In the mature central nervous system (CNS), oligodendrocytes provide support and insulation to axons thanks to the production of a myelin sheath. During their maturation to myelinating cells, oligodendroglial precursors (OPCs) follow a very precise differentiation program, which is finely orchestrated by transcription factors, epigenetic factors and microRNAs (miRNAs), a class of small non-coding RNAs involved in post-transcriptional regulation. Any alterations in this program can potentially contribute to dysregulated myelination, impaired remyelination and neurodegenerative conditions, as it happens in multiple sclerosis (MS). Here, we identify miR-125a-3p, a developmentally regulated miRNA, as a new actor of oligodendroglial maturation, that, in the mammalian CNS regulates the expression of myelin genes by simultaneously acting on several of its already validated targets. In cultured OPCs, over-expression of miR-125a-3p by mimic treatment impairs while its inhibition with an antago-miR stimulates oligodendroglial maturation. Moreover, we show that miR-125a-3p levels are abnormally high in the cerebrospinal fluid of MS patients bearing active demyelinating lesions, suggesting that its pathological upregulation may contribute to MS development, at least in part by blockade of OPC differentiation leading to impaired repair of demyelinated lesions.
In previous studies, we have demonstrated that exposure of astroglial cells to A3 adenosine receptor agonists results in dual actions on cell survival, with “trophic” and antiapoptotic effects at nanomolar concentrations and induction of cell death at micromolar agonist concentrations. The protective actions of A3 agonists have been associated with a reinforcement of the actin cytoskeleton, which likely results in increased resistance of cells to cytotoxic stimuli. The molecular mechanisms at the basis of this effect and the signalling pathway(s) linking the A3 receptor to the actin cytoskeleton have never been elucidated. Based on previous literature data suggesting that the actin cytoskeleton is controlled by small GTP-binding proteins of the Rho family, in the study reported here we investigated the involvement of these proteins in the effects induced by A3 agonists on human astrocytoma ADF cells. The presence of the A3 adenosine receptor in these cells has been confirmed by immunoblotting analysis. As expected, exposure of human astrocytoma ADF cells to nanomolar concentrations of the selective A3 agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA) resulted in formation of thick actin positive stress fibers. Preexposure of cells to the C3B toxin that inactivates Rho-proteins completely prevented the actin changes induced by Cl-IB-MECA. Exposure to the A3 agonist also resulted in significant reduction of Rho-GDI, an inhibitory protein known to maintain Rho proteins in their inactive state, suggesting a potentiation of Rho-mediated effects. This effect was fully counteracted by the concomitant exposure to the selective A3 receptor antagonist MRS1191. These results suggest that the reinforcement of the actin cytoskeleton induced by A3 receptor agonists is mediated by an interference with the activation/inactivation cycle of Rho proteins, which may, therefore, represent a biological target for the identification of novel neuroprotective strategies.
Adenosine; A3 receptor; Neuroprotection; Rho proteins
Montelukast and pranlukast are orally active leukotriene receptor antagonists selective for the CysLT1 receptor. Conversely, the hP2Y1,2,4,6,11,12,13,14 receptors represent a large family of GPCRs responding to either adenine or uracil nucleotides, or to sugar-nucleotides. Montelukast and pranlukast were found to inhibit nucleotide-induced calcium mobilization in a human monocyte-macrophage like cell line, DMSO-differentiated U937 (dU937). Montelukast and pranlukast inhibited the effects of UTP with IC50 values of 7.7 and 4.3 μM, respectively, and inhibited the effects of UDP with IC50 values of 4.5 and 1.6 μM, respectively, in an insurmountable manner. Furthermore, ligand binding studies using [3H]LTD4 excluded the possibility of orthosteric nucleotide binding to the CysLT1 receptor. dU937 cells were shown to express P2Y2, P2Y4, P2Y6, P2Y11, P2Y13 and P2Y14 receptors. Therefore, these antagonists were studied functionally in a heterologous expression system for the human P2Y receptors. In 1321N1 astrocytoma cells stably expressing human P2Y1,2,4,6 receptors, CysLT1 antagonists inhibited both the P2Y agonist-induced activation of phospholipase C and intracellular Ca2+ mobilization. IC50 values at P2Y1 and P2Y6 receptors were <1 μM. In control astrocytoma cells expressing an endogenous M3 muscarinic receptor, 10 μM montelukast had no effect on the carbachol-induced rise in intracellular Ca2+. These data demonstrated that CysLT1 receptor antagonists interact functionally with signaling pathways of P2Y receptors, and this should foster the study of possible implications for the clinical use of these compounds in asthma or in other inflammatory conditions.
