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Pannexin 1 (PANX1) is an ion and metabolite membrane channel and scaffold protein enriched in synaptic compartments of neurons in the central nervous system. In addition to a well‐established link between PANX1 and synaptic plasticity, we recently identified a role for PANX1 in the regulation of dendritic spine stability. Notably, PANX1 and its interacting proteins are linked to neurological conditions involving dendritic spine loss. Understanding the dual role of PANX1 in synaptic function and morphology may help to shed light on these links. We explore potential mechanisms, including PANX1's interactions with postsynaptic receptors and cytoskeleton regulating proteins. Finally, we contextualize PANX1's dual role within neurological diseases involving dendritic spine and synapse dysfunction. [ABSTRACT FROM AUTHOR]

Purinergic signaling has emerged as an important paracrine–autocrine intercellular system that regulates physiological and pathological processes in practically all organs of the body. Although this system has been thoroughly defined since the nineties, recent research has made substantial advances regarding its role in aspects of liver physiology. However, most studies have mainly targeted the entire organ, 70% of which is made up of parenchymal cells or hepatocytes. Because of its physiological role, the liver is exposed to toxic metabolites, such as xenobiotics, drugs, and fatty acids, as well as to pathogens such as viruses and bacteria. Under injury conditions, all cell types within the liver undergo adaptive changes. In this context, the concentration of extracellular ATP has the potential to increase dramatically. Indeed, this purinergic response has not been studied in sufficient detail in non-parenchymal liver cells. In the present review, we systematize the physiopathological adaptations related to the purinergic system in chronic liver diseases of non-parenchymal liver cells, such as hepatic stellate cells, Kupffer cells, sinusoidal endothelial cells, and cholangiocytes. The role played by non-parenchymal liver cells in these circumstances will undoubtedly be strategic in understanding the regenerative activities that support the viability of this organ under stressful conditions. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease is the most frequent form of dementia characterized by the deposition of amyloid-beta plaques and neurofibrillary tangles consisting of hyperphosphorylated tau. Targeting amyloid-beta plaques has been a primary direction for developing Alzheimer’s disease treatments in the last decades. However, existing drugs targeting amyloid-beta plaques have not fully yielded the expected results in the clinic, necessitating the exploration of alternative therapeutic strategies. Increasing evidence unravels that astrocyte morphology and function alter in the brain of Alzheimer’s disease patients, with dysregulated astrocytic purinergic receptors, particularly the P2Y1 receptor, all of which constitute the pathophysiology of Alzheimer’s disease. These receptors are not only crucial for maintaining normal astrocyte function but are also highly implicated in neuroinflammation in Alzheimer’s disease. This review delves into recent insights into the association between P2Y1 receptor and Alzheimer’s disease to underscore the potential neuroprotective role of P2Y1 receptor in Alzheimer’s disease by mitigating neuroinflammation, thus offering promising avenues for developing drugs for Alzheimer’s disease and potentially contributing to the development of more effective treatments.

Gefapixant (MK‐7264, RO4926219, AF‐219) is a first‐in‐class P2X3 antagonists being developed to treat refractory or unexplained chronic cough. The initial single‐ and multiple‐dose safety, tolerability, and pharmacokinetics of gefapixant at doses ranging from 7.5 to 1800 mg were assessed in four clinical trials. Following single‐dose administration of 10‐450 mg, the pharmacokinetic (PK) profile of gefapixant in plasma and urine demonstrated low inter‐subject variability and a dose‐proportional exposure. Following administration of multiple doses twice daily, the plasma exposures were dose‐proportional at doses ranging from 7.5 to 50 mg and less than dose‐proportional at doses ranging from 100 to 1800 mg. The time to mean peak drug concentration ranged from 2 to 3 h post‐dose, and steady state was achieved by 7 days after dosing, with an accumulation ratio of approximately 2, comparing data from day 1 to steady state. The mean apparent terminal half‐life ranged from 8.2 to 9.6 h. Gefapixant was primarily excreted unmodified in urine. Gefapixant was well tolerated following single‐dose administration up to 1800 mg and multiple doses up to 1800 mg twice daily; there were no serious adverse events (AEs) reported. The most common AE reported was dysgeusia. The PK profile supports a twice‐daily dosing regimen. [ABSTRACT FROM AUTHOR]

Nucleosides and purine nucleotides serve as transmitter and modulator agents that extend their functions beyond the cell. In this context, purinergic signaling plays a crucial role in regulating energy homeostasis and modulating metabolic alterations in tumor cells. Therefore, it is essential to consider the pharmacological targeting of purinergic receptors (PUR), which encompass the expression and inhibition of P1 receptors (metabotropic adenosine receptors) as well as P2 receptors (extracellular ATP/ADP) comprising P2X and P2Y receptors. Thus, the pharmacological interaction between inhibitors (such as RNA, monoclonal antibodies, and small molecules) and PUR represents a key aspect in facilitating the development of therapeutic interventions. Moreover, this review explores recent advancements in pharmacological inhibitors and the regulation of innate and adaptive immunity of PUR, specifically in relation to immunological and inflammatory responses. These responses encompass the release of pro-inflammatory cytokines (PIC), the production of reactive oxygen and nitrogen species (ROS and RNS), the regulation of T cells, and the activation of inflammasomes in all human leukocytes. [ABSTRACT FROM AUTHOR]

Microglia are endogenous immune cells in the brain, and their pyroptosis and phenotype dichotomy are proved to play roles in neurodegenerative diseases. We investigated whether and how hypoxia affected pyroptosis and phenotype polarization in mouse microglia. Primary mouse microglia and BV2 microglia were exposed to hypoxia. Pyroptosis and M1/M2 phenotype were assessed by measuring gasdermin D truncation and M1/M2 surface marker expression. Mechanisms including purinergic ionotropic receptor (P2XR), peroxisome proliferator‐activated receptor coactivator‐1α (PGC‐1α) and NOD‐like receptor protein 3 (NLRP3) inflammasome were investigated. We reported hypoxia (90% N2, 5% O2 and 5% CO2) induced pyroptosis and promoted M1 phenotype polarization in primary mouse microglia and BV2 microglia, and the effect appeared after 6 h exposure. Although hypoxia (90% N2, 5% O2 and 5% CO2, 6 h) had no effect on P2X1R and P2X7R expression, it increased P2X4R expression and decreased PGC‐1α expression. Interestingly, blockade of P2X4R or P2X7R abolished hypoxia‐modulated PGC‐1α expression, pyroptosis and M1 polarization. PGC‐1α overexpression or overactivation alleviated hypoxia‐induced pyroptosis and M1 polarization, while PGC‐1α knockdown or deactivation promoted pyroptosis and M1 polarization under normoxic situation. Further, hypoxia induced NLRP3 expression and activated caspase‐1 and induced the phosphorylation of NF‐κB and reduced the phosphorylation of STAT3/6. NLRP3 inhibitor and caspase‐1 inhibitor abolished hypoxia‐induced pyroptosis, while NF‐κB inhibitor and STAT phosphorylation inducer ameliorated hypoxia‐induced M1 polarization. In addition, NF‐κB activator and STAT3/6 inhibitor caused microglia M1 polarization under normoxic situation. We concluded in cultured mouse microglia, hypoxia may induce pyroptosis via P2XR/PGC‐1α/NLRP3/caspase‐1 pathway and trigger M1 polarization through P2XR/PGC‐1α/NF‐κB/STAT3/6 pathway. [ABSTRACT FROM AUTHOR]

