P2X7 Receptor Activation Research Articles

Purinergic ionotropic P2X7 and metabotropic glutamate mGlu5 receptors crosstalk influences pro-inflammatory conditions in microglia

Microglia represent the resident immune system in the brain. They mediate neuroinflammatory processes and have been described as important regulators of homeostasis in the central nervous system (CNS). Among several players and mechanisms contributing to microglial function in inflammation, ATP and glutamate have been shown to be involved in microgliosis. In this study, we focused on receptor subtypes that respond to these neurotransmitters, purinergic ionotropic P2X7 receptor and metabotropic glutamate mGlu5 receptor. We found that both receptors are functionally expressed in a murine microglia cell line, BV2 cells, and we performed patch-clamp experiments to measure purinergic ionotropic P2X7 receptor ion flux in control condition and after metabotropic glutamate mGlu5 receptor activation. The selective purinergic ionotropic P2X7 receptor agonist, 2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate (BzATP, 100 μM), elicited a robust current that was prevented by the selective purinergic ionotropic P2X7 receptor antagonist A438079 (10 μM). When BV2 cells were acutely stimulated with the selective metabotropic glutamate mGlu5 agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 200 μM), purinergic ionotropic P2X7 receptor current was increased. This positive modulation was prevented by the selective metabotropic glutamate mGlu5 receptor antagonist 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP, 1 μM). Moreover, nitric oxide synthesis elicited by purinergic ionotropic P2X7 receptor activation was enhanced by metabotropic glutamate mGlu5 receptor co-stimulation. Taken together, our results suggest an important crosstalk between ATP and glutamate in inflammation. Pro-inflammatory effects mediated by purinergic ionotropic P2X7 receptor might be exacerbated by simultaneous exposure of microglia to ATP and glutamate, suggesting new pharmacological targets to modulate neuroinflammation.

Nitrated Hsp90 Supports Glioblastoma Multiforme Cell Survival by Maintaining P2X7 Receptor Activation

Nitrated Hsp90 Supports Glioblastoma Multiforme Cell Survival by Maintaining P2X7 Receptor Activation

Modulation of P2X4 receptor activity by ivermectin and 5-BDBD has no effect on the development of ARPKD in PCK rats.

Autosomal recessive polycystic kidney disease (ARPKD) is an inherited pathology caused mainly by mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene, which usually leads to end-stage renal disease. Previous studies suggested that the P2X purinoreceptor 4 (P2X4 R) may play an important role in the progression of ARPKD. To test this hypothesis, we assessed the chronic effects of ivermectin (P2X4 R allosteric modulator) and 5-BDBD (P2X4 R antagonist) on the development of ARPKD in PCK/CrljCrl-Pkhd1pck/CRL (PCK) rats. Our data indicated that activation of ATP-mediated P2X4 R signaling with ivermectin for 6 weeks in high dose (50 mg/L; water supplementation) decreased the total body weight of PCK rats while the heart and kidney weight remained unaffected. Smaller doses of ivermectin (0.5 or 5mg/L, 6 weeks) or the inhibition of P2X4 R signaling with 5-BDBD (18 mg/kg/day, food supplement for 8 weeks) showed no effect on electrolyte balance or the basic physiological parameters. Furthermore, cystic index analysis for kidneys and liver revealed no effect of smaller doses of ivermectin (0.5 or 5mg/L) and 5-BDBD on the cyst development of PCK rats. We observed a slight increase in the cystic liver index on high ivermectin dose, possibly due to the cytotoxicity of the drug. In conclusion, this study revealed that pharmacological modulation of P2X4 R by ivermectin or 5-BDBD does not affect the development of ARPKD in PCK rats, which may provide insights for future studies on investigating the therapeutic potential of adenosine triphosphate (ATP)-P2 signaling in PKD diseases.

P2X7 is expressed on human innate-like T lymphocytes and mediates susceptibility to ATP-induced cell death.