Montelukast; Pranlukast; Purine receptors; Nucleotides; ATP; UDP
As human life expectancy has improved rapidly in industrialized societies, age-related cognitive impairment presents an increasing challenge. Targeting histopathological processes that correlate with age-related cognitive declines, such as neuroinflammation, low levels of neurogenesis, disrupted blood–brain barrier and altered neuronal activity, might lead to structural and functional rejuvenation of the aged brain. Here we show that a 6-week treatment of young (4 months) and old (20 months) rats with montelukast, a marketed anti-asthmatic drug antagonizing leukotriene receptors, reduces neuroinflammation, elevates hippocampal neurogenesis and improves learning and memory in old animals. By using gene knockdown and knockout approaches, we demonstrate that the effect is mediated through inhibition of the GPR17 receptor. This work illustrates that inhibition of leukotriene receptor signalling might represent a safe and druggable target to restore cognitive functions in old individuals and paves the way for future clinical translation of leukotriene receptor inhibition for the treatment of dementias.
The leukotriene receptor antagonist montelukast is an anti-asthmatic drug. Here, the authors show that montelukast reduces neuroinflammation, promotes hippocampal neurogenesis and restores learning and memory in old rats suffering from ageing-associated cognitive dysfunction.
G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors, and are the target of at least one-third of the current therapeutic drugs on the market. Along their life cycle, GPCRs are accompanied by a range of specialized GPCR-interacting proteins (GIPs), which take part in receptor proper folding, targeting to the appropriate subcellular compartments and in receptor signaling tasks, and also in receptor regulation processes, such as desensitization and internalization. The direction of protein-protein interactions and multi-protein complexes formation is crucial in understanding protein function and their implication in pathological events. Although several methods have been already developed to assay protein complexes, some of them are quite laborious, expensive, and, more important, they do not generate fully quantitative results. Herein, we show a rapid immunoenzymatic assay to quantify GPCR interactionswith its signaling proteins. The recently de-orphanized GPCR, GPR17, was chosen as a GPCR prototype to optimize the assay. In a GPR17 transfected cell line and primary oligodendrocyte precursor cells, GPR17 interaction with proteins involved in the typical GPCR regulation, such as desensitization and internalization machinery, was investigated. The obtained results were validated by co-immunoprecipitation experiments, confirming this new method as a rapid and quantitative assay to study protein-protein interactions.
