Neuron-astrocyte metabolic coupling facilitates spinal plasticity and maintenance of inflammatory pain.

Long-lasting pain stimuli can trigger maladaptive changes in the spinal cord, reminiscent of plasticity associated with memory formation. Metabolic coupling between astrocytes and neurons has been implicated in neuronal plasticity and memory formation in the central nervous system, but neither its involvement in pathological pain nor in spinal plasticity has been tested. Here we report a form of neuroglia signalling involving spinal astrocytic glycogen dynamics triggered by persistent noxious stimulation via upregulation of the Protein Targeting to Glycogen (PTG) in spinal astrocytes. PTG drove glycogen build-up in astrocytes, and blunting glycogen accumulation and turnover by Ptg gene deletion reduced pain-related behaviours and promoted faster recovery by shortening pain maintenance in mice. Furthermore, mechanistic analyses revealed that glycogen dynamics is a critically required process for maintenance of pain by facilitating neuronal plasticity in spinal lamina 1 neurons. In summary, our study describes a previously unappreciated mechanism of astrocyte-neuron metabolic communication through glycogen breakdown in the spinal cord that fuels spinal neuron hyperexcitability.

Cellular and Molecular Mechanisms Underlying Pain Chronicity.

Chronic pain affects a significant amount of the population and is responsible for vast worldwide socio-economic costs [...].

Activation of ß2-Adrenergic Receptors in Microglia Alleviates Neuropathic Hypersensitivity in Mice.

Drugs enhancing the availability of noradrenaline are gaining prominence in the therapy of chronic neuropathic pain. However, underlying mechanisms are not well understood, and research has thus far focused on α2-adrenergic receptors and neuronal excitability. Adrenergic receptors are also expressed on glial cells, but their roles toward antinociception are not well deciphered. This study addresses the contribution of ß2-adrenergic receptors (ß2-ARs) to the therapeutic modulation of neuropathic pain in mice. We report that selective activation of ß2-ARs with Formoterol inhibits pro-inflammatory signaling in microglia ex vivo and nerve injury-induced structural remodeling and functional activation of microglia in vivo. Systemic delivery of Formoterol inhibits behaviors related to neuropathic pain, such as mechanical hypersensitivity, cold allodynia as well as the aversive component of pain, and reverses chronically established neuropathic pain. Using conditional gene targeting for microglia-specific deletion of ß2-ARs, we demonstrate that the anti-allodynic effects of Formoterol are primarily mediated by microglia. Although Formoterol also reduces astrogliosis at late stages of neuropathic pain, these functions are unrelated to ß2-AR signaling in microglia. Our results underline the value of developing microglial ß2-AR agonists for relief from neuropathic pain and clarify mechanistic underpinnings.

Axon Guidance Molecules and Pain.

Chronic pain is a debilitating condition that influences the social, economic, and psychological aspects of patients' lives. Hence, the need for better treatment is drawing extensive interest from the research community. Developmental molecules such as Wnt, ephrins, and semaphorins are acknowledged as central players in the proper growth of a biological system. Their receptors and ligands are expressed in a wide variety in both neurons and glial cells, which are implicated in pain development, maintenance, and resolution. Thereby, it is not surprising that the impairment of those pathways affects the activities and functions of the entire cell. Evidence indicates aberrant activation of their pathways in the nervous system in rodent models of chronic pain. In those conditions, Wnt, ephrin, and semaphorin signaling participate in enhancing neuronal excitability, peripheral sensitization, synaptic plasticity, and the production and release of inflammatory cytokines. This review summarizes the current knowledge on three main developmental pathways and their mechanisms linked with the pathogenesis and progression of pain, considering their impacts on neuronal and glial cells in experimental animal models. Elucidations of the downstream pathways may provide a new mechanism for the involvement of Wnt, ephrin, and semaphorin pathways in pain chronicity.

The Macrophage Iron Signature in Health and Disease.

Throughout life, macrophages are located in every tissue of the body, where their main roles are to phagocytose cellular debris and recycle aging red blood cells. In the tissue niche, they promote homeostasis through trophic, regulatory, and repair functions by responding to internal and external stimuli. This in turn polarizes macrophages into a broad spectrum of functional activation states, also reflected in their iron-regulated gene profile. The fast adaptation to the environment in which they are located helps to maintain tissue homeostasis under physiological conditions.

