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Selective Small Molecule Activators of TREK-2 Channels Stimulate Dorsal Root Ganglion c-Fiber Nociceptor Two-Pore-Domain Potassium Channel Currents and Limit Calcium Influx.
Dadi PK, Vierra NC, Days E, Dickerson MT, Vinson PN, Weaver CD, Jacobson DA
(2017) ACS Chem Neurosci 8: 558-568
MeSH Terms: Action Potentials, Animals, Antibodies, Calcium, Dinoprostone, Electric Stimulation, Fluoxetine, Ganglia, Spinal, HEK293 Cells, Humans, Lectins, Mice, Mice, Inbred C57BL, Mutation, Nociceptors, Potassium Channel Blockers, Potassium Channels, Tandem Pore Domain, Protein Synthesis Inhibitors, Tetracycline
Show Abstract · Added November 13, 2017
The two-pore-domain potassium (K2P) channel TREK-2 serves to modulate plasma membrane potential in dorsal root ganglia c-fiber nociceptors, which tunes electrical excitability and nociception. Thus, TREK-2 channels are considered a potential therapeutic target for treating pain; however, there are currently no selective pharmacological tools for TREK-2 channels. Here we report the identification of the first TREK-2 selective activators using a high-throughput fluorescence-based thallium (Tl) flux screen (HTS). An initial pilot screen with a bioactive lipid library identified 11-deoxy prostaglandin F2α as a potent activator of TREK-2 channels (EC ≈ 0.294 μM), which was utilized to optimize the TREK-2 Tl flux assay (Z' = 0.752). A HTS was then performed with 76 575 structurally diverse small molecules. Many small molecules that selectively activate TREK-2 were discovered. As these molecules were able to activate single TREK-2 channels in excised membrane patches, they are likely direct TREK-2 activators. Furthermore, TREK-2 activators reduced primary dorsal root ganglion (DRG) c-fiber Ca influx. Interestingly, some of the selective TREK-2 activators such as 11-deoxy prostaglandin F2α were found to inhibit the K2P channel TREK-1. Utilizing chimeric channels containing portions of TREK-1 and TREK-2, the region of the TREK channels that allows for either small molecule activation or inhibition was identified. This region lies within the second pore domain containing extracellular loop and is predicted to play an important role in modulating TREK channel activity. Moreover, the selective TREK-2 activators identified in this HTS provide important tools for assessing human TREK-2 channel function and investigating their therapeutic potential for treating chronic pain.
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19 MeSH Terms
Deletion of KCC3 in parvalbumin neurons leads to locomotor deficit in a conditional mouse model of peripheral neuropathy associated with agenesis of the corpus callosum.
Ding J, Delpire E
(2014) Behav Brain Res 274: 128-36
MeSH Terms: Agenesis of Corpus Callosum, Analysis of Variance, Animals, Disease Models, Animal, Exploratory Behavior, Ganglia, Spinal, Mice, Mice, Transgenic, Motor Activity, Movement Disorders, Neurons, Parvalbumins, Peripheral Nervous System Diseases, Phosphopyruvate Hydratase, Psychomotor Performance, Reaction Time, Spinal Cord, Symporters
Show Abstract · Added November 25, 2014
Hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum (HMSN/ACC or ACCPN) is an autosomal recessive disease caused by the disruption of the SLC12A6 gene, which encodes the K-Cl cotransporter-3 (KCC3). A ubiquitous deletion of KCC3 in mice leads to severe locomotor deficits similar to ACCPN patients. However, the underlying pathological mechanism leading to the disease remains unclear. Even though a recent study suggests that the neuropathic features of ACCPN are mostly due to neuronal loss of KCC3, the specific cell type responsible for the disease is still unknown. Here we established four tissue specific KCC3 knockout mouse lines to explore the cell population origin of ACCPN. Our results showed that the loss of KCC3 in parvalbumin-positive neurons led to significant locomotor deficit, suggesting a crucial role of these neurons in the development of the locomotor deficit. Interestingly, mice in which KCC3 deletion was driven by the neuron-specific enolase (NSE) did not develop any phenotype. Furthermore, we demonstrated that nociceptive neurons targeted with Nav1.8-driven CRE and Schwann cells targeted with a desert hedgehog-driven CRE were not involved in the development of ACCPN. Together, these results establish that the parvalbumin-positive neuronal population is an important player in the pathogenic development of ACCPN.
Copyright © 2014 Elsevier B.V. All rights reserved.
