Emerging Molecular and Synaptic Targets for the Management of Chronic Pain Caused by Systemic Lupus Erythematosus.

Patients with systemic lupus erythematosus (SLE) frequently experience chronic pain due to the limited effectiveness and safety profiles of current analgesics. Understanding the molecular and synaptic mechanisms underlying abnormal neuronal activation along the pain signaling pathway is essential for developing new analgesics to address SLE-induced chronic pain. Recent studies, including those conducted by our team and others using the SLE animal model (MRL/lpr lupus-prone mice), have unveiled heightened excitability in nociceptive primary sensory neurons within the dorsal root ganglia and increased glutamatergic synaptic activity in spinal dorsal horn neurons, contributing to the development of chronic pain in mice with SLE. Nociceptive primary sensory neurons in lupus animals exhibit elevated resting membrane potentials, and reduced thresholds and rheobases of action potentials. These changes coincide with the elevated production of TNFα and IL-1ß, as well as increased ERK activity in the dorsal root ganglion, coupled with decreased AMPK activity in the same region. Dysregulated AMPK activity is linked to heightened excitability in nociceptive sensory neurons in lupus animals. Additionally, the increased glutamatergic synaptic activity in the spinal dorsal horn in lupus mice with chronic pain is characterized by enhanced presynaptic glutamate release and postsynaptic AMPA receptor activation, alongside the reduced activity of glial glutamate transporters. These alterations are caused by the elevated activities of IL-1ß, IL-18, CSF-1, and thrombin, and reduced AMPK activities in the dorsal horn. Furthermore, the pharmacological activation of spinal GPR109A receptors in microglia in lupus mice suppresses chronic pain by inhibiting p38 MAPK activity and the production of both IL-1ß and IL-18, as well as reducing glutamatergic synaptic activity in the spinal dorsal horn. These findings collectively unveil crucial signaling molecular and synaptic targets for modulating abnormal neuronal activation in both the periphery and spinal dorsal horn, offering insights into the development of analgesics for managing SLE-induced chronic pain.

Activation of microglial GPR109A alleviates thermal hyperalgesia in female lupus mice by suppressing IL-18 and glutamatergic synaptic activity.

Many patients with systemic lupus erythematosus (SLE) live with chronic pain despite advances in medical management in reducing mortality related to SLE. Few animal studies have addressed mechanisms and treatment for chronic pain caused by SLE. In this study, we provide the first evidence for the analgesic effects of a GPR109A specific agonist (MK1903) and its action mechanisms in thermal hyperalgesia in female MRL/lpr mice, an SLE mouse model. Specifically, we show that MRL/lpr mice had a higher sensitivity to thermal stimuli at age 11-16 weeks, which was accompanied with significantly microglial and astrocytic activation, increases in p38 MAPK and glutamatergic synaptic activities in the spinal dorsal horn. We demonstrate that thermal hyperalgesia in MRL/lpr mice was significantly attenuated by intrathecal injection of MK1903. GPR109A was expressed in spinal microglia but not astrocytes or neurons. Its expression was significantly increased in MRL/lpr mice with thermal hyperalgesia. Activation of GPR109A receptors in microglia attenuated glutamatergic synaptic activity via suppressing production of interleukin-18 (IL-18). We provide evidence that activation of GPR109A attenuated thermal hyperalgesia in the SLE animal model via suppressing p38 MAPK activity and production of IL-18. Our study suggests that targeting the microglial GPR109A is a potent approach for reversing spinal neuroinflammation, abnormal excitatory synaptic activity, and management of thermal hyperalgesia caused by SLE.

Phytohormone abscisic acid ameliorates neuropathic pain via regulating LANCL2 protein abundance and glial activation at the spinal cord.