immunoenzymatic assay; G protein coupled-receptors; protein-protein interactions
The ADP-responsive P2Y12 receptor is expressed on both platelets and microglia. Clinical data show that ticagrelor, a direct-acting, reversibly binding P2Y12-receptor antagonist, reduces total cardiovascular events, including stroke. In our present study, we investigated the expression of P2Y12 receptors and the effects of ticagrelor on brain injury in Sprague-Dawley rats subjected to a permanent middle cerebral artery occlusion (MCAo). Rats were treated per os with ticagrelor 3 mg/kg or vehicle at 10 minutes, 22, and 36 hours after MCAo and killed after 48 hours. Immunofluorescence analysis showed an ischemia-related modulation of the P2Y12 receptor, which is constitutively expressed in Iba1+ resting microglia. After MCAo, activated microglia was mainly concentrated around the lesion, with fewer cells present inside the ischemic core. Ticagrelor significantly attenuated the evolution of ischemic damage—evaluated by magnetic resonance imaging (MRI) at 2, 24, and 48 hours after MCAo—, the number of infiltrating cells expressing the microglia/monocyte marker ED-1, the cerebral expression of proinflammatory mediators (interleukin 1 (IL-1), monocyte chemoattractant protein 1 (MCP-1), nitric oxide synthase (iNOS)) and the associated neurologic impairment. In transgenic fluorescent reporter CX3CR1-green fluorescent protein (GFP) mice, 72 hours after MCAo, ticagrelor markedly reduced GFP+ microglia and both early and late infiltrating blood-borne cells. Finally, in primary cultured microglia, ticagrelor fully inhibited ADP-induced chemotaxis (P<0.01). Our results show that ticagrelor is protective against ischemia-induced cerebral injury and this effect is mediated, at least partly, by inhibition of P2Y12-mediated microglia activation and chemotaxis.
microglia; middle cerebral artery occlusion; P2Y12 receptor; rat; ticagrelor
The effects of novel, selective adenosine (ADO) A3 receptor antagonists of diverse structure on cells of the human HL-60 leukemia and U-937 lymphoma cell lines were examined. Both 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine3,5-dicarboxylate (MRS 1191, 0.5µM) and 6-carboxymethyl-5,9-dihydro-9-methyl-2-phenyl-[1,2,4]-triazolo[5,1-a][2,7]naphthyridine (L-249313, 0.5 µM) induced apoptotic cell death and expression of bak protein. Low concentrations of the A3 receptor agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyluxonamide (Cl-IB-MECA, 10 nM or 1 µM) protected against antagonist-induced cell death. At concentrations ≥ 10 µM, the agonist alone produced apoptosis and bak expression in various cell lines. It is suggested that there exists a tonic low level of A3 receptor activation, possibly induced by release of endogenous adenosine, that results in cell protection.
Blood levels of extracellular nucleotides (e.g. ATP) are greatly increased during heart ischaemia, but, despite the presence of their specific receptors on cardiomyocytes (both P2X and P2Y subtypes), their effects on the subsequent myocardial damage are still unknown. In this study, we aimed at investigating the role of ATP and specific P2 receptors in the appearance of cell injury in a cardiac model of ischaemic/hypoxic stress. Cells were maintained in a modular incubator chamber in a controlled humidified atmosphere of 95% N2 for 16 hrs in a glucose-free medium. In this condition, we detected an early increase in the release of ATP in the culture medium, which was followed by a massive increase in the release of cytoplasmic histone-associated-DNA-fragments, a marker of apoptosis. Addition of either apyrase, which degrades extracellular ATP, or various inhibitors of ATP release via connexin hemichannels fully abolished ischaemic/hypoxic stress-associated apoptosis. To dissect the role of specific P2 receptor subtypes, we used a combined approach: (i) non-selective and, when available, subtype-selective P2 antagonists, were added to cardiomyocytes before ischaemic/hypoxic stress; (ii) selected P2 receptors genes were silenced via specific small interfering RNAs. Both approaches indicated that the P2Y2 and P2χ7 receptor subtypes are directly involved in the induction of cell death during ischaemic/hypoxic stress, whereas the P2Y4 receptor has a protective effect. Overall, these findings indicate a role for ATP and its receptors in modulating cardiomyocyte damage during ischaemic/hypoxic stress.
cardiomyocytes; P2 receptors; ischaemic/hypoxic stress; apoptosis
The pathophysiological role of the adenosine A3 receptor in the central nervous system is largely unknown. We have investigated the effects of the selective A3 receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine, Cl-IB-MECA, in cells of the astroglial lineage (human astrocytoma ADF cells). A marked reorganization of the cytoskeleton, with appearance of stress fibers and numerous cell protrusions, was found following exposure of cells to low (nM) concentrations of Cl-IB-MECA. These “trophic” effects were accompanied by induction of the expression of Rho, a small GTP-binding protein, which was virtually absent in control cells, and by changes of the intracellular distribution of the antiapoptotic protein Bcl-xL, that, in agonist-exposed cells, became specifically associated to cell protrusions. This is the first demonstration that the intracellular organization of Bcl-xL can be modulated by the activation of a G-protein-coupled membrane receptor, such as the A3 adenosine receptor. Moreover, modulation of the astrocytic cytoskeleton by adenosine may have intriguing implications in both nervous system development and in the response of the brain to trauma and ischemia.
