Different localization of P2X4 and P2X7 receptors in native mouse lung - lack of evidence for a direct P2X4-P2X7 receptor interaction.

Introduction: P2X receptors are a family of homo- and heterotrimeric cation channels gated by extracellular ATP. The P2X4 and P2X7 subunits show overlapping expression patterns and have been involved in similar physiological processes, such as pain and inflammation as well as various immune cell functions. While formation of P2X2/P2X3 heterotrimers produces a distinct pharmacological phenotype and has been well established, functional identification of a P2X4/P2X7 heteromer has been difficult and evidence for and against a physical association has been found. Most of this evidence stems, however, from in vitro model systems. Methods: Here, we used a P2X7-EGFP BAC transgenic mouse model as well as P2X4 and P2X7 knock-out mice to re-investigate a P2X4-P2X7 interaction in mouse lung by biochemical and immunohistochemical experiments as well as quantitative expression analysis. Results: No detectable amounts of P2X4 could be co-purified from mouse lung via P2X7-EGFP. In agreement with these findings, immuno-histochemical analysis using a P2X7-specific nanobody revealed only limited overlap in the cellular and subcellular localizations of P2X4 and P2X7 in both the native lung tissue and primary cells. Comparison of P2X4 and P2X7 transcript and protein levels in the respective gene-deficient and wild type mice showed no mutual interrelation between their expression levels in whole lungs. However, a significantly reduced P2rx7 expression was found in alveolar macrophages of P2rx4 -/- mice. Discussion: In summary, our detailed analysis of the cellular and subcellular P2X4 and P2X7 localization and expression does not support a physiologically relevant direct association of P2X4 and P2X7 subunits or receptors in vivo.

Opposing effects of the purinergic P2X7 receptor on seizures in neurons and microglia in male mice.

BACKGROUND: The purinergic ATP-gated P2X7 receptor (P2X7R) is increasingly recognized to contribute to pathological neuroinflammation and brain hyperexcitability. P2X7R expression has been shown to be increased in the brain, including both microglia and neurons, in experimental models of epilepsy and patients. To date, the cell type-specific downstream effects of P2X7Rs during seizures remain, however, incompletely understood. METHODS: Effects of P2X7R signaling on seizures and epilepsy were analyzed in induced seizure models using male mice including the kainic acid model of status epilepticus and pentylenetetrazole model and in male and female mice in a genetic model of Dravet syndrome. RNA sequencing was used to analyze P2X7R downstream signaling during seizures. To investigate the cell type-specific role of the P2X7R during seizures and epilepsy, we generated mice lacking exon 2 of the P2rx7 gene in either microglia (P2rx7:Cx3cr1-Cre) or neurons (P2rx7:Thy-1-Cre). To investigate the protective potential of overexpressing P2X7R in GABAergic interneurons, P2X7Rs were overexpressed using adeno-associated virus transduction under the mDlx promoter. RESULTS: RNA sequencing of hippocampal tissue from wild-type and P2X7R knock-out mice identified both glial and neuronal genes, in particular genes involved in GABAergic signaling, under the control of the P2X7R following seizures. Mice with deleted P2rx7 in microglia displayed less severe acute seizures and developed a milder form of epilepsy, and microglia displayed an anti-inflammatory molecular profile. In contrast, mice lacking P2rx7 in neurons showed a more severe seizure phenotype when compared to epileptic wild-type mice. Analysis of single-cell expression data revealed that human P2RX7 expression is elevated in the hippocampus of patients with temporal lobe epilepsy in excitatory and inhibitory neurons. Functional studies determined that GABAergic interneurons display increased responses to P2X7R activation in experimental epilepsy. Finally, we show that viral transduction of P2X7R in GABAergic interneurons protects against evoked and spontaneous seizures in experimental temporal lobe epilepsy and in mice lacking Scn1a, a model of Dravet syndrome. CONCLUSIONS: Our results suggest a dual and opposing action of P2X7R in epilepsy and suggest P2X7R overexpression in GABAergic interneurons as a novel therapeutic strategy for acquired and, possibly, genetic forms of epilepsy.

Macrophages and glia are the dominant P2X7-expressing cell types in the gut nervous system-No evidence for the role of neuronal P2X7 receptors in colitis.

The blockade or deletion of the pro-inflammatory P2X7 receptor channel has been shown to reduce tissue damage and symptoms in models of inflammatory bowel disease, and P2X7 receptors on enteric neurons were suggested to mediate neuronal death and associated motility changes. Here, we used P2X7-specific antibodies and nanobodies, as well as a bacterial artificial chromosome transgenic P2X7-EGFP reporter mouse model and P2rx7-/- controls to perform a detailed analysis of cell type-specific P2X7 expression and possible overexpression effects in the enteric nervous system of the distal colon. In contrast to previous studies, we did not detect P2X7 in neurons but found dominant expression in glia and macrophages, which closely interact with the neurons. The overexpression of P2X7 per se did not induce significant pathological effects. Our data indicate that macrophages and/or glia account for P2X7-mediated neuronal damage in inflammatory bowel disease and provide a refined basis for the exploration of P2X7-based therapeutic strategies.

