In situ imaging reveals properties of purinergic signalling in trigeminal sensory ganglia in vitro.

Chronic pain is supported by sterile inflammation that induces sensitisation of sensory neurons to ambient stimuli including extracellular ATP acting on purinergic P2X receptors. The development of in vitro methods for drug screening would be useful to investigate cell crosstalk and plasticity mechanisms occurring during neuronal sensitisation and sterile neuroinflammation. Thus, we studied, at single-cell level, membrane pore dilation based on the uptake of a fluorescent probe following sustained ATP-gated P2X receptor function in neurons and non-neuronal cells of trigeminal ganglion cultures from wild-type (WT) and R192Q CaV2.1 knock-in (KI) mice, a model of familial hemiplegic migraine type 1 characterised by neuronal sensitisation and higher release of soluble mediators. In WT cultures, pore responses were mainly evoked by ATP rather than benzoyl-ATP (BzATP) and partly inhibited by the P2X antagonist TNP-ATP. P2X7 receptors were expressed in trigeminal ganglia mainly by non-neuronal cells. In contrast, KI cultures showed higher expression of P2X7 receptors, stronger responses to BzATP, an effect largely prevented by prior administration of CaV2.1 blocker ω-agatoxin IVA, small interfering RNA (siRNA)-based silencing of P2X7 receptors or the P2X7 antagonist A-804598. No cell toxicity was detected with the protocols. Calcitonin gene-related peptide (CGRP), a well-known migraine mediator, potentiated BzATP-evoked membrane permeability in WT as well as R192Q KI cultures, demonstrating its modulatory role on trigeminal sensory ganglia. Our results show an advantageous experimental approach to dissect pharmacological properties potentially relevant to chronic pain and suggest that CGRP is a soluble mediator influencing purinergic P2X pore dilation and regulating inflammatory responses.

ATP-Gated P2X3 Receptors Are Specialised Sensors of the Extracellular Environment.

P2X3 receptors are ion channels expressed by autonomic and sensory nerves and specialised in transducing extracellular ATP signals. Structural data, together with functional and biochemical studies, suggest that conformational changes of P2X3 receptors upon agonist binding influence downstream intracellular molecular mechanisms relevant for neuronal responses. Activity of P2X3 receptors is implicated in pain, itch, asthma, cardiovascular dysfunction and other pathologies. The study of these receptors has therefore a large potential in the field of drug development and interdisciplinary efforts could clarify molecular mechanisms controlling P2X3 receptor function in different physiological or pathological contexts.

The scaffold protein calcium/calmodulin-dependent serine protein kinase controls ATP release in sensory ganglia upon P2X3 receptor activation and is part of an ATP keeper complex.

P2X3 receptors, gated by extracellular ATP, are expressed by sensory neurons and are involved in peripheral nociception and pain sensitization. The ability of P2X3 receptors to transduce extracellular stimuli into neuronal signals critically depends on the dynamic molecular partnership with the calcium/calmodulin-dependent serine protein kinase (CASK). The present work used trigeminal sensory neurons to study the impact that activation of P2X3 receptors (evoked by the agonist α,ß-meATP) has on the release of endogenous ATP and how CASK modulates this phenomenon. P2X3 receptor function was followed by ATP efflux via Pannexin1 (Panx1) hemichannels, a mechanism that was blocked by the P2X3 receptor antagonist A-317491, and by P2X3 silencing. ATP efflux was enhanced by nerve growth factor, a treatment known to potentiate P2X3 receptor function. Basal ATP efflux was not controlled by CASK, and carbenoxolone or Pannexin silencing reduced ATP release upon P2X3 receptor function. CASK-controlled ATP efflux followed P2X3 receptor activity, but not depolarization-evoked ATP release. Molecular biology experiments showed that CASK was essential for the transactivation of Panx1 upon P2X3 receptor activation. These data suggest that P2X3 receptor function controls a new type of feed-forward purinergic signaling on surrounding cells, with consequences at peripheral and spinal cord level. Thus, P2X3 receptor-mediated ATP efflux may be considered for the future development of pharmacological strategies aimed at containing neuronal sensitization. P2X3 receptors are involved in sensory transduction and associate to CASK. We have studied in primary sensory neurons the molecular mechanisms downstream P2X3 receptor activation, namely ATP release and partnership with CASK or Panx1. Our data suggest that CASK and P2X3 receptors are part of an ATP keeper complex, with important feed-forward consequences at peripheral and central level.