The NLRP3-inflammasome is a cytosolic multiprotein complex that triggers an inflammatory response to certain danger signals. Recently adenosine diphosphate (ADP) was found to activate the NLRP3-inflammasome in murine macrophages via the P2Y1 receptor. Blockade of this signaling pathway reduced disease severity in a murine colitis-model. However, the role of the ADP/P2Y1-axis has not yet been studied in humans. This present study confirmed ADP-dependent NLRP3-inflammasome activation in murine macrophages, but found no evidence for a role of ADP in inflammasome activation in humans. We investigated the THP1 cell line as well as primary monocytes and further looked at macrophages. Although all cells express the three human ADP-receptors P2Y1, P2Y12 and P2Y13, independent of priming, neither increased ASC-speck formation could be detected with flow cytometry nor additional IL-1β release be found in the culture supernatant of ADP stimulated cells. We now show for the first time that the responsiveness of monocytes and macrophages to ADP as well as the regulation of its purinergic receptors is very much dependent on the species. Therefore the signaling pathway found to contribute to colitis in mice is likely not applicable to humans. [ABSTRACT FROM AUTHOR]

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of invasive non-Hodgkin lymphoma. 60–70% of patients are curable with current chemoimmunotherapy, whereas the rest are refractory or relapsed. Understanding of the interaction between DLBCL cells and tumor microenvironment raises the hope of improving overall survival of DLBCL patients. P2X7, a member of purinergic receptors P2X family, is activated by extracellular ATP and subsequently promotes the progression of various malignancies. However, its role in DLBCL has not been elucidated. In this study, the expression level of P2RX7 in DLBCL patients and cell lines was analyzed. MTS assay and EdU incorporation assay were carried out to study the effect of activated/inhibited P2X7 signaling on the proliferation of DLBCL cells. Bulk RNAseq was performed to explore potential mechanism. The results demonstrated high level expression of P2RX7 in DLBCL patients, typically in patients with relapse DLBCL. 2′(3′)-O-(4-benzoylbenzoyl) adenosine 5-triphosphate (Bz-ATP), an agonist of P2X7, significantly accelerated the proliferation of DLBCL cells, whereas delayed proliferation was detected when administrated with antagonist A740003. Furthermore, a urea cycle enzyme named CPS1 (carbamoyl phosphate synthase 1), which up-regulated in P2X7-activated DLBCL cells while down-regulated in P2X7-inhibited group, was demonstrated to involve in such process. Our study reveals the role of P2X7 in the proliferation of DLBCL cells and implies that P2X7 may serve as a potential molecular target for the treatment of DLBCL. [ABSTRACT FROM AUTHOR]

The role of extracellular nucleotides as modulators of inflammation and cell stress is well established. One of the main actions of these molecules is mediated by the activation of purinergic receptors (P2) of the plasma membrane. P2 receptors can be classified according to two different structural families: P2X ionotropic ion channel receptors, and P2Y metabotropic G protein-coupled receptors. During inflammation, damaged cells release nucleotides and purinergic signaling occurs along the temporal pattern of the synthesis of pro-inflammatory and pro-resolving mediators by myeloid and lymphoid cells. In macrophages under pro-inflammatory conditions, the expression and activity of cyclooxygenase 2 significantly increases and enhances the circulating levels of prostaglandin E2 (PGE2), which exerts its effects both through specific plasma membrane receptors (EP1-EP4) and by activation of intracellular targets. Here we review the mechanisms involved in the crosstalk between PGE2 and P2Y receptors on macrophages, which is dependent on several isoforms of protein kinase C and protein kinase D1. Due to this crosstalk, a P2Y-dependent increase in calcium is blunted by PGE2 whereas, under these conditions, macrophages exhibit reduced migratory capacity along with enhanced phagocytosis, which contributes to the modulation of the inflammatory response and tissue repair. [ABSTRACT FROM AUTHOR]

The individual and global burden of migraine is of such significance that there are accelerated efforts to develop new therapies. New migraine therapeutics are needed to address the current deficiencies that exist in the efficacy and adherence rate of approved anti-migraine medications. The recent discovery of the calcitonin gene related peptide as an add-on to the role of serotonin has markedly increased the range of new treatment options for acute and chronic migraine. Despite this, tackling the complexity of migraine disorders requires a complete understanding of its pathophysiology. Preclinical animal models can shed light on disease-related pathophysiology, including migraine. Indeed, the use of animal models has been instrumental in developing many therapeutics. However, an animal model is limited by the predictive and face validity of that model, and this extends to preclinical migraine models. In this review, a summary of the current understanding of the pathophysiology of migraine is given from both a preclinical and clinical perspective, and an emphasis is placed on the animal models of migraine. We will discuss the strengths and pitfalls of common preclinical migraine models as well as experimental research areas to explore further. [ABSTRACT FROM AUTHOR]

Oligodendrocytes (OLs) play a crucial role in myelination during the development and repair of the central nervous system. ATP serves not only as an important signaling molecule involving in the intercellular communications, but also as an energetic molecule, with its purinergic receptor subtypes widely present in neurons and glial cells. These subtypes are composed of two purinergic receptors: P1 and P2: The former are primarily activated by adenosine, and the latter mainly by ATP, ADP, and UTP. The two receptors paly their respective role in various regions of the CNS under physiological or pathological conditions through distinct mechanisms. In this paper, we review recent literature on the roles and mechanisms of the purinergic receptors in OL development, myelination, and myelin repair. It may be of great significance for further understanding the role of purinergic signaling in demyelinating diseases and myelin dysplasia and exploring potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Allogeneic haematopoietic stem cell transplantation (HSCT) leads to the establishment of graft-versus-leukaemia (GVL) immunity, but in many cases also results in the development of graft-versus-host disease (GVHD). This study aimed to determine if P2X7 antagonism using Brilliant Blue G (BBG) could improve the beneficial effects of post-transplant cyclophosphamide (PTCy) in a humanised mouse model of GVHD, without comprising GVL immunity. NOD.Cg-Prkdcscid Il2rgtm1Wjl (NSG) mice were injected with human peripheral blood mononuclear cells (PBMCs) (Day 0), then with cyclophosphamide (33 mg/kg) on Days 3 and 4, and with BBG (50 mg/kg) (or saline) on Days 0–10. PTCy with BBG reduced clinical GVHD development like that of PTCy alone. However, histological analysis revealed that the combined treatment reduced liver GVHD to a greater extent than PTCy alone. Flow cytometric analyses revealed that this reduction in liver GVHD by PTCy with BBG corresponded to an increase in human splenic CD39+ Tregs and a decrease in human serum interferon-γ concentrations. In additional experiments, humanised NSG mice, following combined treatment, were injected with human THP-1 acute myeloid leukaemia cells on Day 14. Flow cytometric analyses of liver CD33+ THP-1 cells showed that PTCy with BBG did not mitigate GVL immunity. In summary, PTCy combined with BBG can reduce GVHD without compromising GVL immunity. Future studies investigating P2X7 antagonism in combination with PTCy may lead to the development of novel treatments that more effectively reduce GVHD in allogeneic HSCT patients without promoting leukaemia relapse. [ABSTRACT FROM AUTHOR]