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18 MeSH Terms
Prostaglandin metabolite induces inhibition of TRPA1 and channel-dependent nociception.
Weng Y, Batista-Schepman PA, Barabas ME, Harris EQ, Dinsmore TB, Kossyreva EA, Foshage AM, Wang MH, Schwab MJ, Wang VM, Stucky CL, Story GM
(2012) Mol Pain 8: 75
MeSH Terms: Animals, Ganglia, Spinal, Male, Mice, Mice, Knockout, Mustard Plant, Nociception, Plant Oils, Prostaglandin D2, Prostaglandins, TRPA1 Cation Channel, Transient Receptor Potential Channels
Show Abstract · Added May 15, 2015
BACKGROUND - The Transient Receptor Potential (TRP) ion channel TRPA1 is a key player in pain pathways. Irritant chemicals activate ion channel TRPA1 via covalent modification of N-terminal cysteines. We and others have shown that 15-Deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2) similarly activates TRPA1 and causes channel-dependent nociception. Paradoxically, 15d-PGJ2 can also be anti-nociceptive in several pain models. Here we hypothesized that activation and subsequent desensitization of TRPA1 in dorsal root ganglion (DRG) neurons underlies the anti-nociceptive property of 15d-PGJ2. To investigate this, we utilized a battery of behavioral assays and intracellular Ca2+ imaging in DRG neurons to test if pre-treatment with 15d-PGJ2 inhibited TRPA1 to subsequent stimulation.
RESULTS - Intraplantar pre-injection of 15d-PGJ2, in contrast to mustard oil (AITC), attenuated acute nocifensive responses to subsequent injections of 15d-PGJ2 and AITC, but not capsaicin (CAP). Intraplantar 15d-PGJ2-administered after the induction of inflammation-reduced mechanical hypersensitivity in the Complete Freund's Adjuvant (CFA) model for up to 2 h post-injection. The 15d-PGJ2-mediated reduction in mechanical hypersensitivity is dependent on TRPA1, as this effect was absent in TRPA1 knockout mice. Ca2+ imaging studies of DRG neurons demonstrated that 15d-PGJ2 pre-exposure reduced the magnitude and number of neuronal responses to AITC, but not CAP. AITC responses were not reduced when neurons were pre-exposed to 15d-PGJ2 combined with HC-030031 (TRPA1 antagonist), demonstrating that inhibitory effects of 15d-PGJ2 depend on TRPA1 activation. Single daily doses of 15d-PGJ2, administered during the course of 4 days in the CFA model, effectively reversed mechanical hypersensitivity without apparent tolerance or toxicity.
CONCLUSIONS - Taken together, our data support the hypothesis that 15d-PGJ2 induces activation followed by persistent inhibition of TRPA1 channels in DRG sensory neurons in vitro and in vivo. Moreover, we demonstrate novel evidence that 15d-PGJ2 is analgesic in mouse models of pain via a TRPA1-dependent mechanism. Collectively, our studies support that TRPA1 agonists may be useful as pain therapeutics.
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12 MeSH Terms
Jedi-1 and MEGF10 signal engulfment of apoptotic neurons through the tyrosine kinase Syk.
Scheib JL, Sullivan CS, Carter BD
(2012) J Neurosci 32: 13022-31
MeSH Terms: Amino Acid Motifs, Animals, Apoptosis, Arabidopsis Proteins, Cell Count, Cells, Cultured, Coculture Techniques, Embryo, Mammalian, Enzyme Inhibitors, Female, Ganglia, Spinal, Gene Expression Regulation, Green Fluorescent Proteins, Humans, Immunoprecipitation, Immunoreceptor Tyrosine-Based Activation Motif, Intracellular Signaling Peptides and Proteins, Intramolecular Transferases, Male, Membrane Proteins, Mice, Microglia, Mutagenesis, Site-Directed, Mutation, Neurons, Niacinamide, Phagocytosis, Phosphorylation, Protein-Tyrosine Kinases, Pyrimidines, RNA, Small Interfering, Signal Transduction, Staurosporine, Syk Kinase, Transfection
Show Abstract · Added March 5, 2014
During the development of the peripheral nervous system there is extensive apoptosis, and these neuronal corpses need to be cleared to prevent an inflammatory response. Recently, Jedi-1 and MEGF10, both expressed in glial precursor cells, were identified in mouse as having an essential role in this phagocytosis (Wu et al., 2009); however, the mechanisms by which they promote engulfment remained unknown. Both Jedi-1 and MEGF10 are homologous to the Drosophila melanogaster receptor Draper, which mediates engulfment through activation of the tyrosine kinase Shark. Here, we identify Syk, the mammalian homolog of Shark, as a signal transducer for both Jedi-1 and MEGF10. Syk interacted with each receptor independently through the immunoreceptor tyrosine-based activation motifs (ITAMs) in their intracellular domains. The interaction was enhanced by phosphorylation of the tyrosines in the ITAMs by Src family kinases (SFKs). Jedi association with Syk and activation of the kinase was also induced by exposure to dead cells. Expression of either Jedi-1 or MEGF10 in HeLa cells facilitated engulfment of carboxylated microspheres to a similar extent, and there was no additive effect when they were coexpressed. Mutation of the ITAM tyrosines of Jedi-1 and MEGF10 prevented engulfment. The SFK inhibitor PP2 or a selective Syk inhibitor (BAY 61-3606) also blocked engulfment. Similarly, in cocultures of glial precursors and dying sensory neurons from embryonic mice, addition of PP2 or knock down of endogenous Syk decreased the phagocytosis of apoptotic neurons. These results indicate that both Jedi-1 and MEGF10 can mediate phagocytosis independently through the recruitment of Syk.
