Role of purinergic P2X4 receptors in regulating striatal dopamine homeostasis and dependent behaviors.
ABSTRACT: Purinergic P2X4 receptors (P2X4Rs) belong to the P2X superfamily of ion channels regulated by ATP. We recently demonstrated that P2X4R knockout (KO) mice exhibited deficits in sensorimotor gating, social interaction, and ethanol drinking behavior. Dopamine (DA) dysfunction may underlie these behavioral changes, but there is no direct evidence for P2X4Rs' role in DA neurotransmission. To test this hypothesis, we measured markers of DA function and dependent behaviors in P2X4R KO mice. P2X4R KO mice exhibited altered density of pre-synaptic markers including tyrosine hydroxylase, dopamine transporter; post-synaptic markers including dopamine receptors and phosphorylation of downstream targets including dopamine and cyclic-AMP regulated phosphoprotein of 32 kDa and cyclic-AMP-response element binding protein in different parts of the striatum. Ivermectin, an allosteric modulator of P2X4Rs, significantly affected dopamine and cyclic AMP regulated phosphoprotein of 32 kDa and extracellular regulated kinase1/2 phosphorylation in the striatum. Sensorimotor gating deficits in P2X4R KO mice were rescued by DA antagonists. Using the 6-hydroxydopamine model of DA depletion, P2X4R KO mice exhibited an attenuated levodopa (L-DOPA)-induced motor behavior, whereas ivermectin enhanced this behavior. Collectively, these findings identified an important role for P2X4Rs in maintaining DA homeostasis and illustrate how this association is important for CNS functions including motor control and sensorimotor gating. We propose that P2X4 receptors (P2X4Rs) regulate dopamine (DA) homeostasis and associated behaviors. Pre-synaptic and post-synaptic DA markers were significantly altered in the dorsal and ventral striatum of P2X4R KO mice, implicating altered DA neurotransmission. Sensorimotor gating deficits in P2X4R KO mice were rescued by DA antagonists. Ivermectin (IVM), a positive modulator of P2X4Rs, enhanced levodopa (L-DOPA)-induced motor behavior. These studies highlight potential interactions between P2X4Rs and DA system.
Project description:Sensorimotor gating refers to the ability to filter incoming sensory information in a stimulus-laden environment and disruption of this physiological process has been documented in psychiatric disorders characterized by cognitive aberrations. The effectiveness of current pharmacotherapies for treatment of sensorimotor gating deficits in the patient population still remains controversial. These challenges emphasize the need to better understand the biological underpinnings of sensorimotor gating which could lead to discovery of novel drug targets for therapeutic intervention. Notably, we recently reported a role for purinergic P2X4 receptors (P2X4Rs) in regulation of sensorimotor gating using prepulse inhibition (PPI) of acoustic startle reflex. P2X4Rs are ion channels gated by adenosine-5'-triphosphate (ATP). Ivermectin (IVM) induced PPI deficits in C57BL/6J mice in a P2X4R-specific manner. Furthermore, mice deficient in P2X4Rs [P2X4R knockout (KO)] exhibited PPI deficits that were alleviated by dopamine (DA) receptor antagonists demonstrating an interaction between P2X4Rs and DA receptors in PPI regulation. On the basis of these findings, we hypothesized that increased DA neurotransmission underlies IVM-mediated PPI deficits. To test this hypothesis, we measured the effects of D1 and D2 receptor antagonists, SCH 23390 and raclopride respectively and D1 agonist, SKF 82958 on IVM-mediated PPI deficits. To gain mechanistic insights, we investigated the interaction between IVM and dopaminergic drugs on signaling molecules linked to PPI regulation in the ventral striatum. SCH 23390 significantly attenuated the PPI disruptive effects of IVM to a much greater degree than that of raclopride. SKF 82958 failed to potentiate IVM-mediated PPI disruption. At the molecular level, modulation of D1 receptors altered IVM's effects on dopamine and cyclic-AMP regulated phosphoprotein of 32 kDa (DARPP-32) phosphorylation. Additionally, IVM interacted with the DA receptors antagonists and SKF 82958 in phosphorylation of Ca2+/calmodulin kinase II? (CaMKII?) and its downstream target, neuronal nitric oxide synthase (nNOS). Current findings suggest an involvement for D1 and D2 receptors in IVM-mediated PPI disruption via modulation of DARPP-32, CaMKII? and nNOS. Taken together, the findings suggest that stimulation of P2X4Rs can lead to DA hyperactivity and disruption of information processing, implicating P2X4Rs as a novel drug target for treatment of psychiatric disorders characterized by sensorimotor gating deficits.