Extracellular ATP and adenosine are neuromodulators that regulate numerous neuronal functions in the brain. Neuronal activity and brain insults such as ischemic and traumatic injury upregulate these neuromodulators, which exert their effects by activating purinergic receptors. In addition, extracellular ATP/adenosine signaling plays a pivotal role in the pathogenesis of neurological diseases. Virtually every cell type in the brain contributes to the elevation of ATP/adenosine, and various mechanisms underlying this increase have been proposed. Extracellular adenosine is thought to be mainly produced via the degradation of extracellular ATP. However, adenosine is also released from neurons and glia in the brain. Therefore, the regulation of extracellular ATP/adenosine in physiological and pathophysiological conditions is likely far more complex than previously thought. To elucidate the complex mechanisms that regulate extracellular ATP/adenosine levels, accurate methods of assessing their spatiotemporal dynamics are needed. Several novel techniques for acquiring spatiotemporal information on extracellular ATP/adenosine, including fluorescent sensors, have been developed and have started to reveal the mechanisms underlying the release, uptake and degradation of ATP/adenosine. Here, we review methods for analyzing extracellular ATP/adenosine dynamics as well as the current state of knowledge on the spatiotemporal dynamics of ATP/adenosine in the brain. We focus on the mechanisms used by neurons and glia to cooperatively produce the activity-dependent increase in ATP/adenosine and its physiological and pathophysiological significance in the brain. [ABSTRACT FROM AUTHOR]

Neurodegenerative diseases (NDDs) continue to be a significant healthcare problem. The economic and social implications of NDDs increase with longevity. NDDs are linked to neuroinflammation and activated microglia and astrocytes play a central role. There is a growing interest for stem cell-based therapy to deliver genes, and for tissue regeneration. The promise of mesenchymal stem cells (MSC) is based on their availability as off-the-shelf source, and ease of expanding from discarded tissues. We tested the hypothesis that MSC have a major role of resetting activated microglial cells. We modeled microglial cell lines by using U937 cell-derived M1 and M2 macrophages. We studied macrophage types, alone, or in a non-contact culture with MSCs. MSCs induced significant release of exosomes from both types of macrophages, but significantly more of the M1 type. RNA sequencing showed enhanced gene expression within the exosomes with the major changes linked to the inflammatory response, including cytokines and the purinergic receptors. Computational analyses of the transcripts supported the expected effect of MSCs in suppressing the inflammatory response of M1 macrophages. The inflammatory cargo of M1 macrophage-derived exosomes revealed involvement of cytokines and purinergic receptors. At the same time, the exosomes from MSC-M2 macrophages were able to reset the classical M2 macrophages to more balanced inflammation. Interestingly, we excluded transfer of purinergic receptor transcripts from the co-cultured MSCs by analyzing these cells for the identified purinergic receptors. Since exosomes are intercellular communicators, these findings provide insights into how MSCs may modulate tissue regeneration and neuroinflammation. [ABSTRACT FROM AUTHOR]

We examined the involvement of the P2 × 7 receptor and the canonical and noncanonical inflammasomes in the control of single-species or dual-species infection by the periodontal bacteria Porphyromonas gingivalis and Fusobacterium nucleatum in cells and mice. Stimulation of the P2 × 7 receptor leads to activation of the canonical NLRP3 inflammasome and activation of caspase-1, which leads to cleavage of pro-IL-1β to IL-1β, a key cytokine in the host inflammatory response in periodontal disease. The non-canonical inflammasome pathway involves caspase-11. Thus, wildtype (WT), P2 × 7-/-, caspase-11-/- and caspase-1/11-/- mice were co-infected with both bacterial species. In parallel, bone marrow-derived macrophages (BMDMs) from WT mice and the different knockout mice were infected with P. gingivalis and/or F. nucleatum, and treated or not with extracellular ATP, which is recognized by P2 × 7. F. nucleatum infection alone promoted secretion of IL-1β in BMDMs. Conversely, the canonical pathway involving P2 × 7 and caspase-1 was necessary for secretion of IL-1β in BMDMs infected with P. gingivalis and in the mandible of mice coinfected with P. gingivalis and F. nucleatum. The P2 × 7 pathway can limit bacterial load in single-species and dual-species infection with P. gingivalis and F. nucleatum in BMDMs and in mice. The non-canonical pathway involving caspase-11 was required for secretion of IL-1β induced by F. nucleatum infection in BMDMs, without treatment with ATP. Caspase-11 was also required for induction of cell death during infection with F. nucleatum and contributed to limiting bacterial load during F. nucleatum infection in BMDMs and in the gingival tissue of mice coinfected with P. gingivalis and F. nucleatum. Together, these data suggest that the P2 × 7-caspase-1 and caspase-11 pathways are involved in the immune response against infection by P. gingivalis and F. nucleatum, respectively.

The individual and global burden of migraine is of such significance that there are accelerated efforts to develop new therapies. New migraine therapeutics are needed to address the current deficiencies that exist in the efficacy and adherence rate of approved anti-migraine medications. The recent discovery of the calcitonin gene related peptide as an add-on to the role of serotonin has markedly increased the range of new treatment options for acute and chronic migraine. Despite this, tackling the complexity of migraine disorders requires a complete understanding of its pathophysiology. Preclinical animal models can shed light on disease-related pathophysiology, including migraine. Indeed, the use of animal models has been instrumental in developing many therapeutics. However, an animal model is limited by the predictive and face validity of that model, and this extends to preclinical migraine models. In this review, a summary of the current understanding of the pathophysiology of migraine is given from both a preclinical and clinical perspective, and an emphasis is placed on the animal models of migraine. We will discuss the strengths and pitfalls of common preclinical migraine models as well as experimental research areas to explore further.

Extracellular adenosine triphosphate (ATP) is one of the most abundant biochemical constitutes within the stem cell microenvironment and is postulated to play critical roles in cell migration. However, it is unclear whether ATP regulates the cell migration of CD34+ vascular wall-resident stem/progenitor cells (VW-SCs) and participates in angiogenesis. Therefore, the biological mechanisms of cell migration mediated by ATP was determined by in vivo subcutaneous matrigel plug assay, ex vivo aortic ring assay, in vitro transwell migration assay, and other molecular methods. In the present study, ATP dose-dependently promoted CD34+ VW-SCs migration, which was more obviously attenuated by inhibiting or knocking down P2Y2 than P2Y6. Furthermore, it was confirmed that ATP potently promoted the migration of resident CD34+ cells from cultured aortic artery rings and differentiation into endothelial cells in matrigel plugs by using inducible lineage tracing Cd34-CreERT2; R26-tdTomato mice, whereas P2Y2 and P2Y6 blocker greatly inhibited the effect of ATP. In addition, ATP enhanced the protein expression of stromal interaction molecule 1 (STIM1) on cell membrane, blocking the calcium release-activated calcium (CRAC) channel with shSTIM1 or BTP2 apparently inhibited ATP-evoked intracellular Ca2+ elevation and channel opening, thereby suppressing ATP-driven cell migration. Moreover, extracellular signal-regulated protein kinase (ERK) inhibitor PD98059 and p38 inhibitor SB203580 remarkably inhibited ERK and p38 phosphorylation, cytoskeleton rearrangement, and subsequent cell migration. Unexpectedly, it was found that knocking down STIM1 greatly inhibited ATP-triggered ERK/p38 activation. Taken together, it was suggested that P2Y2 signaled through the CRAC channel mediated Ca2+ influx and ERK/p38 pathway to reorganize the cytoskeleton and promoted the migration of CD34+ VW-SCs. [ABSTRACT FROM AUTHOR]