Locus revealed: Painlessness via loss of NaV1.7 at central terminals of sensory neurons.

How genetic loss of the sodium channel NaV1.7 results in painlessness is puzzling. MacDonald et al. (2021) demonstrate that instead of impairing peripheral excitability, NaV1.7 channels at central terminals of pain-sensing afferents play a pivotal role in the balance between pain and analgesia.

The impact of Semaphorin 4C/Plexin-B2 signaling on fear memory via remodeling of neuronal and synaptic morphology.

Aberrant fear is a cornerstone of several psychiatric disorders. Consequently, there is large interest in elucidation of signaling mechanisms that link extracellular cues to changes in neuronal function and structure in brain pathways that are important in the generation and maintenance of fear memory and its behavioral expression. Members of the Plexin-B family of receptors for class 4 semaphorins play important roles in developmental plasticity of neurons, and their expression persists in some areas of the adult nervous system. Here, we aimed to elucidate the role of Semaphorin 4C (Sema4C) and its cognate receptor, Plexin-B2, in the expression of contextual and cued fear memory, setting a mechanistic focus on structural plasticity and exploration of contributing signaling pathways. We observed that Plexin-B2 and Sema4C are expressed in forebrain areas related to fear memory, such as the anterior cingulate cortex, amygdala and the hippocampus, and their expression is regulated by aversive stimuli that induce fear memory. By generating forebrain-specific Plexin-B2 knockout mice and analyzing fear-related behaviors, we demonstrate that Sema4C-PlexinB2 signaling plays a crucial functional role in the recent and remote recall of fear memory. Detailed neuronal morphological analyses revealed that Sema4C-PlexinB2 signaling largely mediates fear-induced structural plasticity by enhancing dendritic ramifications and modulating synaptic density in the adult hippocampus. Analyses on signaling-related mutant mice showed that these functions are mediated by PlexinB2-dependent RhoA activation. These results deliver important insights into the mechanistic understanding of maladaptive plasticity in fear circuits and have implications for novel therapeutic strategies against fear-related disorders.

Spinal Wnt5a Plays a Key Role in Spinal Dendritic Spine Remodeling in Neuropathic and Inflammatory Pain Models and in the Proalgesic Effects of Peripheral Wnt3a.

Wnt signaling represents a highly versatile signaling system, which plays critical roles in developmental morphogenesis as well as synaptic physiology in adult life and is implicated in a variety of neural disorders. Recently, we demonstrated that Wnt3a is able to recruit multiple noncanonical signaling pathways to alter peripheral sensory neuron function in a nociceptive modality-specific manner. Furthermore, several studies recently reported an important role for Wnt5a acting via canonical and noncanonical signaling in spinal processing of nociception in a number of pathologic pain disorders. Here, using diverse molecular, genetic, and behavioral approaches in mouse models of pain in vivo, we report a novel role for Wnt5a signaling in nociceptive modulation at the structural level. In models of chronic pain, using male and female mice, we found that Wnt5a is released spinally from peripheral sensory neurons, where it recruits the tyrosine kinase receptors Ror2 and Ryk to modulate dendritic spine rearrangement. Blocking the Wnt5a-Ryk/Ror2 axis in spinal dorsal horn neurons prevented activity-dependent dendritic spine remodeling and significantly reduced mechanical hypersensitivity induced by peripheral injury as well as inflammation. Moreover, we observed that peripheral Wnt3a signaling triggers the release of Wnt5a in the spinal cord, and inhibition of spinal Wnt5a signaling attenuates the functional impact of peripheral Wnt3a on nociceptive sensitivity. In conclusion, this study reports a novel role for the Wnt signaling axis in coordinating peripheral and spinal sensitization and shows that targeting Wnt5a-Ryk/ROR2 signaling alleviates both structural and functional mechanisms of nociceptive hypersensitivity in models of chronic pain in vivoSIGNIFICANCE STATEMENT There is a major need to elucidate molecular mechanisms underlying chronic pain disorders to develop novel therapeutic approaches. Wnt signaling represents a highly versatile signaling system, which plays critical roles during development and adult physiology, and it was implicated in several diseases, including chronic pain conditions. Using mouse models, our study identifies a novel role for Wnt5a signaling in nociceptive modulation at the spinal cord level. We observed that Wnt5a recruits Ror2 and Ryk receptors to enhance dendritic spine density, leading to nociceptive sensitization. Blocking the Wnt5a-Ryk/Ror2 interaction in the spinal dorsal horn prevented spine remodeling and significantly reduced inflammatory and neuropathic hypersensitivity. These findings provide proof-of-concept for targeting spinal Wnt signaling for alleviating nociceptive hypersensitivity in vivo.

