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1.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 55(1): 132-138, 2024 Jan 20.
Article in Chinese | MEDLINE | ID: mdl-38322530

ABSTRACT

Objective: To investigate the effects of long-term administration of tacrolimus (also known as FK506) on the pain-related behaviors in mice and to study the underlying mechanism of pain induced by FK506 via measuring the effect of FK506 on the synaptic expression and phosphorylation of alpha-amino-3-hyroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the spinal cord dorsal horn of mice. Methods: 1) A total of 24 mice were evenly and randomly assigned to two groups, a FK506 group and a Saline group. The FK506 group was given daily intraperitoneal injection of FK506 and the Saline group received normal saline. Both groups received injection once a day for 7 days in a row. Some of the mice ( n=6 in each group) were monitored for the changes in the paw withdrawal threshold (PWT), the paw withdrawal latency (PWL), and the spontaneous pain behaviors to establish the pain model. The other mice ( n=6 in each group) of each group underwent isolation of the dorsal horn when obvious pain symptoms were induced on day 7 of injection. Then, immunoblotting was performed to determine the synaptic expression and phosphorylation levels of GluA1 and GluA2 subunits of AMPA receptors. 2) The mice were randomly divided into two groups, FK506+calcineurin (CaN) group and FK506+Saline group ( n=6 in each group). After the pain model was constructed, the mice were given intrathecal injection of recombinant CaN (also know as 33 U) or normal saline. Then, 60 minutes later, the PWT and the PWL of the mice were measured to investigate the role of CaN in FK506-induced pain. 3) Another18 mice were selected. The mice were randomly and evenly assigned to three groups, a control group (receiving intraperitoneal injection of normal saline followed by intrathecal injection of normal saline), FK506+Saline group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of normal saline) and FK506+CaN group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of CaN). Then, 60 minutes later, the spinal cords were isolated and subjected to immunoblotting assay to determine the role of CaN in FK506-induced AMPA receptor modification. Results: 1) After 7 consecutive days of intraperitoneal injection of FK506, the PWT and PWL of mice dropped significantly, reaching on day 7 as low as 22.3%±0.05% and 66.6%±0.05% of the control group, respectively ( P<0.01). The FK506-treated mice displayed evident spontaneous pain behavior, presenting significantly increased licking activities ( P<0.01). These results indicated that FK506-induced pain model was successfully established. Immunoblotting assay showed that the total expressions of GluA1 and GluA2 subunits in the spinal dorsal horn of the FK506 group remained unchanged in comparison with those of the Saline group. However, FK506 specifically induced an increase in the synaptic expression of GluA1. In addition, the phosphorylation levels of GluA1 at Ser845 and Ser831 in FK506-treated mice were significantly increased in comparison with those of the control group ( P<0.05). 2) Compared with those of the mice in the FK506+Saline group, the PWT and the PWL of mice in the FK506+CaN group were significantly increased ( P<0.05). 3) Compared with those of the FK506+Saline group, the synaptic expression of GluA1 were decreased in FK506+CaN group ( P<0.01) and the phosphorylation levels of GluA1 at Ser845 and Ser831 were significantly downregulated ( P<0.001). Conclusion: The hyper-expression and hyperphosphorylation of GluA1 subunit in the spinal cord dorsal horn resulting from CaN inhibition contributes to the FK506-induced pain syndrome. FK506 induces the synaptic hyper-expression and hyperphosphorylation of GluA1 in the dorsal horn of the spinal cord through CaN inhibition, thereby inducing pain.


Subject(s)
Receptors, AMPA , Tacrolimus , Mice , Animals , Tacrolimus/metabolism , Tacrolimus/pharmacology , Receptors, AMPA/metabolism , Saline Solution/pharmacology , Spinal Cord Dorsal Horn/metabolism , Spinal Cord , Pain/metabolism
2.
Sci Adv ; 10(5): eadj3808, 2024 Feb 02.
Article in English | MEDLINE | ID: mdl-38306424

ABSTRACT

G protein-coupled receptor 39 (GPR39) senses the change of extracellular divalent zinc ion and signals through multiple G proteins to a broad spectrum of downstream effectors. Here, we found that GPR39 was prevalent at inhibitory synapses of spinal cord somatostatin-positive (SOM+) interneurons, a mechanosensitive subpopulation that is critical for the conveyance of mechanical pain. GPR39 complexed specifically with inhibitory glycine receptors (GlyRs) and helped maintain glycinergic transmission in a manner independent of G protein signalings. Targeted knockdown of GPR39 in SOM+ interneurons reduced the glycinergic inhibition and facilitated the excitatory output from SOM+ interneurons to spinoparabrachial neurons that engaged superspinal neural circuits encoding both the sensory discriminative and affective motivational domains of pain experience. Our data showed that pharmacological activation of GPR39 or augmenting GPR39 interaction with GlyRs at the spinal level effectively alleviated the sensory and affective pain induced by complete Freund's adjuvant and implicated GPR39 as a promising therapeutic target for the treatment of inflammatory mechanical pain.