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35 MeSH Terms
Fig4 expression in the rodent nervous system and its potential role in preventing abnormal lysosomal accumulation.
Guo J, Ma YH, Yan Q, Wang L, Zeng YS, Wu JL, Li J
(2012) J Neuropathol Exp Neurol 71: 28-39
MeSH Terms: Animals, Cells, Cultured, Female, Flavoproteins, Ganglia, Spinal, Gene Expression Regulation, Developmental, Lysosomes, Male, Mice, Mice, Inbred C57BL, Neurodegenerative Diseases, Neuroglia, Neurons, Phosphoinositide Phosphatases, Rats, Rats, Sprague-Dawley, Spinal Cord Injuries
Show Abstract · Added May 20, 2014
The phosphatase FIG4 regulates the concentration of phosphatidylinositol 3,5-diphosphate (PI3,5P2), a molecule critical for endosomal/lysosomal membrane trafficking and neuron function. We investigated Fig4 expression in the developing CNS of mice and rats using Western blot, real-time polymerase chain reaction, and morphological techniques in situ and in vitro and after spinal cord injury. Fig4 was expressed at a high levels throughout development in myelinating cells, particularly Schwann cells, and dorsal root ganglia sensory neurons. Fig4 protein and mRNA in CNS neurons were markedly diminished in adult versus embryonal animals. Spinal cord hemisection induced upregulation of Fig4 in adult spinal cord tissues that was associated with accumulation of lysosomes in neurons and glia. This accumulation appeared similar to the abnormal lysosomal storage observed in dorsal root ganglia of young fig4-null mice. The results suggest that Fig4 is involved in normal neural development and the maintenance of peripheral nervous system myelin. We speculate that adequate levels of Fig4 may be required to prevent neurons and glia from excessive lysosomal accumulation after injury and in neurodegeneration.
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17 MeSH Terms
Glial precursors clear sensory neuron corpses during development via Jedi-1, an engulfment receptor.
Wu HH, Bellmunt E, Scheib JL, Venegas V, Burkert C, Reichardt LF, Zhou Z, Fariñas I, Carter BD
(2009) Nat Neurosci 12: 1534-41
MeSH Terms: Animals, Apoptosis, Cells, Cultured, Female, Fibroblasts, Ganglia, Spinal, Gene Expression Regulation, Developmental, Green Fluorescent Proteins, Humans, Kidney, Membrane Proteins, Mice, Mice, Transgenic, Nerve Growth Factor, Neuroglia, Phagocytosis, Pregnancy, Sensory Receptor Cells, Stem Cells
Show Abstract · Added March 5, 2014
During the development of peripheral ganglia, 50% of the neurons that are generated undergo apoptosis. How the massive numbers of corpses are removed is unknown. We found that satellite glial cell precursors are the primary phagocytic cells for apoptotic corpse removal in developing mouse dorsal root ganglia (DRG). Confocal and electron microscopic analysis revealed that glial precursors, rather than macrophages, were responsible for clearing most of the dead DRG neurons. Moreover, we identified Jedi-1, an engulfment receptor, and MEGF10, a purported engulfment receptor, as homologs of the invertebrate engulfment receptors Draper and CED-1 expressed in the glial precursor cells. Expression of Jedi-1 or MEGF10 in fibroblasts facilitated binding to dead neurons, and knocking down either protein in glial cells or overexpressing truncated forms lacking the intracellular domain inhibited engulfment of apoptotic neurons. Together, these results suggest a cellular and molecular mechanism by which neuronal corpses are culled during DRG development.