Spinal neuroinflammation plays a critical role in the genesis of neuropathic pain. Accumulating data suggest that abscisic acid (ABA), a phytohormone, regulates inflammatory processes in mammals. In this study, we found that reduction of the LANCL2 receptor protein but not the agonist ABA in the spinal cord is associated with the genesis of neuropathic pain. Systemic or intrathecal administration of ABA ameliorates the development and pre-existence of mechanical allodynia and heat hyperalgesia in animals with partial sciatic nerve ligation (pSNL). LANCL2 is expressed only in microglia in the spinal dorsal horn. Pre-emptive treatment with ABA attenuates activation of microglia and astrocytes, ERK activity, and TNFα protein abundance in the dorsal horn in rats with pSNL. These are accompanied by restoration of spinal LANCL2 protein abundance. Spinal knockdown of LANCL2 gene with siRNA recapitulates the behavioral and spinal molecular changes induced by pSNL. Activation of spinal toll-like receptor 4 (TLR4) with lipopolysaccharide leads to activation of microglia, and over production of TNFα, which are concurrently accompanied by suppression of protein levels of LANCL2 and peroxisome proliferator activated-receptor γ. These changes are ameliorated when ABA is added with LPS. The anti-inflammatory effects induced by ABA do not requires Gi protein activity. Our study reveals that the ABA/LANCL2 system is a powerful endogenous system regulating spinal neuroinflammation and nociceptive processing, suggesting the potential utility of ABA as the management of neuropathic pain.

Interleukin-1beta released by microglia initiates the enhanced glutamatergic activity in the spinal dorsal horn during paclitaxel-associated acute pain syndrome.

Patients receiving paclitaxel for cancer treatment often develop an acute pain syndrome (paclitaxel-associated acute pain syndrome, P-APS), which occurs immediately after paclitaxel treatment. Mechanisms underlying P-APS remain largely unknown. We recently reported that rodents receiving paclitaxel develop acute pain and activation of spinal microglial toll like receptor 4 (TLR4) by paclitaxel penetrating into the spinal cord is a critical event in the genesis of P-APS. Our current study dissected cellular and molecular mechanisms underlying the P-APS. We demonstrated that bath-perfusion of paclitaxel, at a concentration similar to that found in the cerebral spinal fluid in animals receiving i.v. paclitaxel (2 mg/kg), resulted in increased calcium activity in microglia instantly, and in astrocytes with 6 min delay. TLR4 activation in microglia by paclitaxel caused microglia to rapidly release interleukin-1ß (IL-1ß) but not tumor necrosis factor α, IL-6, or interferon-γ. IL-1ß release from microglia depended on capthepsin B. IL-1ß acted on astrocytes, leading to elevated calcium activity and suppressed glutamate uptake. IL-1ß also acted on neurons to increase presynaptic glutamate release and postsynaptic AMPA receptor activity in the spinal dorsal horn. Knockout of IL-1 receptors prevented the development of acute pain induced by paclitaxel in mice. Our study indicates that IL-1ß is a crucial molecule used by microglia to alter functions in astrocytes and neurons upon activation of TLR4 in the genesis of P-APS, and targeting the signaling pathways regulating the production and function of IL-1ß from microglia is a potential avenue for the development of analgesics for the treatment of P-APS.

Regulator of G Protein Signaling 10 (Rgs10) Expression Is Transcriptionally Silenced in Activated Microglia by Histone Deacetylase Activity.

RGS10 has emerged as a key regulator of proinflammatory cytokine production in microglia, functioning as an important neuroprotective factor. Although RGS10 is normally expressed in microglia at high levels, expression is silenced in vitro following activation of TLR4 receptor. Given the ability of RGS10 to regulate inflammatory signaling, dynamic regulation of RGS10 levels in microglia may be an important mechanism to tune inflammatory responses. The goals of the current study were to confirm that RGS10 is suppressed in an in vivo inflammatory model of microglial activation and to determine the mechanism for activation-dependent silencing of Rgs10 expression in microglia. We demonstrate that endogenous RGS10 is present in spinal cord microglia, and RGS10 protein levels are suppressed in the spinal cord in a nerve injury-induced neuropathic pain mouse model. We show that the histone deacetylase (HDAC) enzyme inhibitor trichostatin A blocks the ability of lipopolysaccharide (LPS) to suppress Rgs10 transcription in BV-2 and primary microglia, demonstrating that HDAC enzymes are required for LPS silencing of Rgs10 Furthermore, we used chromatin immunoprecipitation to demonstrate that H3 histones at the Rgs10 proximal promoter are deacetylated in BV-2 microglia following LPS activation, and HDAC1 association at the Rgs10 promoter is enhanced following LPS stimulation. Finally, we have shown that sphingosine 1-phosphate, an endogenous microglial signaling mediator that inhibits HDAC activity, enhances basal Rgs10 expression in BV-2 microglia, suggesting that Rgs10 expression is dynamically regulated in microglia in response to multiple signals.

