Tonic NMDAR Currents of NR2A-Containing NMDARs Represent Altered Ambient Glutamate Concentration in the Supraoptic Nucleus.

NMDA receptors (NMDARs) modulate glutamatergic excitatory tone in the brain via two complementary modalities: a phasic excitatory postsynaptic current and a tonic extrasynaptic modality. Here, we demonstrated that the tonic NMDAR-current (I NMDA) mediated by NR2A-containing NMDARs is an efficient biosensor detecting the altered ambient glutamate level in the supraoptic nucleus (SON). I NMDA of magnocellular neurosecretory cells (MNCs) measured by nonselective NMDARs antagonist, AP5, at holding potential (V holding) -70 mV in low concentration of ECF Mg2+ ([Mg2+]o) was transiently but significantly increased 1-week post induction of a DOCA salt hypertensive model rat which was compatible with that induced by a NR2A-selective antagonist, PEAQX (I PEAQX) in both DOCA-H2O and DOCA-salt groups. In agreement, NR2B antagonist, ifenprodil, or NR2C/D antagonist, PPDA, did not affect the holding current (I holding) at V holding -70 mV. Increased ambient glutamate by exogenous glutamate (10 mM) or excitatory amino acid transporters (EAATs) antagonist (TBOA, 50 mM) abolished the I PEAQX difference between two groups, suggesting that attenuated EAATs activity increased ambient glutamate concentration, leading to the larger I PEAQX in DOCA-salt rats. In contrast, only ifenprodil but not PEAQX and PPDA uncovered I NMDA at V holding +40 mV under 1.2 mM [Mg2+]o condition. I ifenprodil was not different in DOCA-H2O and DOCA-salt groups. Finally, NR2A, NR2B, and NR2D protein expression were not different in the SON of the two groups. Taken together, NR2A-containing NMDARs efficiently detected the increased ambient glutamate concentration in the SON of DOCA-salt hypertensive rats due to attenuated EAATs activity.

Tonic Activation of NR2D-Containing NMDARs Exacerbates Dopaminergic Neuronal Loss in MPTP-Injected Parkinsonian Mice.

NR2D subunit-containing NMDA receptors (NMDARs) gradually disappear during brain maturation but can be recruited by pathophysiological stimuli in the adult brain. Here, we report that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication recruited NR2D subunit-containing NMDARs that generated an Mg2+-resistant tonic NMDA current (INMDA) in dopaminergic (DA) neurons in the midbrain of mature male mice. MPTP selectively generated an Mg2+-resistant tonic INMDA in DA neurons in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA). Consistently, MPTP increased NR2D but not NR2B expression in the midbrain regions. Pharmacological or genetic NR2D interventions abolished the generation of Mg2+-resistant tonic INMDA in SNpc DA neurons, and thus attenuated subsequent DA neuronal loss and gait deficits in MPTP-treated mice. These results show that extrasynaptic NR2D recruitment generates Mg2+-resistant tonic INMDA and exacerbates DA neuronal loss, thus contributing to MPTP-induced Parkinsonism. The state-dependent NR2D recruitment could be a novel therapeutic target for mitigating cell type-specific neuronal death in neurodegenerative diseases.SIGNIFICANCE STATEMENT NR2D subunit-containing NMDA receptors (NMDARs) are widely expressed in the brain during late embryonic and early postnatal development, and then downregulated during brain maturation and preserved at low levels in a few regions of the adult brain. Certain stimuli can recruit NR2D subunits to generate tonic persistent NMDAR currents in nondepolarized neurons in the mature brain. Our results show that MPTP intoxication recruits NR2D subunits in midbrain dopaminergic (DA) neurons, which leads to tonic NMDAR current-promoting dopaminergic neuronal death and consequent abnormal gait behavior in the MPTP mouse model of Parkinson's disease (PD). This is the first study to indicate that extrasynaptic NR2D recruitment could be a target for preventing neuronal death in neurodegenerative diseases.

MAO-B Inhibitor, KDS2010, Alleviates Spinal Nerve Ligation-induced Neuropathic Pain in Rats Through Competitively Blocking the BDNF/TrkB/NR2B Signaling.

