Giulia Magni

Calcitonin Gene-Related Peptide-Mediated Enhancement of Purinergic Neuron/Glia Communication by the Algogenic Factor Bradykinin in Mouse Trigeminal Ganglia from Wild-Type and R192Q Cav2.1 Knock-In Mice: Implications for Basic Mechanisms of Migraine Pain

Within the trigeminal ganglion, crosstalk between neurons and satellite glial cells (SGCs) contributes to neuronal sensitization and transduction of painful stimuli, including migraine pain, at least partly through activation of purinergic receptor mechanisms. We previously showed that the algogenic mediator bradykinin (BK) potentiates purinergic P2Y receptors on SGCs in primary trigeminal cultures. Our present study investigated the molecular basis of this effect in wild-type (WT) mice and CaV2.1 α1 R192Q mutant knock-in (KI) mice expressing a human mutation causing familial hemiplegic migraine type 1. Single-cell calcium imaging of WT cultures revealed functional BK receptors in neurons only, suggesting a paracrine action by BK to release a soluble mediator responsible for its effects on SGCs. We identified this mediator as the neuropeptide calcitonin gene-related peptide (CGRP), whose levels were markedly increased by BK, while the CGRP antagonist CGRP8-37 and the anti-migraine drug sumatriptan inhibited BK actions. Unlike CGRP, BK was ineffective in neuron-free SGC cultures, confirming the CGRP neuronal source. P2Y receptor potentiation induced by CGRP in SGCs was mediated via activation of the extracellular signal-regulated kinase 1/2 pathways, and after exposure to CGRP, a significant release of several cytokines was detected. Interestingly, both basal and BK-stimulated CGRP release was higher in KI mouse cultures, where BK significantly upregulated the number of SGCs showing functional UTP-sensitive P2Y receptors. Our findings suggest that P2Y receptors on glial cells might be considered as novel players in the cellular processes underlying migraine pathophysiology and might represent new targets for the development of innovative therapeutic agents against migraine pain.

Temporomandibular joint inflammation activates glial and immune cells in both the trigeminal ganglia and in the spinal trigeminal nucleus

BackgroundGlial cells have been shown to directly participate to the genesis and maintenance of chronic pain in both the sensory ganglia and the central nervous system (CNS). Indeed, glial cell activation has been reported in both the dorsal root ganglia and the spinal cord following injury or inflammation of the sciatic nerve, but no data are currently available in animal models of trigeminal sensitization. Therefore, in the present study, we evaluated glial cell activation in the trigeminal-spinal system following injection of the Complete Freunds Adjuvant (CFA) into the temporomandibular joint, which generates inflammatory pain and trigeminal hypersensitivity.ResultsCFA-injected animals showed ipsilateral mechanical allodynia and temporomandibular joint edema, accompanied in the trigeminal ganglion by a strong increase in the number of GFAP-positive satellite glial cells encircling neurons and by the activation of resident macrophages. Seventy-two hours after CFA injection, activated microglial cells were observed in the ipsilateral trigeminal subnucleus caudalis and in the cervical dorsal horn, with a significant up-regulation of Iba1 immunoreactivity, but no signs of reactive astrogliosis were detected in the same areas. Since the purinergic system has been implicated in the activation of microglial cells during neuropathic pain, we have also evaluated the expression of the microglial-specific P2Y12 receptor subtype. No upregulation of this receptor was detected following induction of TMJ inflammation, suggesting that any possible role of P2Y12 in this paradigm of inflammatory pain does not involve changes in receptor expression.ConclusionsOur data indicate that specific glial cell populations become activated in both the trigeminal ganglia and the CNS following induction of temporomandibular joint inflammation, and suggest that they might represent innovative targets for controlling pain during trigeminal nerve sensitization.

Expression of the new P2Y-like receptor GPR17 during oligodendrocyte precursor cell maturation regulates sensitivity to ATP-induced death.