Chronic pain and impaired glial glutamate transporter function in lupus-prone mice are ameliorated by blocking macrophage colony-stimulating factor-1 receptors.

Systemic lupus erythematosus (SLE) is a multi-organ disease of unknown etiology in which the normal immune responses are directed against the body's own healthy tissues. Patients with SLE often suffer from chronic pain. Currently, no animal studies have been reported about the mechanisms underlying pain in SLE. In this study, the development of chronic pain in MRL lupus-prone (MRL/lpr) mice, a well-established lupus mouse model, was characterized for the first time. We found that female MRL/lpr mice developed thermal hyperalgesia at the age of 13 weeks, and mechanical allodynia at the age of 16 weeks. MRL/lpr mice with chronic pain had activation of microglia and astrocytes, over-expression of macrophage colony-stimulating factor-1 (CSF-1) and interleukin-1 beta (IL-1ß), as well as suppression of glial glutamate transport function in the spinal cord. Intrathecal injection of either the CSF-1 blocker or IL-1 inhibitor attenuated thermal hyperalgesia in MRL/lpr mice. We provide evidence that the suppressed activity of glial glutamate transporters in the spinal dorsal horn in MRL/lpr mice is caused by activation of the CSF-1 and IL-1ß signaling pathways. Our findings suggest that targeting the CSF-1 and IL-1ß signaling pathways or the glial glutamate transporter in the spinal cord is an effective approach for the management of chronic pain caused by SLE.

Blocking the GABA transporter GAT-1 ameliorates spinal GABAergic disinhibition and neuropathic pain induced by paclitaxel.

Paclitaxel is a chemotherapeutic agent widely used for treating carcinomas. Patients receiving paclitaxel often develop neuropathic pain and have a reduced quality of life which hinders the use of this life-saving drug. In this study, we determined the role of GABA transporters in the genesis of paclitaxel-induced neuropathic pain using behavioral tests, electrophysiology, and biochemical techniques. We found that tonic GABA receptor activities in the spinal dorsal horn were reduced in rats with neuropathic pain induced by paclitaxel. In normal controls, tonic GABA receptor activities were mainly controlled by the GABA transporter GAT-1 but not GAT-3. In the spinal dorsal horn, GAT-1 was expressed at presynaptic terminals and astrocytes while GAT-3 was only expressed in astrocytes. In rats with paclitaxel-induced neuropathic pain, the protein expression of GAT-1 was increased while GAT-3 was decreased. This was concurrently associated with an increase in global GABA uptake. The paclitaxel-induced attenuation of GABAergic tonic inhibition was ameliorated by blocking GAT-1 but not GAT-3 transporters. Paclitaxel-induced neuropathic pain was significantly attenuated by the intrathecal injection of a GAT-1 inhibitor. These findings suggest that targeting GAT-1 transporters for reversing disinhibition in the spinal dorsal horn may be a useful approach for treating paclitaxel-induced neuropathic pain. Patients receiving paclitaxel for cancer therapy often develop neuropathic pain and have a reduced quality of life. In this study, we demonstrated that animals treated with paclitaxel develop neuropathic pain, have enhancements of GABA transporter-1 protein expression and global GABA uptake, as well as suppression of GABAergic tonic inhibition in the spinal dorsal horn. Pharmacological inhibition of GABA transporter-1 ameliorates the paclitaxel-induced suppression of GABAergic tonic inhibition and neuropathic pain. Thus, targeting GAT-1 transporters for reversing GABAergic disinhibition in the spinal dorsal horn could be a useful approach for treating paclitaxel-induced neuropathic pain.

Paclitaxel induces acute pain via directly activating toll like receptor 4.

