Robert W. Gereau

MAP kinase and pain

Mitogen-activated protein kinases (MAPKs) are important for intracellular signal transduction and play critical roles in regulating neural plasticity and inflammatory responses. The MAPK family consists of three major members: extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK), which represent three separate signaling pathways. Accumulating evidence shows that all three MAPK pathways contribute to pain sensitization after tissue and nerve injury via distinct molecular and cellular mechanisms. Activation (phosphorylation) of MAPKs under different persistent pain conditions results in the induction and maintenance of pain hypersensitivity via non-transcriptional and transcriptional regulation. In particular, ERK activation in spinal cord dorsal horn neurons by nociceptive activity, via multiple neurotransmitter receptors, and using different second messenger pathways plays a critical role in central sensitization by regulating the activity of glutamate receptors and potassium channels and inducing gene transcription. ERK activation in amygdala neurons is also required for inflammatory pain sensitization. After nerve injury, ERK, p38, and JNK are differentially activated in spinal glial cells (microglia vs astrocytes), leading to the synthesis of proinflammatory/pronociceptive mediators, thereby enhancing and prolonging pain. Inhibition of all three MAPK pathways has been shown to attenuate inflammatory and neuropathic pain in different animal models. Development of specific inhibitors for MAPK pathways to target neurons and glial cells may lead to new therapies for pain management. Although it is well documented that MAPK pathways can increase pain sensitivity via peripheral mechanisms, this review will focus on central mechanisms of MAPKs, especially ERK.

cAMP-Dependent Protein Kinase Regulates Desensitization of the Capsaicin Receptor (VR1) by Direct Phosphorylation

The capsaicin receptor, VR1 (also known as TRPV1), is a ligand-gated ion channel expressed on nociceptive sensory neurons that responds to noxious thermal and chemical stimuli. Capsaicin responses in sensory neurons exhibit robust potentiation by cAMP-dependent protein kinase (PKA). In this study, we demonstrate that PKA reduces VR1 desensitization and directly phosphorylates VR1. In vitro phosphorylation, phosphopeptide mapping, and protein sequencing of VR1 cytoplasmic domains delineate several candidate PKA phosphorylation sites. Electrophysiological analysis of phosphorylation site mutants clearly pinpoints Ser116 as the residue responsible for PKA-dependent modulation of VR1. Given the significant roles of VR1 and PKA in inflammatory pain hypersensitivity, VR1 phosphorylation at Ser116 by PKA may represent an important molecular mechanism involved in the regulation of VR1 function after tissue injury.

Protein kinase C phosphorylation sensitizes but does not activate the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1)

Protein kinase C (PKC) modulates the function of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). This modulation manifests as increased current when the channel is activated by capsaicin. In addition, studies have suggested that phosphorylation by PKC might directly gate the channel, because PKC-activating phorbol esters induce TRPV1 currents in the absence of applied ligands. To test whether PKC both modulates and gates the TRPV1 function by direct phosphorylation, we used direct sequencing to determine the major sites of PKC phosphorylation on TRPV1 intracellular domains. We then tested the ability of the PKC-activating phorbol 12-myristate 13-acetate (PMA) to potentiate capsaicin-induced currents and to directly gate TRPV1. We found that mutation of S800 to alanine significantly reduced the PMA-induced enhancement of capsaicin-evoked currents and the direct activation of TRPV1 by PMA. Mutation of S502 to alanine reduced PMA enhancement of capsaicin-evoked currents, but had no effect on direct activation of TRPV1 by PMA. Conversely, mutation of T704 to alanine had no effect on PMA enhancement of capsaicin-evoked currents but dramatically reduced direct activation of TRPV1 by PMA. These results, combined with pharmacological studies showing that inactive phorbol esters also weakly activate TRPV1, suggest that PKC-mediated phosphorylation modulates TRPV1 but does not directly gate the channel. Rather, currents induced by phorbol esters result from the combination of a weak direct ligand-like activation of TRPV1 and the phosphorylation-induced enhancement of the TRPV1 function. Furthermore, modulation of the TRPV1 function by PKC appears to involve distinct phosphorylation sites depending on the mechanism of channel activation.

