No abstract

No abstract

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.

BackgroundThe R192Q mutation of the CACNA1A gene, encoding for the α1 subunit of voltage-gated P/Q Ca2+ channels (Cav2.1), 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.ResultsUsing 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 Cav2.1 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.ConclusionsThe 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.

Summary Persistent pain induced by noxious stimuli is characterized by the transition from normosensitivity to hypersensitivity. Underlying mechanisms are not well understood, although gene expression is considered important. Here we show that persistent nociceptive-like activity triggers calcium transients in neuronal nuclei within the superficial spinal dorsal horn, and that nuclear calcium is necessary for the development of long-term inflammatory hypersensitivity. Using a nucleus-specific calcium signal perturbation strategy in vivo complemented by gene profiling, bioinformatics and functional analyses, we discovered a pain-associated, nuclear calcium-regulated gene program in spinal excitatory neurons. This includes C1q, a novel modulator of synaptic spine morphogenesis, which we found to contribute to activity-dependent spine remodelling on spinal neurons in a manner functionally associated with inflammatory hypersensitivity. Thus, nuclear calcium integrates synapse-to-nucleus communication following noxious stimulation and controls a spinal genomic response that mediates the transition between acute and long-term nociceptive sensitization by modulating functional and structural plasticity.

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 P2X 3 receptors natively coexpressed with NGF receptors on cultured mouse trigeminal neurons. Treatment with an NGF antibody (24 h) decreased P2X 3 receptor-mediated currents and Ca 2ϩ 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 P2X 3 subunits, presumably accounting for their reduced responses and slower recovery. Anti-NGF treatment could also increase the residual current typical of heteromeric P2X 2/3 receptors, consistent with enhanced membrane location of P2X 2 subunits. This possibility was confirmed with cross-linking and immunoprecipitation studies. NGF neutralization also led to increased P2X 2e splicing variant at mRNA and membrane protein levels. These data suggest that NGF controlled plasticity of P2X 3 subunits and their membrane assembly with P2X 2 subunits. Despite anti-NGF treatment, CGRP could still enhance P2X 3 receptor activity, indicating separate NGF-or CGRP-mediated mechanisms to upregulate P2X 3 receptors. In an in vivo model of mouse trigeminal pain, anti-NGF pretreatment suppressed responses evoked by P2X 3 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.

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 generelated 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.Sensitization of sensory neurons represents the key phenomenon for generating chronic pain that remains relatively refractory to standard treatment (1). Current theories predict that chronic pain is brought about by a gain in the expression and function of sensory nociceptors that enhance their signal flow to the brain to ensure the long lasting nature of this process (2). Clarification of the novel gene expression profiles associated to pain should, therefore, be useful to target emerging molecules (and mechanisms) and for designing new analgesics. Previous studies (3) aimed at detecting changes in transcriptome patterns during pain conditions have provided only a limited description of the global alterations because of the complexity of pain pathologies and signal pathways.Migraine is a classical example of chronic, relapsing pain triggered by local release of endogenous pain mediators, such as the neuropeptide calcitonin gene-related peptide (CGRP) 2 (4, 5) that induces long lasting trigeminal neuronal sensitization (6) and novel gene transcription in sensory neurons (7-9). CGRPmediated signals are transduced at the cell membrane by activation of a G-protein-coupled receptor complex (composed b...

Wnt signaling represents a highly versatile signaling system, which plays diverse and critical roles in various aspects of neural development. Sensory neurons of the dorsal root ganglia require Wnt signaling for initial cell-fate determination as well as patterning and synapse formation. Here we report that Wnt signaling pathways persist in adult sensory neurons and play a functional role in their sensitization in a pathophysiological context. We observed that Wnt3a recruits the Wnt-calcium signaling pathway and the Wnt planar cell polarity pathway in peripheral nerves to alter pain sensitivity in a modality-specific manner and we elucidated underlying mechanisms. In contrast, biochemical, pharmacological, and genetic studies revealed lack of functional relevance for the classical canonical β-catenin pathway in peripheral sensory neurons in acute modulation of nociception. Finally, this study provides proof-of-concept for a translational potential for Wnt3a-Frizzled3 signaling in alleviating disease-related pain hypersensitivity in cancer-associated pain in vivo.