GPR17 is a Gi-coupled dual receptor activated by uracil-nucleotides and cysteinyl-leukotrienes. These mediators are massively released into hypoxic tissues. In the normal heart, GPR17 expression has been reported. By contrast, its role in myocardial ischaemia has not yet been assessed. In the present report, the expression of GPR17 was investigated in mice before and at early stages after myocardial infarction by using immunofluorescence, flow cytometry and RT-PCR. Before induction of ischaemia, results indicated the presence of the receptor in a population of stromal cells expressing the stem-cell antigen-1 (Sca-1). At early stages after ligation of the coronary artery, the receptor was expressed in Sca-1+ cells, and cells stained with Isolectin-B4 and anti-CD45 antibody. GPR17+ cells also expressed mesenchymal marker CD44. GPR17 function was investigated in vitro in a Sca-1+/CD31− cell line derived from normal hearts. These experiments showed a migratory function of the receptor by treatment with UDP-glucose and leukotriene LTD4, two GPR17 pharmacological agonists. The GPR17 function was finally assessed in vivo by treating infarcted mice with Cangrelor, a pharmacological receptor antagonist, which, at least in part, inhibited early recruitment of GPR17+ and CD45+ cells. These findings suggest a regulation of heart-resident mesenchymal cells and blood-borne cellular species recruitment following myocardial infarction, orchestrated by GPR17.
myocardial ischaemia; cardiac stromal cell; GPR17; Cysteinyl-Leukotrienes; myofibroblasts
We previously showed that the human heart expresses all known P2X and P2Y receptors activated by extra-cellular adenine or uracil nucleotides. Despite evidence that, both in humans and rodents, plasma levels of ATP and UTP markedly increase during myocardial infarction, the differential effects mediated by the various adenine- and uracil-preferring myocardial P2 receptors are still largely unknown. Here, we studied the effects of adenine and uracil nucleotides on murine HL-1 cardiomyocytes. RT-PCR analysis showed that HL-1 cardiomyocytes express all known P2X receptors (except for P2X2), as well as the P2Y2,4,6,14 subtypes. Exposure of cardiomyocytes to adenine nucleotides (ATP, ADP or BzATP) induced apoptosis and necrosis, as determined by flow-cytometry. Cell death was exacerbated by tumour necrosis factor (TNF)-α, a cytokine implicated in chronic heart failure progression. Conversely, uracil nucleotides (UTP, UDP and UDPglucose) had no effect ‘per se’, but fully counteracted the deleterious effects induced by adenine nucleotides and TNF-α, even if added to cardiomyocytes after beginning exposure to these cell death-inducing agents. Thus, exposure of cardiomyocytes to elevated concentrations of ATP or ADP in the presence of TNF-α contributes to cell death, an effect which is counteracted by uracil-preferring P2 receptors. Cardiomyocytes do not need to be ‘primed’ by uracil nucleotides to become insensitive to adenine nucleotides-induced death, suggesting the existence of a possible ‘therapeutic’ window for uracil nucleotides-mediated protection. Thus, release of UTP during cardiac ischaemia and in chronic heart failure may protect against myocardial damage, setting the basis for developing novel cardioprotective agents that specifically target uracil-preferring P2Y receptors.