Brain natriuretic peptide constitutively downregulates P2X3 receptors by controlling their phosphorylation state and membrane localization.

BACKGROUND: ATP-gated P2X3 receptors are important transducers of nociceptive stimuli and are almost exclusively expressed by sensory ganglion neurons. In mouse trigeminal ganglion (TG), P2X3 receptor function is unexpectedly enhanced by pharmacological block of natriuretic peptide receptor-A (NPR-A), outlining a potential inhibitory role of endogenous natriuretic peptides in nociception mediated by P2X3 receptors. Lack of change in P2X3 protein expression indicates a complex modulation whose mechanisms for downregulating P2X3 receptor function remain unclear. RESULTS: To clarify this process in mouse TG cultures, we suppressed NPR-A signaling with either siRNA of the endogenous agonist BNP, or the NPR-A blocker anantin. Thus, we investigated changes in P2X3 receptor distribution in the lipid raft membrane compartment, their phosphorylation state, as well as their function with patch clamping. Delayed onset of P2X3 desensitization was one mechanism for the anantin-induced enhancement of P2X3 activity. Anantin application caused preferential P2X3 receptor redistribution to the lipid raft compartment and decreased P2X3 serine phosphorylation, two phenomena that were not interdependent. An inhibitor of cGMP-dependent protein kinase and siRNA-mediated knockdown of BNP mimicked the effect of anantin. CONCLUSIONS: We demonstrated that in mouse trigeminal neurons endogenous BNP acts on NPR-A receptors to determine constitutive depression of P2X3 receptor function. Tonic inhibition of P2X3 receptor activity by BNP/NPR-A/PKG pathways occurs via two distinct mechanisms: P2X3 serine phosphorylation and receptor redistribution to non-raft membrane compartments. This novel mechanism of receptor control might be a target for future studies aiming at decreasing dysregulated P2X3 receptor activity in chronic pain.

Calcium/calmodulin-dependent serine protein kinase (CASK) is a new intracellular modulator of P2X3 receptors.

ATP-gated P2X3 receptors of sensory ganglion neurons are important transducers of painful stimuli and are modulated by extracellular algogenic substances, via changes in the receptor phosphorylation state. The present study investigated the role of calcium/calmodulin-dependent serine protein kinase (CASK) in interacting and controlling P2X3 receptor expression and function in mouse trigeminal ganglia. Most ganglion neurons in situ or in culture co-expressed P2X3 and CASK. CASK was immunoprecipitated with P2X3 receptors from trigeminal ganglia and from P2X3/CASK-cotransfected human embryonic kidney (HEK) cells. Recombinant P2X3/CASK expression in HEK cells increased serine phosphorylation of P2X3 receptors, typically associated with receptor upregulation. CASK deletion mutants also enhanced P2X3 subunit expression. After silencing CASK, cell surface P2X3 receptor expression was decreased, which is consistent with depressed P2X3 currents. The reduction in P2X3 expression levels was reversed by the proteasomal inhibitor MG-132. Moreover, neuronal CASK/P2X3 interaction was up-regulated by nerve growth factor (NGF) signaling and down-regulated by P2X3 agonist-induced desensitization. These data suggest a novel interaction between CASK and P2X3 receptors with positive outcome for receptor stability and function. As CASK-mediated control of P2X3 receptors was dependent on the receptor activation state, CASK represents an intracellular gateway to regulate purinergic nociceptive signaling.

Mutated CaV2.1 channels dysregulate CASK/P2X3 signaling in mouse trigeminal sensory neurons of R192Q Cacna1a knock-in mice.

BACKGROUND: ATP-gated P2X3 receptors of sensory ganglion neurons are important transducers of pain as they adapt their expression and function in response to acute and chronic nociceptive signals. The present study investigated the role of calcium/calmodulin-dependent serine protein kinase (CASK) in controlling P2X3 receptor expression and function in trigeminal ganglia from Cacna1a R192Q-mutated knock-in (KI) mice, a genetic model for familial hemiplegic migraine type-1. RESULTS: KI ganglion neurons showed more abundant CASK/P2X3 receptor complex at membrane level, a result that likely originated from gain-of-function effects of R192Q-mutated CaV2.1 channels and downstream enhanced CaMKII activity. The selective CaV2.1 channel blocker ω-Agatoxin IVA and the CaMKII inhibitor KN-93 were sufficient to return CASK/P2X3 co-expression to WT levels. After CASK silencing, P2X3 receptor expression was decreased in both WT and KI ganglia, supporting the role of CASK in P2X3 receptor stabilization. This process was functionally observed as reduced P2X3 receptor currents. CONCLUSIONS: We propose that, in trigeminal sensory neurons, the CASK/P2X3 complex has a dynamic nature depending on intracellular calcium and related signaling, that are enhanced in a transgenic mouse model of genetic hemiplegic migraine.

