Functional characterization of P2Y1 and P2X4 receptors in human neuroblastoma SK-N-MC cells / Caracterización funcional de receptores P2Y1 y P2X4 en células SK-N-MC de neuroblastoma humano

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 line

Los 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 α,β- 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-MC

In vivo P2X7 inhibition reduces amyloid plaques in Alzheimer's disease through GSK3ß and secretases.

ß-Amyloid (Aß) peptide production from amyloid precursor protein (APP) is essential in the formation of the ß-amyloid plaques characteristic of Alzheimer's disease. However, the extracellular signals that maintain the balance between nonpathogenic and pathologic forms of APP processing, mediated by α-secretase and ß-secretase respectively, remain poorly understood. In the present work, we describe regulation of the processing of APP via the adenosine triphosphate (ATP) receptor P2X7R. In 2 different cellular lines, the inhibition of either native or overexpressed P2X7R increased α-secretase activity through inhibition of glycogen synthase kinase 3 (GSK-3). In vivo inhibition of the P2X7R in J20 mice, transgenic for mutant human APP, induced a significant decrease in the number of hippocampal amyloid plaques. This reduction correlated with a decrease in glycogen synthase kinase 3 activity in J20 mice, increasing the proteolytic processing of APP through an increase in α-secretase activity. The in vivo findings presented here demonstrate for the first time the therapeutic potential of P2X7R antagonism in the treatment of familiar Alzheimer's disease (FAD).

Opposite effects of P2X7 and P2Y2 nucleotide receptors on α-secretase-dependent APP processing in Neuro-2a cells.

The amyloid precursor protein (APP) is proteolytically processed by ß- and γ-secretases to release amyloid-ß peptide (Aß), the main component found in senile plaques of Alzheimer's disease (AD) patient brains. Alternatively, APP can be cleaved within the Aß sequence by α-secretase, thus precluding the generation of Aß. We have demonstrated that activation of the P2X7 receptor leads to a reduction of α-secretase activity in Neuro-2a cells. Moreover, the P2X7 ligand 2'(3')-O-(4-benzoylbenzoyl) ATP (BzATP) can also activate a different P2 receptor in these cells. This receptor, whose pharmacology resembles that of the P2Y(2) receptor, has an opposite effect, leading to increases in α-secretase activity. Our study suggests that P2X7R and P2Y(2)R could be novel therapeutic targets in AD.

Altered nucleotide receptor expression in a murine model of cerebral malaria.

In cerebral malaria, the most severe complication of malaria, both neurotransmission mechanisms and energy metabolism are affected. To understand how metabolic changes modify neurotransmission, we examined P2 receptor expression in a murine model of cerebral malaria. Quantitative polymerase chain reaction experiments revealed that parasite deposition was greatest in the cerebellum, compared with other areas of the brain, suggesting a correlation between brain parasitemia and loss of control of movement. Infected mice showed modified patterns of expression of P2 receptor subtype messenger RNA (mRNA), depending on both the specific purinergic receptor and the cerebral region analyzed. Immunohistochemical studies indicated altered levels of protein expression by these receptors in infected brains and, in some cases, a pattern of expression different from that noted in control mice. These differences in both the amount of mRNA and the protein distribution of P2 receptors observed in the different brain sites in infected mice suggest an important role for P2 receptors in either provoking cerebral damage or conferring neuroprotection.

Axodendritic fibres of mouse cerebellar granule neurons exhibit a diversity of functional P2X receptors.

Distribution and functional expression of P2X receptors were analyzed in mouse cerebellum axodendritic fibres, using different experimental approaches such as RT-PCR, western blot, immunochemistry, microfluorimetric experiments and exocytotic studies. RT-PCR and western blot demonstrated the presence of P2X1-4,7 subunits in both whole cerebellum and mouse cerebellar granule cultured neurons. Immunochemistry analysis of tissular and cellular location of P2X1-4,7 receptors confirmed their presence and unequal distribution between somas and axodendritic prolongations. Microfluorimetric experiments using a variety of modulators of the P2X subunits revealed the presence of different functional P2X receptors in the axodendritic fibres. The use of the synthetic agonist alpha,beta-meATP and the antagonist Ip(5)I revealed the activation of functional P2X1 and P2X3 receptors. Responses mediated by P2X1 subunits were also confirmed by using ZnSO(4). Activation of functional P2X4 receptors is observed when stimulated in the presence of ivermectin. Exocytotic studies confirmed the role of most P2X subunits in the activation of neurotransmitter release in axodendritic fibres from mouse cerebellar granule neurons.