apoptosis; heart failure; P2Y receptors; cytokines; signal transduction
Unveiling the mechanisms participating in the damage and repair of traumatic brain injury (TBI) is fundamental to develop new therapies. The P2Y-like GPR17 receptor has recently emerged as a sensor of damage and a key actor in lesion remodeling/repair in the rodent brain, but its role in humans is totally unknown. Here, we characterized GPR17 expression in brain specimens from seven intensive care unit TBI patients undergoing neurosurgery for contusion removal and from 28 autoptic TBI cases (and 10 control subjects of matched age and gender) of two university hospitals. In both neurosurgery and autoptic samples, GPR17 expression was strong inside the contused core and progressively declined distally according to a spatio-temporal gradient. Inside and around the core, GPR17 labeled dying neurons, reactive astrocytes, and activated microglia/macrophages. In peri-contused parenchyma, GPR17 decorated oligodendrocyte precursor cells (OPCs) some of which had proliferated, indicating re-myelination attempts. In autoptic cases, GPR17 expression positively correlated with death for intracranial complications and negatively correlated with patients’ post-traumatic survival. Data indicate lesion-specific sequential involvement of GPR17 in the (a) death of irreversibly damaged neurons, (b) activation of microglia/macrophages remodeling the lesion, and (c) activation/proliferation of multipotent parenchymal progenitors (both reactive astrocytes and OPCs) starting repair processes. Data validate GPR17 as a target for neurorepair and are particularly relevant to setting up new therapies for TBI patients.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-013-9366-3) contains supplementary material, which is available to authorized users.
Activated microglia; Adult neural precursors; Human brain injury; Lesion repair; Reactive astrocytes
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Xanthine and adenosine derivatives, known to bind to recombinant rat A3 adenosine receptors stably expressed in Chinese hamster ovary cells, were characterized in a functional assay consisting of activation of A3 receptor-stimulated binding of [35S]GTPγS in rat RBL-2H3 cell membranes. 1,3-Dibutylxanthine-7-riboside-5′-N-methylcarboxamide (DBXRM, 7b), previously shown to inhibit adenylyl cyclase via rat A3 receptors with full efficacy, appeared to be a partial agonist at the rat A3 receptor of RBL-2H3 cells. Full agonists, such as Cl-IB-MECA or I-AB-MECA, were more potent and effective than the partial agonist DBXRM in causing desensitization of rat A3 receptors, as indicated by loss of [35S]GTPγS binding. At A1 receptors, antagonism of agonist-elicited inhibition of rat adipocyte adenylyl cyclase was observed for several xanthine-7-riboside derivatives that had been shown to be full agonists at rat A3 receptors. A new xanthine riboside (3′-deoxyDBXRM, 7c) was synthesized and found to be a partial agonist at rat A3 receptors and an antagonist at rat A1 receptors. Thus, it is possible for the same compound to stimulate one adenosine receptor subtype (A3) and block another subtype (A1) within the same species.
xanthines; adenosine derivatives; nucleosides; adenylyl cyclase; guanine nucleotides
There have been many advances in our knowledge about different aspects of P2Y receptor signaling since the last review published by our International Union of Pharmacology subcommittee. More receptor subtypes have been cloned and characterized and most orphan receptors deorphanized, so that it is now possible to provide a basis for a future subdivision of P2Y receptor subtypes. More is known about the functional elements of the P2Y receptor molecules and the signaling pathways involved, including interactions with ion channels. There have been substantial developments in the design of selective agonists and antagonists to some of the P2Y receptor subtypes. There are new findings about the mechanisms underlying nucleotide release and ectoenzymatic nucleotide breakdown. Interactions between P2Y receptors and receptors to other signaling molecules have been explored as well as P2Y-mediated control of gene transcription. The distribution and roles of P2Y receptor subtypes in many different cell types are better understood and P2Y receptor-related compounds are being explored for therapeutic purposes. These and other advances are discussed in the present review.