Extracellular ATP activates the P2X7 receptor, leading to inflammasome activation and release of pro-inflammatory cytokines in monocytes. However, a detailed analysis of P2X7 receptor expression and function in the human T cell compartment has not been reported. Here, we used a P2X7-specific nanobody to assess cell membrane expression and function of P2X7 on peripheral T lymphocyte subsets. The results show that innate-like T cells, which effectively react to innate stimuli by secreting high amounts of pro-inflammatory cytokines, have the highest expression of P2X7 in the human T cell compartment. Using Tγδ cells as example for an innate-like lymphocyte population, we demonstrate that these cells are more sensitive to P2X7 receptor activation than conventional T cells, affecting fundamental cellular mechanisms like calcium signaling and ATP-induced cell death. The increased susceptibility of innate-like T cells to P2X7-mediated cell death provides a mechanism to control their homeostasis under inflammatory conditions. Understanding the expression and function of P2X7 on human immune cells is essential to assume the benefits and consequences of newly developed P2X7-based therapeutic approaches.

P2X7 receptor activation impairs antitumour activity of natural killer cells.

A high number of intratumoural infiltrating natural killer (NK) cells is associated with better survival in several types of cancer, constituting an important first line of defence against tumours. Hypoxia in the core of solid tumours induces cellular stress and ATP release into the extracellular space where it triggers purinergic receptor activation on tumour-associated immune cells. The aim of this study was to assess whether activation of the purinergic receptor P2X7 by extracellular ATP plays a role in the NK cells antitumour activity. We carried out in vitro experiments using purified human NK cells triggered through P2X7 by extracellular ATP. NK cell killing activity against the tumour target cells K562 was studied by means of NK cytotoxicity assays. Likewise, we designed a subcutaneous solid tumour in vivo mouse model. In this study we found that human NK cells, expressing a functional plasma membrane P2X7, acquired an anergic state after ATP treatment, which impaired their antitumour activity and decreased IFN-γ secretion. This effect was reversed by specific P2X7 antagonists and pretreatment with either IL-2 or IL-15. Furthermore, genetic P2rx7 knockdown resulted in improved control of tumour size by NK cells. In addition, IL-2 therapy restored the ability of NK cells to diminish the size of tumours. Our results show that P2X7 activation represents a new mechanism whereby NK cells may lose antitumour effectiveness, opening the possibility of generating modified NK cells lacking P2X7 but with improved antitumour capacity.

Purinergic P2X7 receptor antagonist inhibits methamphetamine-induced reward, hyperlocomotion, and cortical IL-7A levels in mice: A role for P2X7/IL-17A crosstalk in methamphetamine behaviors?

P2X7 receptors are dysregulated during psychostimulant exposure. Furthermore, P2X7 receptors enhance endogenous systems (e.g., cytokines, dopamine, and glutamate) that facilitate psychostimulant addiction. Therefore, using mouse locomotor, conditioned place preference (CPP), and intracranial self-stimulation (ICSS) assays, we tested the hypothesis that methamphetamine (METH) reward and acute locomotor activation requires P2X7 receptor activity. We also investigated effects of P2X7 blockade on METH-induced changes in cytokine levels in brain reward regions. A438079 (5, 10, 50 mg/kg), a P2X7 antagonist, did not affect spontaneous locomotor activity but reduced hyperlocomotion caused by acute METH (1 mg/kg) exposure. A438079 (10 mg/kg) also prevented expression of METH CPP without causing aversive or rewarding effects. For ICSS experiments, METH (1 mg/kg) facilitated brain reward function as interpreted from reductions in baseline threshold. In the presence of A438079 (50 mg/kg), METH-induced facilitation of ICSS was reduced. Repeated METH exposure (1 mg/kg × 7 d) caused enhancement of IL-17A levels in the prefrontal cortex (PFC) that was normalized by A438070 (10 mg/kg × 7 d). The present data suggest that P2X7 receptor activity contributes to rewarding and locomotor-stimulant effects of METH through a potential mechanism involving IL-17A, which has recently been implicated in anxiety.

TNF-α enhances sensory DRG neuron excitability through modulation of P2X3 receptors in an acute colitis model.