Oxidized linoleic acid metabolites maintain mechanical and thermal hypersensitivity during sub-chronic inflammatory pain.

Inflammatory pain serves as a protective defense mechanism which becomes pathological when it turns into chronic inflammatory pain. This transition is mediated by a variety of peripheral mediators that sensitize nociceptors and increase pain perception in sensory neurons. Besides cytokines, chemokines and growth factors, accumulating evidence shows that oxidized lipids, such as eicosanoids and oxidized linoleic acid metabolites, contribute to this sensitization process. Most notably, the oxidized linoleic acid metabolite and partial TRPV1 agonist 9-HODE (hydroxyoctadecadienoic acid) was shown to be involved in this sensitization process. However, it is still unknown how some of the oxidized linoleic acid metabolites are synthesized in the inflammatory environment and in which phase of inflammation they become relevant. Here we show that the concentrations of oxidized linoleic acid metabolites, especially 9-HODE and 13-HODE, are significantly increased in inflamed paw tissue and the corresponding dorsal root ganglia in the sub-chronic phase of inflammation. Surprisingly, classical inflammatory lipid markers, such as prostaglandins were at basal levels in this phase of inflammation. Moreover, we revealed the cell type specific synthesis pathways of oxidized linoleic acid metabolites in primary macrophages, primary neutrophils and dorsal root ganglia. Finally, we show that blocking the most elevated metabolites 9-HODE and 13-HODE at the site of inflammation in the sub-chronic phase of inflammation, leads to a significant relief of mechanical and thermal hypersensitivity in vivo. In summary, these data offer an approach to specifically target oxidized linoleic acid metabolites in the transition of acute inflammatory pain to chronic inflammatory pain.

Increased expression of the ATP-gated P2X7 receptor reduces responsiveness to anti-convulsants during status epilepticus in mice.

BACKGROUND AND PURPOSE: Refractory status epilepticus is a clinical emergency associated with high mortality and morbidity. Increasing evidence suggests neuroinflammation contributes to the development of drug-refractoriness during status epilepticus. Here, we have determined the contribution of the ATP-gated P2X7 receptor, previously linked to inflammation and increased hyperexcitability, to drug-refractory status epilepticus and its therapeutic potential. EXPERIMENTAL APPROACH: Status epilepticus was induced via a unilateral microinjection of kainic acid into the amygdala in adult mice. Severity of status epilepticus was compared in animals with overexpressing or knock-out of the P2X7 receptor, after inflammatory priming by pre-injection of bacterial lipopolysaccharide (LPS) and in mice treated with P2X7 receptor-targeting and anti-inflammatory drugs. KEY RESULTS: Mice overexpressing P2X7 receptors were unresponsive to several anticonvulsants (lorazepam, midazolam, phenytoin and carbamazepine) during status epilepticus. P2X7 receptor expression increased in microglia during status epilepticus, at times when responses to anticonvulsants were reduced. Overexpression of P2X7 receptors induced a pro-inflammatory phenotype in microglia during status epilepticus and the anti-inflammatory drug minocycline restored normal responses to anticonvulsants in mice overexpressing P2X7 receptors. Pretreatment of wild-type mice with LPS increased P2X7 receptor levels in the brain and reduced responsiveness to anticonvulsants during status epilepticus, which was overcome by either genetic deletion of P2X7 receptors or treatment with the P2X7 receptor antagonists, AFC-5128 or ITH15004. CONCLUSION AND IMPLICATIONS: Our results demonstrate that P2X7 receptor-induced pro-inflammatory effects contribute to resistance to pharmacotherapy during status epilepticus. Therapies targeting P2X7 receptors could be novel adjunctive treatments for drug-refractory status epilepticus.

The Lipid Receptor G2A (GPR132) Mediates Macrophage Migration in Nerve Injury-Induced Neuropathic Pain.

Nerve injury-induced neuropathic pain is difficult to treat and mechanistically characterized by strong neuroimmune interactions, involving signaling lipids that act via specific G-protein coupled receptors. Here, we investigated the role of the signaling lipid receptor G2A (GPR132) in nerve injury-induced neuropathic pain using the robust spared nerve injury (SNI) mouse model. We found that the concentrations of the G2A agonist 9-HODE (9-Hydroxyoctadecadienoic acid) are strongly increased at the site of nerve injury during neuropathic pain. Moreover, G2A-deficient mice show a strong reduction of mechanical hypersensitivity after nerve injury. This phenotype is accompanied by a massive reduction of invading macrophages and neutrophils in G2A-deficient mice and a strongly reduced release of the proalgesic mediators TNFα, IL-6 and VEGF at the site of injury. Using a global proteome analysis to identify the underlying signaling pathways, we found that G2A activation in macrophages initiates MyD88-PI3K-AKT signaling and transient MMP9 release to trigger cytoskeleton remodeling and migration. We conclude that G2A-deficiency reduces inflammatory responses by decreasing the number of immune cells and the release of proinflammatory cytokines and growth factors at the site of nerve injury. Inhibiting the G2A receptor after nerve injury may reduce immune cell-mediated peripheral sensitization and may thus ameliorate neuropathic pain.