In the last decades, the discovery that glial cells do not only fill in the empty space among neurons or furnish them with trophic support but are rather essential participants to the various activities of the central and peripheral nervous system has fostered the search for the signalling pathways controlling their functions. Since the early 1990s, purines were foreseen as some of the most promising candidate molecules. Originally just a hypothesis, this has become a certainty as experimental evidence accumulated over years, as demonstrated by the exponentially growing number of articles related to the role of extracellular nucleotides and nucleosides in controlling glial cell functions. Indeed, as new functions for already known glial cells (for example, the ability of parenchymal astrocytes to behave as stem cells) or new subtypes of glial cells (for example, NG2+ cells, also called polydendrocytes) are discovered also, new actions and new targets for the purinergic system are identified. Thus, glial purinergic receptors have emerged as new possible pharmacological targets for various acute and chronic pathologies, such as stroke, traumatic brain and spinal cord injury, demyelinating diseases, trigeminal pain and migraine, and retinopathies. In this article, we will summarize the most important and promising actions mediated by extracellular purines and pyrimidines in controlling the functions, survival, and differentiation of the various “classical” types of glial cells (i.e., astrocytes, oligodendrocytes, microglial cells, Müller cells, satellite glial cells, and enteric glial cells) but also of some rather new members of the family (e.g., polydendrocytes) and of other cells somehow related to glial cells (e.g., pericytes and spinal cord ependymal cells).
Astrocytes; Microglial cells; Oligodendrocytes; Müller cells; Reactive gliosis; Myelination
Glial cells have been shown to directly participate to the genesis and maintenance of chronic pain in both the sensory ganglia and the central nervous system (CNS). Indeed, glial cell activation has been reported in both the dorsal root ganglia and the spinal cord following injury or inflammation of the sciatic nerve, but no data are currently available in animal models of trigeminal sensitization. Therefore, in the present study, we evaluated glial cell activation in the trigeminal-spinal system following injection of the Complete Freund's Adjuvant (CFA) into the temporomandibular joint, which generates inflammatory pain and trigeminal hypersensitivity.
CFA-injected animals showed ipsilateral mechanical allodynia and temporomandibular joint edema, accompanied in the trigeminal ganglion by a strong increase in the number of GFAP-positive satellite glial cells encircling neurons and by the activation of resident macrophages. Seventy-two hours after CFA injection, activated microglial cells were observed in the ipsilateral trigeminal subnucleus caudalis and in the cervical dorsal horn, with a significant up-regulation of Iba1 immunoreactivity, but no signs of reactive astrogliosis were detected in the same areas. Since the purinergic system has been implicated in the activation of microglial cells during neuropathic pain, we have also evaluated the expression of the microglial-specific P2Y12 receptor subtype. No upregulation of this receptor was detected following induction of TMJ inflammation, suggesting that any possible role of P2Y12 in this paradigm of inflammatory pain does not involve changes in receptor expression.
Our data indicate that specific glial cell populations become activated in both the trigeminal ganglia and the CNS following induction of temporomandibular joint inflammation, and suggest that they might represent innovative targets for controlling pain during trigeminal nerve sensitization.
GPR17 is a hybrid G-protein-coupled receptor (GPCR) activated by two unrelated ligand families, extracellular nucleotides and cysteinyl-leukotrienes (cysteinyl-LTs), and involved in brain damage and repair. Its exploitment as a target for novel neuro-reparative strategies depends on the elucidation of the molecular determinants driving binding of purinergic and leukotrienic ligands. Here, we applied docking and molecular dynamics simulations (MD) to analyse the binding and the forced unbinding of two GPR17 ligands (the endogenous purinergic agonist UDP and the leukotriene receptor antagonist pranlukast from both the wild-type (WT) receptor and a mutant model, where a basic residue hypothesized to be crucial for nucleotide binding had been mutated (R255I) to Ile.
MD suggested that GPR17 nucleotide binding pocket is enclosed between the helical bundle and extracellular loop (EL) 2. The driving interaction involves R255 and the UDP phosphate moiety. To support this hypothesis, steered MD experiments showed that the energy required to unbind UDP is higher for the WT receptor than for R255I. Three potential binding sites for pranlukast where instead found and analysed. In one of its preferential docking conformations, pranlukast tetrazole group is close to R255 and phenyl rings are placed into a subpocket highly conserved among GPCRs. Pulling forces developed to break polar and aromatic interactions of pranlukast were comparable. No differences between the WT receptor and the R255I receptor were found for the unbinding of pranlukast.