Effects of LPS on P2X3 receptors of trigeminal sensory neurons and macrophages from mice expressing the R192Q Cacna1a gene mutation of familial hemiplegic migraine-1.

A knockin (KI) mouse model with the R192Q missense mutation in the Cacna1a gene commonly detected in familial hemiplegic migraine was used to study whether trigeminal ganglia showed a basal inflammatory profile that could be further enhanced by the lipopolysaccharide (LPS) toxin. Adenosine-5'-triphosphate (ATP)-gated purinergic ionotropic receptor 3 (P2X3) currents expressed by the large majority of trigeminal sensory neurons were taken as functional readout. Cultured R192Q KI trigeminal ganglia showed higher number of active macrophages, basal release of tumor necrosis factor alpha (TNFα), and larger P2X3 receptor currents with respect to wild type (WT) cells. After 5 h application of LPS in vitro, both WT and R192Q KI cultures demonstrated significant increase in macrophage activation, very large rise in TNFα mRNA content, and ambient protein levels together with fall in TNFα precursor, suggesting potent release of this inflammatory mediator. Notwithstanding the unchanged expression of P2X3 receptor protein in WT or R192Q KI cultures, LPS evoked a large rise in WT neuronal currents that recovered faster from desensitization. Basal R192Q KI currents were larger than WT ones and could not be further augmented by LPS. These data suggest that KI cultures had a basal neuroinflammatory profile that might facilitate the release of endogenous mediators (including ATP) to activate constitutively hyperfunctional P2X3 receptors and amplify nociceptive signaling by trigeminal sensory neurons.

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 Ca(V)2.1 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 CGRP(8-37) 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.

Functional differences between ATP-gated human and rat P2X3 receptors are caused by critical residues of the intracellular C-terminal domain.

ATP-activated P2X3 receptors of sensory ganglion neurons contribute to pain transduction and are involved in chronic pain signaling. Although highly homologous (97%) in rat and human species, it is unclear whether P2X3 receptors have identical function. Studying human and rat P2X3 receptors expressed in patch-clamped human embryonic kidney (HEK) cells, we investigated the role of non-conserved tyrosine residues in the C-terminal domain (rat tyrosine-393 and human tyrosine-376) as key determinants of receptor function. In comparison with rat P2X3 receptors, human P2X3 receptors were more expressed and produced larger responses with slower desensitization and faster recovery. In general, desensitization was closely related to peak current amplitude for rat and human receptors. Downsizing human receptor expression to the same level of the rat one still yielded larger responses retaining slower desensitization and faster recovery. Mutating phenylalanine-376 into tyrosine in the rat receptor did not change current amplitude; yet, it retarded desensitization onset, demonstrating how this residue was important to functionally link these two receptor states. Conversely, removing tyrosine from position 376 strongly down-regulated human receptor function. The different topology of tyrosine residues in the C-terminal domain has contrasting functional consequences and is sufficient to account for species-specific properties of this pain-transducing channel.

Functional crosstalk in culture between macrophages and trigeminal sensory neurons of a mouse genetic model of migraine.

BACKGROUND: Enhanced activity of trigeminal ganglion neurons is thought to underlie neuronal sensitization facilitating the onset of chronic pain attacks, including migraine. Recurrent headache attacks might establish a chronic neuroinflammatory ganglion profile contributing to the hypersensitive phenotype. Since it is difficult to study this process in vivo, we investigated functional crosstalk between macrophages and sensory neurons in primary cultures from trigeminal sensory ganglia of wild-type (WT) or knock-in (KI) mice expressing the Cacna1a gene mutation (R192Q) found in familial hemiplegic migraine-type 1. After studying the number and morphology of resident macrophages in culture, the consequences of adding host macrophages on macrophage phagocytosis and membrane currents mediated by pain-transducing P2X3 receptors on sensory neurons were examined. RESULTS: KI ganglion cultures constitutively contained a larger number of active macrophages, although no difference in P2X3 receptor expression was found. Co-culturing WT or KI ganglia with host macrophages (active as much as resident cells) strongly stimulated single cell phagocytosis. The same protocol had no effect on P2X3 receptor expression in WT or KI co-cultures, but it largely enhanced WT neuron currents that grew to the high amplitude constitutively seen for KI neurons. No further potentiation of KI neuronal currents was observed. CONCLUSIONS: Trigeminal ganglion cultures from a genetic mouse model of migraine showed basal macrophage activation together with enhanced neuronal currents mediated by P2X3 receptors. This phenotype could be replicated in WT cultures by adding host macrophages, indicating an important functional crosstalk between macrophages and sensory neurons.