Project description:INTRODUCTION:Acute ischemic injury leads to severe neuronal loss. One of the key mechanisms responsible for this effect is inflammation, which is characterized by the activation of myeloid cells, including resident microglia and infiltrating monocytes/macrophages. P2X4 receptors (P2X4Rs) present on these immune cells modulate the inflammatory response. For example, excessive release of adenosine triphosphate during acute ischemic stroke triggers stimulation of P2X4Rs, leading to myeloid cell activation and proliferation and further exacerbating post-ischemic inflammation. In contrast, during recovery P2X4Rs activation on microglia leads to the release of brain-derived neurotrophic factor (BDNF), which alleviate depression, maintain synaptic plasticity and hasten post-stroke behavioral recovery. Therefore, we hypothesized that deletion of the P2X4R specifically from myeloid cells would have differential effects on acute versus chronic recovery following stroke. METHODS:We subjected global or myeloid-specific (MS) P2X4R knock-out (KO) mice and wild-type littermates of both sexes to right middle cerebral artery occlusion (60min). We performed histological, behavioral (sensorimotor and depressive), and biochemical (quantitative PCR and flow cytometry) analyses to determine the acute (three days after occlusion) and chronic (30days after occlusion) effects of receptor deletion. RESULTS:Global P2X4R deletion led to reduced infarct size in both sexes. In MS P2X4R KO mice, only females showed reduced infarct size, an effect that did not change with ovariectomy. MS P2X4R KO mice of both sexes showed swift recovery from sensorimotor deficits during acute recovery but exhibited a more pronounced post-stroke depressive behavior phenotype that was independent of infarct size. Quantitative PCR analysis of whole cell lysate as well as flow-sorted myeloid cells from the perilesional cortex showed increased cellular interleukin 1 beta (IL-1?), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-?) mRNA levels but reduced plasma levels of these cytokines in MS P2X4R KO mice after stroke. The expression levels of BDNF and other depression-associated genes were reduced in MS P2X4R KO mice after stroke. CONCLUSIONS:P2X4R deletion protects against stroke acutely but predisposes to depression-like behavior chronically after stroke. Thus, a time-sensitive approach should be considered when targeting P2X4Rs after stroke.
Project description:Purinergic P2X receptors are a family of ligand-gated ion channels gated by extracellular adenosine 5'-triphosphate (ATP). Of the seven P2X subtypes, P2X4 receptors (P2X4Rs) are richly expressed in the brain, yet their role in behavioral organization remains poorly understood. In this study, we examined the behavioral responses of P2X4R heterozygous (HZ) and knockout (KO) mice in a variety of testing paradigms designed to assess complementary aspects of sensory functions, emotional reactivity, and cognitive organization. P2X4R deficiency did not induce significant alterations of locomotor activity and anxiety-related indices in the novel open field and elevated plus-maze tests. Conversely, P2X4R KO mice displayed marked deficits in acoustic startle reflex amplitude, as well as significant sensorimotor gating impairments, as assessed by the prepulse inhibition of the startle. In addition, P2X4R KO mice displayed enhanced tactile sensitivity, as signified by a lower latency in the sticky-tape removal test. Moreover, both P2X4R HZ and KO mice showed significant reductions in social interaction and maternal separation-induced ultrasonic vocalizations in pups. Notably, brain regions of P2X4R KO mice exhibited significant brain-regional alterations in the subunit composition of glutamate ionotropic receptors. These results collectively document that P2X4-deficient mice exhibit a spectrum of phenotypic abnormalities partially akin to those observed in other murine models of autism-spectrum disorder. In conclusion, our findings highlight a putative role of P2X4Rs in the regulation of perceptual and sociocommunicative functions and point to these receptors as putative targets for disturbances associated with neurodevelopmental disorders.