MAO-B inhibitors have been implicated to reverse neuropathic pain behaviors. Our previous study has demonstrated that KDS2010 (KDS), a newly developed reversible MAO-B inhibitor, could attenuate Paclitaxel (PTX)-induced tactile hypersensitivity in mice through suppressing reactive oxidant species (ROS)-decreased inhibitory GABA synaptic transmission in the spinal cord. In this study, we evaluated the analgesic effect of KDS under a new approach, in which KDS acts on dorsal horn sensory neurons to reduce excitatory transmission. Oral administration of KDS effectively enhanced mechanical thresholds in the spinal nerve ligation (SNL) induced neuropathic pain in rats. Moreover, we discovered that although treatment with KDS increased brain-derived neurotrophic factor (BDNF) levels, KDS inhibited Tropomyosin receptor kinase B (TrkB) receptor activation, suppressing increased p-NR2B-induced hyperexcitability in spinal dorsal horn sensory neurons after nerve injury. In addition, KDS showed its anti-inflammatory effects by reducing microgliosis and astrogliosis and the activation of MAPK and NF-ᴋB inflammatory pathways in these glial cells. The levels of ROS production in the spinal cords after the SNL procedure were also decreased with KDS treatment. Taken together, our results suggest that KDS may represent a promising therapeutic option for treating neuropathic pain. PERSPECTIVE: Our study provides evidence suggesting the mechanisms by which KDS, a novel MAO-B inhibitor, can be effective in pain relief. KDS, by targeting multiple mechanisms involved in BDNF/TrkB/NR2B-related excitatory transmission and neuroinflammation, may represent the next future of pain medicine.

Targeting spinal microglia with fexofenadine-loaded nanoparticles prolongs pain relief in a rat model of neuropathic pain.

Targeting microglial activation is emerging as a clinically promising drug target for neuropathic pain treatment. Fexofenadine, a histamine receptor 1 antagonist, is a clinical drug for the management of allergic reactions as well as pain and inflammation. However, the effect of fexofenadine on microglial activation and pain behaviors remains elucidated. Here, we investigated nanomedicinal approach that targets more preferentially microglia and long-term analgesics. Fexofenadine significantly abolished histamine-induced microglial activation. The fexofenadine-encapsulated poly(lactic-co-glycolic acid) nanoparticles (Fexo NPs) injection reduced the pain sensitivity of spinal nerve ligation rats in a dose-dependent manner. This alleviation was sustained for 4 days, whereas the effective period by direct fexofenadine injection was 3 h. Moreover, Fexo NPs inhibited microglial activation, inflammatory signaling, cytokine release, and a macrophage phenotype shift towards the alternative activated state in the spinal cord. These results show that Fexo NPs exhibit drug repositioning promise as a long-term treatment modality for neuropathic pain.

Antiallodynic effects of KDS2010, a novel MAO-B inhibitor, via ROS-GABA inhibitory transmission in a paclitaxel-induced tactile hypersensitivity model.

Monoamine oxidase (MAO) inhibitors have been investigated for the treatment of neuropathic pain. Here, we assessed the antiallodynic effects of a novel MAO-B inhibitor, KDS2010, on paclitaxel (PTX)-induced mechanical hypersensitivity. Oral administration of KDS2010 effectively relieved PTX-induced mechanical hypersensitivity in a dose-dependent manner. KDS2010 (25 mg/Kg) significantly prevented and suppressed PTX-induced pain responses with minimal effects on the body weight, motor activity, and working memory. KDS2010 significantly reduced reactive astrocytosis and reactive oxygen species (ROS) level in the L4-L6 spinal cord of PTX-treated mice. Furthermore, KDS2010 reversed the attenuation of GABAergic spontaneous inhibitory postsynaptic current (sIPSC) frequency in spinal dorsal horn neurons, although it failed to restore the reduced tonic GABAA inhibition nor the increased GABA transporter 1 (GAT1) expression in PTX-treated mice. In addition, bath application of a reactive oxygen species (ROS) scavenger (PBN) restored the sIPSC frequency in PTX-treated mice but not in control and PTX + KDS2010-treated mice. These results indicated that the antiallodynic effect of KDS2010 is not due to a MAO-B-dependent GABA production. Finally, PBN alone also exerted a similar analgesic effect as KDS2010, but a co-treatment of PBN with KDS2010 showed no additive effect, suggesting that inhibition of MAO-B-dependent ROS production is responsible for the analgesic effect by KDS2010 on PTX-induced allodynia. Overall, KDS2010 attenuated PTX-induced pain behaviors by restoring the altered ROS level and GABAergic inhibitory signaling in the spinal cord, suggesting that KDS2010 is a promising therapeutic strategy for chemotherapy-induced peripheral neuropathy.