Previous studies have demonstrated that acute colonic inflammation leads to an increase in dorsal root ganglia (DRG) neuronal excitability. However, the signaling elements implicated in this hyperexcitability have yet to be fully unraveled. Extracellular adenosine 5’-triphosphate (ATP) is a well-recognized sensory signaling molecule that enhances the nociceptive response after inflammation through activation of P2X3 receptors, which are expressed mainly by peripheral sensory neurons. The aim of this study is to continue investigating how P2X3 affects neuronal hypersensitivity in an acute colitis animal model. To achieve this, DNBS (Dinitrobenzene sulfonic acid; 200 mg/kg) was intrarectally administered to C57BL/6 mice, and inflammation severity was assessed according to the following parameters: weight loss, macroscopic and microscopic scores. Perforated patch clamp technique was used to evaluate neuronal excitability via measuring changes in rheobase and action potential firing in T8-L1 DRG neurons. A-317491, a well-established potent and selective P2X3 receptor antagonist, served to dissect their contribution to recorded responses. Protein expression of P2X3 receptors in DRG was evaluated by western blotting and immunofluorescence. Four days post-DNBS administration, colons were processed for histological analyses of ulceration, crypt morphology, goblet cell density, and immune cell infiltration. DRG neurons from DNBS-treated mice were significantly more excitable compared with controls; these changes correlated with increased P2X3 receptor expression. Furthermore, TNF-α mRNA expression was also significantly higher in inflamed colons compared to controls. Incubation of control DRG neurons with TNF-α resulted in similar cell hyperexcitability as measured in DNBS-derived neurons. The selective P2X3 receptor antagonist, A-317491, blocked the TNF-α-induced effect. These results support the hypothesis that TNF-α enhances colon-innervating DRG neuron excitability via modulation of P2X3 receptor activity.

Single channel properties of pannexin-1 and connexin-43 hemichannels and P2X7 receptors in astrocytes cultured from rodent spinal cords.

Astrocytes express surface channels involved in purinergic signaling. Among these channels, pannexin-1 (Px1) and connexin-43 (Cx43) hemichannels (HCs) release ATP that acts directly, or through its derivatives, on neurons and glia via purinergic receptors. Although HCs are functional, that is, open and close under physiological and pathological conditions, single channel properties of Px1 HCs in astrocytes have not been defined. Here, we developed a dual voltage clamp technique in HeLa cells expressing human Px1-YFP, and then applied this system to rodent spinal astrocytes to compare their single channel properties with other surface channels, that is, Cx43 HCs and P2X7 receptors (P2X7Rs). Channels were recorded in cell attached patches and evoked with ramp cycles applied through another pipette in whole cell voltage clamp. The mean unitary conductances of Px1 HCs were comparable in HeLa Px1-YFP cells and spinal astrocytes, ~42 and ~48 pS, respectively. Based on their unitary conductance, voltage-dependence, and unitary activity after pharmacological and gene silencing, Px1 HCs in astrocytes could be distinguished from Cx43 HCs and P2X7Rs. Channel activity of Px1 HCs and P2X7Rs was greater than that of Cx43 HCs in control astrocytes during ramps. Unitary activity of Px1 HCs was decreased and that of Cx43 HCs and P2X7Rs increased in astrocytes treated with fibroblast growth factor 1 (FGF-1). In summary, we resolved single channel properties of three different surface channels involved in purinergic signaling in spinal astrocytes, which were differentially modulated by FGF-1, a growth factor involved in neurodevelopment, inflammation and repair.

Simulated hypoxia modulates P2X7 receptor function in mice peritoneal macrophages

The inflammatory focus is similar to the tumor microenvironment, which contains a complex milieu with immune cells and macrophages. The accumulation of cells promotes local pH and O2 tension decline (hypoxia). Local O2 tension decline activates hypoxia-inducible factor α and β (HIF-1α and HIF-1β adenosine triphosphate (ATP) release. ATP activates the P2X7 receptor and modulates ischemic/hypoxic conditions. Similarly, α1α may regulate P2X7 receptor expression in the hypoxic microenvironment. Therefore, we investigated P2X7 receptor function under simulated hypoxic conditions by pretreating peritoneal macrophages with mitochondrial electron transport chain complex inhibitors (simulated hypoxia). Treatment with mitochondrial electron transport chain complex inhibitors until three hours of exposure did not cause LDH release. Additionally, mitochondrial electron transport chain complex inhibitors increased ATP-induced P2X7 receptor function without being able to directly activate this receptor. Other P2 receptor subtypes do not appear to participate in this mechanism. Simulated hypoxia augmented HIF-1α levels and suppressed HIF-1α and P2X7 receptor antagonists. Similarly, simulated hypoxia increased ATP-induced dye uptake and inhibited HIF-1α antagonists. Another factor activated in simulated hypoxic conditions was the intracellular P2X7 receptor regulator PIP2. Treatment with HIF-1α agonists increased PIP2 levels and reversed the effects of HIF-1α and P2X7 receptor antagonists. Additionally, the improved ATP-induced dye uptake caused by the simulated hypoxia stimulus was inhibited by P2X7 receptor and PIP2 antagonists. Therefore, simulated hypoxia may augment P2X7 receptor activity for a pathway dependent on HIF-1α and PIP2 activation.