Subject(s)
Pain , Receptors, G-Protein-Coupled , Humans , Neurons/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Glycine/metabolism , Signal Transduction , Spinal Cord/metabolism
3.
Neurosci Lett ; 775: 136538, 2022 04 01.
Article in English | MEDLINE | ID: mdl-35189316

ABSTRACT

The calcium/calmodulin-dependent protein phosphase calcineurin (CaN) regulates synaptic plasticity by controlling the phosphorylation of synaptic proteins including AMPA type glutamate receptors. The regulator of calcineurin 1 (RCAN1) is characterized as an endogenous inhibitor of CaN and its dysregulation is implicated in multiple neurological disorders. However, whether RCAN1 is engaged in nociceptive processing in the spinal dorsal horn remains unrevealed. In this study, we found that RCAN1 was predominately expressed in pain-related neurons in the superficial dorsal horn of the spinal cord. Intraplantar injection of complete Freund's adjuvant (CFA) specifically increased the total and synaptic expression of the RCAN1.4 isoform in spinal dorsal horn. The CFA-induced inflammation also caused an increased binding of RCAN1.4 to CaN. Overexpression of RCAN1.4 in spinal dorsal horn of intact mice produced both mechanical allodynia and thermal hyperalgesia, which were accompanied by increased synaptic expression and phosphorylation of GluA1 subunit. Furthermore, the siRNA-mediated knockdown of RCAN1.4 significantly attenuated the development of pain hypersensitivity, meanwhile, decreased the synaptic expression of GluA1 in mice with peripheral inflammation. These data suggested that the increased expression of RCAN1.4 contributed to the development of inflammatory pain hypersensitivity, at least in part by promoting the synaptic recruitment of GluA1-containing AMPA receptor.


Subject(s)
Calcineurin , Spinal Cord Dorsal Horn , Animals , Calcineurin/metabolism , Freund's Adjuvant/metabolism , Freund's Adjuvant/toxicity , Hyperalgesia/metabolism , Inflammation/metabolism , Mice , Pain/metabolism , Posterior Horn Cells/metabolism , Receptors, AMPA/metabolism , Spinal Cord/metabolism , Spinal Cord Dorsal Horn/metabolism , Up-Regulation
4.
Mol Neurobiol ; 58(12): 6505-6519, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34559357

ABSTRACT

The A-kinase anchoring protein 150 (AKAP150) organizes kinases and phosphatases to regulate AMPA receptors (AMPARs) that are pivotal for synaptic plasticity. AKAP150 itself undergoes S-palmitoylation. However, the roles of AKAP150 and its palmitoylation in spinal nociceptive processing remain unknown. In this study, we found that intraplantar injection of complete Freund's adjuvant (CFA) significantly increased the synaptic expression of AKAP150 and caused a reorganization of AKAP150 signaling complex in spinal dorsal horn. Knockdown of AKAP150 or interruption of its interactions with kinases effectively suppressed the CFA-induced synaptic expression of GluA1 subunit of AMPARs. Our data also showed that an upregulation of AKAP150 palmitoylation was involved in the synaptic redistribution of AKAP150. Inhibition of AKAP150 palmitoylation by expression of palmitoylation-defective mutant AKAP150 (C36, 123S) effectively repressed the CFA-induced phosphorylation and synaptic expression of GluA1 subunit, meanwhile, attenuated the development of mechanical allodynia and thermal hyperalgesia. Furthermore, we found that an increased expression of palmitoyl acyltransferase ZDHHC2 contributed to the upregulation of AKAP150 palmitoylation and GluA1 accumulation in inflamed mouse. These data indicated that AKAP150 and its palmitoylation were involved in AMPA receptor-dependent modification of nociceptive transmission, and the manipulations of AKAP150 signaling complex and palmitoylation might serve as potential therapeutic strategies for persistent pain after inflammation.


Subject(s)
A Kinase Anchor Proteins/metabolism , Hyperalgesia/metabolism , Neurons/metabolism , Receptors, AMPA/metabolism , Spinal Cord Dorsal Horn/metabolism , Synapses/metabolism , Animals , Lipoylation , Male , Mice , Neurons/drug effects , Palmitates/pharmacology , Phosphorylation , Spinal Cord Dorsal Horn/drug effects , Synapses/drug effects
5.
Eur J Pharmacol ; 906: 174205, 2021 Sep 05.
Article in English | MEDLINE | ID: mdl-34048740

ABSTRACT

The K+-Cl- co-transporter 2 (KCC2) is a neuron-specific Cl- extruder in the dorsal horn of spinal cord. The low intracellular Cl- concentration established by KCC2 is critical for GABAergic and glycinergic systems to generate synaptic inhibition. Peripheral nerve lesions have been shown to cause KCC2 dysfunction in adult spinal cord through brain-derived neurotrophic factor (BDNF) signaling, which switches the hyperpolarizing inhibitory transmission to be depolarizing and excitatory. However, the mechanisms by which BDNF impairs KCC2 function remain to be elucidated. Here we found that BDNF treatment enhanced KCC2 ubiquitination in the dorsal horn of adult mice, a post-translational modification that leads to KCC2 degradation. Our data showed that spinal BDNF application promoted KCC2 interaction with Casitas B-lineage lymphoma b (Cbl-b), one of the E3 ubiquitin ligases that are involved in the spinal processing of nociceptive information. Knockdown of Cbl-b expression decreased KCC2 ubiquitination level and attenuated the pain hypersensitivity induced by BDNF. Spared nerve injury significantly increased KCC2 ubiquitination, which could be reversed by inhibition of TrkB receptor. Our data implicated that KCC2 was one of the important pain-related substrates of Cbl-b and that ubiquitin modification contributed to BDNF-induced KCC2 hypofunction in the spinal cord.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Brain-Derived Neurotrophic Factor/metabolism , Hyperalgesia/pathology , Proto-Oncogene Proteins c-cbl/metabolism , Spinal Cord Dorsal Horn/pathology , Symporters/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Disease Models, Animal , Gene Knockdown Techniques , Humans , Hyperalgesia/etiology , Male , Mice , Posterior Horn Cells/metabolism , Proteolysis , Proto-Oncogene Proteins c-cbl/genetics , Signal Transduction , Spinal Cord Dorsal Horn/cytology , Ubiquitination , K Cl- Cotransporters
6.
Eur J Pharmacol ; 899: 174034, 2021 May 15.
Article in English | MEDLINE | ID: mdl-33727056