Paclitaxel, a powerful anti-neoplastic drug, often causes pathological pain, which significantly reduces the quality of life in patients. Paclitaxel-induced pain includes pain that occurs immediately after paclitaxel treatment (paclitaxel-associated acute pain syndrome, P-APS) and pain that persists for weeks to years after cessation of paclitaxel treatment (paclitaxel induced chronic neuropathic pain). Mechanisms underlying P-APS remain unknown. In this study, we found that paclitaxel causes acute pain in rodents in a dose-dependent manner. The paclitaxel-induced acute pain occurs within 2 hrs after a single intravenous injection of paclitaxel. This is accompanied by low levels of paclitaxel penetrating into the cerebral spinal fluid and spinal dorsal horn. We demonstrated that an intrathecal injection of paclitaxel induces mechanical allodynia in a dose-dependent manner. Paclitaxel causes activation of toll like receptor 4 (TLR4) in the spinal dorsal horn and dorsal root ganglions. Through activating TLR4, paclitaxel increases glutamatergic synaptic activities and reduces glial glutamate transporter activities in the dorsal horn. Activations of TLR4 are necessary in the genesis of paclitaxel-induced acute pain. The cellular and molecular signaling pathways revealed in this study could provide rationales for the development of analgesics and management strategies for P-APS in patients.

Activation of toll like receptor 4 attenuates GABA synthesis and postsynaptic GABA receptor activities in the spinal dorsal horn via releasing interleukin-1 beta.

Toll like receptor 4 (TLR4) is an innate immune pattern recognition receptor, expressed predominantly on microglia in the CNS. Activation of spinal TLR4 plays a critical role in the genesis of pathological pain induced by nerve injury, bone cancer, and tissue inflammation. Currently, it remains unknown how synaptic activities in the spinal dorsal horn are regulated by TLR4 receptors. Through recording GABAergic currents in neurons and glial glutamate transporter currents in astrocytes in rodent spinal slices, we determined whether and how TLR4 modulates GABAergic synaptic activities in the superficial spinal dorsal horn. We found that activation of TLR4 by lipopolysaccharide (LPS) reduces GABAergic synaptic activities through both presynaptic and postsynaptic mechanisms. Specifically, LPS causes the release of IL-1ß from microglia. IL-1ß in turn suppresses GABA receptor activities at the postsynaptic site through activating protein kinase C (PKC) in neurons. GABA synthesis at the presynaptic site is reduced upon activation of TLR4. Glial glutamate transporter activities are suppressed by IL-1ß and PKC activation induced by LPS. The suppression of glial glutamate transporter activities leads to a deficiency of glutamine supply, which results in an attenuation of the glutamate-glutamine cycle-dependent GABA synthesis. These findings shed light on understanding synaptic plasticity induced by activation of TLR4 under neuroinflammation and identify GABA receptors, glial glutamate transporters, IL-1ß and PKC as therapeutic targets to abrogate abnormal neuronal activities following activation of TLR4 in pathological pain conditions.

Adenosine Monophosphate-activated Protein Kinase Regulates Interleukin-1ß Expression and Glial Glutamate Transporter Function in Rodents with Neuropathic Pain.

BACKGROUND: Neuroinflammation and dysfunctional glial glutamate transporters (GTs) in the spinal dorsal horn are implicated in the genesis of neuropathic pain. The authors determined whether adenosine monophosphate-activated protein kinase (AMPK) in the spinal dorsal horn regulates these processes in rodents with neuropathic pain. METHODS: Hind paw withdrawal responses to radiant heat and mechanical stimuli were used to assess nociceptive behaviors. Spinal markers related to neuroinflammation and glial GTs were determined by Western blotting. AMPK activities were manipulated pharmacologically and genetically. Regulation of glial GTs was determined by measuring protein expression and activities of glial GTs. RESULTS: AMPK activities were reduced in the spinal dorsal horn of rats (n = 5) with thermal hyperalgesia induced by nerve injury, which were accompanied with the activation of astrocytes, increased production of interleukin-1ß and activities of glycogen synthase kinase 3ß, and suppressed protein expression of glial glutamate transporter-1. Thermal hyperalgesia was reversed by spinal activation of AMPK in neuropathic rats (n = 10) and induced by inhibiting spinal AMPK in naive rats (n = 7 to 8). Spinal AMPKα knockdown (n = 6) and AMPKα1 conditional knockout (n = 6) induced thermal hyperalgesia and mechanical allodynia. These genetic alterations mimicked the changes of molecular markers induced by nerve injury. Pharmacological activation of AMPK enhanced glial GT activity in mice with neuropathic pain (n = 8) and attenuated glial glutamate transporter-1 internalization induced by interleukin-1ß (n = 4). CONCLUSIONS: These findings suggest that enhancing spinal AMPK activities could be an effective approach for the treatment of neuropathic pain.