Project description:Alcohol use disorders (AUDs) have a staggering socioeconomic impact. Few therapeutic options are available, and they are largely inadequate. These shortcomings highlight the urgent need to develop effective medications to prevent and/or treat AUDs. A critical barrier is the lack of information regarding the molecular target(s) by which ethanol (EtOH) exerts its pharmacological activity. This review highlights findings implicating P2X4 receptors (P2X4Rs) as a target for the development of therapeutics to treat AUDs and discusses the use of ivermectin (IVM) as a potential clinical tool for treatment of AUDs. P2XRs are a family of ligand-gated ion channels (LGICs) activated by extracellular ATP. Of the P2XR subtypes, P2X4Rs are expressed the most abundantly in the CNS. Converging evidence suggests that P2X4Rs are involved in the development and progression of AUDs. First, in vitro studies report that pharmacologically relevant EtOH concentrations can negatively modulate ATP-activated currents. Second, P2X4Rs in the mesocorticolimbic dopamine system are thought to play a role in synaptic plasticity and are located ideally to modulate brain reward systems. Third, alcohol-preferring (P) rats have lower functional expression of the p2rx4 gene than alcohol-non-preferring (NP) rats suggesting an inverse relationship between alcohol intake and P2X4R expression. Similarly, whole brain p2rx4 expression has been shown to relate inversely to innate 24 h alcohol preference across 28 strains of rats. Fourth, mice lacking the p2rx4 gene drink more EtOH than wildtype controls. Fifth, IVM, a positive modulator of P2X4Rs, antagonizes EtOH-mediated inhibition of P2X4Rs in vitro and reduces EtOH intake and preference in vivo. These findings suggest that P2X4Rs contribute to EtOH intake. The present review summarizes recent findings focusing on the P2X4R as a molecular target of EtOH action, its role in EtOH drinking behavior and modulation of its activity by IVM as a potential therapy for AUDs.
Project description:The macrophage is a major phagocytic cell type, and its impaired function is a primary cause of immune paralysis, organ injury, and death in sepsis. An incomplete understanding of the endogenous molecules that regulate macrophage bactericidal activity is a major barrier for developing effective therapies for sepsis. Using an in vitro killing assay, we report here that the endogenous purine ATP augments the killing of sepsis-causing bacteria by macrophages through P2X4 receptors (P2X4Rs). Using newly developed transgenic mice expressing a bioluminescent ATP probe on the cell surface, we found that extracellular ATP levels increase during sepsis, indicating that ATP may contribute to bacterial killing in vivo. Studies with P2X4R-deficient mice subjected to sepsis confirm the role of extracellular ATP acting on P2X4Rs in killing bacteria and protecting against organ injury and death. Results with adoptive transfer of macrophages, myeloid-specific P2X4R-deficient mice, and P2rx4 tdTomato reporter mice indicate that macrophages are essential for the antibacterial, antiinflammatory, and organ protective effects of P2X4Rs in sepsis. Pharmacological targeting of P2X4Rs with the allosteric activator ivermectin protects against bacterial dissemination and mortality in sepsis. We propose that P2X4Rs represent a promising target for drug development to control bacterial growth in sepsis and other infections.
Project description:ATP-gated purinergic P2X4 receptors (P2X4Rs) are the most alcohol-sensitive P2XR subtype. We recently reported that ivermectin (IVM), an antiparasitic used in animals and humans, antagonized ethanol inhibition of P2X4Rs. Furthermore, IVM reduced ethanol intake in mice. The first molecular model of the rat P2X4R, built onto the X-ray crystal structure of zebrafish P2X4R, revealed an action pocket for both ethanol and IVM formed by Asp331, Met336 in TM2 and Trp46, and Trp50 in TM1 segments. The role of Asp331 and Met336 was experimentally confirmed. The present study tested the hypothesis that Trp46 plays a role in ethanol and IVM modulation of P2X4Rs. Trp46 was mutated to residues with different physicochemical properties and the resultant mutants tested for ethanol and IVM responses using Xenopus oocyte expression system and two-electrode voltage clamp. Nonaromatic substitutions at position 46 reduced ethanol inhibition at higher concentrations and switched IVM potentiation to inhibition. Simultaneous substitution of alanine at positions Trp46 and Met336 also resulted in similar changes in ethanol and IVM responses. Furthermore, a new molecular model based on the open pore conformation of zebrafish P2X4R suggested a role for Tyr42 that was further supported experimentally. Our previous and current findings, combined with our preliminary evidence of increased ethanol consumption in P2X4R knockout mice, suggest that the ethanol and IVM action pocket in P2X4Rs formed by positions 42, 46, 331, and 336 presents a potential target for medication development for alcohol use disorders.