The oxidized linoleic acid metabolite 12,13-DiHOME mediates thermal hyperalgesia during inflammatory pain.

Eicosanoids play a crucial role in inflammatory pain. However, there is very little knowledge about the contribution of oxidized linoleic acid metabolites in inflammatory pain and peripheral sensitization. Here, we identify 12,13-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME), a cytochrome P450-derived linoleic acid metabolite, as crucial mediator of thermal hyperalgesia during inflammatory pain. We found 12,13-DiHOME in increased concentrations in peripheral nervous tissue during acute zymosan- and complete Freund's Adjuvant-induced inflammatory pain. 12,13-DiHOME causes calcium transients in sensory neurons and sensitizes the transient receptor potential vanilloid 1 (TRPV1)-mediated intracellular calcium increases via protein kinase C, subsequently leading to enhanced TRPV1-dependent CGRP-release from sensory neurons. Peripheral injection of 12,13-DiHOME in vivo causes TRPV1-dependent thermal pain hypersensitivity. Finally, application of the soluble epoxide hydrolase (sEH)-inhibitor TPPU reduces 12,13-DiHOME concentrations in nervous tissue and reduces zymosan- and CFA-induced thermal hyperalgesia in vivo. In conclusion, we identify a novel role for the lipid mediator 12,13-DiHOME in mediating thermal hyperalgesia during inflammatory pain and propose a novel mechanism that may explain the antihyperalgesic effects of sEH inhibitors in vivo.

Targeting CYP2J to reduce paclitaxel-induced peripheral neuropathic pain.

Chemotherapy-induced peripheral neuropathic pain (CIPNP) is a severe dose- and therapy-limiting side effect of widely used cytostatics that is particularly difficult to treat. Here, we report increased expression of the cytochrome-P450-epoxygenase CYP2J6 and increased concentrations of its linoleic acid metabolite 9,10-EpOME (9,10-epoxy-12Z-octadecenoic acid) in dorsal root ganglia (DRGs) of paclitaxel-treated mice as a model of CIPNP. The lipid sensitizes TRPV1 ion channels in primary sensory neurons and causes increased frequency of spontaneous excitatory postsynaptic currents in spinal cord nociceptive neurons, increased CGRP release from sciatic nerves and DRGs, and a reduction in mechanical and thermal pain hypersensitivity. In a drug repurposing screen targeting CYP2J2, the human ortholog of murine CYP2J6, we identified telmisartan, a widely used angiotensin II receptor antagonist, as a potent inhibitor. In a translational approach, administration of telmisartan reduces EpOME concentrations in DRGs and in plasma and reverses mechanical hypersensitivity in paclitaxel-treated mice. We therefore suggest inhibition of CYP2J isoforms with telmisartan as a treatment option for paclitaxel-induced neuropathic pain.

Progranulin overexpression in sensory neurons attenuates neuropathic pain in mice: Role of autophagy.

Peripheral or central nerve injury is a frequent cause of chronic pain and the mechanisms are not fully understood. Using newly generated transgenic mice we show that progranulin overexpression in sensory neurons attenuates neuropathic pain after sciatic nerve injury and accelerates nerve healing. A yeast-2-hybrid screen revealed putative interactions of progranulin with autophagy-related proteins, ATG12 and ATG4b. This was supported by colocalization and proteomic studies showing regulations of ATG13 and ATG4b and other members of the autophagy network, lysosomal proteins and proteins involved in endocytosis. The association of progranulin with the autophagic pathway was functionally confirmed in primary sensory neurons. Autophagy and survival were impaired in progranulin-deficient neurons and improved in progranulin overexpressing neurons. Nerve injury in vivo caused an accumulation of LC3b-EGFP positive bodies in neurons of the dorsal root ganglia and nerves suggesting an impairment of autophagic flux. Overexpression of progranulin in these neurons was associated with a reduction of the stress marker ATF3, fewer protein aggregates in the injured nerve and enhanced stump healing. At the behavioral level, further inhibition of the autophagic flux by hydroxychloroquine intensified cold and heat nociception after sciatic nerve injury and offset the pain protection provided by progranulin. We infer that progranulin may assist in removal of protein waste and thereby helps to resolve neuropathic pain after nerve injury.