cultures and adult rat synaptosomes leads to the inhibition of GLYT2 and the stimulation of GLYT1 by a paracrine regulation. These effects are mainly mediated by the ADP-preferring subtypes P2Y(1) and P2Y(13) because the effects are partially reversed by the specific antagonists N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate and pyridoxal-5'-phosphate-6-azo(2-chloro-5-nitrophenyl)-2,4-disulfonate and are totally blocked by suramin. P2Y(12) receptor is additionally involved in GLYT1 stimulation. Using pharmacological approaches and siRNA-mediated protein knockdown methodology, we elucidate the molecular mechanisms of GLYT regulation. Modulation takes place through a signaling cascade involving phospholipase C activation, inositol 1,4,5-trisphosphate production, intracellular Ca(2+) mobilization, protein kinase C stimulation, nitric oxide formation, cyclic guanosine monophosphate production, and protein kinase G-I (PKG-I) activation. GLYT1 and GLYT2 are differentially sensitive to NO/cGMP/PKG-I both in brain-derived preparations and in heterologous systems expressing the recombinant transporters and P2Y(1) receptor. Sensitivity to 2-methylthioadenosine 5'-diphosphate by GLYT1 and GLYT2 was abolished by small interfering RNA (siRNA)-mediated knockdown of nitric-oxide synthase. Our data may help define the role of GLYTs in nociception and pain sensitization.
Asunto(s)
Tronco Encefálico/metabolismo , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Glicina/metabolismo , Neurotransmisores/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Receptores Purinérgicos P2/metabolismo , Médula Espinal/metabolismo , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/farmacología , Animales , Tronco Encefálico/citología , Células Cultivadas , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Neuronas , Dolor/metabolismo , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Médula Espinal/citología , Fosfolipasas de Tipo C/metabolismoRESUMEN
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.
RESUMEN
Glycogen synthase kinase-3 (GSK3) is a key player in the regulation of neuronal survival. Herein, we report evidence of an interaction between P2X7 receptors with NMDA and BDNF receptors at the level of GSK3 signalling and neuroprotection. The activation of these receptors in granule neurons led to a sustained pattern of GSK3 phosphorylation that was mainly PKC-dependent. BDNF was the most potent at inducing GSK3 phosphorylation, which was also dependent on PI3K. The P2X7 agonist, BzATP, exhibited additive effects with both NMDA and BDNF to rescue granule neurons from cell death induced by PI3K inhibition. This survival effect was mediated by the PKC-dependent GSK3 pathway. In addition, ERK1/2 proteins were also involved in BDNF protective effect. These results show the function of ATP in amplifying neuroprotective actions of glutamate and neurotrophins, and support the role of GSK3 as an important convergence point for these survival promoting factors in granule neurons.
Asunto(s)
Gránulos Citoplasmáticos/enzimología , Glucógeno Sintasa Quinasa 3/metabolismo , Neuronas/citología , Neuronas/enzimología , Receptor trkB/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Factor Neurotrófico Derivado del Encéfalo/farmacología , Supervivencia Celular/efectos de los fármacos , Cerebelo/citología , Cromonas/farmacología , Gránulos Citoplasmáticos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Modelos Biológicos , Morfolinas/farmacología , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , Receptores Purinérgicos P2X7RESUMEN
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.
Asunto(s)
Sistema Nervioso Central/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Sistema Nervioso Central/efectos de los fármacos , Humanos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Agonistas del Receptor Purinérgico P2X/administración & dosificación , Antagonistas del Receptor Purinérgico P2X/administración & dosificación , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
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).
Asunto(s)
Cerebelo/citología , Técnicas Citológicas/métodos , Células-Madre Neurales/citología , Animales , Células Cultivadas , Femenino , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Protein kinase D (PKD) is a family of serine/threonine kinases that can be activated by many stimuli via protein kinase C in a variety of cells. This is the first report where PKD activation and localization is studied in glial cells. Herein, we demonstrate that P2Y(2) and P2X7 receptor stimulation of primary rat cerebellar astrocytes rapidly increases PKD1/2 phosphorylation and activity. P2Y(2) receptor response evokes a PKD1/2 activation that is dependent on a pertussis toxin-insensitive G protein, phospholipase C (PLC)-mediated generation of diacylglycerol, and protein kinase C. This mechanism is similar to the one described for other G-protein coupled receptors. In contrast, the way the ionotropic P2X7 receptor activates PKD1/2 is significantly different. Importantly, this response is not dependent on calcium entry, but depends on the activity of several phospholipases, including phosphoinositide-phospholipase C (PI-PLC), phosphatidylcholine-phospholipase C (PC-PLC) and also phospholipase D (PLD). Immunoblot and confocal microscopy analysis show that PKD1/2 activation by nucleotides is transient. The active kinase first moves to and concentrates in certain plasma membrane domains. Then, phosphorylated-PKD1/2 translocates to intracellular vesicles, where it remains active. All together, our results open the perspective of PKD1/2 being involved in many physiological functions where nucleotides play important roles not only in astrocytes but in other cell types bearing these receptors.
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Astrocitos/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Proteína Quinasa C/metabolismo , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Astrocitos/efectos de los fármacos , Cerebelo/citología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleótidos/farmacología , Fosforilación/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacología , Ratas , Ratas Wistar , Receptores Purinérgicos P2/deficiencia , Receptores Purinérgicos P2X7 , Receptores Purinérgicos P2Y2 , Serina/metabolismo , Transfección/métodosRESUMEN
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.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Fura-2/metabolismo , Microscopía por Video/instrumentación , Microscopía por Video/métodos , Receptores Purinérgicos/metabolismo , Animales , Células Cultivadas , Colorantes Fluorescentes/metabolismo , Humanos , Microscopía FluorescenteRESUMEN
A comprehensive understanding of the mechanisms controlling the behavior of cell populations with regenerative potential is the first step to design effective therapeutic strategies for many diseases. However, a precise description of the biological events involved, such as proliferation, differentiation, cell fate decisions, migration, or viability, may be hampered by the classical use of experiments based on end-point analysis. By contrast, live imaging and single cell tracking provides researchers with an accurate readout of these features in cells throughout an experiment. Here, we describe a protocol to apply time-lapse video microscopy and post-processing of the data to study critical aspects of the biology and the lineage progression of multiple neural populations.
Asunto(s)
Rastreo Celular , Microscopía por Video , Neuronas/citología , Análisis de la Célula Individual , Imagen de Lapso de Tiempo/métodos , Animales , Linaje de la Célula , Supervivencia Celular , Células Cultivadas , Procesamiento de Imagen Asistido por ComputadorRESUMEN
Little is known about the intrinsic specification of postnatal cerebellar neural stem cells (NSCs) and to what extent they depend on information from their local niche. Here, we have used an adapted cell preparation of isolated postnatal NSCs and live imaging to demonstrate that cerebellar progenitors maintain their neurogenic nature by displaying hallmarks of NSCs. Furthermore, by using this preparation, all the cell types produced postnatally in the cerebellum, in similar relative proportions to those observed in vivo, can be monitored. The fact that neurogenesis occurs in such organized manner in the absence of signals from the local environment, suggests that cerebellar lineage progression is to an important extent governed by cell-intrinsic or pre-programmed events. Finally, we took advantage of the absence of the niche to assay the influence of the vesicular nucleotide transporter inhibition, which dramatically reduced the number of NSCs in vitro by promoting their progression toward neurogenesis.