P2X3 receptor expression by HEK cells conditions their survival.

On nociceptive neurons, one important mechanism to generate pain signals is the activation of P2X3 receptors, which are membrane proteins gated by extracellular ATP. In this work, we have studied the recovery of recombinant P2X3 receptor expression in human embryonic kidney (HEK) cells. Our data demonstrated that HEK cells were not permissive for stable P2X3 expression, since the significant time-dependent cell loss. In vivo treatment with P2X3 receptor antagonist limited the effect. The expression of a single P2X3 point mutant Y393A, also largely accelerated cell death. We suggest the requirements of a permissive intracellular molecular machinery for appropriate receptor expression. The present report suggests that despite HEK cells are often used as recombinant expression system for the study a variety of receptors function, they represent a limiting permissive environment for P2X3 receptors.

Lipid rafts control P2X3 receptor distribution and function in trigeminal sensory neurons of a transgenic migraine mouse model.

BACKGROUND: A genetic knock-in mouse model expressing the R192Q mutation of the α1-subunit of the Ca(V)2.1 channels frequently found in patients with familial hemiplegic migraine shows functional upregulation of ATP-sensitive P2X3 receptors of trigeminal sensory neurons that transduce nociceptive inputs to the brainstem. In an attempt to understand the basic mechanisms linked to the upregulation of P2X3 receptor activity, we investigated the influence of the lipid domain of these trigeminal sensory neurons on receptor compartmentalization and function. RESULTS: Knock-in neurons were strongly enriched with lipid rafts containing a larger fraction of P2X3 receptors at membrane level. Pretreatment with the Ca(V)2.1 channel blocker ω-agatoxin significantly decreased the lipid raft content of KI membranes. After pharmacologically disrupting the cholesterol component of lipid rafts, P2X3 receptors became confined to non-raft compartments and lost their functional potentiation typically observed in KI neurons with whole-cell patch-clamp recording. Following cholesterol depletion, all P2X3 receptor currents decayed more rapidly and showed delayed recovery indicating that alteration of the lipid raft milieu reduced the effectiveness of P2X3 receptor signalling and changed their desensitization process. Kinetic modeling could reproduce the observed data when slower receptor activation was simulated and entry into desensitization was presumed to be faster. CONCLUSIONS: The more abundant lipid raft compartment of knock-in neurons was enriched in P2X3 receptors that exhibited stronger functional responses. These results suggest that the membrane microenvironment of trigeminal sensory neurons is an important factor in determining sensitization of P2X3 receptors and could contribute to a migraine phenotype by enhancing ATP-mediated responses.

Amyloid-ß Impairs Dendritic Trafficking of Golgi-Like Organelles in the Early Phase Preceding Neurite Atrophy: Rescue by Mirtazapine.

Neurite atrophy with loss of neuronal polarity is a pathological hallmark of Alzheimer's disease (AD) and other neurological disorders. While there is substantial agreement that disruption of intracellular vesicle trafficking is associated with axonal pathology in AD, comparatively less is known regarding its role in dendritic atrophy. This is a significant gap of knowledge because, unlike axons, dendrites are endowed with the complete endomembrane system comprising endoplasmic reticulum (ER), ER-Golgi intermediate compartment (ERGIC), Golgi apparatus, post-Golgi vesicles, and a recycling-degradative route. In this study, using live-imaging of pGOLT-expressing vesicles, indicative of Golgi outposts and satellites, we investigate how amyloid-ß (Aß) oligomers affect the trafficking of Golgi-like organelles in the different dendritic compartments of cultured rat hippocampal neurons. We found that short-term (4 h) treatment with Aß led to a decrease in anterograde trafficking of Golgi vesicles in dendrites of both resting and stimulated (with 50 mM KCl) neurons. We also characterized the ability of mirtazapine, a noradrenergic and specific serotonergic tetracyclic antidepressant (NaSSA), to rescue Golgi dynamics in dendrites. Mirtazapine treatment (10 µM) increased the number and both anterograde and retrograde motility, reducing the percentage of static Golgi vesicles. Finally, mirtazapine reverted the neurite atrophy induced by 24 h treatment with Aß oligomers, suggesting that this drug is able to counteract the effects of Aß by improving the dendritic trafficking of Golgi-related vesicles.

