Understanding the role of P2X7 in affective disorders—are glial cells the major players?

Pathophysiology associated with several psychiatric disorders has been linked to inflammatory biomarkers. This has generated a theory of major depressive disorders as an inflammatory disease. The idea of pro-inflammatory cytokines altering behavior is now well accepted however many questions remain. Microglia can produce a plethora of inflammatory cytokines and these cells appear to be critical in the link between inflammatory changes and depressive disorders. Microglia play a known role in sickness behavior which has many components of depressive-like behavior such as social withdrawal, sleep alterations, and anorexia. Numerous candidate genes have been identified for psychiatric disorders in the last decade. Single nucleotide polymorphisms (SNPs) in the human P2X7 gene have been linked to bipolar disorder, depression, and to the severity of depressive symptoms. P2X7 is a ligand-gated cation channel expressed on microglia with lower levels found on astrocytes and on some neuronal populations. In microglia P2X7 is a major regulator of pro-inflammatory cytokines of the interleukin-1 family. Genetic deletion of P2X7 in mice is protective for depressive behavior in addition to inflammatory responses. P2X7−/− mice have been shown to demonstrate anti-depressive-like behavior in forced swim and tail suspension behavioral tests and stressor-induced behavioral responses were blunted. Both neurochemical (norepinephrine, serotonin, and dopamine) and inflammatory changes have been observed in the brains of P2X7−/− mice. This review will discuss the recent evidence for involvement of P2X7 in the pathophysiology of depressive disorders and propose mechanisms by which altered signaling through this ion channel may affect the inflammatory state of the brain

Revisiting the idea that amyloid-β peptide acts as an agonist for P2X7

The P2X7 receptor (P2X7) is a cell surface ligand-gated ion channel, activated by its physiological nucleotide agonist ATP and a synthetic analog (BzATP). However, it has also been suggested that there may be structurally unrelated, non-nucleotide agonists such as the amyloidogenic β peptide. Here we aimed to reassess the effect of amyloid β peptides in various in vitro cell models, namely HEK293 overexpressing human P2X7, the microglial BV-2 cell line, and BV-2 cells lacking P2X7. We measured YO-PRO-1 dye uptake in response to full-length amyloid β peptide (1–42) or the shorter amyloid β peptide (25–35) and there was a concentration-dependent increase in YO-PRO-1 dye uptake in HEK-hP2X7 cells. However, these amyloid β peptide-induced increases in YO-PRO-1 dye uptake were also identical in non-transfected HEK-293 cells. We could observe small transient increases in [Ca2+]i induced by amyloid β peptides in BV-2 cells, however these were identical in BV-2 cells lacking P2X7. Furthermore, our metabolic viability and LDH release experiments suggest no significant change in viability or cell membrane damage in HEK-hP2X7 cells. In the BV-2 cells we found that high concentrations of amyloid β peptides (1–42) and (25–35) could reduce cell viability by up to 35% but this was also seen in BV-2 cells lacking P2X7. We found no evidence of LDH release by amyloid β peptides. In summary, we found no evidence that amyloid β peptides act as agonists of P2X7 in our in vitro models. Our study raises the possibility that amyloid β peptides simply mimic features of P2X7 activation

To Inhibit or Enhance? Is There a Benefit to Positive Allosteric Modulation of P2X Receptors?

The family of ligand-gated ion channels known as P2X receptors were discovered several decades ago. Since the cloning of the seven P2X receptors (P2X1-P2X7), a huge research effort has elucidated their roles in regulating a range of physiological and pathophysiological processes. Transgenic animals have been influential in understanding which P2X receptors could be new therapeutic targets for disease. Furthermore, understanding how inherited mutations can increase susceptibility to disorders and diseases has advanced this knowledge base. There has been an emphasis on the discovery and development of pharmacological tools to help dissect the individual roles of P2X receptors and the pharmaceutical industry has been involved in pushing forward clinical development of several lead compounds. During the discovery phase, a number of positive allosteric modulators have been described for P2X receptors and these have been useful in assigning physiological roles to receptors. This review will consider the major physiological roles of P2X1-P2X7 and discuss whether enhancement of P2X receptor activity would offer any therapeutic benefit. We will review what is known about identified compounds acting as positive allosteric modulators and the recent identification of drug binding pockets for such modulators

Development of high-throughput fluorescent-based screens to accelerate discovery of P2X inhibitors from animal venoms

Animal venoms can play an important role in drug discovery, as they are a rich source of evolutionarily tuned compounds that target a variety of ion channels and receptors. To date, there are six FDA-approved drugs derived from animal venoms, with recent work using high-throughput platforms providing a variety of new therapeutic candidates. However, high-throughput methods for screening animal venoms against purinoceptors, one of the oldest signaling receptor families, have not been reported. Here, we describe a variety of quantitative fluorescent-based high-throughput screening (HTS) cell-based assays for screening animal venoms against ligand-gated P2X receptors. A diverse selection of 180 venoms from arachnids, centipedes, hymenopterans, and cone snails were screened, analyzed, and validated, both analytically and pharmacologically. Using this approach, we performed screens against human P2X3, P2X4, and P2X7 using three different fluorescent-based dyes on stable cell lines and isolated the active venom components. Our HTS assays are performed in 96-well format and allow simultaneous screening of multiple venoms on multiple targets, improving testing characteristics while minimizing costs, specimen material, and testing time. Moreover, utilizing our assays and applying them to the other natural product libraries, rather than venoms, might yield other novel natural products that modulate P2X activity

