Resveratrol engages AMPK to attenuate ERK and mTOR signaling in sensory neurons and inhibits incision-induced acute and chronic pain

<p>Abstract</p> <p>Background</p> <p>Despite advances in our understanding of basic mechanisms driving post-surgical pain, treating incision-induced pain remains a major clinical challenge. Moreover, surgery has been implicated as a major cause of chronic pain conditions. Hence, more efficacious treatments are needed to inhibit incision-induced pain and prevent the transition to chronic pain following surgery. We reasoned that activators of AMP-activated protein kinase (AMPK) may represent a novel treatment avenue for the local treatment of incision-induced pain because AMPK activators inhibit ERK and mTOR signaling, two important pathways involved in the sensitization of peripheral nociceptors.</p> <p>Results</p> <p>To test this hypothesis we used a potent and efficacious activator of AMPK, resveratrol. Our results demonstrate that resveratrol profoundly inhibits ERK and mTOR signaling in sensory neurons in a time- and concentration-dependent fashion and that these effects are mediated by AMPK activation and independent of sirtuin activity. Interleukin-6 (IL-6) is thought to play an important role in incision-induced pain and resveratrol potently inhibited IL-6-mediated signaling to ERK in sensory neurons and blocked IL-6-mediated allodynia in vivo through a local mechanism of action. Using a model of incision-induced allodynia in mice, we further demonstrate that local injection of resveratrol around the surgical wound strongly attenuates incision-induced allodynia. Intraplantar IL-6 injection and plantar incision induces persistent nociceptive sensitization to PGE₂injection into the affected paw after the resolution of allodynia to the initial stimulus. We further show that resveratrol treatment at the time of IL-6 injection or plantar incision completely blocks the development of persistent nociceptive sensitization consistent with the blockade of a transition to a chronic pain state by resveratrol treatment.</p> <p>Conclusions</p> <p>These results highlight the importance of signaling to translation control in peripheral sensitization of nociceptors and provide further evidence for activation of AMPK as a novel treatment avenue for acute and chronic pain states.</p

Targeting adenosine monophosphate-activated protein kinase (AMPK) in preclinical models reveals a potential mechanism for the treatment of neuropathic pain

Neuropathic pain is a debilitating clinical condition with few efficacious treatments, warranting development of novel therapeutics. We hypothesized that dysregulated translation regulation pathways may underlie neuropathic pain. Peripheral nerve injury induced reorganization of translation machinery in the peripheral nervous system of rats and mice, including enhanced mTOR and ERK activity, increased phosphorylation of mTOR and ERK downstream targets, augmented eIF4F complex formation and enhanced nascent protein synthesis. The AMP activated protein kinase (AMPK) activators, metformin and A769662, inhibited translation regulation signaling pathways, eIF4F complex formation, nascent protein synthesis in injured nerves and sodium channel-dependent excitability of sensory neurons resulting in a resolution of neuropathic allodynia. Therefore, injury-induced dysregulation of translation control underlies pathology leading to neuropathic pain and reveals AMPK as a novel therapeutic target for the potential treatment of neuropathic pain

The MNK - eIF4E signaling axis contributes to injury-induced nociceptive plasticity and the development of chronic pain