Familial hemiplegic migraine Ca(v)2.1 channel mutation R192Q enhances ATP-gated P2X3 receptor activity of mouse sensory ganglion neurons mediating trigeminal pain.

BACKGROUND: The R192Q mutation of the CACNA1A gene, encoding for the α1 subunit of voltage-gated P/Q Ca2+ channels (Ca(v)2.1), is associated with familial hemiplegic migraine-1. We investigated whether this gain-of-function mutation changed the structure and function of trigeminal neuron P2X3 receptors that are thought to be important contributors to migraine pain. RESULTS: Using in vitro trigeminal sensory neurons of a mouse genetic model knockin for the CACNA1A R192Q mutation, we performed patch clamp recording and intracellular Ca2+ imaging that showed how these knockin ganglion neurons generated P2X3 receptor-mediated responses significantly larger than wt neurons. These enhanced effects were reversed by the Ca(v)2.1 blocker ω-agatoxin. We, thus, explored intracellular signalling dependent on kinases and phosphatases to understand the molecular regulation of P2X3 receptors of knockin neurons. In such cells we observed strong activation of CaMKII reversed by ω-agatoxin treatment. The CaMKII inhibitor KN-93 blocked CaMKII phosphorylation and the hyperesponsive P2X3 phenotype. Although no significant difference in membrane expression of knockin receptors was found, serine phosphorylation of knockin P2X3 receptors was constitutively decreased and restored by KN-93. No change in threonine or tyrosine phosphorylation was detected. Finally, pharmacological inhibitors of the phosphatase calcineurin normalized the enhanced P2X3 receptor responses of knockin neurons and increased their serine phosphorylation. CONCLUSIONS: The present results suggest that the CACNA1A mutation conferred a novel molecular phenotype to P2X3 receptors of trigeminal ganglion neurons via CaMKII-dependent activation of calcineurin that selectively impaired the serine phosphorylation state of such receptors, thus potentiating their effects in transducing trigeminal nociception.

The Cdk5 kinase downregulates ATP-gated ionotropic P2X3 receptor function via serine phosphorylation.

Cdk5 is an endogenous kinase activated by the neuronal-specific protein p35 and implicated in multiple neuronal functions, including modulation of certain pain responses. We investigated whether Cdk5 could regulate ATP-gated P2X(3) receptors that are members of the family of membrane proteins expressed by sensory neurons to transduce nociception in baseline and chronic pain. To study the potential P2X(3) receptor modulation by Cdk5, we co-transfected rat P2X(3) receptors and Cdk5 into HEK cells and observed increased P2X(3) receptor serine phosphorylation together with downregulation of receptor currents only when these genes were transfected together with the gene of the Cdk5 activator p35. The changes in receptor responses were limited to depressed current amplitude as desensitization and recovery were not altered. Transfection of p35 with P2X(3) similarly downregulated receptor responses, suggesting that this phenomenon could be observed even with constitutive Cdk5. The present data indicate a novel target to express the action of Cdk5 on membrane proteins involved in pain perception.

P2X3 receptors are transducers of sensory signals.

Peripheral stimuli are transduced by specific receptors expressed by sensory neurons and are further processed in the dorsal horn of spinal cord before to be transmitted to the brain. While relative few receptor subtypes mediate the initial depolarisation of sensory neurons, an impressive number of molecules and ion channels integrate these inputs into coded signals. Soluble mediators and ambient conditions further shape these processes, potentially triggering peripheral and central sensitisation, or sensory downregulation. Extracellular ATP is a major signaling molecule that acts via purinergic receptors and is a powerful modulator of cell communication as well as a neurotransmitter at peripheral/central synapses. In particular, ATP-mediated signals are transduced by P2X3 receptors expressed mainly by peripheral sensory neurons. Recent evidence suggests that P2X3 receptor function not only induces neuron depolarisation and firing with consequent neurotransmitter release, but it also triggers intracellular molecular changes that amplify purinergic signaling with important consequences.