Background: As a member of the purinergic receptor family, divalent cation-regulated ionotropic P2X7 (P2rx7) plays a role in the pathophysiology of psychiatric disorders. This study aimed to investigate whether the effects of acute zinc administration and long-term zinc deprivation on depression-like behaviors in mice are mediated by P2X7 receptors. Methods: The antidepressant-like effect of elevated zinc level was studied using a single acute intraperitoneal injection in C57BL6/J wild-type and P2rx7 gene-deficient (P2rx7 -/-) young adult and elderly animals in the tail suspension test (TST) and the forced swim test (FST). In the long-term experiments, depression-like behavior caused by zinc deficiency was investigated with the continuous administration of zinc-reduced and control diets for 8 weeks, followed by the same behavioral tests. The actual change in zinc levels owing to the treatments was examined by assaying serum zinc levels. Changes in monoamine and brain-derived neurotrophic factor (BDNF) levels were measured from the hippocampus and prefrontal cortex brain areas by enzyme-linked immunosorbent assay and high-performance liquid chromatography, respectively. Results: A single acute zinc treatment increased the serum zinc level evoked antidepressant-like effect in both genotypes and age groups, except TST in elderly P2rx7 -/- animals, where no significant effect was detected. Likewise, the pro-depressant effect of zinc deprivation was observed in young adult mice in the FST and TST, which was alleviated in the case of the TST in the absence of functional P2X7 receptors. Among elderly mice, no pro-depressant effect was observed in P2rx7 -/- mice in either tests. Treatment and genotype changes in monoamine and BDNF levels were also detected in the hippocampi. Conclusion: Changes in zinc intake were associated with age-related changes in behavior in the TST and FST. The antidepressant-like effect of zinc is partially mediated by the P2X7 receptor. [ABSTRACT FROM AUTHOR]

Extracellular nucleotides are important mediators of activation, triggering various responses through plasma membrane P2 and P1 receptors. P2 receptors are further subdivided into ionotropic P2X receptors and G protein-coupled P2Y receptors. P2X4 is an ATP-gated cation channel broadly expressed in most tissues of the body. Within the P2X family, P2X4 has a unique subcellular distribution, being preferentially localized in lysosomes. In these organelles, high ATP concentrations do not trigger P2X4 because of the low pH. However, when the pH increases to 7.4, P2X4 can be stimulated by intra-lysosomal ATP, which is in its active, tetra-anionic form. Elucidation of P2X4, P2X3 and P2X7 structures has shed some light on the functional differences between these purinergic receptors. The potential interaction between P2X4 and P2X7 has been extensively studied. Despite intensive effort, it has not been possible yet to determine whether P2X4 and P2X7 interact as heterotrimers or homotrimers at the plasma membrane. However, several publications have shown that functional interactions between P2X4 and P2X7 do occur. Importantly, these studies indicate that P2X4 potentiates P2X7-dependent activation of inflammasomes, leading to increased release of IL-1β and IL-18. The role of P2X4 in various diseases could be beneficial or deleterious even though the pathophysiological mechanisms involved are still poorly defined. However, in diseases whose physiopathology involves activation of the NLRP3 inflammasome, P2X4 was found to exacerbate severity of disease. The recent production of monoclonal antibodies specific for the human and mouse P2X4, some of which are endowed with agonist or antagonist properties, raises the possibility that they could be used therapeutically. Analysis of single nucleotide polymorphisms of the human P2RX4 gene has uncovered the association of P2RX4 gene variants with susceptibility to several human diseases.

Background: As a member of the purinergic receptor family, divalent cation-regulated ionotropic P2X7 (P2rx7) plays a role in the pathophysiology of psychiatric disorders. This study aimed to investigate whether the effects of acute zinc administration and long-term zinc deprivation on depression-like behaviors in mice are mediated by P2X7 receptors.Methods: The antidepressant-like effect of elevated zinc level was studied using a single acute intraperitoneal injection in C57BL6/J wild-type and P2rx7 gene-deficient (P2rx7 −/−) young adult and elderly animals in the tail suspension test (TST) and the forced swim test (FST). In the long-term experiments, depression-like behavior caused by zinc deficiency was investigated with the continuous administration of zinc-reduced and control diets for 8 weeks, followed by the same behavioral tests. The actual change in zinc levels owing to the treatments was examined by assaying serum zinc levels. Changes in monoamine and brain-derived neurotrophic factor (BDNF) levels were measured from the hippocampus and prefrontal cortex brain areas by enzyme-linked immunosorbent assay and high-performance liquid chromatography, respectively.Results: A single acute zinc treatment increased the serum zinc level evoked antidepressant-like effect in both genotypes and age groups, except TST in elderly P2rx7 −/− animals, where no significant effect was detected. Likewise, the pro-depressant effect of zinc deprivation was observed in young adult mice in the FST and TST, which was alleviated in the case of the TST in the absence of functional P2X7 receptors. Among elderly mice, no pro-depressant effect was observed in P2rx7 −/− mice in either tests. Treatment and genotype changes in monoamine and BDNF levels were also detected in the hippocampi.Conclusion: Changes in zinc intake were associated with age-related changes in behavior in the TST and FST. The antidepressant-like effect of zinc is partially mediated by the P2X7 receptor.

Vagus nerve innervates several organs including the heart, stomach, and pancreas among others. Somas of sensory neurons that project through the vagal nerve are located in the nodose ganglion. The presence of purinergic receptors has been reported in neurons and satellite glial cells in several sensory ganglia. In the nodose ganglion, calcium depletion-induced increases in neuron activity can be partly reversed by P2X7 blockers applied directly into the ganglion. The later suggest a possible role of P2X7 receptors in the modulation of neuronal activity within this sensory ganglion. We aimed to characterize the response to P2X7 activation in nodose ganglion neurons under physiological conditions. Using an ex vivo preparation for electrophysiological recordings of the neural discharges of nodose ganglion neurons, we found that treatments with ATP induce transient neuronal activity increases. Also, we found a concentration-dependent increase in neural activity in response to Bz-ATP (ED50 = 0.62 mM, a selective P2X7 receptor agonist), with a clear desensitization pattern when applied every ~ 30 s. Electrophysiological recordings from isolated nodose ganglion neurons reveal no differences in the responses to Bz-ATP and ATP. Finally, we showed that the P2X7 receptor was expressed in the rat nodose ganglion, both in neurons and satellite glial cells. Additionally, a P2X7 receptor negative allosteric modulator decreased the duration of Bz-ATP-induced maximal responses without affecting their amplitude. Our results show the presence of functional P2X7 receptors under physiological conditions within the nodose ganglion of the rat, and suggest that ATP modulation of nodose ganglion activity may be in part mediated by the activation of P2X7 receptors. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by impaired episodic memory and two pathological lesions: amyloid plaques and neurofibrillary tangles. In AD, damaged neurons and the accumulation of amyloid β (Aβ) peptides cause a significant release of high amounts of extracellular ATP, which acts as a danger signal. The purinergic receptor P2X7 is the main sensor of high concentrations of ATP, and P2X7 has been shown to be upregulated in the brains of AD patients, contributing to the disease's pathological processes. Further, there are many polymorphisms of the P2X7 gene that impact the risk of developing AD. P2X7 can directly modulate Aβ plaques and Tau protein lesions as well as the inflammatory response by regulating NLRP3 inflammasome and the expression of several chemokines. The significant role of microglial P2X7 in AD has been well established, although other cell types may also be important in P2X7-mediated mechanisms. In this review, we will discuss the different P2X7-dependent pathways involved in the development of AD. [ABSTRACT FROM AUTHOR]

The primary role of microglia is to maintain homeostasis by effectively responding to various disturbances. Activation of transcriptional programs determines the microglia's response to external stimuli. In this study, we stimulated murine neonatal microglial cells with benzoyl ATP (bzATP) and lipopolysaccharide (LPS), and monitored their ability to release pro-inflammatory cytokines. When cells are exposed to bzATP, a purinergic receptor agonist, a short-lived wave of transcriptional changes, occurs. However, only combining bzATP and LPS led to a sustainable and robust response. The transcriptional profile is dominated by induced cytokines (e.g., IL-1α and IL-1β), chemokines, and their membrane receptors. Several abundant long noncoding RNAs (lncRNAs) are induced by bzATP/LPS, including Ptgs2os2, Bc1, and Morrbid, that function in inflammation and cytokine production. Analyzing the observed changes through TNF (Tumor necrosis factor) and NF-κB (nuclear factor kappa light chain enhancer of activated B cells) pathways confirmed that neonatal glial cells exhibit a distinctive expression program in which inflammatory-related genes are upregulated by orders of magnitude. The observed capacity of the microglial culture to activate a robust inflammatory response is useful for studying neurons under stress, brain injury, and aging. We propose the use of a primary neonatal microglia culture as a responsive in vitro model for testing drugs that may interact with inflammatory signaling and the lncRNA regulatory network. [ABSTRACT FROM AUTHOR]