Brain Damage-linked ATP Promotes P2X7 Receptors Mediated Pineal N-acetylserotonin Release

The pineal gland is a key player in surveillance and defense responses. In healthy conditions, nocturnal circulating melatonin (MEL) impairs the rolling and adhesion of leukocytes to the endothelial layer. Fungi, bacteria, and pro-inflammatory cytokines block nocturnal pineal MEL synthesis, facilitating leukocyte migration to injured areas. ATP is a cotransmitter of the noradrenergic signal and potentiates noradrenaline (NAd)-induced MEL synthesis via P2Y1 receptor (P2Y1R) activation. Otherwise, ATP low-affinity P2X7 receptor (P2X7R) activation impairs N-acetylserotonin (NAS) into MEL conversion in NAd incubated pineals. Here we mimicked a focal increase of ATP by injecting low (0.3 and 1.0 µg) and high (3.0 µg) ATP in the right lateral ventricle of adult rats. Nocturnal pineal activity mimicked the in culture data. Low ATP doses increased MEL output, while high ATP dose and the P2X7R agonist BzATP (15.0–50.0 ng) increased NAS pineal and blood content. In the brain, the response was structure-dependent. There was an increase in cortical and no change in cerebellar MEL. These effects were mediated by changes in the expression of coding genes to synthetic and metabolizing melatonergic enzymes. Thus, the pineal gland plays a role as a first-line structure to respond to the death of cells inside the brain by turning NAS into the darkness hormone.

Evaluation of Placental Toxicity of Five Essential Oils and Their Potential Endocrine-Disrupting Effects.

Pregnant women may use EOs in case of morning sickness, nausea, stress management, etc. Little is known about the potential danger that EOs represent for the placenta and therefore for the pregnancy. Our aim was to explore and compare the placental toxicity and potential endocrine disrupting effects of niaouli, orange, tea tree, wintergreen and ylang-ylang EOs, and their key compounds: 4-terpineol, 1,8-cineol, limonene, methyl salicylate and benzyl salicylate. We studied the release of four hormones and the activation of P2X7 receptor in JEG-Tox human placental cells as key biomarkers for endocrine toxicity. We observed that niaouli, orange, tea tree, wintergreen and ylang-ylang EOs and their key components disrupted at least one of the studied hormones but none of them activated the P2X7 cell death receptor. The tested EOs appear then to be more hormonal modulators rather than EDCs in human placental cells. The hormonal effects observed with the key components were very different from those observed with the EOs. EOs are very complex mixtures, and it is essential to study whole EOs rather than their components individually in safety assessment.

Abstract 321: Inhibition of CD39 results in elevated ATP and activation of myeloid cells to promote anti-tumor immunity

Abstract CD39 (ENTPD1) is an ecto-nucleoside triphosphate diphosphohydrolase expressed widely in the tumor microenvironment (TME) responsible for catalyzing the conversion of ATP to AMP. Inhibition of CD39 enzymatic activity can promote anti-tumor immune responses by increasing the immunostimulatory substrate ATP and decreasing the formation of the product AMP, a precursor to immunoinhibitory adenosine. CD39 inhibition has been shown to have an anti-tumor effect by activating dendritic cells, macrophages, and NK cells in the TME to promote antigen presentation and inflammatory cytokine release. AB598 is a novel antibody, highly specific for human and cynomolgus CD39. AB598 potently binds to CD39 expressed on human primary myeloid cells and tumor cells and potently inhibits both soluble and membrane-bound CD39 enzymatic activity. CD39 inhibition results in increased extracellular ATP (eATP) in the TME and subsequent activation of P2X and P2Y receptors. Myeloid cells highly express both CD39 and P2X7, the P2X receptor with the weakest ligand affinity for ATP (greater than 100 μM), and most likely to be specifically activated by very high levels of eATP resulting from CD39 inhibition. Thus, the myeloid compartment has emerged as a key target associated with the therapeutic effects of CD39 inhibition. AB598 retains the ability to bind and inhibit membrane bound CD39 in the presence of high ATP (400 μM). AB598 promotes maturation of human dendritic cells in the presence of ATP, as determined by increased expression of CD83 and CD86 and decreased expression of CD14. In human macrophages treated with ATP, AB598 activates the inflammasome, resulting in the secretion of pro-inflammatory IL-18 and IL-1β. The ATP-dependent effects of AB598 support a therapeutic rationale of combining it with immunogenic therapies that induce release of ATP, such as chemotherapy and radiation, which might represent a new paradigm in the treatment of advanced solid tumors. Citation Format: Christine E. Bowman, Ada Chen, Damie Juat, Kaustubh Parashar, Julie Clor, Hema Singh, Ritu Kushwaha, Bryan Handlos, Suan Liu, Janine Kline, Xiaoning Zhao, Hyock Joo Kwon, David Green, Stephen W. Young, Ester Fernandez-Salas, Matthew J. Walters, Nigel P. Walker. Inhibition of CD39 results in elevated ATP and activation of myeloid cells to promote anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 321.