Acute p38-Mediated Modulation of Tetrodotoxin-Resistant Sodium Channels in Mouse Sensory Neurons by Tumor Necrosis Factor-α

Tumor necrosis factor-α (TNFα) is a proinflammatory cytokine involved in the development and maintenance of inflammatory and neuropathic pain conditions. TNFα can have long-lasting effects by regulating the expression of a variety of inflammatory mediators, including other cytokines and TNFα itself. However, the speed with which TNFα induces tactile and thermal hypersensitivity suggests that transcriptional regulation cannot fully account for its sensitizing effects, and some recent findings suggest that TNFα may act directly on primary afferent neurons to induce pain hypersensitivity. In the present study, we show that peripheral administration of TNFα induces thermal hypersensitivity in wild-type mice but not in transient receptor potential vanilloid receptor TRPV1–/– mice. In contrast, TNFα produced equivalent mechanical hypersensitivity in TRPV1–/– mice and wild-type littermates, suggesting a role for TRPV1 in TNFα-induced thermal, but not mechanical, hypersensitivity. Because tetrodotoxin (TTX)-resistant Na+ channels are a critical site of modulation underlying mechanical hypersensitivity in inflammatory and neuropathic pain conditions, we tested the effects of TNFα on these channels in isolated mouse dorsal root ganglion (DRG) neurons. We report that acute application of TNFα rapidly enhances TTX-resistant Na+ currents in isolated DRG neurons. This potentiation of TTX-resistant currents by TNFα is dramatically reduced in DRG neurons from TNF receptor 1 (TNFR1) knock-out mice and is blocked by the p38 mitogen-activated protein kinase inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole]. Mechanical hypersensitivity induced by peripherally applied TNFα is also significantly reduced by SB202190. These results suggest that TNFα may induce acute peripheral mechanical sensitization by acting directly on TNFR1 in primary afferent neurons, resulting in p38-dependent modulation of TTX-resistant Na+ channels.

Peripheral group I metabotropic glutamate receptors modulate nociception in mice

The metabotropic glutamate receptors (mGluRs) are found throughout the central nervous system, where they modulate neuronal excitability and synaptic transmission. Here we report the presence of phospholipase C-coupled group I mGluRs (mGluR1 and mGluR5) outside the central nervous system on peripheral unmyelinated sensory afferents. Given their localization on predominantly nociceptive afferents, we investigated whether these receptors modulate nociceptive signaling, and found that agonist-induced activation of peripheral group I mGluRs leads to increased sensitivity to noxious heat, a phenomenon termed thermal hyperalgesia. Furthermore, group I mGluR antagonists not only prevent, but also attenuate established formalin-induced pain. Taken together, these results suggest that peripheral mGluRs mediate a component of hyperalgesia and may be therapeutically targeted to prevent and treat inflammatory pain.

Soft, stretchable, fully implantable miniaturized optoelectronic systems for wireless optogenetics

Optogenetics allows rapid, temporally specific control of neuronal activity by targeted expression and activation of light-sensitive proteins. Implementation typically requires remote light sources and fiber-optic delivery schemes that impose considerable physical constraints on natural behaviors. In this report we bypass these limitations using technologies that combine thin, mechanically soft neural interfaces with fully implantable, stretchable wireless radio power and control systems. The resulting devices achieve optogenetic modulation of the spinal cord and peripheral nervous system. This is demonstrated with two form factors; stretchable film appliqués that interface directly with peripheral nerves, and flexible filaments that insert into the narrow confines of the spinal epidural space. These soft, thin devices are minimally invasive, and histological tests suggest they can be used in chronic studies. We demonstrate the power of this technology by modulating peripheral and spinal pain circuitry, providing evidence for the potential widespread use of these devices in research and future clinical applications of optogenetics outside the brain.