Endogenous interleukin-1ß in neuropathic rats enhances glutamate release from the primary afferents in the spinal dorsal horn through coupling with presynaptic N-methyl-D-aspartic acid receptors.

Excessive activation of glutamate receptors and overproduction of proinflammatory cytokines, including interleukin-1ß (IL-1ß) in the spinal dorsal horn, are key mechanisms underlying the development and maintenance of neuropathic pain. In this study, we investigated the mechanisms by which endogenous IL-1ß alters glutamatergic synaptic transmission in the spinal dorsal horn in rats with neuropathic pain induced by ligation of the L5 spinal nerve. We demonstrated that endogenous IL-1ß in neuropathic rats enhances glutamate release from the primary afferent terminals and non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Myeloid differentiation primary response protein 88 (MyD88) is a mediator used by IL-1ß to enhance non-NMDA glutamate receptor activities in postsynaptic neurons in the spinal dorsal horn. Presynaptic NMDA receptors are effector receptors used by the endogenous IL-1ß to enhance glutamate release from the primary afferents in neuropathic rats. This is further supported by the fact that NMDA currents recorded from small neurons in the dorsal root ganglion of normal rats are potentiated by exogenous IL-1ß. Furthermore, we provided evidence that functional coupling between IL-1ß receptors and presynaptic NMDA receptors at the primary afferent terminals is mediated by the neutral sphingomyelinase/ceramide signaling pathway. Hence, functional coupling between IL-1ß receptors and presynaptic NMDA receptors at the primary afferent terminals is a crucial mechanism leading to enhanced glutamate release and activation of non-NMDA receptors in the spinal dorsal horn neurons in neuropathic pain conditions. Interruption of such functional coupling could be an effective approach for the treatment of neuropathic pain.

Interleukin-1 beta enhances endocytosis of glial glutamate transporters in the spinal dorsal horn through activating protein kinase C.

Excessive activation of glutamate receptors in spinal dorsal horn neurons is a key mechanism leading to abnormal neuronal activation in pathological pain conditions. Previous studies have shown that activation of glutamate receptors in the spinal dorsal horn is enhanced by impaired glial glutamate transporter functions and proinflammatory cytokines including interleukin-1 beta (IL-1ß). In this study, we for the first time revealed that spinal glial glutamate transporter activities in the neuropathic animals are attenuated by endogenous IL-1ß. Specifically, we demonstrated that nerve injury results in an increased expression of IL-1ß and activation of PKC in the spinal dorsal horn as well as suppression of glial glutamate uptake activities. We provided evidence that the nerve-injury induced suppression of glial glutamate uptake is at least in part ascribed to endogenous IL-1ß and activation of PKC in the spinal dorsal horn. IL-1ß reduces glial glutamate transporter activities through enhancing the endocytosis of both GLT-1 and GLAST glial glutamate transporters. The IL-1ß induced trafficking of glial glutamate transporters is through the calcium/PKC signaling pathway, and the dynamin-dependent endocytosis, which is dependent on the integrity of actin filaments. The signaling pathway regulating glial glutamate transporters revealed in this study provides novel targets to attenuate aberrant activation of glutamate receptors in the spinal dorsal horn, which could ultimately help the development of analgesics.

Endogenous activation of presynaptic NMDA receptors enhances glutamate release from the primary afferents in the spinal dorsal horn in a rat model of neuropathic pain.