Physiological role of extracellular nucleotides at the central nervous system: signalling through P2X and P2Y receptors

En los últimos años los receptores de nucleótidos, receptores ionotrópicosP2X1-7 y metabotrópicos P2Y1, 2, 4, 6, 11, 12, 13, 14, han adquirido una importancia excepcionaldebido a su localización estratégica en órganos y tejidos, a su gran variedadjunto con la complejidad de vías de señalización a las que están asociados y a lasprimeras evidencias de importantes alteraciones debidas a su mal funcionamiento.Nuestro grupo ha sido pionero en la caracterización estos receptores en el sistemanervioso, donde definimos su localización y su funcionalidad. La abundante presencia,a nivel presináptico, de las subunidades P2X3 y P2X7 debe ser resaltada,donde gracias a la entrada de calcio inducen la exocitosis de varios neurotransmisores,como glutamato, GABA, catecolaminas y acetilcolina entre otros, como hasido descrito por nuestro grupo en trabajos previos. Además, estos receptores inducenuna profunda remodelación del citoesqueleto de las terminales nerviosas yde los mecanismos exocitóticos a través de la CaMKII y pueden interactuar conotros receptores ionotrópicos y metabotrópicos co-existentes en sus cercanías. Lamayoría de los receptores P2Y también están presentes en las células nerviosas,activando vías de señalización a través de una gran variedad de cascadas intracelulares.Recientemente hemos demostrado que los receptores metabotrópicos P2Ypertenecientes a la sub-familia de receptores activados por ADP, especialmente elP2Y13, están conectados con la señalización hacia GSK3 y β-catenina, lo que abrenuevas vías para la comprensión de la función de los nucleótidos en la supervivenciay el mantenimiento de las células nerviosas. Además, tanto los receptores P2Xcomo los P2Y juegan un papel en los estadíos iniciales del desarrollo y en lamaduración neuronal donde su función aún ha de ser plenamente comprendida.Los receptores de nucleótidos son también muy abundantes en las células gliales, y nuestro grupo ha demostrado que la mayoría de los receptores P2Y están presentesy son plenamente funcionales en astrocitos en cultivo, donde, dependiendo delsubtipo de receptor, activan una gran variedad de cascadas de señalización

In the last few years nucleotide receptors, the ionotropic P2X1-7 subunits andthe metabotropic P2Y1, 2, 4, 6, 11, 12, 13, 14, have acquired an excepcional importance dueto their strategic location in organs and tissues, their great variety along with thecomplexity of the associated signalling pathways and the first evidence of theserious alterations entailed in their dysfunctions. Our group has been pioneer inthe characterization of these receptors in the nervous system, where we definedtheir location and functionality. The abundant presence, at a presynaptic level, ofP2X3 and P2X7 should be emphasized, where by means of calcium intake theyinduce neurotransmitter exocytosis, such as glutamate, GABA, catecholamines andacetylcholine among others, as described in previous works by our group. In addition, they induce an extensive remodeling of the terminal’s cytoskeleton and exocytoticmechanisms through CaMKII and they can interact widely with other ionotropicand metabotropic receptors co-existing in nearby areas. Neural cells alsoexhibit the presence of most P2Y receptors signalling through a large variety ofintracellular cascades. Recently we have demostrated that P2Y metabotropic receptorsof the sub-family activated by ADP, especially P2Y13, are connected withthe signalling towards GSK3 and Beta-catenin, opening new ways of understading thenucleotide function in survival and maintenance of neural cells. In addition bothP2X and P2Y receptors play a role in early developmental stages and neural maturationwhere their function has to be fully understanded. Nucleotide receptorsare also very abundant in glial cells, and our group has shown that most P2Yreceptors are present and fully functional in cultured astrocytes, where, dependingon the subtype receptor they activate a large variety of signalling cascades