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19 MeSH Terms
The Ste20 kinases Ste20-related proline-alanine-rich kinase and oxidative-stress response 1 regulate NKCC1 function in sensory neurons.
Geng Y, Hoke A, Delpire E
(2009) J Biol Chem 284: 14020-8
MeSH Terms: Animals, Calibration, Cell Line, Cell Separation, Ganglia, Spinal, Gene Expression Profiling, Gene Expression Regulation, Gene Knockdown Techniques, Mice, Mice, Knockout, Oxidative Stress, Protein Kinases, Protein-Serine-Threonine Kinases, RNA, Small Interfering, Rats, Reverse Transcriptase Polymerase Chain Reaction, Sensory Receptor Cells, Sodium-Potassium-Chloride Symporters, Solute Carrier Family 12, Member 2, Thallium
Show Abstract · Added August 13, 2010
NKCC1 is highly expressed in dorsal root ganglion neurons, where it is involved in gating sensory information. In a recent study, it was shown that peripheral nerve injury results in increased NKCC1 activity, not due to an increase in cotransporter expression, but to increased phosphorylation of the cotransporter (Pieraut, S., Matha, V., Sar, C., Hubert, T., Méchaly, I., Hilaire, C., Mersel, M., Delpire, E., Valmier, J., and Scamps, F. (2007) J. Neurosci. 27, 6751-6759). Our laboratory has also identified two Ste20-like kinases that bind and phosphorylate NKCC1: Ste20-related proline-alanine-rich kinase (SPAK) and oxidative-stress response 1 (OSR1). In this study, we show that both kinases are expressed at similar expression levels in spinal cord and dorsal root ganglion neurons, and that both kinases participate equally in the regulation of NKCC1. Using a novel fluorescence method to assay NKCC1 activity in single cells, we show a 50% reduction in NKCC1 activity in DRG neurons isolated from SPAK knockout mice, indicating that another kinase, e.g. OSR1, is present to phosphorylate and activate the cotransporter. Using a nociceptive dorsal root ganglion sensory neuronal cell line, which expresses the same cation-chloride cotransporters and kinases as native DRG neurons, and gene silencing via short hairpin RNA, we demonstrate a direct relationship between kinase expression and cotransporter activity. We show that inactivation of either kinase significantly affects NKCC1 activity, whereas inactivation of both kinases results in an additive effect. In summary, our study demonstrates redundancy of kinases in the regulation of NKCC1 in dorsal root ganglion neurons.
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20 MeSH Terms
Protein kinase A-induced phosphorylation of the p65 subunit of nuclear factor-kappaB promotes Schwann cell differentiation into a myelinating phenotype.
Yoon C, Korade Z, Carter BD
(2008) J Neurosci 28: 3738-46
MeSH Terms: Animals, Animals, Newborn, Cell Differentiation, Cells, Cultured, Coculture Techniques, Cyclic AMP, Cyclic AMP-Dependent Protein Kinases, Electrophoretic Mobility Shift Assay, Enzyme Inhibitors, Ganglia, Spinal, Gene Expression Regulation, Mice, Mice, Transgenic, Mutation, Myelin Sheath, Neurons, Phosphorylation, Rats, Schwann Cells, Sciatic Nerve, Serine, Transcription Factor RelA
Show Abstract · Added March 5, 2014
Axon-Schwann cell interactions are critical for myelin formation during peripheral nerve development and regeneration. Axonal contact promotes Schwann cell precursors to differentiate into a myelinating phenotype, and cAMP-elevating agents can mimic this; however, the mechanisms underlying this differentiation are poorly understood. We demonstrated previously that the transcription factor nuclear factor-kappaB (NF-kappaB) is required for myelin formation by Schwann cells (Nickols et al., 2003), although how it is activated during this process remained to be determined. Here, we report that culturing Schwann cells with sensory neurons results in the activation of cAMP-dependent protein kinase (PKA), and this kinase phosphorylates the p65 subunit of NF-kappaB at S276. The phosphorylation was also induced in cultured Schwann cells by treatment with forskolin, dibutyryl-cAMP, or by overexpression of a catalytic subunit of PKA, and this increased the transcriptional activity of NF-kappaB. In developing perinatal rat sciatic nerve, the kinetics of p65 phosphorylation at S276 paralleled that of PKA and NF-kappaB activation. To elucidate the role of p65 phosphorylation in myelin formation, we overexpressed an S276A mutant of p65 in cultured Schwann cells, which blocked PKA-mediated transcriptional activation of NF-kappaB. When the Schwann cells expressing the mutant were cocultured with sensory neurons, there was a 45% reduction in the number of myelinated fibers relative to controls, demonstrating a requirement for p65 phosphorylation by PKA during myelin formation.
