María Teresa Miras-Portugal

Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family

The cloning of a human G-protein-coupled receptor (GPCR) that specifically responds to UDP-glucose and related sugar-nucleotides has been reported recently. This receptor has important structural similarities to known members of the P2Y receptor family but also shows a distinctly different pharmacological response profile. Here, the IUPHAR Subcommittee for P2Y receptor nomenclature and classification review the current knowledge of this receptor and present their reasons for including this receptor in the P2Y receptor family as the P2Y(14) receptor.

A purinergic component of the excitatory postsynaptic current mediated by P2X receptors in the CA1 neurons of the rat hippocampus

The pyramidal neurons in the CA1 area of hippocampal slices from 17‐ to 19‐day‐old rats have been investigated by means of patch clamp. Excitatory postsynaptic currents (EPSCs) were elicited by stimulating the Schaffer collateral at a frequency below 0.2 Hz. It was found that inhibition of glutamatergic transmission by 20 μm 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and 100 μm 2‐amino‐5‐phosphonovaleric acid (D‐APV) left a small component of the EPSC uninhibited. The amplitude of this residual EPSC (rEPSC) comprised 25 ± 11% of the total EPSC when measured at a holding potential of −50 mV. The rEPSC was blocked by selective P2 blocker pyridoxal phosphate‐6‐azophenyl‐2′‐4′‐disulphonic acid (PPADS) 10 μm and bath incubation with non‐hydrolysable ATP analogues, ATP‐γ‐S and α,β‐methylene‐ATP at 50 and 20 μm, respectively. The rEPSC was dramatically potentiated by external Zn2+ (10 μm). In another series of experiments exogenous ATP was applied to the CA1 neurons in situ. An inward current evoked by ATP was inhibited by PPADS to the same extent as the rEPSC. It is concluded that, depending on membrane voltage, about one‐fifth to one‐quarter of the EPSC generated by the excitatory synaptic input to the hippocampal CA1 neurons of rat is due to the activity of P2X receptors.

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 Huntingtons 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.—Diaz‐Hernandez, M., Diez‐Zaera, M., Sanchez‐Nogueiro, J., Gomez‐Villafuertes, R., Canals, J.M., Alberch, J., Miras‐Portugal, M.T., Lucas, J.J. Altered P2X7‐receptor level and function in mouse models of Huntingtons disease and therapeutic efficacy of antagonist administration. FASEB J. 23, 1893–1906 (2009)

Extracellular tau promotes intracellular calcium increase through M1 and M3 muscarinic receptors in neuronal cells.

Extracellular tau promotes an increase in the level of intracellular calcium in cultured neuronal cells. We have found that such increase is impaired in the presence of antagonists of muscarinic receptors. In order to identify the nature of those receptors, we have tested the effect of different specific muscarinic receptor antagonists on tau promoted calcium increase. Our results indicate that the increase does not take place in the presence of antagonists of muscarinic (mainly M1 and M3) receptors. A similar increase in intracellular calcium was found in non-neuronal cells transfected with cDNA of M1 and M3 muscarinic receptors when tau was added. These results suggest that observed effect of tau protein on neuronal (neuroblastoma and primary cultures of hippocampal and cortical neurons) cells is through M1 and M3 muscarinic receptors. Therefore blocking M1 and for M3 receptors, by using specific receptor antagonists, can prevent that tau toxic effect that could take place in tauopathies.

Rapid Desensitization of the Metabotropic Glutamate Receptor that Facilitates Glutamate Release in Rat Cerebrocortical Nerve Terminals

The metabotropic autoreceptor of glutamatergic nerve terminals from the cerebral cortex of adult rats has been characterized. Receptor activation involves a rapid and transient increase in diacylglycerol, which is sensitive to l‐2‐amino‐3‐phosphonopropionate (l‐AP3) and l‐2‐amino‐4‐phosphonobutanoic acid (l‐AP4) and is partially blocked by pertussis toxin. Protein kinase C (PKC) has a negative feedback control in this transduction pathway because the activation of the kinase, either by phorbol esters or by the endogenous diacylglycerol produced by the receptor, results in a reversible receptor desensitization, with loss of the ability to further facilitate glutamate release. It is concluded that the facilitatory metabotropic receptor located at the glutamatergic nerve endings belongs to the subclass coupled to phosphoinositide hydrolysis and that the rapid and use‐dependent desensitization of the facilitatory pathway may underlie a mechanism to prevent its permanent activation and thereby to avoid neurotoxicity.

Functional switch from facilitation to inhibition in the control of glutamate release by metabotropic glutamate receptors.

We have investigated the role of metabotropic glutamate receptors linked to phosphoinositide hydrolysis in the control of glutamate release in cerebrocortical nerve terminals. The activation of these receptors with the agonist 3,5-dihydroxyphenylglycine enhanced intrasynaptosomal diacylglycerol and facilitated both the depolarization-induced increase in the cytosolic free Ca2+ concentration and the release of glutamate. However, 5 min after receptor activation, a second stimulation of the pathway with the agonist failed to produce diacylglycerol and to facilitate glutamate release. Interestingly, during the period in which the diacylglycerol response was desensitized, a strong agonist-induced inhibition of Ca2+entry and glutamate release was observed. This change in the presynaptic effects of 3,5-dihydroxyphenylglycine is reversible since 30 min after the first stimulation, the agonist-induced inhibition of release disappeared, whereas both the production of diacylglycerol and the facilitation of glutamate release were recovered. The tonic elevation of the extracellular glutamate concentration from basal levels (0.8 μm) up to 5 μm also produced the switch from facilitation to inhibition in the receptor response. The existence of this activity-dependent switch in the presynaptic control of glutamate release suggests that release facilitation is limited to conditions under which an appropriate clearance of synaptic glutamate exists, probably to prevent the neurotoxic accumulation of glutamate in the synapse.