Injury-induced sensitization of nociceptors contributes to pain states and the development of chronic pain. Inhibiting activity-dependent mRNA translation through mechanistic target of rapamycin and mitogen-activated protein kinase (MAPK) pathways blocks the development of nociceptor sensitization. These pathways convergently signal to the eukaryotic translation initiation factor (eIF) 4F complex to regulate the sensitization of nociceptors, but the details of this process are ill defined. Here we investigated the hypothesis that phosphorylation of the 5' cap-binding protein eIF4E by its specific kinase MAPK interacting kinases (MNKs) 1/2 is a key factor in nociceptor sensitization and the development of chronic pain. Phosphorylation of ser209 on eIF4E regulates the translation of a subset of mRNAs. We show that pronociceptive and inflammatory factors, such as nerve growth factor (NGF), interleukin-6 (IL-6), and carrageenan, produce decreased mechanical and thermal hypersensitivity, decreased affective pain behaviors, and strongly reduced hyperalgesic priming in mice lacking eIF4E phosphorylation (eIF4E(S209A)). Tests were done in both sexes, and no sex differences were found. Moreover, in patch-clamp electrophysiology and Ca2+ imaging experiments on dorsal root ganglion neurons, NGF-and IL-6-induced increases in excitability were attenuated in neurons from eIF4ES209A mice. These effects were recapitulated in Mnk1/2(-/-) mice and with the MNK1/2 inhibitor cercosporamide. We also find that cold hypersensitivity induced by peripheral nerve injury is reduced in eIF4ES209A and Mnk1/2 (-/-) mice and following cercosporamide treatment. Our findings demonstrate that the MNK1/2-eIF4E signaling axis is an important contributing factor to mechanisms of nociceptor plasticity and the development of chronic pain.National Institutes of Health [R01-NS-065926, R01-GM-102575, R01-NS-073664]; University of Texas STARS program; postdoctoral Consejo Nacional de Ciencia y Tecnologia fellowship program [274414]6 month embargo; published: 2 August 2017.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Spinal Sensitization Mechanisms Promoting Pain: Gabaergic Disinhibition and Pkmζ-Mediated Plasticity

As a major public health problem affecting more that 76.5 million Americans, chronic pain is one main reason why people seek medical attention. It is a pathological nervous system disorder that persists for months or years. Sensitization of nociceptive neurons in the dorsal horn of the spinal cord is crucial in the development of allodynia and hyperalgesia. The work presented in this thesis will focus on spinal protein kinase M zeta (PKMζ)-mediated plasticity and GABAergic disinhibition as spinal amplification mechanisms that orchestrate persistent changes in the dorsal horn of the spinal cord. As a result of central sensitization following peripheral nerve injruy, GABAergic disinhibition occurs due to an alteration in Cl- homeostasis via reduced KCC2 expression and function. Intrathecal administration of acetazolamide (ACT), a carbonic anhydrase inhibitor, attenuated neuropathic allodynia and spinal co-adminitation of ACT and midazolam (MZL), an allosteric modulator of the benzodiazepine class of GABAA receptors, synergistically inhibited neuropathic allodynia. Further studies concerning the impact of altered Cl-homeostasis via reduced KCC2-mediated Cl-extrusion capacity on the analgesic efficacy and potency of GABAA receptor agonist and allosteric modulators revealed that there is a differential regulation of the agonists and allosteric modulators at the GABAA receptor complex when Cl-homeostasis is altered. Another spinal amplification mechanism leading to central sensitization is PKMζ-mediated spinal LTP. In model of persistent nociceptive sensitization, allodynia induced by IL-6 injection or plantar incision was abolished by both the inhibition of protein translation machinery and PKMζ inhibitor, ZIP. However, only PKMζ inhibition prevented the enhanced pain hypersensitivity precipitated by a subsequent stimulus after the initial hypersensitivity had resolved, asserting that spinal PKMζ underlies the maintenance mechanisms of persistent nociceptive sensitization. Also, these results confirmed that the initiation mechanisms of persistent sensitization parallel LTP initiation mechanisms and the maintenance mechanisms of persistent sensitization parallel LTP maintenance mechanisms. Taken together, these results indicate that these amplification mechanisms drive a chronic persistent state in these models such that inhibition of these spinal amplication mechanisms will serve as an effective approach in the quenching chronic pain hypersensitivity in chronic pain models

eIF4E Phosphorylation Influences Bdnf mRNA Translation in Mouse Dorsal Root Ganglion Neurons

