The Therapeutic Potential of Purinergic Receptors in Alzheimer's Disease and Promising Therapeutic Modulators.

Recent studies have proven that the purinergic signaling pathway plays a key role in neurotransmission and neuromodulation, and is involved in various neurodegenerative diseases and psychiatric disorders. With the characterization of the subtypes of receptors in purinergic signaling, i.e. the P1 (adenosine), P2X (ion channel) and P2Y (G protein-coupled), more attention has been paid to the pathophysiology and therapeutic potential of purinergic signaling in the central nervous system disorders. Alzheimer's disease (AD) is a progressive and deadly neurodegenerative disease that is characterized by memory loss, cognitive impairment and dementia. However, as drug development aimed to prevent or control AD has series of failures in recent years, more researchers have focused on the neuroprotection-related mechanisms such as purinergic signaling in AD patients to find a potential cure. This article reviews the recent discoveries of purinergic signaling in AD, and summarizes the potential agents as modulators for the receptors of purinergic signaling in AD-related research and treatments. Thus, our paper provides an insight into purinergic signaling in the development of anti- AD therapies.

Lysophosphatidic Acid Receptor 4 Is Transiently Expressed during Cardiac Differentiation and Critical for Repair of the Damaged Heart.

Efficient differentiation of pluripotent stem cells (PSCs) into cardiac cells is essential for the development of new therapeutic modalities to repair damaged heart tissue. We identified a novel cell surface marker, the G protein-coupled receptor lysophosphatidic acid receptor 4 (LPAR4), specific to cardiac progenitor cells (CPCs) and determined its functional significance and therapeutic potential. During in vitro differentiation of mouse and human PSCs toward cardiac lineage, LPAR4 expression peaked after 3-7 days of differentiation in cardiac progenitors and then declined. In vivo, LPAR4 was specifically expressed in the early stage of embryonal heart development, and as development progressed, LPAR4 expression decreased and was non-specifically distributed. We identified the effective agonist octadecenyl phosphate and a p38 MAPK blocker as the downstream signal blocker. Sequential stimulation and inhibition of LPAR4 using these agents enhanced the in vitro efficiency of cardiac differentiation from mouse and human PSCs. Importantly, in vivo, this sequential stimulation and inhibition of LPAR4 reduced the infarct size and rescued heart dysfunction in mice. In conclusion, LPAR4 is a novel CPC marker transiently expressed only in heart during embryo development. Modulation of LPAR4-positive cells may be a promising strategy for repairing myocardium after myocardial infarction.

Agonists and Antagonists for Purinergic Receptors.

Membrane receptors that are activated by the purine nucleoside adenosine (adenosine receptors) or by purine or pyrimidine nucleotides (P2Y and P2X receptors) transduce extracellular signals to the cytosol. They play important roles in physiology and disease. The G protein-coupled adenosine receptors comprise four subtypes: A1, A2A, A2B, and A3. The G-protein-coupled P2Y receptors are subdivided into eight subtypes: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14, while the P2X receptors represent ATP-gated homomeric or heteromeric ion channels consisting of three subunits; the most important subunits are P2X1, P2X2, P2X3, P2X4, and P2X7. This chapter provides guidance for selecting suitable tool compounds for studying these large and important purine receptor families.

Using RNA Interference for Purinoceptor Knockdown In Vivo.

RNA interference (RNAi) is a powerful post-transcriptional gene silencing (PTGS) induced by small double-stranded RNA (dsRNA). The method allows silencing of genes of interest by translation inhibition or by mRNA degradation. In this chapter, we provide a brief overview of the mechanisms involved in each step of gene silencing. A nonviral infusion of short siRNA into ventricular system of rats was used to study purinoceptor in the rat brain.

Multimeric Ionotropic Purinoceptor Detection by Protein Cross-Linking.

P2X receptor subunits (P2X1 to P2X7) assemble to form trimeric homomers or heteromers. Here, we describe the use of protein cross-linking to study the composition of P2X receptor complexes. This simple protocol is useful for determining the stoichiometry of P2X heteromeric receptors as well as for assessing the effect of point mutation, truncation, or concatenation on the quaternary architecture of these receptors.

Molecular characterization of purinergic receptor P2X4 involved in Japanese flounder (Paralichthys olivaceus) innate immune response and its interaction with ATP release channel Pannexin1.