Platelets and the Role of P2X Receptors in Nociception, Pain, Neuronal Toxicity and Thromboinflammation.

P2X receptors belong to a family of cation channel proteins, which respond to extracellular adenosine 5′-triphosphate (ATP). These receptors have gained increasing attention in basic and translational research, as they are central to a variety of important pathophysiological processes such as the modulation of cardiovascular physiology, mediation of nociception, platelet and macrophage activation, or neuronal–glial integration. While P2X1 receptor activation is long known to drive platelet aggregation, P2X7 receptor antagonists have recently been reported to inhibit platelet activation. Considering the role of both P2X receptors and platelet-mediated inflammation in neuronal diseases such as multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, and stroke, targeting purinergic receptors may provide a valuable novel therapeutic approach in these diseases. Therefore, the present review illuminates the role of platelets and purinergic signaling in these neurological conditions to evaluate potential translational implications.

Activation of the P2X7 receptor in the dental pulp tissue contributes to the pain in rats with acute pulpitis.

Treatment of acute pulpitis (AP) is beneficial for pain relief and pulp regeneration. The purinergic P2X7 receptor activation is responsible for the formation and maintenance of inflammation and pain. This study aims to determine the role of the pulp tissue P2X7 receptor to activate the mechanisms of the AP in rats. The Sprague-Dawley rats were divided into groups, namely, normal, normal saline (NS), and lipopolysaccharide (LPS) groups. Alterations in pain behavior were detected through head-withdrawal thresholds (HWTs), and the pathological changes in pulp tissue were studied through hematoxylin and eosin staining. The expression of the P2X7 receptor in pulp tissue was observed through immunohistochemistry and Western Blotting. The effect of the P2X7 receptor antagonist A-740003 on HWTs was also observed. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the pulp tissue of rats were analyzed through enzyme-linked immunosorbent assay. The HWTs were reduced in the rats with AP. Inflammation is formed but was found more severe in the LPS group than the NS group, and the expression levels of the P2X7 receptors in the NS and LPS groups were higher than in the normal group. The periodontal ligament injection of the A-740003 dose-dependant increases the HWTs in rats with AP. The IL-6 and TNF-α levels in the pulp in the NS and LPS groups were increased but reversed by A-740003 injection. In rats with AP, the expression level of the P2X7 receptor and IL-6/TNF-α release was upregulated. The A-740003 can relieve pain and reduce the inflammation progression in rats with AP.

REDUCTION OF HEART RATE VIA P2X4 RECEPTOR ACTIVATION AS A PROMISING TREATMENT STRATEGY IN PULMONARY ARTERIAL HYPERTENSION