Plasticity in dorsal root ganglion (DRG) neurons that promotes pain requires activity-dependent mRNA translation. Protein synthesis inhibitors block the ability of many pain-promoting molecules to enhance excitability in DRG neurons and attenuate behavioral signs of pain plasticity. In line with this, we have recently shown that phosphorylation of the 5′ cap-binding protein, eIF4E, plays a pivotal role in plasticity of DRG nociceptors in models of hyperalgesic priming. However, mRNA targets of eIF4E phosphorylation have not been elucidated in the DRG. Brain-derived neurotrophic factor (BDNF) signaling from nociceptors in the DRG to spinal dorsal horn neurons is an important mediator of hyperalgesic priming. Regulatory mechanisms that promote pain plasticity via controlling BDNF expression that is involved in promoting pain plasticity have not been identified. We show that phosphorylation of eIF4E is paramount for Bdnf mRNA translation in the DRG. Bdnf mRNA translation is reduced in mice lacking eIF4E phosphorylation (eIF4ES209A) and pro-nociceptive factors fail to increase BDNF protein levels in the DRGs of these mice despite robust upregulation of Bdnf-201 mRNA levels. Importantly, bypassing the DRG by giving intrathecal injection of BDNF in eIF4ES209A mice creates a strong hyperalgesic priming response that is normally absent or reduced in these mice. We conclude that eIF4E phosphorylation-mediated translational control of BDNF expression is a key mechanism for nociceptor plasticity leading to hyperalgesic priming

eIF4E Phosphorylation Influences Bdnf mRNA Translation in Mouse Dorsal Root Ganglion Neurons

Plasticity in dorsal root ganglion (DRG) neurons that promotes pain requires activity-dependent mRNA translation. Protein synthesis inhibitors block the ability of many pain-promoting molecules to enhance excitability in DRG neurons and attenuate behavioral signs of pain plasticity. In line with this, we have recently shown that phosphorylation of the 5' cap-binding protein, eIF4E, plays a pivotal role in plasticity of DRG nociceptors in models of hyperalgesic priming. However, mRNA targets of eIF4E phosphorylation have not been elucidated in the DRG. Brain-derived neurotrophic factor (BDNF) signaling from nociceptors in the DRG to spinal dorsal horn neurons is an important mediator of hyperalgesic priming. Regulatory mechanisms that promote pain plasticity via controlling BDNF expression that is involved in promoting pain plasticity have not been identified. We show that phosphorylation of eIF4E is paramount for Bdnf mRNA translation in the DRG. Bdnf mRNA translation is reduced in mice lacking eIF4E phosphorylation (eIF4E(S209A)) and pro-nociceptive factors fail to increase BDNF protein levels in the DRGs of these mice despite robust upregulation of Bdnf-201 mRNA levels. Importantly, bypassing the DRG by giving intrathecal injection of BDNF in eIF4E(S209A) mice creates a strong hyperalgesic priming response that is normally absent or reduced in these mice. We conclude that eIF4E phosphorylation-mediated translational control of BDNF expression is a key mechanism for nociceptor plasticity leading to hyperalgesic priming.NIH [R01NS065926, R01GM102575, R01NS098826]; University of Texas STARS programOpen access journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

The AMPK Activator A769662 Blocks Voltage-Gated Sodium Channels: Discovery of a Novel Pharmacophore with Potential Utility for Analgesic Development

Voltage-gated sodium channels (VGSC) regulate neuronal excitability by governing action potential (AP) generation and propagation. Recent studies have revealed that AMP-activated protein kinase (AMPK) activators decrease sensory neuron excitability, potentially by preventing sodium (Na+) channel phosphorylation by kinases such as ERK or via modulation of translation regulation pathways. The direct positive allosteric modulator A769662 displays substantially greater efficacy than other AMPK activators in decreasing sensory neuron excitability suggesting additional mechanisms of action. Here, we show that A769662 acutely inhibits AP firing stimulated by ramp current injection in rat trigeminal ganglion (TG) neurons. PT1, a structurally dissimilar AMPK activator that reduces nerve growth factor (NGF) -induced hyperexcitability, has no influence on AP firing in TG neurons upon acute application. In voltage-clamp recordings, application of A769662 reduces VGSC current amplitudes. These findings, based on acute A769662 application, suggest a direct channel blocking effect. Indeed, A769662 dose-dependently blocks VGSC in rat TG neurons and in Na(v)1.7-transfected cells with an IC50 of similar to 10 mu M. A769662 neither displayed use-dependent inhibition nor interacted with the local anesthetic (LA) binding site. Popliteal fossa administration of A769662 decreased noxious thermal responses with a peak effect at 5 mins demonstrating an analgesic effect. These data indicate that in addition to AMPK activation, A769662 acts as a direct blocker/modulator of VGSCs, a potential mechanism enhancing the analgesic property of this compound.NIH [R01GM102575]; University of Texas; Medical Research Service and Rehabilitation Research Service, Dept. of Veterans AffairsOpen Access Journal.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Development and evaluation of small peptidomimetic ligands to protease-activated receptor-2 (PAR2) through the use of lipid tethering.

