Role of calcitonin gene-related peptide and brain natriuretic peptide to modulate the excitability state of trigeminal neurons : relevance to migraine pathology and treatment

Sandra Vilotti,1 Elsa Fabbretti,2 Andrea Nistri1 1Neuroscience Department, International School for Advanced Studies (SISSA), Trieste, Italy; 2Center for Biomedical Sciences and Engineering, University of Nova Gorica, Nova Gorica, Slovenia Abstract: Hyperactivity of trigeminal sensory neurons is a major process to generate recurrent headache, typical of migraine attacks. How physiological nociception is converted into strong pathological pain remains, however, poorly understood. In recent years, certain neuropeptides and their receptors have been shown to modulate sensory neuron nociception and to contribute to the persistent hyperalgesia due to the sensory stimulus sensitization that defines the clinical experience of chronic pain syndromes, including migraine. Using calcitonin gene-related peptide (CGRP) and brain natriuretic peptide (BNP) as examples, this review addresses the mechanisms through which neuropeptides might modulate nociceptor activity. One attractive notion is that pain signaling by trigeminal sensory neurons is potently regulated by the ambient levels of these peptides: CGRP is thought to facilitate neuronal firing responsible for trigeminal sensitization necessary to trigger headache, whereas BNP is proposed to act as a negative regulator of trigeminal neuron activity. For either peptide, the key target appears to be the ATP-gated P2X3 receptor that, widely expressed by trigeminal sensory neurons, generates fast, large excitation to release glutamate onto second-order brain neurons. The fine balance between the activities of these peptides is suggested to ultimately determine whether nociception is perceived at higher center as a physiological or pathological response. Hence, the clinical goal of CGRP antagonism using either pharmacological receptor blockers or monoclonal antibodies (to sequester this peptide or to directly inhibit its receptor) is currently considered a novel approach for migraine prophylaxis and to treat acute headache attacks. Keywords: trigeminal ganglion, headache, sensory neurons, P2X3, TRPV

Mechanisms Mediating the Enhanced Gene Transcription of P2X3 Receptor by Calcitonin Gene-related Peptide in Trigeminal Sensory Neurons

The molecular mechanisms underlying migraine pain remain unclear and probably require sustained facilitation in pain-sensing P2X(3) receptors gated by extracellular ATP in nociceptive sensory neurons. The major migraine mediator calcitonin gene-related peptide (CGRP) is known to sensitize P2X(3) receptors to increase impulse flow to brainstem trigeminal nuclei. This process is mediated via changes in the expression and function of P2X(3) receptors initially through enhanced trafficking and, later, perhaps through augmented synthesis of P2X(3) receptors. To clarify the mechanisms responsible for CGRP-evoked long lasting alterations in P2X(3) receptors, we used as a model mouse trigeminal ganglion neurons in culture. CGRP activated Ca(2+)-calmodulin-dependent kinase II, which became localized to the perimembrane region and neuronal processes, a phenomenon already apparent after 30 min and accompanied by a parallel increase in cAMP-response element-binding protein (CREB) phosphorylation and nuclear translocation. These effects triggered increased P2X(3) receptor transcription and were prevented by expressing a dominant negative form of CREB. Increased P2X(3) receptor synthesis was partly mediated by endogenous brain-derived neurotrophic factor (BDNF) because of its block by anti-BDNF antibodies and mimicry by exogenous BDNF. Immunocytochemistry experiments indicated distinct subpopulations of BDNF- or CGRP-sensitive trigeminal neurons with only partial overlap. The present data indicate a novel mechanism for enhancing P2X(3) receptor expression and function in trigeminal sensory neurons by CGRP via CREB phosphorylation. BDNF was an intermediate to extend the sensitizing effect of CGRP also to CGRP-insensitive neurons. This combinatorial action could serve as a powerful process to amplify and prolong pain mediated by P2X(3) receptors

Comparison of P2X and TRPV1 receptors in ganglia or primary culture of trigeminal neurons and their modulation by NGF or serotonin

BACKGROUND: Cultured sensory neurons are a common experimental model to elucidate the molecular mechanisms of pain transduction typically involving activation of ATP-sensitive P2X or capsaicin-sensitive TRPV1 receptors. This applies also to trigeminal ganglion neurons that convey pain inputs from head tissues. Little is, however, known about the plasticity of these receptors on trigeminal neurons in culture, grown without adding the neurotrophin NGF which per se is a powerful algogen. The characteristics of such receptors after short-term culture were compared with those of ganglia. Furthermore, their modulation by chronically-applied serotonin or NGF was investigated. RESULTS: Rat or mouse neurons in culture mainly belonged to small and medium diameter neurons as observed in sections of trigeminal ganglia. Real time RT-PCR, Western blot analysis and immunocytochemistry showed upregulation of P2X(3 )and TRPV1 receptors after 1–4 days in culture (together with their more frequent co-localization), while P2X(2 )ones were unchanged. TRPV1 immunoreactivity was, however, lower in mouse ganglia and cultures. Intracellular Ca(2+ )imaging and whole-cell patch clamping showed functional P2X and TRPV1 receptors. Neurons exhibited a range of responses to the P2X agonist α, β-methylene-adenosine-5'-triphosphate indicating the presence of homomeric P2X(3 )receptors (selectively antagonized by A-317491) and heteromeric P2X(2/3 )receptors. The latter were observed in 16 % mouse neurons only. Despite upregulation of receptors in culture, neurons retained the potential for further enhancement of P2X(3 )receptors by 24 h NGF treatment. At this time point TRPV1 receptors had lost the facilitation observed after acute NGF application. Conversely, chronically-applied serotonin selectively upregulated TRPV1 receptors rather than P2X(3 )receptors. CONCLUSION: Comparing ganglia and cultures offered the advantage of understanding early adaptive changes of nociception-transducing receptors of trigeminal neurons. Culturing did not prevent differential receptor upregulation by algogenic substances like NGF or serotonin, indicating that chronic application led to distinct plastic changes in the molecular mechanisms mediating pain on trigeminal nociceptors

Familial hemiplegic migraine CaV2.1 channel mutation R192Q enhances ATP-gated P2X3 receptor activity of mouse sensory ganglion neurons mediating trigeminal pain

<p>Abstract</p> <p>Background</p> <p>The R192Q mutation of the CACNA1A gene, encoding for the α1 subunit of voltage-gated P/Q Ca²⁺channels (Ca_v2.1), is associated with familial hemiplegic migraine-1. We investigated whether this gain-of-function mutation changed the structure and function of trigeminal neuron P2X₃receptors that are thought to be important contributors to migraine pain.</p> <p>Results</p> <p>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 P2X₃receptor-mediated responses significantly larger than wt neurons. These enhanced effects were reversed by the Ca_v2.1 blocker ω-agatoxin. We, thus, explored intracellular signalling dependent on kinases and phosphatases to understand the molecular regulation of P2X₃receptors 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 P2X₃phenotype. Although no significant difference in membrane expression of knockin receptors was found, serine phosphorylation of knockin P2X₃receptors 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 P2X₃receptor responses of knockin neurons and increased their serine phosphorylation.</p> <p>Conclusions</p> <p>The present results suggest that the CACNA1A mutation conferred a novel molecular phenotype to P2X₃receptors 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.</p

Exocytotic release of ATP from cultured astrocytes.

Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca(2+)](i) also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca(2+)](i) attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca(2+)-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X(3) receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca(2+). Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared

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. \ua9 2015 Marchenkova et al