These data thus suggest that, in contrast to which has been hypothesized for nucleotides, the lack of the R255 residue doesn't affect the binding of pranlukast a crucial role for R255 in binding of nucleotides to GPR17. Aromatic interactions are instead likely to play a predominant role in the recognition of pranlukast, suggesting that two different binding subsites are present on GPR17.
Deciphering the mechanisms regulating the generation of new neurons and new oligodendrocytes, the myelinating cells of the central nervous system, is of paramount importance to address new strategies to replace endogenous damaged cells in the adult brain and foster repair in neurodegenerative diseases. Upon brain injury, the extracellular concentrations of nucleotides and cysteinyl-leukotrienes (cysLTs), two families of endogenous signaling molecules, are markedly increased at the site of damage, suggesting that they may act as “danger signals” to alert responses to tissue damage and start repair. Here we show that, in brain telencephalon, GPR17, a recently deorphanized receptor for both uracil nucleotides and cysLTs (e.g., UDP-glucose and LTD4), is normally present on neurons and on a subset of parenchymal quiescent oligodendrocyte precursor cells. We also show that induction of brain injury using an established focal ischemia model in the rodent induces profound spatiotemporal-dependent changes of GPR17. In the lesioned area, we observed an early and transient up-regulation of GPR17 in neurons expressing the cellular stress marker heat shock protein 70. Magnetic Resonance Imaging in living mice showed that the in vivo pharmacological or biotechnological knock down of GPR17 markedly prevents brain infarct evolution, suggesting GPR17 as a mediator of neuronal death at this early ischemic stage. At later times after ischemia, GPR17 immuno-labeling appeared on microglia/macrophages infiltrating the lesioned area to indicate that GPR17 may also acts as a player in the remodeling of brain circuitries by microglia. At this later stage, parenchymal GPR17+ oligodendrocyte progenitors started proliferating in the peri-injured area, suggesting initiation of remyelination. To confirm a specific role for GPR17 in oligodendrocyte differentiation, the in vitro exposure of cortical pre-oligodendrocytes to the GPR17 endogenous ligands UDP-glucose and LTD4 promoted the expression of myelin basic protein, confirming progression toward mature oligodendrocytes. Thus, GPR17 may act as a “sensor” that is activated upon brain injury on several embryonically distinct cell types, and may play a key role in both inducing neuronal death inside the ischemic core and in orchestrating the local remodeling/repair response. Specifically, we suggest GPR17 as a novel target for therapeutic manipulation to foster repair of demyelinating wounds, the types of lesions that also occur in patients with multiple sclerosis.
GPR17 is a G-protein-coupled receptor located at intermediate phylogenetic position between two distinct receptor families: the P2Y and CysLT receptors for extracellular nucleotides and cysteinyl-LTs, respectively. We previously showed that GPR17 can indeed respond to both classes of endogenous ligands and to synthetic compounds active at the above receptor families, thus representing the first fully characterized non-peptide "hybrid" GPCR. In a rat brain focal ischemia model, the selective in vivo knock down of GPR17 by anti-sense technology or P2Y/CysLT antagonists reduced progression of ischemic damage, thus highlighting GPR17 as a novel therapeutic target for stroke. Elucidation of the structure of GPR17 and of ligand binding mechanisms are the necessary steps to obtain selective and potent drugs for this new potential target. On this basis, a 3-D molecular model of GPR17 embedded in a solvated phospholipid bilayer and refined by molecular dynamics simulations has been the first aim of this study. To explore the binding mode of the "purinergic" component of the receptor, the endogenous agonist UDP and two P2Y receptor antagonists demonstrated to be active on GPR17 (MRS2179 and cangrelor) were then modeled on the receptor.