Objective: Mounting evidence suggest that the ATP-sensitive ionotropic P2X4 receptor (P2X4R) improves cardiac performance and optimizes myocardial energy demand by reducing chronotropy. Here, we set to investigate whether the digitalis-like effect of the P2X4R in the sinus node was preserved in rats with right heart failure due to pulmonary arterial hypertension (PAH). Design and method: Male Wistar rats received a single subcutaneous injection of either monocrotaline (MCT; PAH group) or vehicle (CTRL group). Right heart failure associated to PAH normally occurs in Wistar rats 21 to 25 days after MCT administration. Myographic recordings were performed in spontaneously beating atria (chronotropic effect) isolated from both animal groups. Results: Baseline spontaneous atrial rate was 250 ± 5 (n = 26) bpm in CTRL and 237 ± 7 bpm (n = 23) in PAH group (p > 0.05). ATP (0.01–1 mM) concentration-dependently reduced the spontaneous atrial rate of both animal groups. The negative chronotropic effect of ATP (100 M) was smaller in PAH rats (-9 ± 2%; n = 14 vs. -22 ± 4%; n = 25, p < 0.05), but this divergence was attenuated upon increasing the concentration of ATP to 1 mM (CTRL: -54 ± 5%; n = 15 vs. MCT: -50 ± 7%; n = 8; p > 0.05). The P2X4R antagonist, 5-BDBD (10 M), attenuated the negative chronotropic action of ATP (100 M) in CTRL (from -19 ± 5% to -13 ± 3%; n = 5, p > 0.05) and PAH (from -8 ± 3% to 1 ± 8%; n = 4, p > 0.05) rats. The positive allosteric modulator of P2X4R, ivermectin (30 M), potentiated the negative chronotropic effect of ATP (100 M) in CTRL (from -31 ± 5% to -56 ± 14%; n = 5, p > 0.05) and PAH (from -12 ± 6% to -24 ± 9%; n = 4, p < 0.05). The non-selective P2XR agonist, CTP (1 mM), which contrary to ATP does not breakdown into adenosine, also decreased atrial chronotropy in CTRL (-24.31 ± 6.73%; n = 6) and PAH (-11.82 ± 1.98%; n = 6) rats. Conclusions: Data suggest that the negative chronotropic effect of P2X4R activation is preserved in atria of rats with heart failure associated to PAH, thus strengthening the hypothesis that heart rate reductions using this ATP-sensitive receptor may increase cardiac performance in patients with PAH-induced heart failure.

The P2X4 Receptor: Cellular and Molecular Characteristics of a Promising Neuroinflammatory Target.

The adenosine 5′-triphosphate-gated P2X4 receptor channel is a promising target in neuroinflammatory disorders, but the ability to effectively target these receptors in models of neuroinflammation has presented a constant challenge. As such, the exact role of P2X4 receptors and their cell signalling mechanisms in human physiology and pathophysiology still requires further elucidation. To this end, research into the molecular mechanisms of P2X4 receptor activation, modulation, and inhibition has continued to gain momentum in an attempt to further describe the role of P2X4 receptors in neuroinflammation and other disease settings. Here we provide an overview of the current understanding of the P2X4 receptor, including its expression and function in cells involved in neuroinflammatory signalling. We discuss the pharmacology of P2X4 receptors and provide an overview of P2X4-targeting molecules, including agonists, positive allosteric modulators, and antagonists. Finally, we discuss the use of P2X4 receptor modulators and antagonists in models of neuroinflammatory cell signalling and disease.

Structural Changes Induced in Hsp90 by Nitration lead to a Pathological Gain‐of‐function