P2X7 Receptors in Rat Brain: Presence in Synaptic Terminals and Granule Cells

ATP stimulates [Ca2+]i increases in midbrain synaptosomes via specific ionotropic receptors (P2X receptors). Previous studies have demonstrated the implication of P2X3 subunits in these responses, but additional P2X subunits must be involved. In the present study, ATP and BzATP proved to be able to induce intrasynaptosomal calcium transients in the midbrain synaptosomes, their effects being potentiated when assayed in a Mg2+-free medium. Indeed, BzATP was shown to be more potent than ATP, and their effects could be inhibited by PPADS and KN-62, but not by suramin. This activity profile is consistent with the presence of functional P2X7 receptors in the midbrain terminals. The existence of presynaptic responses to selective P2X7 agonists could be confirmed by means of a microfluorimetric technique allowing [Ca2+]i measurements in single synaptic terminals. Additionally, the P2X7 receptor protein could be identified in the midbrain synaptosomes and in axodendritic prolongations of cerebellar granule cells by immunochemical staining.

Diinosine pentaphosphate (IP5I) is a potent antagonist at recombinant rat P2X1 receptors

The antagonist activity of a series of diinosine polyphosphates (IpnI, where n=3, 4, 5) was assessed against ATP‐activated inward currents at rat P2X1–4 receptors expressed in Xenopus oocytes and studied under voltage‐clamp conditions. Diinosine polyphosphates were prepared by the enzymatic degradation of their corresponding diadenosine polyphosphates (e.g., Ap5A into Ip5I) using 5′‐adenylic deaminase, and purified using reverse‐phase chromatography. Against ATP‐responses at rP2X1 receptors, the potency order for antagonism was (pIC50): Ip5I (8.5)>Ip4I (6.3)>Ip3I (>4.5). Ip5I (10–100 nM) caused a concentration‐dependent rightwards displacement of the ATP concentration‐response curve without reducing the maximum ATP effect. However, the Schild plot was non‐linear which indicated Ip5I is not a competitive antagonist. Blockade by micromolar concentrations of Ip5I was not surmountable. Ip4I also behaved as a non‐surmountable antagonist. Against ATP‐responses at rP2X3 receptors, the potency order for antagonism was (pIC50): Ip4I (6.0)>Ip5I (5.6)>Ip3I (>4.5). Blockade by Ip4I (pA2, 6.75) and Ip5I (pA2, 6.27) was surmountable at micromolar concentrations. Diinosine polyphosphates failed to inhibit ATP‐responses at rP2X2 receptors, whereas agonist responses at rP2X4 were reversibly potentiated by Ip4I and Ip5I. None of the parent diadenosine polyphosphates behave as antagonists at rP2X1–4 receptors. Thus, Ip5I acted as a potent and relatively‐selective antagonist at the rP2X1 receptor. This dinucleotide pentaphosphate represents a high‐affinity antagonist for the P2X1 receptor, at which it acts in a competitive manner at low (100 nM) concentrations but has more complex actions at higher (>100 nM) concentrations.

In vivo P2X7 inhibition reduces amyloid plaques in Alzheimer's disease through GSK3β and secretases

β-Amyloid (Aβ) peptide production from amyloid precursor protein (APP) is essential in the formation of the β-amyloid plaques characteristic of Alzheimers disease. However, the extracellular signals that maintain the balance between nonpathogenic and pathologic forms of APP processing, mediated by α-secretase and β-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 Alzheimers disease (FAD).

Inhibition of the ATP-gated P2X7 receptor promotes axonal growth and branching in cultured hippocampal neurons

During the establishment of neural circuits, the axons of neurons grow towards their target regions in response to both positive and negative stimuli. Because recent reports show that Ca2+ transients in growth cones negatively regulate axonal growth, we studied how ionotropic ATP receptors (P2X) might participate in this process. Our results show that exposing cultured hippocampal neurons to ATP induces Ca2+ transients in the distal domain of the axon and the concomitant inhibition of axonal growth. This effect is mediated by the P2X7 receptor, which is present in the growth cone of the axon. Pharmacological inhibition of P2X7 or its silencing by shRNA interference induces longer and more-branched axons, coupled with morphological changes to the growth cone. Our data suggest that these morphological changes are induced by a signalling cascade in which CaMKII and FAK activity activates PI3-kinase and modifies the activity of its downstream targets. Thus, in the absence or inactivation of P2X7 receptor, axons grow more rapidly and form more branches in cultured hippocampal neurons, indicative that ATP exerts a negative influence on axonal growth. These data suggest that P2X7 antagonists have therapeutic potential to promote axonal regeneration.