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Peripheral nerves and Schwann cells have to sustain constant mechanical constraints, caused by developmental growth as well as stretches associated with movements of the limbs and mechanical compressions from daily activities. In Schwann cells, signaling molecules sensitive to stiffness or stretch of the extracellular matrix, such as YAP/TAZ, have been shown to be critical for Schwann cell development and peripheral nerve regeneration. YAP/TAZ have also been suggested to contribute to tumorigenesis, neuropathic pain, and inherited disorders. Yet, the role of mechanosensitive ion channels in myelinating Schwann cells is vastly unexplored. Here we comprehensively assessed the expression of mechanosensitive ion channels in Schwann cells and identified that PIEZO1 and PIEZO2 are among the most abundant mechanosensitive ion channels expressed by Schwann cells. Using classic genetic ablation studies, we show that PIEZO1 is a transient inhibitor of radial and longitudinal myelination in Schwann cells. Contrastingly, we show that PIEZO2 may be required for myelin formation, as the absence of PIEZO2 in Schwann cells delays myelin formation. We found an epistatic relationship between PIEZO1 and PIEZO2, at both the morphological and molecular levels. Finally, we show that PIEZO1 channels affect the regulation of YAP/TAZ activation in Schwann cells. Overall, we present here the first demonstration that PIEZO1 and PIEZO2 contribute to mechanosensation in Schwann cells as well myelin development in the peripheral nervous system.
Learn More >The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.
Learn More >Glucosylsphingosine (GS) is an endogenous sphingolipid that specifically accumulates in the skin of patients with atopic dermatitis (AD). Notably, it was recently found that GS can induce itch sensation by activating serotonin receptor 2A and TRPV4 ion channels. However, it is still uncertain whether other molecules are involved in GS-induced itch sensation. Therefore, by using the calcium imaging technique, we investigated whether serotonin receptor 2 – specifically 2A and 2B – can interact with TRPV1 and TRPA1, because these are representative ion channels in the transmission of itch. As a result, it was found that GS did not activate TRPV1 or TRPA1 per se. Moreover, cells expressing both serotonin receptor 2 and TRPV1 did not show any changes in calcium responses. However, enhanced calcium responses were observed in cells expressing serotonin receptor 2 and TRPA1, suggesting a possible interaction between these two molecules. Similar synergistic effects were also observed in cells expressing serotonin receptor 2 and TRPA1, but not TRPV1. Furthermore, a phospholipase C inhibitor (U73122) and a store-operated calcium entry blocker (SKF96365) significantly reduced GS-induced responses in cells expressing both serotonin receptor 2 and TRPA1, but not with pre-treatment with a Gβγ-complex blocker (gallein). Therefore, we propose a putative novel pathway for GS-induced itch sensation, such that serotonin receptor 2 could be coupled to TRPA1 but not TRPV1 in sensory neurons.
Learn More >TREK2 is a member of the 2-pore domain family of K+ channels (K2P) preferentially expressed by unmyelinated, slow-conducting and non-peptidergic isolectin B4-binding (IB4+) primary sensory neurons of the dorsal root ganglia (DRG). IB4+ neurons depend on the glial-derived neurotrophic factor (GDNF) family of ligands (GFL's) to maintain their phenotype. In our previous work, we demonstrated that 7 days after spinal nerve axotomy (SNA) of the L5 DRG, TREK2 moves away from the cell membrane resulting in a more depolarised resting membrane potential (Em). Given that axotomy deprives DRG neurons from peripherally-derived GFL's, we hypothesized that they might control the expression of TREK2. Using a combination of immunohistochemistry, immunocytochemistry, western blotting, in vivo pharmacological manipulation and behavioral tests we examined the ability of the GFL's (GDNF, neurturin and artemin) and their selective receptors (GFRα1, GFRα2 and GFRα3) to regulate the expression and function of TREK2 in the DRG. We found that TREK2 correlated strongly with the three receptors normally and ipsilaterally for all GFR's after SNA. GDNF, but not NGF, neurturin or artemin up-regulated the expression of TREK2 in cultured DRG neurons. In vivo continuous, subcutaneous administration of GDNF restored the subcellular distribution of TREK2 ipsilaterally and reversed mechanical and cold allodynia 7 days after SNA. This is the first demonstration that GDNF controls the expression of a K2P channel in nociceptors. As TREK2 controls the Em of C-nociceptors affecting their excitability, our finding has therapeutic potential in the treatment of chronic pain.