ABSTRACT

Glycine receptor is one of the chloride-permeable ion channels composed of combinations of four α subunits and one ß subunit. In adult spinal cord, the glycine receptor α1 subunit is crucial for the generation of inhibitory neurotransmission. The reduced glycinergic inhibition is regarded as one of the key spinal mechanisms underlying pathological pain symptoms. However, the expression and function of glycine receptors in the peripheral system are largely unknown as yet. Here we found that glycine receptor α1 subunit was prevalent in the dorsal root ganglia (DRG) neurons as well as in the sciatic nerves of adult mice. Intraganglionar or intraplantar injection of glycine receptor antagonist strychnine caused the hypersensitivity to mechanical, thermal and cold stimuli, suggesting the functional importance of peripheral glycine receptors in the control of nociceptive signal transmission. Our data showed that peripheral inflammation induced by formalin decreased the expression of glycine receptor α1 subunit on the plasma membrane of DRG neurons, which was attributed to the activation of protein kinase C signaling. Intraplantar application of glycine receptor agonist glycine or positive modulator divalent zinc ion alleviated the first-phase painful behaviors induced by formalin. These data suggested that peripheral glycine receptor might serve as an effective target for pain therapy.


Subject(s)
Ganglia, Spinal/metabolism , Neural Inhibition , Nociceptive Pain/metabolism , Receptors, Glycine/metabolism , Analgesics/pharmacology , Animals , Behavior, Animal , Disease Models, Animal , Formaldehyde , Ganglia, Spinal/drug effects , Ganglia, Spinal/physiopathology , Glycine Agents/pharmacology , Male , Mice , Motor Activity , Neural Inhibition/drug effects , Nociception , Nociceptive Pain/chemically induced , Nociceptive Pain/physiopathology , Nociceptive Pain/prevention & control , Pain Threshold/drug effects , Protein Kinase C/metabolism , Receptors, Glycine/antagonists & inhibitors , Signal Transduction
7.
Sci Signal ; 13(638)2020 06 30.
Article in English | MEDLINE | ID: mdl-32606037

ABSTRACT

N-methyl-d-aspartate (NMDA) glutamate receptors (NMDARs) containing GluN2B subunits are prevalent early after birth in most brain regions in rodents. Upon synapse maturation, GluN2B is progressively removed from synapses, which affects NMDAR function and synaptic plasticity. Aberrant recruitment of GluN2B into mature synapses has been implicated in several neuropathologies that afflict adults. We found that the E3 ubiquitin ligase Cbl-b was enriched in the spinal cord dorsal horn neurons of mice and rats and suppressed GluN2B abundance during development and inflammatory pain. Cbl-b abundance increased from postnatal day 1 (P1) to P14, a critical time period for synapse maturation. Through its N-terminal tyrosine kinase binding domain, Cbl-b interacted with GluN2B. Ubiquitination of GluN2B by Cbl-b decreased the synaptic transmission mediated by GluN2B-containing NMDARs. Knocking down Cbl-b in vivo during P1 to P14 led to sustained retention of GluN2B at dorsal horn synapses, suggesting that Cbl-b limits the synaptic abundance of GluN2B in adult mice. However, peripheral inflammation induced by intraplantar injection of complete Freund's adjuvant resulted in the dephosphorylation of Cbl-b at Tyr363, which impaired its binding to and ubiquitylation of GluN2B, enabling the reappearance of GluN2B-containing NMDARs at synapses. Expression of a phosphomimic Cbl-b mutant in the dorsal horn suppressed both GluN2B-mediated synaptic currents and manifestations of pain induced by inflammation. The findings indicate a ubiquitin-mediated developmental switch in NMDAR subunit composition that is dysregulated by inflammation, which can enhance nociception.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Nociception , Proto-Oncogene Proteins c-cbl/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Spinal Cord Dorsal Horn/metabolism , Synapses/metabolism , Ubiquitination , Animals , Male , Mice , Pain/metabolism , Pain/pathology , Rats , Rats, Sprague-Dawley , Spinal Cord Dorsal Horn/pathology , Synapses/pathology
8.
Neuropharmacology ; 176: 108219, 2020 10 01.
Article in English | MEDLINE | ID: mdl-32579955