Activation of N-methyl-D-aspartate (NMDA) receptors (NMDARs) is a crucial mechanism underlying the development and maintenance of pain. Traditionally, the role of NMDARs in the pathogenesis of pain is ascribed to their activation and signalling cascades in postsynaptic neurons. In this study, we determined if presynaptic NMDARs in the primary afferent central terminals play a role in synaptic plasticity of the spinal first sensory synapse in a rat model of neuropathic pain induced by spinal nerve ligation. Excitatory postsynaptic currents (EPSCs) were recorded from superficial dorsal horn neurons of spinal slices taken from young adult rats. We showed that increased glutamate release from the primary afferents contributed to the enhanced amplitudes of EPSCs evoked by input from the primary afferents in neuropathic rats. Endogenous activation of presynaptic NMDARs increased glutamate release from the primary afferents in neuropathic rats. Presynaptic NMDARs in neuropathic rats were mainly composed of NR2B receptors. The action of presynaptic NMDARs in neuropathic rats was enhanced by exogenous D-serine and/or NMDA and dependent on activation of protein kinase C. In contrast, glutamate release from the primary afferents in sham-operated rats was not regulated by presynaptic NMDARs. We demonstrated that the lack of NMDAR-mediated regulation of glutamate release in sham-operated rats was not attributable to low extracellular levels of the NMDAR agonist and/or coagonist (D-serine), but rather was due to the insufficient function and/or number of presynaptic NMDARs. This was supported by an increase of NR2B receptor protein expression in both the dorsal root ganglion and spinal dorsal horn ipsilateral to the injury site in neuropathic rats. Hence, suppression of the presynaptic NMDAR activity in the primary sensory afferents is an effective approach to attenuate the enhanced glutamatergic response in the spinal first sensory synapse induced by peripheral nerve injury, and presynaptic NMDARs might be a novel target for the development of analgesics.

A rapid analytical method for the quantification of paclitaxel in rat plasma and brain tissue by high-performance liquid chromatography and tandem mass spectrometry.

RATIONALE: Paclitaxel, an antitumor agent for the treatment of several types of cancers, has recently been reported to cause impaired cognitive function and neuropathic pain in humans. To assess the effects of paclitaxel on the central nervous system, a sensitive and accurate method is required to quantify paclitaxel concentrations in plasma and brain tissue obtained from rodents receiving paclitaxel. METHODS: The biological samples were prepared by liquid-liquid extraction and separated by a 3.5 min reversed-phase liquid chromatography (RPLC) method using a BDS Hypersil C8 column under isocratic conditions. Paclitaxel was quantified using multiple reaction monitoring (MRM) with a triple quadrupole tandem mass spectrometer working in the positive electrospray ionization (ESI+) mode. A stable isotope labeled analogue of paclitaxel was used as the internal standard (IS). RESULTS: The method was validated to be precise and accurate within the dynamic range of 0.5-100 ng/mL based on 100 µL plasma and 1.5-300 ng/g based on 33 mg of brain tissue in homogenate. This method was applied to samples from 2 mg/kg intravenously dosed rats. The plasma concentrations were observed to be 26.62 ± 8.93 ng/mL and brain concentrations 11.08 ± 4.18 ng/g when measured 4 h post-dose. CONCLUSIONS: This rapid LC/MS/MS method was validated to be sensitive, specific, precise and accurate for the quantification of paclitaxel in rat plasma and brain tissue homogenate. Application of the method to study samples provided sufficient proof of blood-brain barrier penetration of paclitaxel, allowing further investigation of its influence on the central nervous system.

Deficient AMPK activity contributes to hyperexcitability in peripheral nociceptive sensory neurons and thermal hyperalgesia in lupus mice.