P2X4 receptor (P2X4R) is a member of trimeric ATP-gated receptor channel family. Despite the importance of P2X4R in innate immunity has been addressed in mammals, the immunological significance of P2X4R has not been characterized in fish. In the present study we identified a full-length P2X4R cDNA sequence from Japanese flounder Paralichthys olivaceus (termed poP2X4R) by RT-PCR and RACE approaches and analyzed its gene expression patterns under normal and immune challenge conditions. Qualitative RT-PCR analyses revealed that poP2X4R has a widespread distribution in all examined tissues but dominantly expressed in hepatopancreas. In Japanese flounder head kidney macrophages and peripheral blood lymphocytes, poP2X4R was rapidly and significantly up-regulated by the immune challenges of LPS, poly(I:C) and zymosan. In addition, poP2X4R was up-regulated in spleen, head kidney and gill tissues by Edwardsiella tarda infections. Furthermore, we showed that poP2X4R is a membrane glycoprotein which could interact with ATP release channel Pannexin1, an important component in extracellular ATP-activated purinergic signaling pathways involved in Japanese flounder innate immune response. From a comparative immunological point of view, our results have provided new evidence for the involvement of extracellular ATP-gated P2XRs in fish innate immunity.

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

End-to-end conformational communication through a synthetic purinergic receptor by ligand-induced helicity switching.

The long-range communication of information, exemplified by signal transduction through membrane-bound receptors, is a central biochemical function. Reversible binding of a messenger ligand induces a local conformational change that is relayed through the receptor, inducing a chemical effect typically several nanometres from the binding site. We report a synthetic receptor mimic that transmits structural information from a boron-based ligand binding site to a spectroscopic reporter located more than 2 nm away. Reversible binding of a diol ligand to the N-terminal binding site induces a screw-sense preference in a helical oligo(aminoisobutyric acid) foldamer, which is relayed to a reporter group at the remote C-terminus, communicating information about the structure and stereochemistry of the ligand. The reversible nature of boronate esterification was exploited to switch the receptor sequentially between left- and right-handed helices, while the exquisite conformational sensitivity of the helical relay allowed the reporter to differentiate even between purine and pyrimidine nucleosides as ligands.

Characterization of new G protein-coupled adenine receptors in mouse and hamster.

The nucleobase adenine has previously been reported to activate G protein-coupled receptors in rat and mouse. Adenine receptors (AdeR) thus constitute a new family of purine receptors, for which the designation "P0-receptors" has been suggested. We now describe the cloning and characterization of two new members of the AdeR family from mouse (MrgA10, termed mAde1R) and hamster (cAdeR). Both receptors were expressed in Sf9 insect cells, and radioligand binding studies were performed using [(3)H]adenine. Specific binding of the radioligand was detected in transfected, but not in untransfected cells, and K D values of 286 nM (mAde1R, B max 1.18 pmol/mg protein) and 301 nM (cAdeR, B max 17.7 pmol/mg protein), respectively, were determined. A series of adenine derivatives was investigated in competition binding assays. Minor structural modifications generally led to a reduction or loss of affinity, with one exception: 2-fluoroadenine was at least as potent as adenine itself at the cAdeR. Structure-activity relationships at all AdeR orthologs and subtypes investigated so far were similar, but not identical. For functional analyses, the cAdeR was homologously expressed in Chinese hamster ovary (CHO) cells, while the mAde1R was heterologously expressed in 1321N1 astrocytoma cells. Like the previously described AdeRs from rat (rAdeR) and mouse (mAde2R), the mAde1R (EC50 9.77 nM) and the cAdeR (EC50 51.6 nM) were coupled to inhibition of adenylate cyclase. In addition, the cAdeR from hamster expressed in CHO cells produced an increase in intracellular calcium concentrations (EC50 6.24 nM) and was found to be additionally coupled to Gq proteins.

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site.

The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe110(3.24), Asn115(3.29), Asn173(4.60), Phe179(45.39), Asn194(5.40), Phe195(5.41), Leu201(5.47), His252(6.54), and Tyr268(7.32)) for alanine and expressed them in Spodoptera frugiperda (Sf9) insect cells. Membrane preparations subjected to [(3)H]adenine binding studies revealed only minor effects indicating that none of the exchanged amino acids is part of the ligand binding pocket, at least in the inactive state of the receptor. Furthermore, we coexpressed the rAdeR and its mutants with mammalian Gi proteins in Sf9 insect cells to probe receptor activation. Two amino acid residues, Asn194(5.40) and Leu201(5.47), were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that-in contrast to most other rhodopsin-like GPCRs-the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenine-induced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors.

Metabotropic purinergic receptors in lipid membrane microdomains.