ABSTRACT

Glycine receptor α1ins subunit is located at inhibitory synapses in the superficial dorsal horn of adult spinal cord and is engaged in the glycinergic inhibition of nociceptive neuronal excitability and transmission. The α1ins phosphorylation at Ser380 by extracellular signal-regulated kinase (ERK) has been shown to decrease glycinergic synaptic currents and contribute to spinal disinhibition. Here we found that peripheral inflammation induced by Complete Freund's Adjuvant increased Ser380 phosphorylation in spinal cord dorsal horn of mice, which was repressed by specific activation of adenosine A1 receptor (A1R). Protein phosphatase-1 (PP1), a ubiquitously-distributed serine/threonine phosphatase, was required for A1R to reduce Ser380 phosphorylation. Our data showed that Gßγ dimer, when released after activation of Gi protein-coupled A1R, interacted with PP1 and directed this phosphatase to α1ins, allowing for the full dephosphorylation of Ser380 residue. Sequestration of Gßγ dimer by viral expression of the C-terminal tail of ß-adrenergic receptor kinase (ßARKct) dissociated PP1 from α1ins complex, leading to robust Ser380 phosphorylation. Meanwhile, Gßγ inhibition compromised the ability of A1R to alleviate inflammatory pain. The inhibitory effect of A1R on Ser380 phosphorylation was also attributed to the inactivation of ERK in CFA mice. Our data thus identified glycine receptor α1ins subunit as an important target for adenosinergic suppression of inflammatory pain.


Subject(s)
Analgesia/methods , Receptor, Adenosine A1/metabolism , Receptors, Glycine/metabolism , Spinal Cord Dorsal Horn/metabolism , Adenosine/pharmacology , Adenosine A1 Receptor Agonists/pharmacology , Animals , Dose-Response Relationship, Drug , Freund's Adjuvant/toxicity , HEK293 Cells , Humans , Male , Mice , Pain/chemically induced , Pain/metabolism , Pain Measurement/drug effects , Pain Measurement/methods , Phosphorylation/drug effects , Phosphorylation/physiology , Spinal Cord Dorsal Horn/chemistry , Spinal Cord Dorsal Horn/drug effects
9.
Neuroscience ; 429: 203-212, 2020 03 01.
Article in English | MEDLINE | ID: mdl-31962145

ABSTRACT

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have been implicated in the trafficking of postsynaptic glutamate receptors, including N-methyl-d-aspartate (NMDA)-subtype glutamate receptors (NMDARs) that are critical for nociceptive plasticity and behavioral sensitization. However, the components of SNAREs complex involved in spinal nociceptive processing remain largely unknown. Here we found that SNAP25, syntaxin4, VAMP2 and Munc18-1 were localized at postsynaptic sites and formed the complex in the superficial lamina of spinal cord dorsal horn of rats. The complex formation between these SNAREs components were accelerated after intraplantar injection of complete Freund's adjuvant (CFA), pharmacological removal of GABAergic inhibition or activation of NMDAR in intact rats. The increased SNAP25/syntaxin4/VAMP2/Munc18-1 interaction facilitated the surface delivery and synaptic accumulation of NMDAR during inflammatory pain. Disruption of the molecular interaction between SNAP25 with its SNARE partners by using a blocking peptide derived from the C-terminus of SNAP25 effectively repressed the surface and synaptic accumulation of GluN2B-containing NMDARs in CFA-injected rats. This peptide also alleviated inflammatory mechanical allodynia and thermal hypersensitivity. These data suggested that SNAREs complex assembly in spinal cord dorsal horn was involved in the inflammatory pain hypersensitivity through promoting NMDAR synaptic trafficking.


Subject(s)
Spinal Cord Dorsal Horn , Vesicle-Associated Membrane Protein 2 , Animals , Freund's Adjuvant/toxicity , Hyperalgesia , Pain , Posterior Horn Cells , Rats , Receptors, N-Methyl-D-Aspartate , Spinal Cord
10.
Eur J Pharmacol ; 863: 172700, 2019 Nov 15.
Article in English | MEDLINE | ID: mdl-31563651

ABSTRACT

Protein phosphatase-1 (PP1) is ubiquitously distributed in the nervous system and catalyzes the dephosphorylation of numerous substrates. The specificity and efficacy of PP1-mediated dephosphorylation depend on scaffolding proteins that anchor PP1 to the close vicinity of substrates. Spinophilin is one of the scaffolding proteins which are able to direct PP1 into postsynaptic density and regulate the synaptic transmission and plasticity. Here we found that spinophilin was enriched in dorsal root ganglia (DRG) neurons and engaged in the modification of nociceptive signaling processing. Disturbing spinophilin/PP1 interaction in DRG neurons led to the enhanced sensitivity of mice to heat and mechanical stimuli. The transient receptor potential vanilloid 1 (TRPV1) was identified as an important target for spinophilin modification. Our data showed that spinophilin physically interacted with TRPV1 and facilitated PP1 dephosphorylation of TRPV1 at Ser502. Disruption of spinophilin/PP1 complex enhanced Ser502 phosphorylation and boosted TRPV1 expression on plasma membrane. Peripheral inflammation induced by formalin disturbed spinophilin/PP1 interaction, which removed PP1-mediated inhibition and caused a marked increase of TRPV1 phosphorylation. Viral expression of wild-type spinophilin in DRG neurons repressed TRPV1 phosphorylation and alleviated formalin-induced inflammatory pain. These data suggested that spinophilin/PP1 complex negatively controlled TRPV1 function in DRG neurons.