Rac1 plays a role in CXCL12 but not CCL3-induced chemotaxis and Rac1 GEF inhibitor NSC23766 has off target effects on CXCR4

Cell mi­gra­tion to­wards a chemo­tac­tic stim­u­lus re­lies on the re-arrange­ment of the cy­toskele­ton, which is trig­gered by ac­ti­va­tion of small G pro­teins RhoA, Rac1 and Cd­c42, and leads to for­ma­tion of lamel­lopo­dia and actin poly­meri­sa­tion amongst other ef­fects. Here we show that Rac1 is im­por­tant for CX­CR4 in­duced chemo­taxis but not for CCR1/​CCR5 in­duced chemo­taxis. For CX­CL12-in­duced mi­gra­tion via CX­CR4, breast can­cer MCF-7 cells are re­liant on Rac1, sim­i­larly to THP-1 mono­cytes and Ju­rkat T-cells. For CCL3-in­duced mi­gra­tion via CCR1 and/​or CCR5, Rac1 sig­nalling does not reg­u­late cell mi­gra­tion in ei­ther sus­pen­sion or ad­her­ent cells. We have con­firmed the in­volve­ment of Rac1 with the use of a spe­cific Rac1 block­ing pep­tide. We also used a Rac1 in­hibitor EHT 1864 and a Rac1-GEF in­hibitor NSC23766 to probe the im­por­tance of Rac1 in chemo­taxis. Both in­hibitors did not block CCL3-in­duced chemo­taxis, but they were able to block CX­CL12-in­duced chemo­taxis. This con­firms that Rac1 ac­ti­va­tion is not es­sen­tial for CCL3-in­duced mi­gra­tion, how­ever NSC23766 might have sec­ondary ef­fects on CX­CR4. This small mol­e­cule ex­hibits ag­o­nis­tic fea­tures in in­ter­nal­i­sa­tion and cAMP as­says, whereas it acts as an an­tag­o­nist for CX­CR4 in mi­gra­tion and cal­cium re­lease as­says. Our find­ings strongly sug­gest that Rac1 ac­ti­va­tion is not nec­es­sary for CCL3 sig­nalling, and re­veal that NSC23766 could be a novel CX­CR4 re­cep­tor lig­and

Bug Off Pain: An Educational Virtual Reality Game on Spider Venoms and Chronic Pain for Public Engagement

Virtual reality (VR) technology has been capturing the public imagination for decades. VR software applications that allow for interactive immersion are emerging as a renowned medium in many areas, including educating the public in biochemistry-related subjects via public engagement events. This report provides information about an immersive, interactive and educational virtual reality (VR) game named Bug Off Pain that increases scientific literacy about chronic pain and spider venoms among the public and high school students. Here, VR was shown to be an innovative and fun approach to learning and public engagement in biochemistry. Bug Off Pain places the viewer inside the brain and shows the molecular system that allows people to sense pain. After securing three (learning) points via the multimedia-based clips, this experience translates to the interactive game. Here, a player has to choose a venom that shuts down the pain until that results in “pain over”. Bug Off Pain can be played (free of charge) on two different VR platforms: Oculus Rift and Android devices

Mapping a novel positive allosteric modulator binding site in the central vestibule region of human P2X7

P2X7 receptors are important in the regulation of inflammatory responses and immune responses to intracellular pathogens such as Mycobacterium tuberculosis and Toxoplasma gondii. Enhancement of P2X7 receptor responses may be useful in pathogen clearance particularly in individuals with defective microbial killing mechanisms. Ginsenosides from Panax ginseng have been discovered to act as positive allosteric modulators of P2X7. Here we describe a novel modulator binding site identified by computational docking located in the central vestibule of P2X7 involving S60, D318, and L320 in the lower body β-sheets lining the lateral portals. Potentiation of ATP-mediated responses by ginsenosides CK and Rd caused enhanced ionic currents, Ca2+ influx and YOPRO-1 uptake in stably transfected HEK-293 cells (HEK-hP2X7) plus enhanced cell death responses. Potentiation of ATP responses by CK and Rd was markedly reduced by mutations S59A, S60A, D318L and L320A supporting the proposed allosteric modulator binding site. Furthermore, mutation of the conserved residues S60 and D318 led to alterations in P2X7 response and a higher sensitivity to ATP in the absence of modulators suggesting residues in the connecting rods play an important role in regulating P2X7 gating. Identification of this novel binding site location in the central vestibule may also be relevant for structurally similar channels

Insights into the structure-activity relationship of glycosides as positive allosteric modulators acting on P2X7 receptors