Patients with systemic lupus erythematosus (SLE) often suffer from chronic pain. Little is known about the peripheral mechanisms underlying the genesis of chronic pain induced by SLE. The aim of this study was to investigate whether and how membrane properties in nociceptive neurons in the dorsal root ganglions (DRGs) are altered by SLE. We found elevation of resting membrane potentials, smaller capacitances, lower action potential thresholds and rheobases in nociceptive neurons in the DRGs from MRL/lpr mice (an SLE mouse model) with thermal hyperalgesia. DRGs from MRL/lpr mice had increased protein expressions in TNFα, IL-1ß, and phosphorylated ERK but suppressed AMPK activity, and no changes in sodium channel 1.7 protein expression. We showed that intraplantar injection of Compound C (an AMPK inhibitor) induced thermal hyperalgesia in normal mice while intraplantar injection of AICAR (an AMPK activator) reduced thermal hyperalgesia in MRL/Lpr mice. Upon inhibition of AMPK membrane properties in nociceptive neurons from normal control mice could be rapidly switched to those found in SLE mice with thermal hyperalgesia. Our study indicates that increased excitability in peripheral nociceptive sensory neurons contributes to the genesis of thermal hyperalgesia in mice with SLE, and AMPK regulates membrane properties in nociceptive sensory neurons as well as thermal hyperalgesia in mice with SLE. Our study provides a basis for targeting signaling pathways regulating membrane properties of peripheral nociceptive neurons as a means for conquering chronic pain caused by SLE.

Emerging roles of GPR109A in regulation of neuroinflammation in neurological diseases and pain.

Neuroinflammation plays a critical role in the pathological process of multiple neurological disorders and pathological pain conditions. GPR109A, a Gi protein-coupled receptor, has emerged as an important therapeutic target for controlling inflammation in various tissues and organs. In this review, we summarized current data about the role of GPR109A in neuroinflammation. Specifically, we focused on the pharmacological features of GPR109A and signaling pathways used by GPR109A to ameliorate neuroinflammation and symptoms in Alzheimer's disease, Parkinson's disease, multiple sclerosis, stroke, and pathological pain conditions.

EZH2 Methyltransferase Regulates Neuroinflammation and Neuropathic Pain.

Recent studies by us and others have shown that enhancer of zeste homolog-2 (EZH2), a histone methyltransferase, in glial cells regulates the genesis of neuropathic pain by modulating the production of proinflammatory cytokines and chemokines. In this review, we summarize recent advances in this research area. EZH2 is a subunit of polycomb repressive complex 2 (PRC2), which primarily serves as a histone methyltransferase to catalyze methylation of histone 3 on lysine 27 (H3K27), ultimately resulting in transcriptional repression. Animals with neuropathic pain exhibit increased EZH2 activity and neuroinflammation of the injured nerve, spinal cord, and anterior cingulate cortex. Inhibition of EZH2 with DZNep or GSK-126 ameliorates neuroinflammation and neuropathic pain. EZH2 protein expression increases upon activation of Toll-like receptor 4 and calcitonin gene-related peptide receptors, downregulation of miR-124-3p and miR-378 microRNAs, or upregulation of Lncenc1 and MALAT1 long noncoding RNAs. Genes suppressed by EZH2 include suppressor of cytokine signaling 3 (SOCS3), nuclear factor (erythroid-derived 2)-like-2 factor (NrF2), miR-29b-3p, miR-146a-5p, and brain-specific angiogenesis inhibitor 1 (BAI1). Pro-inflammatory mediators facilitate neuronal activation along pain-signaling pathways by sensitizing nociceptors in the periphery, as well as enhancing excitatory synaptic activities and suppressing inhibitory synaptic activities in the CNS. These studies collectively reveal that EZH2 is implicated in signaling pathways known to be key players in the process of neuroinflammation and genesis of neuropathic pain. Therefore, targeting the EZH2 signaling pathway may open a new avenue to mitigate neuroinflammation and neuropathic pain.

Glial glutamate transporter and glutamine synthetase regulate GABAergic synaptic strength in the spinal dorsal horn.