Project description:ATP-gated purinergic P2X4 receptors (P2X4Rs) are expressed in the central nervous system and are sensitive to ethanol at intoxicating concentrations. P2XRs are trimeric; each subunit consists of two transmembrane (TM) alpha-helical segments, a large extracellular domain, and intracellular amino and carboxyl terminals. Recent work indicates that position 336 (Met336) in the TM2 segment is critical for ethanol modulation of P2X4Rs. The anthelmintic medication ivermectin (IVM) positively modulates P2X4Rs and is believed to act in the same region as ethanol. The present study tested the hypothesis that IVM can antagonize ethanol action. We investigated IVM and ethanol effects in wild-type and mutant P2X4Rs expressed in Xenopus oocytes by using a two-electrode voltage clamp. IVM antagonized ethanol-induced inhibition of P2X4Rs in a concentration-dependent manner. The size and charge of substitutions at position 336 affected P2X4R sensitivity to both ethanol and IVM. The first molecular model of the rat P2X4R, built onto the X-ray crystal structure of zebrafish P2X4R, revealed a pocket formed by Asp331, Met336, Trp46, and Trp50 that may play a role in the actions of ethanol and IVM. These findings provide the first evidence for IVM antagonism of ethanol effects in P2X4Rs and suggest that the antagonism results from the ability of IVM to interfere with ethanol action on the putative pocket at or near position 336. Taken with the building evidence supporting a role for P2X4Rs in ethanol intake, the present findings suggest that the newly identified alcohol pocket is a potential site for development of medication for alcohol use disorders.
Project description:The P2X4 receptor (P2X4R) is a member of a family of ATP-gated cation channels that are composed of three subunits. Each subunit has two transmembrane (TM) domains linked by a large extracellular loop and intracellularly located N- and C-termini. The receptors are expressed in excitable and non-excitable cells and have been implicated in the modulation of membrane excitability, calcium signaling, neurotransmitter and hormone release, and pain physiology. P2X4Rs activate rapidly and desensitize within the seconds of agonist application, both with the rates dependent on ATP concentrations, and deactivate rapidly and independently of ATP concentration. Disruption of conserved cysteine ectodomain residues affects ATP binding and gating. Several ectodomain residues of P2X4R were identified as critical for ATP binding, including K67, K313, and R295. Ectodomain residues also account for the allosteric regulation of P2X4R; H140 is responsible for copper binding and H286 regulates receptor functions with protons. Ivermectin sensitized receptors, amplified the current amplitude, and slowed receptor deactivation by binding in the TM region. Scanning mutagenesis of TMs revealed the helical topology of both domains, and suggested that receptor function is critically dependent on the conserved Y42 residue. In this brief article, we summarize this study and re-interpret it using a model based on crystallization of the zebrafish P2X4.1 receptor.
Project description:Microglia survey the brain microenvironment for signals of injury or infection and are essential for the initiation and resolution of pathogen- or tissue damage-induced inflammation. Understanding the mechanism of microglia responses during pathology is hence vital to promote regenerative responses. Here, we analyzed the role of purinergic receptor P2X4 (P2X4R) in microglia/macrophages during autoimmune inflammation. Blockade of P2X4R signaling exacerbated clinical signs in the experimental autoimmune encephalomyelitis (EAE) model and also favored microglia activation to a pro-inflammatory phenotype and inhibited myelin phagocytosis. Moreover, P2X4R blockade in microglia halted oligodendrocyte differentiation in vitro and remyelination after lysolecithin-induced demyelination. Conversely, potentiation of P2X4R signaling by the allosteric modulator ivermectin (IVM) favored a switch in microglia to an anti-inflammatory phenotype, potentiated myelin phagocytosis, promoted the remyelination response, and ameliorated clinical signs of EAE Our results provide evidence that P2X4Rs modulate microglia/macrophage inflammatory responses and identify IVM as a potential candidate among currently used drugs to promote the repair of myelin damage.
Project description:P2X receptors (P2XRs) are ion channels gated by synaptically released ATP. The P2X4 is the most abundant P2XR subtype expressed in the central nervous system and to date is the most ethanol-sensitive. In addition, genomic findings suggest that P2X4Rs may play a role in alcohol intake/preference. However, little is known regarding how ethanol causes the inhibition of ATP-gated currents in P2X4Rs. We begin to address this issue by investigating the effects of ethanol in wild-type and mutant D331A and M336A P2X4Rs expressed in human embryonic kidney (HEK) 293 cells using whole-cell patch-clamp methods. The results suggest that residues D331 and M336 play a role in P2X4R gating and ethanol inhibits channel functioning via a mechanism different from that in other P2XRs. Key findings from the study include: 1) ethanol inhibits ATP-gated currents in a rapid manner; 2) ethanol inhibition of ATP-gated currents does not depend on voltage and ATP concentration; 3) residues 331 and 336 slow P2X4 current deactivation and regulate the inhibitory effects of ethanol; and 4) ethanol effects are similar in HEK293 cells transfected with P2X4Rs and cultured rat hippocampal neurons transduced with P2X4Rs using a recombinant lentiviral system. Overall, these findings provide key information regarding the mechanism of ethanol action on ATP-gated currents in P2X4Rs and provide new insights into the biophysical properties of P2X4Rs.