P2X7 is an important ligand-gated ion channel expressed in multiple immune cell populations. This study aimed to investigate the chemical requirements of triterpenoid glycosides within a new binding pocket to characterize the structure-activity relationship. A set of glycosides were screened for positive modulator activity at human P2X7 using a YO-PRO-1 dye uptake assay in HEK-293 cells stably expressing the wild-type human P2X7 variant (HEK-hP2X7 cells). The highest positive modulator activity was with ginsenoside-compound K (CK), containing a monosaccharide (glucose) attached at carbon-20. Ginsenoside-20(S)-Rg3, containing a disaccharide group (glucose-glucose) at carbon-3, displayed positive modulator activity with a reduced EC50 for ATP and increased maximal response at human P2X7. The epimer 20(R)-Rg3 was inactive. A similar stereo-specific pattern was observed for 20(S)-Rh2. Ginsenoside-F1, highly similar to ginsenoside-CK but containing a single additional hydroxyl group, was also inactive at P2X7. Computational docking suggests hydrophobic residues in the pocket are involved in steric discrimination between triterpenoids, whereas the position and identity of the carbohydrate group are important for positive modulator activity at human P2X7. Ginsenosides containing monosaccharide attachments perform better than di- or trisaccharide glycosides. Additional modifications to the triterpenoid scaffold at carbon-6 are not tolerated. Gypenosides from plant sources other than Panax ginseng (gypenoside XVII, gypenoside XLIX, stevenleaf) can also act as positive allosteric modulators of P2X7. We also investigated the effect of positive allosteric modulators on endogenous P2X7 in THP-1 monocytes and confirmed our findings in a calcium response assay. A cell viability assay showed potentiation of ATP-induced cell death with ginsenoside-CK in THP-1 and HEK-hP2X7 cells. SIGNIFICANCE STATEMENT: Ginsenosides are active as positive allosteric modulators at P2X7, and this study determines the chemical features important for mediating this effect. The position and identity of the sugar group is important for activity, as is the position of a number of hydroxyl groups on the triterpenoid scaffold. Diastereomers of ginsenoside-Rg3 and ginsenoside-Rh2 demonstrate the importance of the location of hydroxyl groups relative to the hydrophobic face of the predicted binding pocket

Bombesin receptor-targeted liposomes for enhanced delivery to lung cancer cells

Background: Gastrin-releasing peptide is a member of the bombesin family of peptides. Its cognate receptor, GRPR, is widely expressed in cancers of the lung, pancreas and ovaries. GRP is an autocrine growth factor in small cell lung cancer, which has very poor patient outcomes. High affinity antagonist peptides have been developed for in vivo cancer imaging. In this report we decorated pegylated liposomes with a GRPR antagonist peptide and studied its interaction with and accumulation within lung cancer cells. Results: An N-terminally cysteine modified GRPR antagonist (termed cystabn) was synthesised and shown to inhibit cell growth in vitro. Cystabn was used to prepare a targeted 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000) lipid conjugate that was formulated into liposomes. The liposomes displayed desirable colloidal properties and good stability under storage conditions. Flow cytometric and microscopic studies showed that fluorescently labelled cystabn-decorated liposomes accumulated more extensively in GRPR over-expressing cells than matched liposomes that contained no cystabn targeting motif. Conclusion: The use of GRPR antagonistic peptides for nanoparticle targeting has potential for enhancing drug accumulation in resistant cancer cells

Methods for studying P2X4 receptor ion channels in immune cells

The P2X4 receptor is a trimeric ligand-gated ion channel activated by adenosine 5′-triphosphate (ATP). P2X4 is present in immune cells with emerging roles in inflammation and immunity, and related disorders. This review aims to provide an overview of the methods commonly used to study P2X4 in immune cells, focusing on those methods used to assess P2RX4 gene expression, the presence of the P2X4 protein, and P2X4 ion channel activity in these cells from humans, dogs, mice and rats. P2RX4 gene expression in immune cells is commonly assessed using semi-quantitative and quantitative reverse-transcriptase-PCR. The presence of P2X4 protein in immune cells is mainly assessed using anti-P2X4 polyclonal antibodies with immunoblotting or immunochemistry, but the use of these antibodies, as well as monoclonal antibodies and nanobodies to detect P2X4 with flow cytometry is increasing. Notably, use of an anti-P2X4 monoclonal antibody and flow cytometry has revealed that P2X4 is present on immune cells with a rank order of expression in eosinophils, then neutrophils and monocytes, then basophils and B cells, and finally T cells. P2X4 ion channel activity has been assessed mainly by Ca2+ flux assays using the cell permeable Ca2+-sensitive dyes Fura-2 and Fluo-4 with fluorescence microscopy, spectrophotometry, or flow cytometry. However, other methods including electrophysiology, and fluorescence assays measuring Na+ flux (using sodium green tetra-acetate) and dye uptake (using YO-PRO-12+) have been applied. Collectively, these methods have demonstrated the presence of functional P2X4 in monocytes and macrophages, microglia, eosinophils, mast cells and CD4+ T cells, with other evidence suggestive of functional P2X4 in dendritic cells, neutrophils, B cells and CD8+ T cells