Sigma-1 receptor increases intracellular calcium in cultured astrocytes and contributes to mechanical allodynia in a model of neuropathic pain.

Recent studies have revealed that glial sigma-1 receptor (Sig-1R) in the spinal cord may be a critical factor to mediate sensory function. However, the functional role of Sig-1R in astrocyte has not been clearly elucidated. Here, we determined whether Sig-1Rs modulate calcium responses in primary cultured astrocytes and pathological changes in spinal astrocytes, and whether they contribute to pain hypersensitivity in naïve mice and neuropathic pain following chronic constriction injury (CCI) of the sciatic nerve in mice. Sig-1R was expressed in glial fibrillary acidic protein (GFAP)-positive cultured astrocytes. Treatment with the Sig-1R agonist, PRE-084 or neurosteroid dehydroepiandrosterone (DHEA) increased intracellular calcium responses in cultured astrocytes, and this increase was blocked by the pretreatment with the Sig-1R antagonist, BD-1047 or neurosteroid progesterone. Intrathecal administration of PRE-084 or DHEA for 10 days induced mechanical and thermal hypersensitivity and increased the number of Sig-1R-immunostained GFAP-positive cells in the superficial dorsal horn (SDH) region of the spinal cord in naïve mice, and these changes were inhibited by administration with BD-1047 or progesterone. In CCI mice, intrathecal administration of BD-1047 or progesterone at post-operative day 14 suppressed the developed mechanical allodynia and the number of Sig-1R-immunostained GFAP-positive cells that were increased in the SDH region of the spinal cord following CCI of the sciatic nerve. These results demonstrate that Sig-1Rs play an important role in the modulation of intracellular calcium responses in cultured astrocytes and pathological changes in spinal astrocytes and that administration of BD-1047 or progesterone alleviates the Sig-1R-induced pain hypersensitivity and the peripheral nerve injury-induced mechanical allodynia.

Burn Injury-Induced Pain and Depression-Like Behavior in Mice.

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the high mortality and sequelae in low- and middle-income countries. Burns frequently induce intense spontaneous pain and persistent allodynia as well as life-threatening problem. More importantly, excessive pain is often accompanied by depression, which may significantly decrease the quality of life. This article shows how to develop an animal model for the study of burn-induced pain and depression-like behavior. After anesthesia, burn injury was induced by dipping one hind paw of the mouse into hot water (65 °C ± 0.5 °C) for 3 s. The von Frey test and automated gait analysis were performed every 2 days after burn injury. In addition, depression-like behavior was examined using the forced swimming test, and the rota-rod test was performed to differentiate the abnormal motor function after burn injury. The main purpose of this study is to describe the development of an animal model for the study of burn injury-induced pain and depression-like behavior in mice.

Electrophysiological Properties of Ion Channels in Ascaris suum Tissue Incorporated into Planar Lipid Bilayers.

Ion channels are important targets of anthelmintic agents. In this study, we identified 3 types of ion channels in Ascaris suum tissue incorporated into planar lipid bilayers using an electrophysiological technique. The most frequent channel was a large-conductance cation channel (209 pS), which accounted for 64.5% of channels incorporated (n=60). Its open-state probability (Po) was ~0.3 in the voltage range of -60~+60 mV. A substate was observed at 55% of the main-state. The permeability ratio of Cl- to K+ (PCl/PK) was ~0.5 and PNa/PK was 0.81 in both states. Another type of cation channel was recorded in 7.5% of channels incorporated (n=7) and discriminated from the large-conductance cation channel by its smaller conductance (55.3 pS). Its Po was low at all voltages tested (~0.1). The third type was an anion channel recorded in 27.9% of channels incorporated (n=26). Its conductance was 39.0 pS and PCl/PK was 8.6±0.8. Po was ~1.0 at all tested potentials. In summary, we identified 2 types of cation and 1 type of anion channels in Ascaris suum. Gating of these channels did not much vary with voltage and their ionic selectivity is rather low. Their molecular nature, functions, and potentials as anthelmintic drug targets remain to be studied further.

Inhibition of angiotensin converting enzyme induces mechanical allodynia through increasing substance P expression in mice.