Microglia-mediated inflammation exerts adverse effects in ischemic stroke and in neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the voltage-gated potassium channel Kv1.3 is required for microglia activation. Both genetic deletion and pharmacological inhibition of Kv1.3 are effective in reducing microglia activation and the associated inflammatory responses, as well as in improving neurological outcomes in animal models of AD and ischemic stroke. Here we sought to elucidate the molecular mechanisms underlying the therapeutic effects of Kv1.3 inhibition, which remain incompletely understood. Using a combination of whole-cell voltage-clamp electrophysiology and quantitative PCR (qPCR), we first characterized a stimulus-dependent differential expression pattern for Kv1.3 and P2X4, a major ATP-gated cationic channel, both in vitro and in vivo. We then demonstrated by whole-cell current-clamp experiments that Kv1.3 channels contribute not only to setting the resting membrane potential but also play an important role in counteracting excessive membrane potential changes evoked by depolarizing current injections. Similarly, the presence of Kv1.3 channels renders microglia more resistant to depolarization produced by ATP-mediated P2X4 receptor activation. Inhibiting Kv1.3 channels with ShK-223 completely nullified the ability of Kv1.3 to normalize membrane potential changes, resulting in excessive depolarization and reduced calcium transients through P2X4 receptors. Our report thus links Kv1.3 function to P2X4 receptor-mediated signaling as one of the underlying mechanisms by which Kv1.3 blockade reduces microglia-mediated inflammation. While we could confirm previously reported differences between males and females in microglial P2X4 expression, microglial Kv1.3 expression exhibited no gender differences in vitro or in vivo. MAIN POINTS: The voltage-gated K+ channel Kv1.3 regulates microglial membrane potential. Inhibition of Kv1.3 depolarizes microglia and reduces calcium entry mediated by P2X4 receptors by dissipating the electrochemical driving force for calcium.

Abstract Despite the rapid development of therapeutic strategies in cancer treatment, metastasis remains the major cause of cancer-related death and scientific challenge. Epithelial-Mesenchymal Transition (EMT) plays a crucial role in cancer invasion and progression, a process by which tumor cells lose cell-cell adhesion and acquire increased invasiveness and metastatic activity. Recent work has uncovered some crucial roles of extracellular adenosine 5’- triphosphate (eATP), a major component of the tumor microenvironment (TME), in promoting tumor growth and metastasis. Intratumoral extracellular ATP (eATP), at levels of 100–700 µM, is 103–104 times higher than in normal tissues. In the current literature, eATP’s function in promoting metastasis has been relatively poorly understood as compared with intracellular ATP (iATP). Recent evidence has shown that cancer cells internalize eATP via macropinocytosis in vitro and in vivo, promoting cell growth and survival, drug resistance, and metastasis. Furthermore, ATP acts as a messenger molecule that activates P2 purinergic receptors expressed on both tumor and host cells, stimulating downstream signaling pathways to enhance the invasive and metastatic properties of tumor cells. Here, we review recent progress in understanding eATP’s role in each step of the metastatic cascade, including initiating invasion, inducing EMT, overcoming anoikis, facilitating intravasation, circulation, and extravasation, and eventually establishing metastatic colonization. Collectively, these studies reveal eATP’s important functions in many steps of metastasis and identify new opportunities for developing more effective therapeutic strategies to target ATP-associated processes in cancer.

The P2X7 receptor is a trimeric ligand-gated cation channel activated by extracellular adenosine 5′-triphosphate. The study of animals has greatly advanced the investigation of P2X7 and helped to establish the numerous physiological and pathophysiological roles of this receptor in human health and disease. Following a short overview of the P2X7 distribution, roles and functional properties, this article discusses how animal models have contributed to the generation of P2X7-specific antibodies and nanobodies (including biologics), recombinant receptors and radioligands to study P2X7 as well as to the pharmacokinetic testing of P2X7 antagonists. This article then outlines how mouse and rat models have been used to study P2X7. These sections include discussions on preclinical disease models, polymorphic P2X7 variants, P2X7 knockout mice (including bone marrow chimeras and conditional knockouts), P2X7 reporter mice, humanized P2X7 mice and P2X7 knockout rats. Finally, this article reviews the limited number of studies involving guinea pigs, rabbits, monkeys (rhesus macaques), dogs, cats, zebrafish, and other fish species (seabream, ayu sweetfish, rainbow trout and Japanese flounder) to study P2X7. [ABSTRACT FROM AUTHOR]

Single-nucleotide polymorphisms are connected with the risk of epilepsy on occurrence, progress, and the individual response to drugs. Progress in genomic technology is exposing the complex genetic architecture of epilepsy. Compelling evidence has demonstrated that purines and adenosine are key mediators in the epileptic process. Our previous study found the interconnection of P2Y12 receptor single-nucleotide polymorphisms and epilepsy. However, little is known about the interaction between the purine nucleoside A2A receptor and rate-limiting enzyme ecto-5'-nucleotidase/CD73 and epilepsy from the genetic polymorphism aspect. The aim of the study is to evaluate the impact of A2AR and CD73 polymorphisms on epilepsy cases. The study group encompassed 181 patients with epilepsy and 55 healthy volunteers. A significant correlation was confirmed between CD73 rs4431401 and epilepsy (p < 0.001), with TT genotype frequency being higher and C allele being lower among epilepsy patients in comparison with healthy individuals, indicating that the presence of the TT genotype is related to an increased risk of epilepsy (OR = 2.742, p = 0.006) while carriers of the C allele demonstrated a decreased risk of epilepsy (OR = 0.304, p < 0.001). According to analysis based on gender, the allele and genotype of rs4431401 in CD73 were associated with both male and female cases (p < 0.0001, p = 0.026, respectively). Of note, we found that A2AR genetic variants rs2267076 T>C (p = 0.031), rs2298383 C>T (p = 0.045), rs4822492 T>G (p = 0.034), and rs4822489 T>G (p = 0.029) were only associated with epilepsy in female subjects instead of male. It is evident that the TT genotype and T allele of rs4431401 in CD73 were genetic risk factors for epilepsy, whereas rs2267076, rs2298383, rs4822492, and rs4822489 polymorphisms of the A2AR were mainly associated with female subjects. [ABSTRACT FROM AUTHOR]

Ecto-5′-nucleotidase (CD73) plays a strategic role in calibrating the magnitude and chemical nature of purinergic signals that are delivered to immune cells. Its primary function is to convert extracellular ATP to adenosine in concert with ectonucleoside triphosphate diphosphohydrolase-1 (CD39) in normal tissues to limit an excessive immune response in many pathophysiological events, such as lung injury induced by a variety of contributing factors. Multiple lines of evidence suggest that the location of CD73, in proximity to adenosine receptor subtypes, indirectly determines its positive or negative effect in a variety of organs and tissues and that its action is affected by the transfer of nucleoside to subtype-specific adenosine receptors. Nonetheless, the bidirectional nature of CD73 as an emerging immune checkpoint in the pathogenesis of lung injury is still unknown. In this review, we explore the relationship between CD73 and the onset and progression of lung injury, highlighting the potential value of this molecule as a drug target for the treatment of pulmonary disease. [ABSTRACT FROM AUTHOR]