Heat Shock Protein (Hsp90) is a pro‐survival molecular chaperone essential for the proper folding of a wide range of cellular proteins. It is highly abundant in most cell types and exists as a dimer that interacts with the client proteins. It is well described that the activity of Hsp90 is regulated by post‐translational modifications. We showed that in pathological conditions in which the powerful oxidant peroxynitrite is produced, Hsp90 undergoes nitration at 5 out of 24 tyrosine residues (Y) in its sequence. Further, nitration at Y33 and/or Y56 induces a pathological gain‐of‐function in Hsp90, leading to decreased mitochondrial activity and activation of the purinergic P2X7 receptor, which in motor neurons induces cell death. This is the first nitrated protein with a pathological function described to date. Nitration of Hsp90 is of particular relevance in amyotrophic lateral sclerosis (ALS), a motor neuron degenerative condition with an elusive pathogenesis and scant treatment options. We discovered that Hsp90 is endogenously nitrated in motor neurons in the spinal cord of ALS patients, the cell type that is compromised in this disease process. Establishing the structural changes induced in Hsp90 by nitration that lead to the pathological toxic function is crucial for the development of novel therapeutic strategies for ALS treatment. Here, using a combination of biophysical approaches we show that nitration of Hsp90 at Y33 and Y56, the residues relevant to the toxic function, had a profound impact on Hsp90 structure. To dissect the contribution of nitration at Y33 and/or Y56 on Hsp90 structural changes, we performed analytical ultracentrifugation sedimentation velocity experiments using recombinant Hsp90, peroxynitrite‐treated Hsp90 (fully oxidized protein), and site‐specific nitrated Hsp90 produced by genetic code expansion, carrying nitrotyrosine at either position 33 or 56, or simultaneously at both positions as the sole modification in the protein. Following peroxynitrite treatment, Hsp90 dimer was destabilized, showing a significant increase in the number of monomers, and the formation of oligomeric species. These results were confirmed by negative stain cryo‐electron microscopy, and size exclusion chromatography followed by multiangle light scattering. Site‐specific nitration at Y33 or Y56 was enough to destabilize the dimer conformation, while simultaneous nitration at Y33 and Y56 most closely resembled the structural changes observed after peroxynitrite treatment. In contrast, replacement of Y33 and Y56 by nitration‐resistant phenylalanine decreased dimer destabilization and prevented oligomer formation after peroxynitrite treatment. Together, these results suggest that nitration at Y33 and Y56 is enough to significantly affect the global structure of Hsp90, and that these changes may be responsible for nitrated Hsp90's toxic gain‐of‐function. Thus, targeting these structural changes may lead to the development of drugs that selectively inhibit nitrated Hsp90's pathological activity in ALS, without affecting the function of the unmodified chaperone in normal tissues.

Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia

SUMMARYActivated microglia release extracellular vesicles (EVs) as modulators of brain homeostasis and innate immunity. However, the molecules critical for regulating EV production from microglia are poorly understood. Here we establish a murine microglial cell model to monitor EV secretion by measuring the fluorescence signal of tdTomato, which is linked to tetraspanin CD63. Stimulation of tdTomato+ cells with ATP induces rapid secretion of EVs and a reduction in cellular tdTomato intensity, reflecting EV secretion. We generate a GFP+ tdTomato+ cell library expressing TurboGFP and barcoded short hairpin RNAs for genome-wide screening using next-generation sequencing. We identify Mcfd2, Sepp1, and Sdc1 as critical regulators of ATP-induced EV secretion from murine microglia. Small interfering RNA (siRNA-based) silencing of each of these genes suppresses lipopolysaccharide- and ATP-induced inflammasome activation, as determined by interleukin-1β release from primary cultured murine microglia. These molecules are critical for microglial EV secretion and are potential therapeutic targets for neuroinflammatory disorders.

Autocrine P2X4 receptor activation in RBCs drives oxygen‐dependent hyperemic responses in mouse skeletal muscle capillaries

Microvascular blood flow is tightly coupled to tissue function and when disturbed, organ insult ensues. Changes in red blood cell (RBC) oxygenation are thought to be key to the initiation of robust microvascular responses, a process mechanistically linked to ATP release from RBCs and conducted signalling. The RBC signaling cascade governing ATP release was the focus of this study, and of note is the role of ATP‐activated P2X4 receptors. Using an in‐vivo mouse muscle preparation (extensor digitorum longus, EDL) and a focally delivered oxygen stimulus via a rapid gas exchange chamber, we probed capillary hemodynamics, prior to and following purinergic receptor antagonism. Dropping oxygen tension from 7% to 2% reduced RBC oxygen saturation in surface capillaries while increasing RBC supply rate. Incubating the EDL with PPADS (non‐selective purinergic receptor antagonist, 30 µM) or 5‐BDBD (selective P2X4 antagonist, 20 µM) reduced baseline RBC supply rate by ≍ 40% and 47%, respectively, and attenuated the capillary responses to the oxygen stimulus by ≍ 5 and 3 RBC/s, respectively. This blunting predictably enhanced the drop in capillary RBC oxygen saturation, as more oxygen was drawn per RBC to meet tissue demand. Immunofluorescence labeling revealed robust expression of P2X4 receptors in RBCs but not endothelial cells. Together, this data begins to elucidate a role for RBC P2X4 receptors, potentially in a positive feedback loop with pannexin channels, to dynamically regulate demand‐supply coupling in skeletal muscle.

P2X7 receptor activation mediates superoxide dismutase 1 (SOD1) release from murine NSC-34 motor neurons

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.