Decreased GABAergic synaptic strength ('disinhibition') in the spinal dorsal horn is a crucial mechanism contributing to the development and maintenance of pathological pain. However, mechanisms leading to disinhibition in the spinal dorsal horn remain elusive. We investigated the role of glial glutamate transporters (GLT-1 and GLAST) and glutamine synthetase in maintaining GABAergic synaptic activity in the spinal dorsal horn. Electrically evoked GABAergic inhibitory post-synaptic currents (eIPSCs), spontaneous IPSCs (sIPSCs) and miniature IPSCs were recorded in superficial spinal dorsal horn neurons of spinal slices from young adult rats. We used (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), to block both GLT-1 and GLAST and dihydrokainic acid to block only GLT-1. We found that blockade of both GLAST and GLT-1 and blockade of only GLT-1 in the spinal dorsal horn decreased the amplitude of GABAergic eIPSCs, as well as both the amplitude and frequency of GABAergic sIPSCs or miniature IPSCs. Pharmacological inhibition of glial glutamine synthetase had similar effects on both GABAergic eIPSCs and sIPSCs. We provided evidence demonstrating that the reduction in GABAergic strength induced by the inhibition of glial glutamate transporters is due to insufficient GABA synthesis through the glutamate-glutamine cycle between astrocytes and neurons. Thus, our results indicate that deficient glial glutamate transporters and glutamine synthetase significantly attenuate GABAergic synaptic strength in the spinal dorsal horn, which may be a crucial synaptic mechanism underlying glial-neuronal interactions caused by dysfunctional astrocytes in pathological pain conditions.

Impaired glial glutamate uptake induces extrasynaptic glutamate spillover in the spinal sensory synapses of neuropathic rats.

Glial cell dysfunction and excessive glutamate receptor activation in spinal dorsal horn neurons are hallmark mechanisms of pathological pain. The way in which glial cell dysfunction leads to excessive glutamate receptor activation in the spinal sensory synapses remains unknown. We and others recently reported the downregulation of glial glutamate transporter (GT) protein expression in the spinal dorsal horn of neuropathic rats. In this study, we showed that excitatory postsynaptic currents originating from N-methyl-d-aspartate receptor activation (NMDA EPSCs) elicited by peripheral synaptic input in the spinal sensory synapses were enhanced in neuropathic rats with mechanical allodynia induced by partial sciatic nerve ligation. The enhanced NMDA EPSCs were accompanied by an increased proportion of NR2B receptor activation. Physically blocking the extrasynaptic glutamate with dextran or chemically scavenging the glutamate with glutamic-pyruvic transaminase ameliorated the abnormal NMDA EPSCs in neuropathic rats. Pharmacological blockade of glial GTs with dihydrokainic acid enhanced NMDA receptor activation elicited by synaptic input or puffed glutamate in normal control rats, but this effect was precluded in neuropathic rats. Thus extrasynaptic glutamate spillover and extrasynaptic NMDA receptor activation induced by deficient glial glutamate uptake in the synapses resulted in the excessive activation of NMDA receptors in neuropathic rats. It is suggested that extrasynaptic glutamate spillover may be a key synaptic mechanism related to phenotypic alterations induced by nerve injury in the spinal dorsal horn and that glial GTs are potential new targets in the development of analgesics.

Bidirectional neuron-glia interactions triggered by deficiency of glutamate uptake at spinal sensory synapses.

Bidirectional interactions between neurons and glial cells are crucial to the genesis of pathological pain. The mechanisms regulating these interactions and the role of this process in relaying synaptic input in the spinal dorsal horn remain to be established. We studied the role of glutamate transporters in the regulation of such interactions. On pharmacological blockade of glutamate transporters, slow inward currents (SICs) appeared spontaneously and/or were evoked by peripheral synaptic input in the spinal superficial dorsal horn neurons, including the spinothalamic tract neurons. We showed that the SICs were induced by the release of glutamate from glial cells. On inhibition of glutamate uptake, the stimulation-induced, synaptically released glutamate activated glial cells and caused glial cells to release glutamate. Glial-derived glutamate acted on extrasynaptic N-methyl-d-aspartate (NMDA) receptors mainly composed of NR2B receptors and generated SICs, which led to depolarization and action potential generation in superficial spinal dorsal horn neurons. Thus glutamate transporters regulate glutamatergic neuron-glia interactions at spinal sensory synapses. When glutamate uptake is impaired, glial cells function like excitatory interneurons-they are activated by peripheral synaptic input and release glutamate to activate postsynaptic neurons in spinal pain pathways.