Background and Purpose: Pharmacological inhibitors of TMEM16A (ANO1), a Ca2+‐activated Cl− channel, are important tools of research and possible therapeutic agents acting on smooth muscle, airway epithelia and cancer cells. We tested a panel of TMEM16A inhibitors, including CaCCinh‐A01, niclosamide, MONNA, Ani9 and niflumic acid, to evaluate their possible effect on intracellular Ca2+. Experimental Approach: We recorded cytosolic Ca2+ increase elicited with UTP, ionomycin or IP3 uncaging. Key Results: Unexpectedly, we found that all compounds, except for Ani9, markedly decreased intracellular Ca2+ elevation induced by stimuli acting on intracellular Ca2+ stores. These effects were similarly observed in cells with and without TMEM16A expression. We investigated in more detail the mechanism of action of niclosamide and CaCCinh‐A01. Acute addition of niclosamide directly increased intracellular Ca2+, an activity consistent with inhibition of the SERCA pump. In contrast to niclosamide, CaCCinh‐A01 did not elevate intracellular Ca2+, thus implying a different mechanism of action, possibly a block of inositol triphosphate receptors. Conclusions and Implications: Most TMEM16A inhibitors are endowed with indirect effects mediated by alteration of intracellular Ca2+ handling, which may in part preclude their use as TMEM16A research tools. [ABSTRACT FROM AUTHOR]

Myocardial remodelling is a molecular, cellular, and interstitial adaptation of the heart in response to altered environmental demands. The heart undergoes reversible physiological remodelling in response to changes in mechanical loading or irreversible pathological remodelling induced by neurohumoral factors and chronic stress, leading to heart failure. Adenosine triphosphate (ATP) is one of the potent mediators in cardiovascular signalling that act on the ligand-gated (P2X) and G-protein-coupled (P2Y) purinoceptors via the autocrine or paracrine manners. These activations mediate numerous intracellular communications by modulating the production of other messengers, including calcium, growth factors, cytokines, and nitric oxide. ATP is known to play a pleiotropic role in cardiovascular pathophysiology, making it a reliable biomarker for cardiac protection. This review outlines the sources of ATP released under physiological and pathological stress and its cell-specific mechanism of action. We further highlight a series of cardiovascular cell-to-cell communications of extracellular ATP signalling cascades in cardiac remodelling, which can be seen in hypertension, ischemia/reperfusion injury, fibrosis, hypertrophy, and atrophy. Finally, we summarize current pharmacological intervention using the ATP network as a target for cardiac protection. A better understanding of ATP communication in myocardial remodelling could be worthwhile for future drug development and repurposing and the management of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Obesity is a public-health challenge resulting from an imbalance between energy expenditure and calorie intake. This health problem exacerbates a variety of metabolic complications worldwide. Adipose tissue is an essential regulator of energy homeostasis, and the functions within it are regulated by purinergic receptors. A1R, P2X7R, and P2YR mainly mediate energy homeostasis primarily through regulating energy storage and adipokines secretion in white adipose tissue (WAT). P2X5R is a novel-specific cell surface marker in brown/beige adipocytes. A2R is a promising therapeutic target for stimulating energy expenditure in brown adipose tissue (BAT) and also mediating WAT browning. Based on these features, purinergic receptors may be an appropriate target in treating obesity. In this review, the role of purinergic receptors in different types of adipose tissue is summarized. An improved understanding of purinergic receptor functions in adipose tissue may lead to more effective treatment interventions for obesity and its related metabolic disorders. [ABSTRACT FROM AUTHOR]

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Despite the rapid development of therapeutic strategies in cancer treatment, metastasis remains the major cause of cancer-related death and scientific challenge. Epithelial-Mesenchymal Transition (EMT) plays a crucial role in cancer invasion and progression, a process by which tumor cells lose cell-cell adhesion and acquire increased invasiveness and metastatic activity. Recent work has uncovered some crucial roles of extracellular adenosine 5'- triphosphate (eATP), a major component of the tumor microenvironment (TME), in promoting tumor growth and metastasis. Intratumoral extracellular ATP (eATP), at levels of 100–700 µM, is 103–104 times higher than in normal tissues. In the current literature, eATP's function in promoting metastasis has been relatively poorly understood as compared with intracellular ATP (iATP). Recent evidence has shown that cancer cells internalize eATP via macropinocytosis in vitro and in vivo, promoting cell growth and survival, drug resistance, and metastasis. Furthermore, ATP acts as a messenger molecule that activates P2 purinergic receptors expressed on both tumor and host cells, stimulating downstream signaling pathways to enhance the invasive and metastatic properties of tumor cells. Here, we review recent progress in understanding eATP's role in each step of the metastatic cascade, including initiating invasion, inducing EMT, overcoming anoikis, facilitating intravasation, circulation, and extravasation, and eventually establishing metastatic colonization. Collectively, these studies reveal eATP's important functions in many steps of metastasis and identify new opportunities for developing more effective therapeutic strategies to target ATP-associated processes in cancer. [ABSTRACT FROM AUTHOR]

Background: Methamphetamine (METH) is a psychostimulant drug that remains a popular and threatening drug of abuse with high abuse liability. There is no established pharmacotherapy to treat METH dependence, but evidence suggests that stimulation of adenosine receptors reduces the reinforcing properties of METH and could be a potential pharmacological target. This study examines the effects of adenosine receptor subtype stimulation on METH seeking using both a cue-induced reinstatement and cue-craving model of relapse. Methods: Male and female rats were trained to self-administer METH during daily 2-h sessions. Cue-induced reinstatement of METH seeking was evaluated after extinction training. A systemic pretreatment of an adenosine A1 receptor (A1R) or A2A receptor (A2AR) agonist was administered prior to an extinction or cue session to evaluate the effects of adenosine receptor subtype stimulation on METH seeking. The effects of a systemic pretreatment of A1R or A2AR agonists were also evaluated in a cue-craving model where the cued-seeking test was conducted after 21 days of forced home-cage abstinence without extinction training. Results: Cue-induced reinstatement was reduced in both male and female rats that received A1R or A2AR agonist pretreatments. Similarly, an A1R or A2AR agonist pretreatment also inhibited cue craving in both male and female rats. Conclusion: Stimulation of either adenosine A1R or A2AR subtypes inhibits METH-seeking behavior elicited by METH-associated cues. These effects may be attributed to the ability of A1R and A2AR stimulation to disrupt cue-induced dopamine and glutamate signaling throughout the brain. [ABSTRACT FROM AUTHOR]

A series of 12 novel polyethylene‐glycol(PEG)‐alkynyl C2‐adenosine(ADN) conjugates were synthesized using a robust Sonogashira coupling protocol and characterized by NMR spectroscopy and mass spectrometry analysis. The ADN‐PEG conjugates showed null to moderate toxicity in murine macrophages and 12c was active against Mycobacterium aurum growth (MIC = 62.5 mg/L). The conjugates were not active against Mycobacterium bovis BCG. Conjugates 10b and 11b exhibited high water solubility with solubility values of 1.22 and 1.18 mg/ml, respectively, in phosphate buffer solutions at pH 6.8. Further, 10b and 11b induced a significant increase in cAMP accumulation in RAW264.7 cells comparable with that induced by adenosine. Analogues 10c, 11c and 12c were docked to the A1, A2A, A2B and A3 adenosine receptors (ARs) using crystal‐structures and homology models. ADN‐PEG‐conjugates bearing chains with up to five ethyleneoxy units could be well accommodated within the binding sites of A1, A2A and A3 ARs. Docking studies showed that compound 10b and 11b were the best A2A receptor binders of the series, whereas 12c was the best binder for A1 AR. In summary, introduction of hydrophilic PEG substituents at the C2 of adenine ring significantly improved water solubility and did not affect AR binding properties of the ADN‐PEG conjugates. [ABSTRACT FROM AUTHOR]