Subject(s)
Ganglia, Spinal/cytology , Microfilament Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , TRPV Cation Channels/metabolism , Animals , Cell Membrane/metabolism , Gene Expression Regulation , Male , Mice , Mice, Inbred C57BL , Neurons/cytology , Phosphorylation , Protein Transport , Time Factors
11.
PLoS Biol ; 17(8): e3000371, 2019 08.
Article in English | MEDLINE | ID: mdl-31433808

ABSTRACT

Inhibitory glycinergic transmission in adult spinal cord is primarily mediated by glycine receptors (GlyRs) containing the α1 subunit. Here, we found that α1ins, a longer α1 variant with 8 amino acids inserted into the intracellular large loop (IL) between transmembrane (TM)3 and TM4 domains, was expressed in the dorsal horn of the spinal cord, distributed at inhibitory synapses, and engaged in negative control over nociceptive signal transduction. Activation of metabotropic glutamate receptor 5 (mGluR5) specifically suppressed α1ins-mediated glycinergic transmission and evoked pain sensitization. Extracellular signal-regulated kinase (ERK) was critical for mGluR5 to inhibit α1ins. By binding to a D-docking site created by the 8-amino-acid insert within the TM3-TM4 loop of α1ins, the active ERK catalyzed α1ins phosphorylation at Ser380, which favored α1ins ubiquitination at Lys379 and led to α1ins endocytosis. Disruption of ERK interaction with α1ins blocked Ser380 phosphorylation, potentiated glycinergic synaptic currents, and alleviated inflammatory and neuropathic pain. These data thus unraveled a novel, to our knowledge, mechanism for the activity-dependent regulation of glycinergic neurotransmission.


Subject(s)
Posterior Horn Cells/metabolism , Receptors, Glycine/metabolism , Animals , Extracellular Signal-Regulated MAP Kinases/metabolism , Glycine/metabolism , MAP Kinase Signaling System/physiology , Male , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 7/metabolism , Phosphorylation , Receptor, Metabotropic Glutamate 5/metabolism , Receptor, Metabotropic Glutamate 5/physiology , Receptors, Glycine/physiology , Signal Transduction/physiology , Spinal Cord/metabolism , Spinal Cord Dorsal Horn/metabolism , Spine/metabolism , Synapses/metabolism , Synaptic Transmission/physiology
12.
Eur J Pharmacol ; 854: 62-69, 2019 Jul 05.
Article in English | MEDLINE | ID: mdl-30951721

ABSTRACT

Src Homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1) interacts specifically with GluN2A subunit of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors in spinal cord dorsal horn. This molecular interaction is involved in the development of GluN2A-dependent spinal sensitization of nociceptive behaviors. Intrathecal application of a GluN2A-derived polypeptide (short for pep-GluN2A) has been shown to disturb spinal GluN2A/SHP1 interaction and inhibit inflammatory pain. Here we found that SHP1 was also located at dorsal root ganglion (DRG) neurons and formed complexes with GluN2A subunit. Peripheral inflammation activated SHP1 in DRG neurons, which promoted GluN2A tyrosine phosphorylation. The SHP1 binding to GluN2A facilitated the glutamate release from primary afferent fibers and exaggerated nociceptive synaptic transmission onto postsynaptic spinal cord neurons. Our data showed that intradermal application of pep-GluN2A disrupted GluN2A/SHP1 interaction in DRG neurons, attenuated the ability of GluN2A subunit-containing NMDA receptors to regulate the presynaptic glutamate release and more importantly, alleviated the pain hypersensitivity caused by carrageenan, complete Freund's adjuvant and formalin. The neuropathic pain induced by spared nerve injury was also ameliorated by intradermal pep-GluN2A application. These data suggested that disruption of GluN2A/SHP1 interaction in DRG neurons generated an effective analgesic action against pathological pain.


Subject(s)
Ganglia, Spinal/drug effects , Neuralgia/drug therapy , Peptides/pharmacology , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Amino Acid Sequence , Animals , Behavior, Animal/drug effects , Ganglia, Spinal/pathology , Male , Neuralgia/metabolism , Neuralgia/pathology , Neuralgia/physiopathology , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Nociception/drug effects , Peptides/chemistry , Peptides/therapeutic use , Protein Binding/drug effects , Rats , Rats, Sprague-Dawley
13.
Neuropharmacology ; 148: 358-365, 2019 04.
Article in English | MEDLINE | ID: mdl-30721695

ABSTRACT

Glycine receptors (GlyRs) are pentameric proteins that consist of α (α1-α4) subunits and/or ß subunit. In the spinal cord of adult animals, the majority of inhibitory glycinergic neurotransmission is mediated by α1 subunit-containing GlyRs. The reduced glycinergic inhibition (disinhibition) is proposed to increase the excitabilities and spontaneous activities of spinal nociceptive neurons during pathological pain. However, the molecular mechanisms by which peripheral lesions impair GlyRs-α1-mediated synaptic inhibition remain largely unknown. Here we found that activity-dependent ubiquitination of GlyRs-α1 subunit might contribute to glycinergic disinhibition after peripheral inflammation. Our data showed that HUWE1 (HECT, UBA, WWE domain containing 1), an E3 ubiquitin ligase, located at spinal synapses and specifically interacted with GlyRs-α1 subunit. By ubiquitinating GlyRs-α1, HUWE1 reduced the surface expression of GlyRs-α1 through endocytic pathway. In the dorsal horn of Complete Freund's Adjuvant-injected mice, shRNA-mediated knockdown of HUWE1 blunted GlyRs-α1 ubiquitination, potentiated glycinergic synaptic transmission and attenuated inflammatory pain. These data implicated that ubiquitin modification of GlyRs-α1 represented an important way for peripheral inflammation to reduce spinal glycinergic inhibition and that interference with HUWE1 activity generated analgesic action by resuming GlyRs-α1-mediated synaptic transmission.