The P2Y‐like receptor GPR17 is expressed by adult neural progenitor cells, suggesting a role in lineage determination. Here, we characterized GPR17 expression and function in mouse cortical primary astrocytes/precursor cell cultures. GPR17 is expressed by a subpopulation of oligodendrocyte precursor cells (OPCs), but not by astrocytes. This expression pattern was also confirmed in vivo. In vitro, GPR17 expression was markedly influenced by culturing conditions. In the presence of growth factors (GFs), no significant GPR17 expression was found. When cultures were shifted to a differentiating medium, a dramatic, time‐dependent increase in the number of highly branched GPR17‐positive cells was observed. Under these conditions, GPR17 was induced in the totality of O4‐positive immature oligodendrocytes. Instead, in cultures originally grown in the absence of GFs, GPR17 was already expressed in morphologically more mature OPCs. Shifting of these cultures to differentiating conditions induced GPR17 only in a subpopulation of O4‐positive cells. Under both culture protocols, appearance of more mature CNPase‐ and MBP‐positive cells was associated to a progressive loss of GPR17. GPR17 expression also sensitized cells to adenine nucleotide‐induced cytotoxicity, whereas activation with uracil nucleotides promoted differentiation towards a more mature phenotype. We suggest that GFs may keep OPCs in a less differentiated stage by restraining GPR17 expression, and that, under permissive conditions, GPR17 contributes to OPCs differentiation. However, upon high extracellular adenine nucleotide concentrations, as during trauma and ischemia, GPR17 sensitizes cells to cytotoxicity. This double‐edged sword role may be exploited to unveil new therapeutic approaches to acute and chronic brain disorders.

Oxygen-glucose deprivation increases the enzymatic activity and the microvesicle-mediated release of ectonucleotidases in the cells composing the blood-brain barrier

The blood-brain barrier (BBB), the dynamic interface between the nervous tissue and the blood, is composed by endothelial cells, pericytes and astrocytes. Extracellular nucleotides and nucleosides and their receptors (the purinergic system) constitute a widely diffused signaling system involved in many pathophysiological processes. However, the role of this system in controlling BBB functions is still largely unknown. By using cultures of these three cell types grown separately and a BBB in vitro model consisting of triple co-cultures, we studied for the first time the expression and distribution of the ecto-enzymes nucleoside triphosphate diphosphohydrolases (NTPDases, the enzymes which hydrolyze extracellular nucleotides) under control and ischemic (oxygen-glucose deprivation in vitro; OGD) conditions. NTPDase1 was detected in all three cell types, whereas NTPDase2 was expressed by astrocytes and pericytes and, to a lesser extent, by endothelial cells. Endothelial cells were extremely susceptible to cell death when OGD was applied to mimic in vitro the cytotoxicity induced by ischemia, whereas astrocytes and pericytes were more resistant. A semi-quantitative assay highlighted markedly increased e-ATPase activity following exposure to OGD in all three cell types, either when grown separately or when co-cultured together to resemble the composition of the BBB. Moreover, electron microscopy analysis showed that both endothelial cells and astrocytes shed microvesicles containing NTPDases from their membrane, which may suggest a novel mechanism to increase the breakdown of ATP released to toxic levels by damaged BBB cells. We hypothesize that this phenomenon could have a protective and/or modulatory effect for brain parenchymal cells. This in vitro model is therefore useful to study the role of extracellular nucleotides in modulating BBB responses to ischemic events, and to develop new effective purinergic-based approaches for brain ischemia.

P2Y2 receptor antagonists as anti-allodynic agents in acute and sub-chronic trigeminal sensitization: Role of satellite glial cells

Trigeminal (TG) pain often lacks a satisfactory pharmacological control. A better understanding of the molecular cross‐talk between TG neurons and surrounding satellite glial cells (SGCs) could help identifying innovative targets for the development of more effective analgesics. We have previously demonstrated that neuronal pro‐algogenic mediators upregulate G protein‐coupled nucleotide P2Y receptors (P2YRs) expressed by TG SGCs in vitro. Here, we have identified the specific P2YR subtypes involved (i.e., the ADP‐sensitive P2Y1R and the UTP‐responsive P2Y2R subtypes), and demonstrated the contribution of neuron‐derived prostaglandins to their upregulation. Next, we have translated these data to an in vivo model of TG pain (namely, rats injected with Complete Freunds adjuvant in the temporomandibular joint), by demonstrating activation of SGCs and upregulation of P2Y1R and P2Y2R in the ipsi‐lateral TG. To unequivocally link P2YRs to the development of facial allodynia, we treated animals with various purinergic antagonists. The selective P2Y2R antagonist AR‐C118925 completely inhibited SGCs activation, exerted a potent anti‐allodynic effect that lasted over time, and was still effective when administration was started 6‐days post induction of allodynia, i.e. under subchronic pain conditions. Conversely, the selective P2Y1R antagonist MRS2179 was completely ineffective. Moreover, similarly to the anti‐inflammatory drug acetylsalicylic acid and the known anti‐migraine agent sumatriptan, the P2X/P2Y nonselective antagonist PPADS was only partially effective, and completely lost its activity under sub‐chronic conditions. Taken together, our results highlight glial P2Y2Rs as potential “druggable” targets for the successful management of TG‐related pain. GLIA 2015;63:1256–1269

The purinergic system and glial cells: emerging costars in nociception.