Molecular dynamics simulations suggest that GPR17 nucleotide binding pocket is similar to that described for the other P2Y receptors, although only one of the three basic residues that have been typically involved in ligand recognition is conserved (Arg255). The binding pocket is enclosed between the helical bundle and covered at the top by EL2. Driving interactions are H-bonds and salt bridges between the 6.55 and 6.52 residues and the phosphate moieties of the ligands. An "accessory" binding site in a region formed by the EL2, EL3 and the Nt was also found.
Nucleotide binding to GPR17 occurs on the same receptor regions identified for already known P2Y receptors. Agonist/antagonist binding mode are similar, but not identical. An accessory external binding site could guide small ligands to the deeper principal binding site in a multi-step mechanism of activation. The nucleotide binding pocket appears to be unable to allocate the leukotrienic type ligands in the same effective way.
Central nervous system glial cells release and respond to nucleotides under both physiological and pathological conditions, suggesting that these molecules play key roles in both normal brain function and in repair after damage. In particular, ATP released from astrocytes activates P2 receptors on astrocytes and other brain cells, allowing a form of homotypic and heterotypic signalling, which also involves microglia, neurons and oligodendrocytes. Multiple P2X and P2Y receptors are expressed by both astrocytes and microglia; however, these receptors are differentially recruited by nucleotides, depending upon specific pathophysiological conditions, and also mediate the long-term trophic changes of these cells during inflammatory gliosis. In astrocytes, P2-receptor-induced gliosis occurs via activation of the extracellular-regulated kinases (ERK) and protein kinase B/Akt pathways and involves induction of inflammatory and anti-inflammatory genes, cyclins, adhesion and antiapoptotic molecules. While astrocytic P2Y1 and P2Y2,4 are primarily involved in short-term calcium-dependent signalling, multiple P2 receptor subtypes seem to cooperate to astrocytic long-term changes. Conversely, in microglia, exposure to inflammatory and immunological stimuli results in differential functional changes of distinct P2 receptors, suggesting highly specific roles in acquisition of the activated phenotype. We believe that nucleotide-induced activation of astrocytes and microglia may originally start as a defence mechanism to protect neurons from cytotoxic and ischaemic insults; dysregulation of this process in chronic inflammatory diseases eventually results in neuronal cell damage and loss. On this basis, full elucidation of the specific roles of P2 receptors in these cells may help exploit the beneficial neuroprotective features of activated glia while attenuating their harmful properties and thus provide the basis for novel neuroprotective strategies that specifically target the purinergic system.
adenine nucleotides; astrocytes; calcium-mediated communication; microglia; neuroprotection; oligodendroglia; P2 receptors; reactive gliosis; sugar nucleotides; uracil nucleotides
Excessive cyclo-oxygenase-2 (COX-2) induction may play a role in chronic neurological diseases characterized by inflammation and astrogliosis. We have previously identified an astroglial receptor for extracellular nucleotides, a P2Y receptor, whose stimulation leads to arachidonic acid (AA) release, followed, 3 days later, by morphological changes resembling reactive astrogliosis. Since COX-2 may be upregulated by AA metabolites, we assessed a possible role for COX-2 in P2Y receptor-mediated astrogliosis. A brief challenge of rat astrocytes with the ATP analogue α,β-methylene ATP (α,βmeATP) resulted, 24 h later, in significantly increased COX-2 expression. The selective COX-2 inhibitor NS-398 completely abolished α,βmeATP-induced astrocytic activation. Constitutive astroglial COX-1 or COX-2 did not play any role in purine-induced reactive astrogliosis. PGE2, a main metabolite of COX-2, also induced astrocytic activation. These data suggest that a P2Y receptor mediates reactive astrogliosis via induction of COX-2. Antagonists selective for this receptor may counteract excessive COX-2 activation in both acute and chronic neurological diseases.
ATP; cyclo-oxygenase-2; inflammation; astrogliosis; P2Y receptors