Subject(s)
Neural Inhibition/physiology , Receptors, Glycine/physiology , Spinal Cord Dorsal Horn/physiopathology , Tumor Suppressor Proteins/physiology , Ubiquitin-Protein Ligases/physiology , Ubiquitination/drug effects , Animals , Cells, Cultured , Humans , Male , Mice , Neural Inhibition/drug effects , Pain/prevention & control , RNA, Small Interfering/pharmacology , Receptors, Glycine/drug effects , Receptors, Glycine/metabolism , Synaptic Transmission/drug effects , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/pharmacology , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/pharmacology
14.
Neuroscience ; 388: 1-10, 2018 09 15.
Article in English | MEDLINE | ID: mdl-30049666

ABSTRACT

Neuroligin 1 (NLGN1), a cell adhesion molecule present at excitatory glutamatergic synapses, has been shown to be critical for synaptic specialization and N-methyl-d-aspartate (NMDA)-subtype glutamate receptor-dependent synaptic plasticity. Whether and how NLGN1 is engaged in nociceptive behavioral sensitization remains largely unknown. In this study, we found an activity-dependent regulation of NLGN1 synaptic expression in pain-related spinal cord dorsal horns of mice. The enhancement of neuronal activity by pharmacological activation of NMDA receptor (NMDAR) or removal of GABAergic inhibition in intact mice significantly increased NLGN1 concentration at synaptosomal membrane fraction. Intraplantar injection of complete Freund's adjuvant (CFA) also increased the NLGN1 expression at synapses. NMDAR might act through Ca2+/calmodulin-dependent protein kinase II (CaMKII) and Src-family protein tyrosine kinase member Fyn to induce the synaptic redistribution of NLGN1. We also found that one of the important roles of NLGN1 was to facilitate the clustering of NMDAR at synapses. The NLGN1-targeting siRNA suppressed the synaptic expression of GluN2B-containing NMDAR in CFA-injected mice and meanwhile, attenuated the inflammatory mechanical allodynia and thermal hypersensitivity. These data suggested that tissue injury-induced synaptic redistribution of NLGN1 was involved in the development of pain hypersensitivity through facilitating the synaptic incorporation of NMDARs.


Subject(s)
Cell Adhesion Molecules, Neuronal/metabolism , Hyperalgesia/metabolism , Inflammation/metabolism , Spinal Cord Dorsal Horn/metabolism , Synapses/metabolism , Synaptic Transmission/physiology , Animals , Cell Adhesion Molecules, Neuronal/genetics , Disease Models, Animal , Freund's Adjuvant , Gene Expression Regulation/physiology , Hot Temperature , Male , Mice, Inbred C57BL , RNA, Small Interfering , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/metabolism , Tissue Culture Techniques , Touch
15.
Neuropharmacology ; 137: 104-113, 2018 07 15.
Article in English | MEDLINE | ID: mdl-29758384

ABSTRACT

Src-homology 2 domain-containing protein tyrosine phosphatase-1 (SHP1) is one of the non-receptor-like phosphatases that are highly enriched in hematopoietic cells. Although accumulating evidence has implicated the protein tyrosine phosphatases in the regulation of nociceptive transmission and plasticity, it is largely unknown whether SHP1 was expressed in pain-related spinal cord dorsal horn and engaged in the synaptic modification of nociceptive signals. Here we found that SHP1 was present in spinal neurons of rats and functionally coupled to GluN2A subunit-containing N-methyl-d-aspartate subtype of glutamate receptors, one of the key players in central sensitization of nociceptive behaviors. SHP1 interacted with a membrane-proximal region within the cytoplasmic tail of GluN2A. This interaction was necessary to stimulate SHP1 activity and more importantly, restrict SHP1 signaling to specifically enhance the tyrosine phosphorylation of GluN2A during inflammatory pain. Electrophysiological and behavioral studies showed that SHP1 binding potentiated GluN2A currents and evoked GluN2A-dependent pain hypersensitivity. The siRNA-mediated knockdown of SHP1 or interference with SHP1/GluN2A interaction by a synthetic peptide alleviated inflammatory pain induced by either Complete Freund's Adjuvant or formalin. Our data implicated that SHP1 was a specific enhancer of GluN2A-mediated nociceptive synaptic transmission in spinal cord dorsal horn, and manipulation of SHP1 activity may serve as an effective strategy for the treatment of inflammatory pain.


Subject(s)
Inflammation/metabolism , Pain/metabolism , Posterior Horn Cells/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Synaptic Transmission/physiology , Analgesics, Non-Narcotic/pharmacology , Animals , Gene Knockdown Techniques , HEK293 Cells , Humans , Inflammation/drug therapy , Male , Pain/drug therapy , Posterior Horn Cells/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , Rats, Sprague-Dawley , Signal Transduction/drug effects , Signal Transduction/physiology , Synaptic Transmission/drug effects , Tissue Culture Techniques , src-Family Kinases/metabolism
16.
Eur J Pharmacol ; 827: 189-197, 2018 May 15.
Article in English | MEDLINE | ID: mdl-29526716