CXCL10 and CCL21 Promote Migration of Pancreatic Cancer Cells Toward Sensory Neurons and Neural Remodeling in Tumors in Mice, Associated With Pain in Patients.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is frequently accompanied by excruciating pain, which has been associated with attraction of cancer cells and their invasion of intrapancreatic sensory nerves. Neutralization of the chemokine CCL2 reduced cancer-associated pain in a clinical trial, but there have been no systematic analyses of the highly diverse chemokine families and their receptors in PDAC. METHODS: We performed an open, unbiased RNA-interference screen of mammalian chemokines in co-cultures of mouse PDAC cells (K8484) and mouse peripheral sensory neurons, and confirmed findings in studies of DT8082 PDAC cells. We studied the effects of chemokines on migration of PDAC cell lines. Orthotopic tumors were grown from K8484 cells in mice, and mice were given injections of neutralizing antibodies against chemokines, antagonists, or control antibodies. We analyzed abdominal mechanical hypersensitivity and collected tumors and analyzed them by histology and immunohistochemistry to assess neural remodeling. We collected PDAC samples and information on pain levels from 74 patients undergoing resection and measured levels of CXCR3 and CCR7 by immunohistochemistry and immunoblotting. RESULTS: Knockdown of 9 chemokines in DRG neurons significantly reduced migration of PDAC cells towards sensory neurons. Sensory neuron-derived CCL21 and CXCL10 promoted migration of PDAC cells via their receptors CCR7 and CXCR3, respectively, which were expressed by cells in orthotopic tumors and PDAC specimens from patients. Neutralization of CCL21 or CXCL10, or their receptors, in mice with orthotopic tumors significantly reduced nociceptive hypersensitivity and nerve fiber hypertrophy and improved behavioral parameters without affecting tumor infiltration by T cells or neutrophils. Increased levels of CXCR3 and CCR7 in human PDAC specimens were associated with increased frequency of cancer-associated pain, determined from patient questionnaires. CONCLUSIONS: In an unbiased screen of chemokines, we identified CCL21 and CXCL10 as proteins that promote migration of pancreatic cancer cells toward sensory neurons. Inhibition of these chemokines or their receptors reduce hypersensitivity in mice with orthotopic tumors, and patients with PDACs with high levels of the chemokine receptors of CXCR3 and CCR7 had increased frequency of cancer-associated pain.

Semaphorin 4C Plexin-B2 signaling in peripheral sensory neurons is pronociceptive in a model of inflammatory pain.

Semaphorins and their transmembrane receptors, Plexins, are key regulators of axon guidance and development of neuronal connectivity. B-type Plexins respond to Class IV semaphorins and mediate a variety of developmental functions. Here we report that the expression of Plexin-B2 and its high-affinity ligand, Sema4C, persists in peripheral sensory neurons in adult life and is markedly increased in states of persistent pain in mice. Genetic deletion of Sema4C as well as adult-onset loss of Plexin-B2 leads to impairment of the development and duration of inflammatory hypersensitivity. Remarkably, unlike the neurodevelopmental functions of Plexin-B2 that solely rely on Ras signaling, we obtained genetic and pharmacological evidence for a requirement of RhoA-ROCK-dependent mechanisms as well as TRPA1 sensitization in pronociceptive functions of Sema4C-Plexin-B2 signaling in adult life. These results suggest important roles for Plexin-B2 signaling in sensory function that may be of therapeutic relevance in pathological pain.Semaphorins and their receptors are involved in neurodevelopment, but their functions in the adult nervous system are not fully understood. This study finds that semaphorin 4C and its receptor Plexin B are expressed in sensory neurons and are pronociceptive in a mouse model of inflammatory pain.