Learn More >Eicosapentaenoic acid (EPA), an omega-3 (ω-3) polyunsaturated fatty acid, is an essential nutrient that exhibits antiinflammatory, neuroprotective, and cardiovascular-protective activities. Although EPA is used as a nutrient-based pharmaceutical agent or dietary supplement, its molecular target(s) is debatable. Here, we showed that EPA and its metabolites strongly and reversibly inhibit vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and release of adenosine triphosphate (ATP) in purinergic chemical transmission. In vitro analysis showed that EPA inhibits human VNUT-mediated ATP uptake at a half-maximal inhibitory concentration (IC) of 67 nM, acting as an allosteric modulator through competition with Cl. EPA impaired vesicular ATP release from neurons without affecting the vesicular release of other neurotransmitters. In vivo, mice showed a delay in the onset of neuropathic pain and resistance to both neuropathic and inflammatory pain. EPA potently attenuated neuropathic and inflammatory pain in wild-type mice but not in mice without affecting the basal nociception. The analgesic effect of EPA was canceled by the intrathecal injection of purinoceptor agonists and was stronger than that of existing drugs used for neuropathic pain treatment, with few side effects. Neuropathic pain impaired insulin sensitivity in previous studies, which was improved by EPA in the wild-type mice but not in the mice. Our results showed that VNUT is a molecular target of EPA that attenuates neuropathic and inflammatory pain and insulin resistance. EPA may represent a unique nutrient-based treatment and prevention strategy for neurological, immunological, and metabolic diseases by targeting purinergic chemical transmission.
Learn More >Chronic neuropathic pain often leads to cognitive impairment, but the exact mechanism remains unclear. Gamma-aminobutyric acid A receptors (GABARs) are the major inhibitory receptors in the brain, of which the α5-containing GABARs (GABARs-α5) are implicated in a range of neuropsychiatric disorders with cognitive deficits. However, whether GABARs-α5 are involved in chronic neuropathic pain-related cognitive impairment remains unknown. In this study, the rats with chronic neuropathic pain induced by right sciatic nerve ligation injury (SNI) exhibited cognitive impairment with declined spontaneous alternation in Y-maze test and discrimination index in novel object recognition test. The GABARs-α5 expressing on parvalbumin and somatostatin interneurons increased remarkably in hippocampus, resulting in decreased mean frequency of spontaneous inhibitory postsynaptic currents in hippocampal pyramidal neurons. Significantly, antagonizing the GABARs-α5 by L655708 rescued weakened inhibitory synaptic transmission and cognitive impairment induced by chronic neuropathic pain. Taken together, these data suggest that the GABARs-α5 play a crucial role in chronic neuropathic pain-induced cognitive impairment by weakening inhibitory synaptic transmission, which may provide insights into the pharmacologic treatment of chronic neuropathic pain-related cognitive impairment.
Learn More >The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A receptors (ARs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the AR-selective agonist, benzyloxy-cyclopentyladenosine (BnOCPA), is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native ARs arises from BnOCPA's unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of β-arrestin recruitment. BnOCPA thus demonstrates a highly-specific Gα-selective activation of the native AR, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism.
Learn More >Peripheral and central nociceptive sensitization is a critical pathogenetic component in osteoarthritis (OA) chronic pain. T-type calcium channel 3.2 (Ca3.2) regulates neuronal excitability and plays important roles in pain processing. We previously identified that enhanced T-type/Ca3.2 activity in the primary sensory neurons (PSNs) of dorsal root ganglia (DRG) is associated with neuropathic pain behavior in a rat model of monosodium iodoacetate (MIA)-induced knee OA. PSN-specific T-type/Ca3.2 may therefore represent an important mediator in OA painful neuropathy. Here, we test the hypothesis that the T-type/Ca3.2 channels in PSNs can be rationally targeted for pain relief in MIA-OA.
Learn More >Gabapentin and pregabalin are drugs to treat neuropathic pain. Several studies highlighted effects on presynaptic terminals of nociceptors. Via binding to α2δ subunits of voltage-gated calcium channels, gabapentinoids modulate the synaptic transmission of nociceptive information. However, recent studies revealed further properties of these substances. Treatment with gabapentin or pregabalin in animal models of neuropathic pain resulted not only in reduced symptoms of hyperalgesia but also in an attenuated activation of glial cells and decreased production of pro-inflammatory mediators in the spinal dorsal horn.
Learn More >The production of specialized pro-resolving mediators (SPMs) during the resolution phase in the inflammatory milieu is key to orchestrating the resolution of the acute inflammatory response. 17-epi-neuroprotectin D1/17-epi-protectin D1 (17-epi-NPD1/17-epi-PD1: 10R,17R-dihydroxy-4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid) is an SPM of the protectin family, biosynthesized from docosahexaenoic acid (DHA), that exhibits both potent anti-inflammatory and neuroprotective functions. Here, we carried out a new commercial-scale synthesis of 17-epi-NPD1/17-epi-PD1 that enabled the authentication and confirmation of its potent bioactions in vivo and determination of its ability to activate human leukocyte phagocytosis. We provide evidence that this new synthetic 17-epi-NPD1/17-epi-PD1 statistically significantly increases human macrophage uptake of E. coli in vitro and confirm that it limits neutrophilic infiltration in vivo in a murine model of peritonitis. The physical properties of the new synthetic 17-epi-NPD1/17-epi-PD1, namely its ultra-violet absorbance, chromatography, and tandem mass spectrometry fragmentation pattern, matched those of the originally synthesized 17-epi-NPD1/17-epi-PD1. In addition, we verified the structure and complete stereochemical assignment of this new synthetic 17-epi-NPD1/17-epi-PD1 using nuclear magnetic resonance (NMR) spectroscopy. Together, these results authenticate this 17-epi-NPD1/17-epi-PD1 for its structure and potent pro-resolving functions.
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