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22 MeSH Terms
Activation of the transcription factor NF-kappaB in Schwann cells is required for peripheral myelin formation.
Nickols JC, Valentine W, Kanwal S, Carter BD
(2003) Nat Neurosci 6: 161-7
MeSH Terms: Animals, Axons, Cell Differentiation, Cells, Cultured, Coculture Techniques, Fetus, Ganglia, Spinal, Gene Expression Regulation, Developmental, Mice, Mutation, Myelin Sheath, NF-kappa B, Neurons, Afferent, Peripheral Nervous System, Protein Subunits, Rats, Schwann Cells, Sciatic Nerve, Transcription Factor RelA, Transcription Factors
Show Abstract · Added March 5, 2014
Peripheral myelin formation is initiated by axonal cues that trigger a differentiation program in associated Schwann cells. Here, we define one essential differentiation signal: activation of the transcription factor NF-kappaB. In rat sciatic nerves, NF-kappaB was highly upregulated in pre-myelinating Schwann cells, and then its expression progressively declined until it was nearly absent in adults. Similarly, in co-cultures of Schwann cells and sensory neurons, NF-kappaB activation paralleled myelination, and blocking its activity or using cells from mice lacking the NF-kappaB subunit p65 markedly attenuated myelination. Inhibiting NF-kappaB also prevented activation of Oct-6, a transcription factor induced by axonal contact and required for proper myelin formation. These results show that the activation of NF-kappaB is an essential signal for the progression of axon-associated Schwann cells into a myelinating phenotype.
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20 MeSH Terms
Abnormal GABAA receptor-mediated currents in dorsal root ganglion neurons isolated from Na-K-2Cl cotransporter null mice.
Sung KW, Kirby M, McDonald MP, Lovinger DM, Delpire E
(2000) J Neurosci 20: 7531-8
MeSH Terms: Animals, Behavior, Animal, Bumetanide, Carrier Proteins, Cells, Cultured, Chlorides, Contraindications, Female, GABA Antagonists, GABA-A Receptor Antagonists, Gait Disorders, Neurologic, Ganglia, Spinal, Gramicidin, Male, Membrane Potentials, Mice, Mice, Knockout, Neurons, Pain Measurement, Patch-Clamp Techniques, Picrotoxin, Potassium, Receptors, GABA-A, Sodium-Potassium-Chloride Symporters, gamma-Aminobutyric Acid
Show Abstract · Added August 13, 2010
We have recently disrupted Slc12a2, the gene encoding the secretory Na-K-2Cl cotransporter in mice (NKCC1) (Delpire et al., 1999). Gramicidin perforated-patch and whole-cell recordings were performed to study GABA-induced currents in dorsal root ganglion (DRG) neurons isolated from wild-type and homozygote NKCC1 knock-out mice. In wild-type DRG neurons, strong GABA-evoked inward current was observed at the resting membrane potential, suggesting active accumulation of Cl(-) in these cells. This GABA-induced current was blocked by picrotoxin, a GABA(A) receptor blocker. The strong Cl(-) accumulation that gives rise to depolarizing GABA responses is caused by Na-K-2Cl cotransport because reduction of external Cl(-) or application of bumetanide induced a decrease in [Cl(-)](i), whereas an increase in external K(+) caused an apparent [Cl(-)](i) accumulation. In contrast to control neurons, little or no net current was observed at the resting membrane potential in homozygote NKCC1 mutant DRG neurons. E(GABA) was significantly more negative, demonstrating the absence of Cl(-) accumulation in these cells. Application of bumetanide induced a positive shift of E(GABA), suggesting the presence of an outward Cl(-) transport mechanism. In agreement with an absence of GABA depolarization in DRG neurons, behavioral analysis revealed significant alterations in locomotion and pain perception in the knock-out mouse. Our results clearly demonstrate that the Na-K-2Cl cotransporter is responsible for [Cl(-)](i) accumulation in DRG neurons and that via regulation of intracellular Cl(-), the Na-K-2Cl cotransporter participates in the modulation of GABA neurotransmission and sensory perception.
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25 MeSH Terms