It is now well established that glial cells not only provide mechanical and trophic support to neurons but can directly contribute to neurotransmission, for example, by release and uptake of neurotransmitters and by secreting pro- and anti-inflammatory mediators. This has greatly changed our attitude towards acute and chronic disorders, paving the way for new therapeutic approaches targeting activated glial cells to indirectly modulate and/or restore neuronal functions. A deeper understanding of the molecular mechanisms and signaling pathways involved in neuron-to-glia and glia-to-glia communication that can be pharmacologically targeted is therefore a mandatory step toward the success of this new healing strategy. This holds true also in the field of pain transmission, where the key involvement of astrocytes and microglia in the central nervous system and satellite glial cells in peripheral ganglia has been clearly demonstrated, and literally hundreds of signaling molecules have been identified. Here, we shall focus on one emerging signaling system involved in the cross talk between neurons and glial cells, the purinergic system, consisting of extracellular nucleotides and nucleosides and their membrane receptors. Specifically, we shall summarize existing evidence of novel “druggable” glial purinergic targets, which could help in the development of innovative analgesic approaches to chronic pain states.

Lipoic-Based TRPA1/TRPV1 antagonist to treat orofacial pain

Inflammation of the trigeminal nerve is considered one of the most painful conditions known to humankind. The diagnosis is often difficult; moreover, safe and effective pharmacological treatments are lacking. A new molecule, ADM_12, formed by a lipoic and omotaurine residues covalently linked, is here reported. In vitro and in vivo tests showed that ADM_12 is a very attractive original compound presenting (i) a remarkable safety profile; (ii) a high binding constant versus TRPA1; (iii) an intriguing behavior versus TRPV1; and (iv) the ability to significantly and persistently reduce mechanical facial allodynia in rats. Noteworthy, by testing ADM_12, we shed light on the unprecedented involvement of TRPA1 and TRPV1 channels in orofacial pain.

Tackling chronic pain and inflammation through the purinergic system

The purinergic system is composed of purine and pyrimidine transmitters, the enzymes that modulate the interconversion of nucleotides and nucleosides, the membrane transporters that control their extracellular concentrations, and the many receptor subtypes that are responsible for their cellular responses. The components of this system are ubiquitously localized in all tissues and organs, and their involvement in several physiological conditions has been clearly demonstrated. Moreover, extracellular purine and pyrimidine concentrations rise several folds under pathological conditions like tissue damage, ischemia, and inflammation, which suggest that this signaling system might contribute both to disease outcome and, possibly, to its tentative resolution. The complexity of this system has greatly impaired the clear identification of the mediators and receptors that are actually involved in a given pathology, also due to the often opposite roles played by the various receptor subtypes. Nevertheless, this knowledge is fundamental for the possible exploitation of these molecular entities as targets for the development of new pharmacological approaches. In this review, we aim at highlighting what is currently known on the role of the purinergic system in various pain conditions and during inflammatory processes. Although some confusion may arise from conflicting results, literature data clearly show that targeting specific purinergic receptors may represent an innovative approach to various pain and inflammatory conditions, and that new purine-based drugs are now very close to reach the market with these indications.

Purinergic neuron/glia communication in trigeminal ganglia : interactions with other algogens and implications for migraine pain

Fields D. NICHD Porter Neuroscience Research Center, Bethesda, 20892-3713 Maryland, USA Release of neurotransmitters outside synapses has broad biological implications, particularly with regard communication between axons and glia. Previous research has shown that ATP and adenosine released from axons firing action potentials can regulate myelination by Schwann cells and oligodendrocytes. Regulation of myelination by impulse activity could contribute to nervous system development and plasticity, and nonvesicular, nonsynaptic release of ATP could also participate in signaling between axons and other glial cells, neurons, and vasculature. We have identified a mechanism for ATP release from axons through volume-activated anion channels (VAAC) that are activated by microscopic axon swelling during action potential firing. A combination of single-photon imaging of ATP release, together with imaging for intrinsic optical signals, intracellular Ca, time-lapse video and confocal microscopy, is used to investigate action potential-induced nonsynaptic release of this neurotransmitter. ATP release from premyelinated DRG axons persists when bafilomycin or botulinum toxin are used to block vesicular release, whereas pharmacological inhibition of VAACs or preventing action potential-induced axon swelling inhibited ATP release and blocked activity-dependent signaling between axons and astrocytes. This nonvesicular, nonsynaptic communication could mediate diverse activity-dependent interactions between axons and nervous system cells in normal conditions, development, and disease.