The membrane-based purinergic 7 receptor (P2X7R) is expressed on activated microglia and the target of the radioligand [11C]SMW139 for in vivo assessment of neuroinflammation. This study investigated the contribution of radiolabelled metabolites which potentially affect its quantification. Ex vivo high-performance liquid chromatography with a radio detector (radioHPLC) was used to evaluate the parent and radiometabolite fractions of [11C]SMW139 in the brain and plasma of eleven mice. Twelve healthy humans underwent 90-min [11C]SMW139 brain PET with arterial blood sampling and radiometabolite analysis. The volume of distribution was estimated by using one- and two- tissue compartment (TCM) modeling with single (V T) and dual (V Tp) input functions. RadioHPLC showed three major groups of radiometabolite peaks with increasing concentrations in the plasma of all mice and humans. Two radiometabolite peaks were also visible in mice brain homogenates and therefore considered for dual input modeling in humans. 2TCM with single input function provided V T estimates with a wide range (0.10–10.74) and high coefficient of variation (COV: 159.9%), whereas dual input function model showed a narrow range of V Tp estimates (0.04–0.24; COV: 33.3%). In conclusion, compartment modeling with correction for brain-penetrant radiometabolites improves the in vivo quantification of [11C]SMW139 binding to P2X7R in the human brain. [ABSTRACT FROM AUTHOR]

Titanium dioxide (TiO 2) nanotubes can improve the osseointegration of pure titanium implants, but this exact mechanism has not been fully elucidated. The purinergic receptor P2Y6 is expressed in bone marrow mesenchymal stem cells (BMSCs) and participates in the regulation of bone metabolism. However, it is unclear as to whether P2Y6 is involved in the osteogenic differentiation of BMSCs induced by TiO 2 nanotubes. TiO 2 nanotubes were prepared on the surface of titanium specimens using the anodizing method and characterized their features. Quantitative reverse transcriptase polymerase chain reaction and western blotting were used to detect the expression of P2Y6, markers of osteogenic differentiation, and PKCα-ERK1/2. A rat femoral defect model was established to evaluate the osseointegration effect of TiO 2 nanotubes combined with P2Y6 agonists. The results showed that the average inner diameter of the TiO 2 nanotubes increased with an increase in voltage (voltage range of 30–90V), and the expression of P2Y6 in BMSCs could be upregulated by TiO 2 nanotubes in osteogenic culture. Inhibition of P2Y6 expression partially inhibited the osteogenic effect of TiO 2 nanotubes and downregulated the activity of the PKCα-ERK1/2 pathway. When using in vitro and in vivo experiments, the osteogenic effect of TiO 2 nanotubes when combined with P2Y6 agonists was more pronounced. TiO 2 nanotubes promoted the P2Y6 expression of BMSCs during osteogenic differentiation and promoted osteogenesis by activating the PKCα-ERK1/2 pathway. The combined application of TiO 2 nanotubes and P2Y6 agonists may be an effective new strategy to improve the osseointegration of titanium implants. Titanium dioxide (TiO 2) nanotubes can improve the osseointegration of pure titanium implants, but this exact mechanism has not been fully elucidated. The purinergic receptor P2Y6 is expressed in bone marrow mesenchymal stem cells (BMSCs) and participates in the regulation of bone metabolism. However, it is unclear as to whether P2Y6 is involved in the osteogenic differentiation of BMSCs induced by TiO 2 nanotubes. For the first time, this study revealed the relationship between TiO 2 nanotubes and purine receptor P2Y6, and further explored its mode of action, which may provide clues as to the regulatory role of TiO 2 nanotubes on osteogenic differentiation of BMSCs. These findings will help to develop novel methods for guiding material design and biosafety evaluation of nano implants. [Display omitted] [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS) that leads to progressive physical disability. Recent evidence has suggested that P2X7 receptor (P2X7R)-mediated purinergic signalling pathways play a role in MS-associated neuroinflammation, possibly contributing to disease pathogenesis. To evaluate possible associations between P2X7R polymorphisms and MS disease severity, we performed an association study of five non-synonymous SNPs coding variants of the P2X7R gene: rs1718119 Ala348Thr, rs2230911 Thr357Ser, rs2230912 Gln460Arg, rs3751143 Glu496Ala, and rs28360457 Arg307Gln, modulating P2X7R expression in 128 MS patients (relapsing remitting MS, RRMS: n = 94; secondary progressive, SPMS: n = 34). All patients were genotyped, and multiple sclerosis severity score (MSSS) was evaluated in every case; 189 healthy subjects were enrolled as well as controls. Results showed that P2X7R rs1718119(A) 348Thr and rs22390912(G) 464Arg, two SNPs of minor allele frequency (MAF) known to confer gain of function to the P2X7R protein, were associated with significantly higher MSSS in RRMS patients alone (SMRR (p < 0.001, p = 0.01, respectively)). Interestingly, two whole haplotypes resulted in having significant association with MSSS in these same patients. Thus: (1) the P2X7R-4 "ACGAG" haplotype, characterized by the co-presence of the rs1718119-rs2230912 AG MAF alleles, was associated with higher MSSS (Beta: 1.11 p = 0.04), and (2) the P2X7R-1 "GCAAG" complementary haplotype, which contains the rs1718119 and rs2230912 GA wild-type alleles, was more frequently carried by patients with lower MSSS and less severe disease (Beta: −1.54 p < 0.001). Although being preliminary and needing confirmation in an ampler cohort, these results suggest that 348Thr and 464Arg variants have a role as modulators of disease severity in RRMS patients. [ABSTRACT FROM AUTHOR]

Mutant superoxide dismutase 1 (SOD1) can be constitutively released from motor neurons and transmitted to naïve motor neurons to promote the progression of amyotrophic lateral sclerosis (ALS). However, the biological impacts of this process and the precise mechanisms of SOD1 release remain to be fully resolved. Using biochemical and fluorescent techniques, this study aimed to determine if P2X7 receptor activation could induce mutant SOD1 release from motor neurons and whether this released SOD1 could be transmitted to motor neurons or microglia to mediate effects associated with neurodegeneration in ALS. Aggregated SOD1G93A, released from murine NSC-34 motor neurons transiently transfected with SOD1G93A, could be transmitted to naïve NSC-34 cells and murine EOC13 microglia to induce endoplasmic reticulum (ER) stress and tumour necrosis factor-alpha (TNFα) release, respectively. Immunoblotting revealed NSC-34 cells expressed P2X7. Extracellular ATP induced cation dye uptake into these cells, which was blocked by the P2X7 antagonist AZ10606120, demonstrating these cells express functional P2X7. Moreover, ATP induced the rapid release of aggregated SOD1G93A from NSC-34 cells transiently transfected with SOD1G93A, a process blocked by AZ10606120 and revealing a role for P2X7 in this process. ATP-induced SOD1G93A release coincided with membrane blebbing. Finally, aggregated SOD1G93A released via P2X7 activation could also be transmitted to NSC-34 and EOC13 cells to induce ER stress and TNFα release, respectively. Collectively, these results identify a novel role for P2X7 in the prion-like propagation of SOD1 in ALS and provide a possible explanation for the therapeutic benefits of P2X7 antagonism previously observed in ALS SOD1G93A mice. [ABSTRACT FROM AUTHOR]