The serine protease inhibitor SerpinA3N attenuates neuropathic pain by inhibiting T cell-derived leukocyte elastase.

Neuropathic pain is a major, intractable clinical problem and its pathophysiology is not well understood. Although recent gene expression profiling studies have enabled the identification of novel targets for pain therapy, classical study designs provide unclear results owing to the differential expression of hundreds of genes across sham and nerve-injured groups, which can be difficult to validate, particularly with respect to the specificity of pain modulation. To circumvent this, we used two outbred lines of rats, which are genetically similar except for being genetically segregated as a result of selective breeding for differences in neuropathic pain hypersensitivity. SerpinA3N, a serine protease inhibitor, was upregulated in the dorsal root ganglia (DRG) after nerve injury, which was further validated for its mouse homolog. Mice lacking SerpinA3N developed more neuropathic mechanical allodynia than wild-type (WT) mice, and exogenous delivery of SerpinA3N attenuated mechanical allodynia in WT mice. T lymphocytes infiltrate the DRG after nerve injury and release leukocyte elastase (LE), which was inhibited by SerpinA3N derived from DRG neurons. Genetic loss of LE or exogenous application of a LE inhibitor (Sivelastat) in WT mice attenuated neuropathic mechanical allodynia. Overall, we reveal a novel and clinically relevant role for a member of the serpin superfamily and a leukocyte elastase and crosstalk between neurons and T cells in the modulation of neuropathic pain.

Wnt-Fzd signaling sensitizes peripheral sensory neurons via distinct noncanonical pathways.

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 ß-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.

Neuronal calcium signaling in chronic pain.

Acute physiological pain, the unpleasant sensory response to a noxious stimulus, is essential for animals and humans to avoid potential injury. Pathological pain that persists after the original insult or injury has subsided, however, not only results in individual suffering but also imposes a significant cost on society. Improving treatments for long-lasting pathological pain requires a comprehensive understanding of the biological mechanisms underlying pain perception and the development of pain chronicity. In this review, we aim to highlight some of the major findings related to the involvement of neuronal calcium signaling in the processes that mediate chronic pain.

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.

Nuclear calcium signaling in spinal neurons drives a genomic program required for persistent inflammatory pain.

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 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.

Cystic fibrosis transmembrane conductance regulator modulates synaptic chloride homeostasis in motoneurons of the rat spinal cord during neonatal development.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated Cl(-) channel functional in neonatal rat spinal motoneurons. The present study investigated the developmental (P1-P8) expression of CFTR, its impact on motoneuron excitability and Cl(-) homeostasis in relation to canonical Cl(-) transporters. The Cl(-) outward transporter KCC2 gene was upregulated in females over males and increased from P1 to P8. The gene activities of the Cl(-) inward transporter NKCC1 and CFTR were positively correlated and grew between P1 and P8. P1 motoneuronal somata were immunopositive for CFTR whose expression later (P8) extended to cell processes. KCC2 immunopositivity outlined somata and cell processes at P1 and P8. Electrophysiological recording with sharp electrodes showed that the CFTR blocker glibenclamide increased motoneuron input resistance, suggesting functional CFTR in P1-P8 motoneurons. Whole cell patch-clamping of spinal motoneurons to study CFTR contribution to postnatal synaptic Cl(-) regulation indicated that glibenclamide or the selective CFTR blocker diphenylamine-2,2'-dicarboxylic acid produced a negative shift in GABA/glycine reversal potential (E(GABA/Gly) ) of spontaneously occurring synaptic events measured after block of excitatory transmission. A similar effect on E(GABA/Gly) was induced by the NKCC1 inhibitor bumetanide. A 3D reconstructed motoneuron model suggested that CFTR activity contributes to set the E(GABA/Gly) positive to the resting potential. The functional outcome of these Cl(-) mediated synaptic events depended not only on the postnatal age of the animal but also on their timing with respect to the excitatory synaptic signals. We propose that CFTR operated together with NKCC1 to produce depolarizing GABA/glycine mediated synaptic events.

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.

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.