Although emerging evidence shows that angiotensin converting enzyme (ACE) is associated with pain, it is not clear whether inhibition of ACE could affect to nociceptive transmission and which mediators are involved in this process. Here we investigated whether administration of the ACE inhibitors, captopril and enalapril increases the expression of substance P (SP) and whether this increase contributes to the induction of mechanical allodynia in mice. ACE was expressed in the lumbar dorsal root ganglion (DRG) and the superficial dorsal horn (SDH) region of the spinal cord in mice. Either intraperitoneal or intrathecal administration of the ACE inhibitors, captopril and enalapril for 10 days significantly increased the paw withdrawal frequency to innocuous mechanical stimuli and the levels of SP in both the lumbar DRG and the SDH region of the spinal cord dorsal horn. In addition, intraperitoneal administration of the SP receptor (neurokinin-1 receptor) antagonist, L-733,060 suppressed mechanical allodynia that was induced by pretreatment of captopril and enalapril. Intraplantar administration of SP for 3 days induces mechanical allodynia, and this effect was reduced by exogenous ACE administration. These findings demonstrate that inhibition of ACE increases the levels of SP in both the lumbar DRG and spinal cord dorsal horn, ultimately contributing to the induction of mechanical allodynia in mice.

Bee venom reduces burn-induced pain via the suppression of peripheral and central substance P expression in mice.

BACKGROUND: Scalding burn injuries can occur in everyday life but occur more frequently in young children. Therefore, it is important to develop more effective burn treatments. OBJECTIVES: This study examined the effects of bee venom (BV) stimulation on scalding burn injury-induced nociception in mice as a new treatment for burn pain. METHODS: To develop a burn injury model, the right hind paw was immersed temporarily in hot water (65°C, 3 seconds). Immediately after the burn, BV (0.01, 0.02, or 0.1 mg/kg) was injected subcutaneously into the ipsilateral knee area once daily for 14 days. A von Frey test was performed to assess the nociceptive response, and the altered walking parameters were evaluated using an automated gait analysis system. In addition, the peripheral and central expression changes in substance P (Sub P) were measured in the dorsal root ganglion and spinal cord by immunofluorescence. RESULTS: Repeated BV treatment at the 2 higher doses used in this study (0.02 and 0.1 mg/kg) alleviated the pain responses remarkably and recovered the gait performances to the level of acetaminophen (200 mg/kg, intraperitoneal, once daily), which used as the positive control group. Moreover, BV stimulation had an inhibitory effect on the increased expression of Sub P in the peripheral and central nervous systems by a burn injury. CONCLUSIONS: These results suggest that a peripheral BV treatment may have positive potency in treating burn-induced pain.

High Salt Intake Recruits Tonic Activation of NR2D Subunit-Containing Extrasynaptic NMDARs in Vasopressin Neurons.

In addition to producing a classical excitatory postsynaptic current via activation of synaptic NMDA receptors (NMDARs), glutamate in the brain also induces a tonic NMDAR current (INMDA) via activation of extrasynaptic NMDARs (eNMDARs). However, since Mg2+ blocks NMDARs in nondepolarized neurons, the potential contribution of eNMDARs to the overall neuronal excitatory/inhibitory (E/I) balance remains unknown. Here, we demonstrate that chronic (7 d) salt loading (SL) recruited NR2D subunit-containing NMDARs to generate an Mg2+-resistant tonic INMDA in nondepolarized [Vh (holding potential) -70 mV] vasopressin (VP; but not oxytocin) supraoptic nucleus (SON) neurons in male rodents. Conversely, in euhydrated (EU) and 3 d SL mice, Mg2+-resistant tonic INMDA was not observed. Pharmacological and genetic intervention of NR2D subunits blocked the Mg2+-resistant tonic INMDA in VP neurons under SL conditions, while an NR2B antagonist unveiled Mg2+-sensitive tonic INMDA but not Mg2+-resistant tonic INMDA In the EU group VP neurons, an Mg2+-resistant tonic INMDA was not generated by increased ambient glutamate or treatment with coagonists (e.g., d-serine and glycine). Chronic SL significantly increased NR2D expression but not NR2B expression in the SON relative to the EU group or after 3 d under SL conditions. Finally, Mg2+-resistant tonic INMDA selectively upregulated neuronal excitability in VP neurons under SL conditions, independent of ionotropic GABAergic input. Our results indicate that the activation of NR2D-containing NMDARs constitutes a novel mechanism that generates an Mg2+-resistant tonic INMDA in nondepolarized VP neurons, thus causing an E/I balance shift in VP neurons to compensate for the hormonal demands imposed by a chronic osmotic challenge.SIGNIFICANCE STATEMENT The hypothalamic supraoptic nucleus (SON) consists of two different types of magnocellular neurosecretory cells (MNCs) that synthesize and release the following two peptide hormones: vasopressin (VP), which is necessary for regulation of fluid homeostasis; and oxytocin (OT), which plays a major role in lactation and parturition. NMDA receptors (NMDARs) play important roles in shaping neuronal firing patterns and hormone release from the SON MNCs in response to various physiological challenges. Our results show that prolonged (7 d) salt loading generated a Mg2+-resistant tonic NMDA current mediated by NR2D subunit-containing receptors, which efficiently activated nondepolarized VP (but not OT) neurons. Our findings support the hypothesis that NR2D subunit-containing NMDARs play an important adaptive role in adult brain in response to a sustained osmotic challenge.