Activation of NMDA receptors reverses desensitization of mGluR5 in native and recombinant systems

The metabotropic glutamate receptor, mGluR5, has a critical role in induction of NMDA-receptor-dependent forms of synaptic plasticity and excitotoxicity. This is likely mediated by a reciprocal positive-feedback interaction between these two glutamate receptor subtypes in which activation of mGluR5 potentiates NMDA receptor currents and NMDA receptor activation potentiates mGluR5-mediated responses. We have investigated the mechanism by which NMDA receptor activation modulates mGluR5 function and find evidence that this response is mediated by activation of a protein phosphatase and a resultant dephosphorylation of protein kinase C phosphorylation sites on mGluR5. This form of neuromodulation may be important in a number of normal and pathological processes that involve activation of the NMDA receptor.

The kv4.2 potassium channel subunit is required for pain plasticity.

A-type potassium currents are important determinants of neuronal excitability. In spinal cord dorsal horn neurons, A-type currents are modulated by extracellular signal-regulated kinases (ERKs), which mediate central sensitization during inflammatory pain. Here, we report that Kv4.2 mediates the majority of A-type current in dorsal horn neurons and is a critical site for modulation of neuronal excitability and nociceptive behaviors. Genetic elimination of Kv4.2 reduces A-type currents and increases excitability of dorsal horn neurons, resulting in enhanced sensitivity to tactile and thermal stimuli. Furthermore, ERK-mediated modulation of excitability in dorsal horn neurons and ERK-dependent forms of pain hypersensitivity are absent in Kv4.2(-/-) mice compared to wild-type littermates. Finally, mutational analysis of Kv4.2 indicates that S616 is the functionally relevant ERK phosphorylation site for modulation of Kv4.2-mediated currents in neurons. These results show that Kv4.2 is a downstream target of ERK in spinal cord and plays a crucial role in pain plasticity.

Role of Protein Kinase C Phosphorylation in Rapid Desensitization of Metabotropic Glutamate Receptor 5

Metabotropic glutamate receptors (mGluRs) coupled to phosphoinositide hydrolysis desensitize in response to prolonged or repeated agonist exposure, and evidence suggests that this involves activation of protein kinase C (PKC). The present studies were undertaken to determine if cloned mGluR5 undergoes similar PKC-mediated desensitization and to investigate the molecular mechanism underlying PKC-induced desensitization. In Xenopus oocytes, both mGluR5a and mGluR5b showed pronounced desensitization in response to a brief activation by glutamate. Pharmacological studies clearly suggest that this desensitization requires PKC-mediated phosphorylation. Analysis of PKC consensus phosphorylation site mutants suggests that PKC phosphorylates mGluR5 at multiple sites to induce a relatively rapid form of desensitization. Because mGluRs play important roles in synaptic plasticity and in excitotoxicity, this desensitization may be involved in the dynamic regulation of these processes.

Lmx1b is required for maintenance of central serotonergic neurons and mice lacking central serotonergic system exhibit normal locomotor activity

Central serotonergic neurons have been implicated in numerous animal behaviors and psychiatric disorders, but the molecular mechanisms underlying their development are not well understood. Here we generated Lmx1b (LIM homeobox transcription factor 1 β) conditional knock-out mice (Lmx1bf/f/p) in which Lmx1b was only deleted in Pet1 (pheochromocytoma 12 ETS factor-1)-expressing 5-HT neurons. In Lmx1bf/f/p mice, the initial generation of central 5-HT neurons appeared normal. However, the expression of both 5-HT-specific and non-5-HT-specific markers was lost in these neurons at later stages of development. The loss of gene expression is concomitant with downregulation of Lmx1b expression, with the exception of serotonin transporter Sert and tryptophan hydroxylase TPH2, whose expression appears to be most sensitive to Lmx1b. Interestingly, the expression of Pet1 is tightly coupled with expression of Lmx1b during later stages of embryonic development, indicating that Lmx1b maintains Pet1 expression. In Lmx1bf/f/p mice, almost all central 5-HT neurons failed to survive. Surprisingly, Lmx1bf/f/p mice survived to adulthood and exhibited normal locomotor activity. These data reveal a critical role of Lmx1b in maintaining the differentiated status of 5-HT neurons. Lmx1bf/f/p mice with normal locomotor function should provide a unique animal model for examining the roles of central 5-HT in a variety of animal behaviors.