The aim of the current study was to determine the effects of cerebral contusion injury with purinergic adenosine triphosphate Y1 (P2Y1) receptor blockers on postinjury inflammatory responses. Adenosine triphosphate (ATP) is released into the extracellular space in several in vivo models, including traumatic brain injury. Released ATP triggers neuroinflammation via activation of microglial cells. P2Y1 receptor blockers were reported to suppress extracellular ATP elevation in several disease models through inhibition of cellular ATP release. In addition to the beneficial effects of inflammation, excess inflammatory reactions cause secondary damage and aggravate outcomes. Here, we assessed the effect of the selective P2Y1 receptor blocker MRS2179 on its potential to prevent posttraumatic inflammation in a rat cerebral contusion model. Cerebral contusion injury was induced in the rat cerebral cortex. Either MRS2179 or artificial cerebral spinal fluid as a control was administered in situ into the center of contused tissue via a subcutaneously implanted osmotic pump. Galectin 3, a marker of microglia and proinflammatory cytokines, was measured 1, 3 and 7 days following injury. Another group of rats was assessed for behavioral performance up to 28 days after injury, including the beam walk test, neurological response test and plus maze test. The Galectin 3 levels in the cortex around the contusion cavity and in the cortex far from the contusion cavity were significantly suppressed by MRS2179 administration on postinjury Days 1 and 3 (p

The large intracellular C-terminus of the pro-inflammatory P2X7 ion channel receptor (P2X7R) is associated with diverse P2X7R-specific functions. Cryo-EM structures of the closed and ATP-bound open full-length P2X7R recently identified a membrane-associated anchoring domain, an open-state stabilizing “cap” domain, and a globular “ballast domain” containing GTP/GDP and dinuclear Zn2+-binding sites with unknown functions. To investigate protein dynamics during channel activation, we improved incorporation of the environment-sensitive fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)–2-aminopropanoic acid (ANAP) into Xenopus laevis oocyte-expressed P2X7Rs and performed voltage clamp fluorometry. While we confirmed predicted conformational changes within the extracellular and the transmembrane domains, only 3 out of 41 mutants containing ANAP in the C-terminal domain resulted in ATP-induced fluorescence changes. We conclude that the ballast domain functions rather independently from the extracellular ATP binding domain and might require activation by additional ligands and/or protein interactions. Novel tools to study these are presented.

A study published in the journal Clinical & Translational Discovery explores the role of the P2X4 receptor in inflammatory bowel disease (IBD). The researchers found that the loss of the P2X4 receptor worsens colitis in mice, leading to increased inflammation and microbiota dysbiosis. They also discovered that the positive P2X4 modulator, ivermectin, can attenuate colitis in mice, but its therapeutic effect is not dependent on P2X4 activation. The study highlights the importance of P2X4 in IBD, although previous studies have yielded conflicting conclusions about its role in promoting or attenuating colitis. [Extracted from the article]

P2X7, an ion channel gated by extracellular ATP, is widely expressed on the plasma membrane of immune cells and plays important roles in inflammation and apoptosis. Several single nucleotide polymorphisms have been identified in the human P2RX7 gene. In contrast to other members of the P2X family, nonsynonymous polymorphisms in P2X7 are common. Three of these occur at overall frequencies of more than 25% and affect residues in the extracellular "head"-domain of P2X7 (155 Y/H), its "lower body" (270 R/H), and its "tail" in the second transmembrane domain (348 T/A). Comparison of the P2X7 orthologues of human and other great apes indicates that the ancestral allele is Y--R--T (at 155-270-348). Interestingly, each single amino acid variant displays lower ATP-sensitivity than the ancestral allele. The originally published reference sequence of human P2X7, often referred to as "wildtype," differs from the ancestral allele at all three positions, i.e. H--H--A. The 1,000 Genome Project determined the sequences of both alleles of 2,500 human individuals, including roughly 500 persons from each of the five major continental regions. This rich resource shows that the ancestral alleles Y155, R270, and T348 occur in all analyzed human populations, albeit at strikingly different frequencies in various subpopulations (e.g., 25%-59% for Y155, 59%-77% for R270, and 13%-47% for T348). BLAST analyses of ancient human genome sequences uncovered several homozygous carriers of variant P2X7 alleles, possibly reflecting a high degree of inbreeding, e.g., H--R--T for a 50.000 year old Neanderthal, H--R--A for a 24.000 year old Siberian, and Y--R--A for a 7,000 year old mesolithic European. In contrast, most present-day individuals co-express two copies of P2X7 that differ in one or more amino acids at positions 155, 270, and 348. Our results improve the understanding of how P2X7 structure affects its function and suggest the importance of considering P2X7 variants of participants when designing clinical trials targeting P2X7. [ABSTRACT FROM AUTHOR]

Gefapixant, a P2X3 receptor antagonist, has demonstrated efficacy in patients with refractory or unexplained chronic cough. We investigated the effect of renal impairment (RI) on the pharmacokinetics (PK) of gefapixant 50 mg in an open‐label, single‐dose study enrolling participants with moderate (n = 6) or severe (n = 6) RI, end‐stage renal disease (ESRD; n = 6) under hemodialysis (HD) and non‐HD conditions, and healthy matched controls (n = 6). Serial plasma and urine samples for gefapixant concentrations were collected at selected time points over 72 and 48 hours after dosing, respectively. Linear regression analysis predicted a 1.87‐, 2.79‐, and 3.76‐fold higher exposure (area under the plasma concentration–time curve) for participants with mild, moderate, and severe RI, respectively, than that for healthy matched control participants. Categorical analysis exhibited a 2.98‐, 4.43‐, and 4.74‐fold higher exposure for participants with moderate RI, severe RI, and ESRD, respectively, than that for healthy matched control participants. Apparent oral clearance and renal clearance was lower in participants with various degrees of RI, by 66% to 90%, compared with healthy matched control participants, explaining the increased gefapixant exposure with increasing degrees of renal impairment. Gefapixant area under the plasma concentration–time curve and maximum plasma concentration decreased by ≈25% under HD conditions compared to non‐HD conditions. Single‐dose administration of gefapixant was generally well tolerated in this study. The data from this trial informed the enrollment of phase 3 clinical trials that evaluated the efficacy and safety of gefapixant in >2000 participants with refractory or unexplained chronic cough. Those efficacy and safety data, combined with analysis of population pharmacokinetics from across the entire development program, will be used to evaluate the magnitude of the renal impairment effect in the refractory or unexplained chronic cough population and to determine any dose adjustment recommendations. [ABSTRACT FROM AUTHOR]

Trigeminal ganglion (TG) is the first station of sensory pathways in the orofacial region. The TG neurons communicate with satellite glial cells (SGCs), macrophages and other cells forming a functional unit that is responsible for processing of orofacial sensory information. Purinergic signaling, one of the most widespread autocrine and paracrine pathways, plays a crucial role in intercellular communication. The multidirectional action of purinergic signaling in different cell types contributes to the neuromodulation and orofacial sensation. To fully understand the purinergic signaling in these processes, it is essential to determine the shared and unique expression patterns of genes associated with purinergic signaling in different cell types. Here, we performed single-cell RNA sequencing of 22,969 cells isolated from normal mouse TGs. We identified 18 distinct cell populations, including 6 neuron subpopulations, 3 glial subpopulations, 7 immune cell subpopulations, fibroblasts, and endothelial cells. We also revealed the transcriptional features of genes associated with purinergic signaling, including purinergic receptors, extracellular adenosine triphosphate (eATP) release channels, eATP metabolism-associated enzymes, and eATP transporters in each cell type. Our results have important implications for understanding and predicting the cell type-specific roles of the purinergic signaling in orofacial signal processing in the trigeminal primary sensory system. [ABSTRACT FROM AUTHOR]