Neutralization of nerve growth factor induces plasticity of ATP-sensitive P2X3 receptors of nociceptive trigeminal ganglion neurons.

The molecular mechanisms of migraine pain are incompletely understood, although migraine mediators such as NGF and calcitonin gene-related peptide (CGRP) are believed to play an algogenic role. Although NGF block is proposed as a novel analgesic approach, its consequences on nociceptive purinergic P2X receptors of trigeminal ganglion neurons remain unknown. We investigated whether neutralizing NGF might change the function of P2X3 receptors natively coexpressed with NGF receptors on cultured mouse trigeminal neurons. Treatment with an NGF antibody (24 h) decreased P2X3 receptor-mediated currents and Ca2+ transients, an effect opposite to exogenously applied NGF. Recovery from receptor desensitization was delayed by anti-NGF treatment without changing desensitization onset. NGF neutralization was associated with decreased threonine phosphorylation of P2X3 subunits, presumably accounting for their reduced responses and slower recovery. Anti-NGF treatment could also increase the residual current typical of heteromeric P2X2/3 receptors, consistent with enhanced membrane location of P2X2 subunits. This possibility was confirmed with cross-linking and immunoprecipitation studies. NGF neutralization also led to increased P2X2e splicing variant at mRNA and membrane protein levels. These data suggest that NGF controlled plasticity of P2X3 subunits and their membrane assembly with P2X2 subunits. Despite anti-NGF treatment, CGRP could still enhance P2X3 receptor activity, indicating separate NGF- or CGRP-mediated mechanisms to upregulate P2X3 receptors. In an in vivo model of mouse trigeminal pain, anti-NGF pretreatment suppressed responses evoked by P2X3 receptor activation. Our findings outline the important contribution by NGF signaling to nociception of trigeminal sensory neurons, which could be counteracted by anti-NGF pretreatment.

Molecular mechanisms of sensitization of pain-transducing P2X3 receptors by the migraine mediators CGRP and NGF.

Migraine headache originates from the stimulation of nerve terminals of trigeminal ganglion neurons that innervate meninges. Characteristic features of migraine pain are not only its delayed onset but also its persistent duration. Current theories propose that endogenous substances released during a migraine attack (the neuropeptide calcitonin gene-related peptide [CGRP] and the neurotrophin nerve growth factor [NGF]) sensitize trigeminal neurons to transmit nociceptive signals to the brainstem, though the mechanisms remain poorly understood. Recent studies indicate that acute, long-lasting sensitization of trigeminal nociceptive neurons occurs via distinct processes involving enhanced expression and function of adenosine triphosphate (ATP)-gated P2X3 receptors known to play a role in chronic pain. In particular, on cultured trigeminal neurons, CGRP (via protein kinase A-dependent signaling) induces a slowly developing upregulation of the ionic currents mediated by P2X3 receptors by enhancing receptor trafficking to the neuronal membrane and activating their gene transcription. Such upregulated receptors acquire the ability to respond repeatedly to extracellular ATP, thus enabling long-lasting signaling of painful stimuli. In contrast, NGF induces rapid, reversible upregulation of P2X3 receptor function via protein kinase C phosphorylation, an effect counteracted by anti-NGF antibodies. The diverse intracellular signaling pathways used by CGRP and NGF show that the sensitization of P2X3 receptor function persists if the action of only one of these migraine mediators is blocked. These findings imply that inhibiting a migraine attack might be most efficient by a combinatorial approach. The different time domains of P2X3 receptor modulation by NGF and CGRP suggest that the therapeutic efficacy of novel antimigraine drugs depends on the time of administration.

Mast cell activation by myelin through scavenger receptor.

A role for mast cells (MC) in the pathogenesis of multiple sclerosis (MS) has been suggested, based on the analysis of human lesions and on an animal model of the disease (EAE). What role MC play in the development of MS is not well understood. We hypothesized that the link connecting MC with demyelinating diseases may be represented by their interaction with myelin. Here we show that myelin can activate mast cells. This process could be a key event in the mast cell function required for inducing EAE in mice and possibly in MS in man.

Author Correction: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel.

The originally published version of this article contained an error in the name of the author Flóra Gölöncsér, which was incorrectly given as Flóra Göröncsér. This has now been corrected in both the PDF and HTML versions of the article.