There is broad evidence that association of transmembrane receptors and signalling molecules with lipid rafts/caveolae provides an enriched environment for protein-protein interactions necessary for signal transduction, and a mechanism for the modulation of neurotransmitter and/or growth factor receptor function. Several receptors translocate into submembrane compartments after ligand binding, while others move in the opposite direction. The role of such a dynamic localization and functional facilitation is signalling modulation and receptor desensitization or internalization. Purine and pyrimidine nucleotides have been viewed as primordial precursors in the evolution of all forms of intercellular communication, and they are now regarded as fundamental extracellular signalling molecules. They propagate the purinergic signalling by binding to ionotropic and metabotropic receptors expressed on the plasma membrane of almost all cell types, tissues and organs. Here, we have illustrated the localization in lipid rafts/caveolae of G protein-coupled P1 receptors for adenosine and P2Y receptors for nucleoside tri- and di-phosphates. We have highlighted that microdomain partitioning of these purinergic GPCRs is cell-specific, as is the overall expression levels of these same receptors. Moreover, we have described that disruption of submembrane compartments can shift the purinergic receptors from raft/caveolar to non-raft/non-caveolar fractions, and then abolish their ability to activate lipid signalling pathways and to integrate with additional lipid-controlled signalling events. This modulates the biological response to purinergic ligands and most of all indicates that the topology of the various purinergic components at the cell surface not only organizes the signal transduction machinery, but also controls the final cellular response.

A Lys49-PLA2 myotoxin of Bothrops asper triggers a rapid death of macrophages that involves autocrine purinergic receptor signaling.

Lys49-PLA(2) myotoxins, an important component of various viperid snake venoms, are a class of PLA(2)-homolog proteins deprived of catalytic activity. Similar to enzymatically active PLA(2) (Asp49) and to other classes of myotoxins, they cause severe myonecrosis. Moreover, these toxins are used as tools to study skeletal muscle repair and regeneration, a process that can be very limited after snakebites. In this work, the cytotoxic effect of different myotoxins, Bothrops asper Lys49 and Asp49-PLA(2), Notechis scutatus notexin and Naja mossambica cardiotoxin, was evaluated on macrophages, cells that have a key role in muscle regeneration. Only the Lys49-myotoxin was found to trigger a rapid asynchronous death of mouse peritoneal macrophages and macrophagic cell lines through a process that involves ATP release, ATP-induced ATP release and that is inhibited by various purinergic receptor antagonists. ATP leakage is induced also at sublytical doses of the Lys49-myotoxin, it involves Ca(2+) release from intracellular stores, and is reduced by inhibitors of VSOR and the maxi-anion channel. The toxin-induced cell death is different from that caused by high concentration of ATP and appears to be linked to localized purinergic signaling. Based on present findings, a mechanism of cell death is proposed that can be extended to other cytolytic proteins and peptides.

Discovery of purinergic signalling, the initial resistance and current explosion of interest.

There has been a remarkable growth of papers published about purinergic signalling via ATP since 1972. I am most grateful to the wonderful PhD students and postdoctoral fellows who have worked with me over the years to pursue the purinergic hypothesis despite early opposition and to the many outstanding scientists around the world who are currently extending the story. Recently, therapeutic approaches to pathological disorders include the development of selective P1 and P2 receptor subtype agonists and antagonists, as well as of inhibitors of extracellular ATP breakdown and of ATP transport enhancers and inhibitors. Medicinal chemists are starting to develop small molecule purinergic drugs that are orally bioavailable and stable in vivo.

Purinergic signalling: Its unpopular beginning, its acceptance and its exciting future.

Adenosine 5'-triphosphate (ATP) was identified in 1970 as the transmitter responsible for non-adrenergic, non-cholinergic neurotransmission in the gut and bladder and the term 'purinergic' was coined. Purinergic cotransmission was proposed in 1976 and ATP is now recognized as a cotransmitter in all nerves in the peripheral and central nervous systems. P1 (adenosine) and P2 (ATP) receptors were distinguished in 1978. Cloning of these receptors in the early 1990s was a turning point in the acceptance of the purinergic signalling hypothesis. There are both short-term purinergic signalling in neurotransmission, neuromodulation and secretion and long-term (trophic) purinergic signalling of cell proliferation, differentiation and death in development and regeneration. Much is known about the mechanisms of ATP release and its breakdown by ectonucleotidases. Recently, there has been emphasis on purinergic pathophysiology, including neurodegenerative and neuropsychiatric disorders. Purinergic therapeutic strategies are being developed for treatment of gut, kidney, bladder, lung, skeletal and reproductive system disorders, pain and cancer.

The importance of alkynyl chain presence for the activity of adenine nucleosides/nucleotides on purinergic receptors.