ABSTRACT

Protein tyrosine phosphatase 1B (PTP1B) has been shown to dephosphorylate and inactivate insulin receptors, which contributes to the pathogenesis of diabetes. Neuropathic pain is one of the severe complications that results from diabetic neuropathy. However, whether PTP1B was involved in the development of diabetic neuropathic pain is largely unknown. The current study illustrated that PTP1B was located in spinal cord dorsal horn neurons of Sprague-Dawley rats. Western blot analysis demonstrated that the diabetic neuropathic pain induced by intraperitoneal injection of streptozotocin was associated with an increased protein expression and a dynamic redistribution of spinal PTP1B into excitatory glutamatergic synapses. We found that PTP1B operated to stimulate Src kinase and enhance the tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. The siRNA-mediated knockdown of PTP1B in streptozotocin-injected rats repressed Src activity, decreased NMDA receptor phosphorylation and alleviated the thermal hyperalgesia and mechanical allodynia. A similar analgesia against diabetic neuropathic pain was also achieved when PTP1B activity was manipulated by a chemical PTP Inhibitor or PTP1B(C215S) mutant. These data revealed a regulated expression of PTP1B in spinal cord dorsal horn of rats after diabetic neuropathy, and demonstrated that inhibition of PTP1B was beneficial for the treatment of pain hypersensitivity related to diabetes.


Subject(s)
Diabetic Neuropathies/complications , Enzyme Inhibitors/pharmacology , Neuralgia/complications , Neuralgia/drug therapy , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Spinal Cord Dorsal Horn/drug effects , Animals , Enzyme Inhibitors/therapeutic use , Gene Expression Regulation, Enzymologic/drug effects , HEK293 Cells , Humans , Male , Neuralgia/pathology , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Phosphorylation/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Rats , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/chemistry , Receptors, N-Methyl-D-Aspartate/metabolism , Tyrosine/metabolism , src-Family Kinases/chemistry , src-Family Kinases/metabolism
17.
Neuroscience ; 371: 155-165, 2018 02 10.
Article in English | MEDLINE | ID: mdl-29229558

ABSTRACT

The δ subunit-containing γ-Aminobutyric acid type A receptors (δ-GABAARs) are located at extrasynaptic sites and persistently active in the control of neuronal excitability. Here we recorded primary afferent C fiber-evoked field potentials in the superficial dorsal horn of rat spinal cords in vivo and investigated the possible influence of δ-GABAARs activities on nociceptive synaptic transmission. We found that δ-GABAARs-preferring agonist 4,5,6,7-tetrahydroisoxazolol [4,5-c] pyridine-3-ol (THIP), when topically applied onto spinal cord dorsum, inhibited the basal synaptic responses in a dose-dependent manner. Low-frequency stimulation (LFS) of sciatic nerves elicited long-term potentiation (LTP) of C fiber transmission, a synaptic correlate of central sensitization. Pretreatment with THIP before LFS delivery blocked the induction of LTP. When applied at 30 min and 180 min post-LFS, THIP reduced the magnitudes of established LTP. Intraplantar injection of formalin naturally evoked LTP in anesthetized rats. Spinal administration of THIP not only reversed formalin-induced LTP, but alleviated the spontaneous painful behaviors and mechanical hyperalgesia. Biochemical analysis demonstrated that δ-GABAARs activation by THIP decreased the synaptic expression and phosphorylation of AMPA receptor GluA1 subunit in formalin-injected rats, and meanwhile, increased synaptic GluA2 content, allowing the switch of GluA2-lacking AMPA receptors to GluA2-containing ones at synapses. THIP also suppressed the synaptic accumulation and phosphorylation of NMDA receptor GluN1 subunit in formalin-injected rats. Our data suggested that enhanced δ-GABAARs activities blunted the initiation and maintenance of spinal LTP, which correlated with the amelioration of central sensitization of nociceptive behaviors.


Subject(s)
Long-Term Potentiation/physiology , Pain/metabolism , Receptors, GABA-A/metabolism , Spinal Cord/metabolism , Animals , Dose-Response Relationship, Drug , Formaldehyde , GABA-A Receptor Agonists/pharmacology , Isoxazoles/pharmacology , Long-Term Potentiation/drug effects , Male , Nerve Fibers, Unmyelinated/drug effects , Nerve Fibers, Unmyelinated/metabolism , Pain/drug therapy , Rats, Sprague-Dawley , Receptors, AMPA/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Sciatic Nerve/metabolism , Sciatic Nerve/physiology , Spinal Cord/drug effects , Synapses/drug effects , Synapses/metabolism
18.
Neuropharmacology ; 126: 158-167, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28882563

ABSTRACT

Adenosine is present at the extracellular space within spinal cord dorsal horn and engaged in the processing of nociceptive sensory signals. Systemic or spinal administration of exogenous adenosine produces a potent analgesia against pathological pain. Here we found that inhibitory glycinergic neurotransmission was an important target for adenosine regulation. In spinal cord slices from intact rats, adenosine increased the inhibitory postsynaptic currents mediated by glycine receptors (GlyRs). In spinal slices from Complete Freund's Adjuvant-injected rats, adenosine potentiated glycinergic transmission to a more degree than in control rats. This synaptic potentiation was dependent on the activation of adenosine A1 receptor (A1R), and attributed to the modification of postsynaptic GlyRs function. The Gi protein-coupled A1R typically signals through Gαi/cAMP-dependent protein kinase (PKA) and Gßγ pathways. We found that blockade of either Gαi/PKA or Gßγ signaling attenuated the ability of adenosine to increase glycinergic synaptic responses in inflamed rats. To identify which GlyRs subunit was subjected to A1R regulation, we recorded glycine-evoked whole-cell currents in HEK293T cells co-transfected with A1R and distinct GlyRs subunit. We found that α1, the most abundant functional GlyRs subunit in adult spinal cord, was insensitive to A1R activation. However, when GlyRs α3 subunit or α1ins subunit, a longer α1 isoform, was co-expressed with A1R, adenosine caused a significant increase of glycinergic currents. Inhibition of PKA and Gßγ abolished the stimulatory effects of A1R on α3 and α1ins, respectively. These data suggested that A1R might potentiate glycinergic transmission through Gαi/PKA/α3 and Gßγ/α1ins pathways in inflamed rat.