Protease-activated receptor-2 (PAR2) is a G-Protein Coupled Receptor (GPCR) activated by proteolytic cleavage to expose an attached, tethered ligand (SLIGRL). We evaluated the ability for lipid-tethered-peptidomimetics to activate PAR2 with in vitro physiological and Ca2+ signaling assays to determine minimal components necessary for potent, specific and full PAR2 activation. A known PAR2 activating compound containing a hexadecyl (Hdc) lipid via three polyethylene glycol (PEG) linkers (2at-LIGRL-PEG3-Hdc) provided a potent agonist starting point (physiological EC50 = 1.4 nM; 95% CI: 1.2-2.3 nM). In a set of truncated analogs, 2at-LIGR-PEG3-Hdc retained potency (EC50 = 2.1 nM; 1.3-3.4 nM) with improved selectivity for PAR2 over Mas1 related G-protein coupled receptor type C11, a GPCR that can be activated by the PAR2 peptide agonist, SLIGRL-NH2. 2at-LIG-PEG3-Hdc was the smallest full PAR2 agonist, albeit with a reduced EC50 (46 nM; 20-100 nM). 2at-LI-PEG3-Hdc retained specific activity for PAR2 with reduced EC50 (310 nM; 260-360 nM) but displayed partial PAR2 activation in both physiological and Ca2+ signaling assays. Further truncation (2at-L-PEG3-Hdc and 2at-PEG3-Hdc) eliminated in vitro activity. When used in vivo, full and partial PAR2 in vitro agonists evoked mechanical hypersensitivity at a 15 pmole dose while 2at-L-PEG3-Hdc lacked efficacy. Minimum peptidomimetic PAR2 agonists were developed with known heterocycle substitutes for Ser1 (isoxazole or aminothiazoyl) and cyclohexylalanine (Cha) as a substitute for Leu2. Both heterocycle-tetrapeptide and heterocycle-dipeptides displayed PAR2 specificity, however, only the heterocycle-tetrapeptides displayed full PAR2 agonism. Using the lipid-tethered-peptidomimetic approach we have developed novel structure activity relationships for PAR2 that allows for selective probing of PAR2 function across a broad range of physiological systems

PAR2 antagonist C391

Background and purposeProteinase-activated receptor-2 (PAR2) is a GPCR linked to diverse pathologies, including acute and chronic pain. PAR2 is one of the four PARs that are activated by proteolytic cleavage of the extracellular amino terminus, resulting in an exposed, tethered peptide agonist. Several peptide and peptidomimetic agonists, with high potency and efficacy, have been developed to probe the functions of PAR2, in vitro and in vivo. However, few similarly potent and effective antagonists have been described.Experimental approachWe modified the peptidomimetic PAR2 agonist, 2-furoyl-LIGRLO-NH2 , to create a novel PAR2 peptidomimetic ligand, C391. C391 was evaluated for PAR2 agonist/antagonist activity to PAR2 across Gq signalling pathways using the naturally expressing PAR2 cell line 16HBE14o-. For antagonist studies, a highly potent and specific peptidomimetic agonist (2-aminothiazo-4-yl-LIGRL-NH2 ) and proteinase agonist (trypsin) were used to activate PAR2. C391 was also evaluated in vivo for reduction of thermal hyperalgesia, mediated by mast cell degranulation, in mice.Key resultsC391 is a potent and specific peptidomimetic antagonist, blocking multiple signalling pathways (Gq -dependent Ca2+ , MAPK) induced following peptidomimetic or proteinase activation of human PAR2. In a PAR2-dependent behavioural assay in mice, C391 dose-dependently (75 μg maximum effect) blocked the thermal hyperalgesia, mediated by mast cell degranulation.Conclusions and implicationsC391 is the first low MW antagonist to block both PAR2 Ca2+ and MAPK signalling pathways activated by peptidomimetics and/or proteinase activation. C391 represents a new molecular structure for PAR2 antagonism and can serve as a basis for further development for this important therapeutic target