The first demonstrations in the early seventies that adenosine had marked effects in the cerebral cortex, which were independent of its role in intermediary metabolism and could be antagonised by methylxanthines, were followed by the observations that other purine derivatives, notably ATP, may also play a critical role in cell function. In 1978 Burnstock first introduced the terms Pl for the nucleoside receptors and P2 for the nucleotide receptors, based on the most fundamental divisions of purine receptors between those for nucleosides such as adenosine and those for nucleotides such as ATP. At present, the P1 (adenosine) receptor family presents 4 subtypes, while the P2 (ATP, ADP and UTP) receptor family has been divided into P2X ionotropic receptors and P2Y metabotropic G protein-coupled receptors (GPCRs). While knowledge on the purinergic receptor pharmacology was increasing, the development of potent and selective ligands for these receptors has been a target of medicinal chemistry research for several decades. In particular, synthesis of 2-substituted adenosines was carried out in many laboratories starting from seventies aimed at finding adenosine derivatives more resistant than the parent nucleoside to rapid uptake into cells, to deamination by adenosine deaminase, and to phosphorylation by adenosine kinase. In the present review the synthesis of alkynyl derivatives of adenine, adenosine, N-alkylcarboxamidoadenosine, and adenine nucleotides, which have been tested on purinergic receptors, will be summarized. Furthermore, the contribution of chemistry, molecular modelling, and pharmacology to the development of structure-activity relationships in this class of purinergic receptor ligands will be outlined.

Desensitization of neurotransmitter-gated ion channels during high-frequency stimulation: a comparative study of Cys-loop, AMPA and purinergic receptors.

Changes in synaptic strength allow synapses to regulate the flow of information in the neural circuits in which they operate. In particular, changes lasting from milliseconds to minutes ('short-term changes') underlie a variety of computational operations and, ultimately, behaviours. Most studies thus far have attributed the short-term type of plasticity to activity-dependent changes in the dynamics of neurotransmitter release (a presynaptic mechanism) while largely dismissing the role of the loss of responsiveness of postsynaptic receptor channels to neurotransmitter owing to entry into desensitization. To better define the response of the different neurotransmitter-gated ion channels (NGICs) to repetitive stimulation without interference from presynaptic variables, we studied eight representative members of all three known superfamilies of NGICs in fast-perfused outside-out patches of membrane. We found that the responsiveness of all tested channels (two nicotinic acetylcholine receptors, two glycine receptors, one GABA receptor, two AMPA-type glutamate receptors and one purinergic receptor) declines along trains of brief neurotransmitter pulses delivered at physiologically relevant frequencies to an extent that suggests that the role of desensitization in the synaptic control of action-potential transmission may be more general than previously thought. Furthermore, our results indicate that a sizable fraction (and, for some NGICs, most) of this desensitization occurs during the neurotransmitter-free interpulse intervals. Clearly, an incomplete clearance of neurotransmitter from the synaptic cleft between vesicle-fusion events need not be invoked to account for NGIC desensitization upon repetitive stimulation.

Regulation of GABA(A) receptor dynamics by interaction with purinergic P2X(2) receptors.

γ-Aminobutyric acid type A receptors (GABA(A)Rs) in the spinal cord are evolving as an important target for drug development against pain. Purinergic P2X(2) receptors (P2X(2)Rs) are also expressed in spinal cord neurons and are known to cross-talk with GABA(A)Rs. Here, we investigated a possible "dynamic" interaction between GABA(A)Rs and P2X(2)Rs using co-immunoprecipitation and fluorescence resonance energy transfer (FRET) studies in human embryonic kidney (HEK) 293 cells along with co-localization and single particle tracking studies in spinal cord neurons. Our results suggest that a significant proportion of P2X(2)Rs forms a transient complex with GABA(A)Rs inside the cell, thus stabilizing these receptors and using them for co-trafficking to the cell surface, where P2X(2)Rs and GABA(A)Rs are primarily located extra-synaptically. Furthermore, agonist-induced activation of P2X(2)Rs results in a Ca(2+)-dependent as well as an apparently Ca(2+)-independent increase in the mobility and an enhanced degradation of GABA(A)Rs, whereas P2X(2)Rs are stabilized and form larger clusters. Antagonist-induced blocking of P2XRs results in co-stabilization of this receptor complex at the cell surface. These results suggest a novel mechanism where association of P2X(2)Rs and GABA(A)Rs could be used for specific targeting to neuronal membranes, thus providing an extrasynaptic receptor reserve that could regulate the excitability of neurons. We further conclude that blocking the excitatory activity of excessively released ATP under diseased state by P2XR antagonists could simultaneously enhance synaptic inhibition mediated by GABA(A)Rs.

Dimerization of G protein-coupled purinergic receptors: increasing the diversity of purinergic receptor signal responses and receptor functions.

It is well accepted that G protein-coupled receptors (GPCRs) arrange into dimers or higher-order oligomers that may modify various functions of GPCRs. GPCR-type purinergic receptors (i.e. adenosine and P2Y receptors) tend to form heterodimers with GPCRs not only of the different families but also of the same purinergic receptor families, leading to alterations in functional properties. In the present review, we focus on current knowledge of the formation of heterodimers between metabotropic purinergic receptors that activate novel functions in response to extracellular nucleosides/nucleotides, revealing that the dimerization seems to be employed for 'fine-tuning' of purinergic signaling. Thus, the relationship between adenosine and adenosine triphosphate is likely to be more and more intimate than simply being a metabolite of the other.