Subject(s)
Inflammation/physiopathology , Inhibitory Postsynaptic Potentials , Receptor, Adenosine A1/physiology , Receptors, Glycine/physiology , Spinal Cord Dorsal Horn/physiology , Adenosine/administration & dosage , Adenosine/physiology , Animals , HEK293 Cells , Humans , Inflammation/metabolism , Male , Rats, Sprague-Dawley , Receptor, Adenosine A1/metabolism , Signal Transduction
19.
Neuroscience ; 352: 97-105, 2017 06 03.
Article in English | MEDLINE | ID: mdl-28389375

ABSTRACT

Striatal-enriched phosphatase 61 (STEP61) is a member of intracellular protein tyrosine phosphatases, which is involved in the regulation of synaptic plasticity and a line of neuropsychiatric disorders. This protein tyrosine phosphatase is also abundant in pain-related spinal cord dorsal horn neurons. However, whether and how this tyrosine phosphatase modulates the nociceptive plasticity and behavioral hypersensitivity remain largely unknown. The present study recorded the long-term potentiation (LTP) of primary afferent C fiber-evoked field potentials in vivo in superficial dorsal horn of rats, and tested the possible role of STEP61 in spinal LTP. We found that LTP induction significantly increased STEP61 phosphorylation at Ser221 residue, a key molecular event that has been shown to impair the phosphatase activity. The STEP61 hypoactivity allowed for the activation of three substrates, GluN2B subunit-containing N-methyl-d-aspartate-subtype glutamate receptors, Src-family protein tyrosine kinase member Fyn and extracellular signal-regulated kinase 1/2, through which the thresholds for LTP induction were noticeably decreased. To reinstate STEP61 activity, we overexpressed wild-type STEP61 [STEP61(WT)] in spinal dorsal horn, finding that STEP61(WT) completely blunted LTP induction. Behavioral tests showed that LTP blockade by STEP61(WT) correlated with a long-lasting alleviation of thermal hypersensitivity and mechanical allodynia induced by chronic constriction injury of sciatic nerves. These data implicated that STEP61 exerted a negative control over spinal nociceptive plasticity, which might have therapeutic benefit in pathological pain.


Subject(s)
Long-Term Potentiation/physiology , Neuralgia/pathology , Protein Tyrosine Phosphatases/metabolism , Sensory Receptor Cells/enzymology , Spinal Cord Dorsal Horn/pathology , Afferent Pathways/physiopathology , Animals , Butadienes/pharmacology , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hyperalgesia/pathology , Long-Term Potentiation/drug effects , Male , Nerve Fibers/physiology , Nitriles/pharmacology , Pain Measurement , Protein Tyrosine Phosphatases/genetics , Proto-Oncogene Proteins c-fyn/metabolism , Rats , Rats, Sprague-Dawley , Sensory Receptor Cells/drug effects , Transduction, Genetic
20.
Pain ; 157(3): 652-665, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26882347

ABSTRACT

The enzymatic activity of protein tyrosine kinase Src is subjected to the regulation by C-terminal Src kinase (CSK) and protein tyrosine phosphatases (PTPs). Aberrant Src activation in the spinal cord dorsal horn is pivotal for the induction and development of nociceptive behavioral sensitization. In this study, we found that paxillin, one of the well-characterized cell adhesion components involved in cell migration and survival, integrated CSK and PTPs' signaling to regulate Src-dependent nociceptive plasticity. Paxillin localized at excitatory glutamatergic synapses in the spinal dorsal horn of mice, and the phosphorylation of Tyr118 on paxillin was necessary to associate with and target CSK at synapses. After peripheral tissue injury, the enhanced neuronal activity stimulated N-methyl-D-aspartate (NMDA) subtype glutamate receptors, which initiated PTPs' signaling to catalyze Tyr118 dephosphorylation. The reduced Tyr118 phosphorylation disrupted paxillin interaction with CSK, leading to the dispersal of CSK out of synapses. With the loss of CSK-mediated inhibition, Src activity was persistently increased. The active Src potentiated the synaptic transmission specifically mediated by GluN2B subunit-containing NMDA receptors. The active Src also facilitated the induction of long-term potentiation of C fiber-evoked field potentials and exaggerated painful responses. In complete Freund's adjuvant-injected mice, viral expression of phosphomimicking paxillin mutant to resume CSK synaptic localization repressed Src hyperactivity. Meanwhile, this phosphomimicking paxillin mutant blunted NMDA receptor-mediated synaptic transmission and alleviated chronic inflammatory pain. These data showed that PTPs-mediated dephosphorylation of paxillin at Tyr118 was involved in the modification of nociceptive plasticity through CSK-Src signaling.


Subject(s)
Neuronal Plasticity/physiology , Pain/metabolism , Paxillin/metabolism , Spinal Cord Dorsal Horn/metabolism , Animals , HEK293 Cells , Humans , Male , Mice , Pain/pathology , Phosphorylation/physiology , Rats , Rats, Sprague-Dawley , Spinal Cord Dorsal Horn/pathology
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