Alpha-Methylacyl-CoA Racemase (AMACR), a Potential New Biomarker for Glioblastoma.

Alpha-Methylacyl-CoA racemase (AMACR), which was initially discovered as a prostate cancer marker, is critical for the chiral inversion mechanism of branched-chain fatty acids. However, the function of AMACR in brain tumors has not been investigated. In this study, AMACR appeared to be involved in glioblastoma. The protein and mRNA levels of AMACR were highly elevated in glioblastoma. Downregulation of AMACR inhibited cell proliferation. Comprehensive analysis of the public REMBRANDT GBM dataset also confirmed that the level of AMACR expression was correlated with the clinical prognosis of glioma patients. In summary, these findings indicate that AMACR expression is increased in a glioblastoma cell line and glioma patients, suggesting that AMACR might be a potential diagnostic marker and therapeutic target for cancer, including glioma.

Positive allosteric modulation of GABAA receptors by a novel antiepileptic drug cenobamate.

Cenobamate is a novel antiepileptic drug under investigation for use in patients with focal (partial-onset) seizures. To understand its potential molecular mechanism of action, the effects of cenobamate on GABAA-mediated currents and GABAA receptors in rodent hippocampal neurons were examined. Cenobamate potentiated GABA-induced currents (IGABA) in acutely isolated CA3 pyramidal cells in a concentration-dependent manner (EC50, 164 µM), which was not affected by flumazenil, a benzodiazepine receptor antagonist. Cenobamate enhanced tonic GABAA currents (Itonic), which is defined as a holding current shift by the GABAA receptor antagonist bicuculline (EC50, 36.63 µM). At therapeutically relevant concentrations, cenobamate induced minimal changes in the frequency, amplitudes, and decay time of spontaneous inhibitory postsynaptic currents in the CA1 neurons. Flumazenil failed to affect cenobamate-potentiated Itonic and Iphasic in CA1 neurons. Cenobamate showed positive allosteric modulation of GABA-induced IGABA mediated by GABAA receptors. This effect was similar for all tested hGABAA receptors containing six different alpha subunits (α1ß2γ2 or α2-6ß3γ2), with EC50 values ranging from 42 to 194 µM. Cenobamate did not displace the binding of flunitrazepam, a benzodiazepine derivative, or flumazenil to GABAA receptors. The results showed that cenobamate, a novel antiepileptic drug, acts as a positive allosteric modulator of high-affinity GABAA receptors, activated by GABA at a site independent of the benzodiazepine binding site and efficiently enhances Itonic inhibition in hippocampal neurons, which could be an underlying molecular mechanism stabilizing neural circuits of the epileptic hippocampus.

Author Correction: CD200R/Foxp3-mediated signalling regulates microglial activation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bestrophin1-mediated tonic GABA release from reactive astrocytes prevents the development of seizure-prone network in kainate-injected hippocampi.

Tonic extrasynaptic GABAA receptor (GABAA R) activation is under the tight control of tonic GABA release from astrocytes to maintain the brain's excitation/inhibition (E/I) balance; any slight E/I balance disturbance can cause serious pathological conditions including epileptic seizures. However, the pathophysiological role of tonic GABA release from astrocytes has not been tested in epileptic seizures. Here, we report that pharmacological or genetic intervention of the GABA-permeable Bestrophin-1 (Best1) channel prevented the generation of tonic GABA inhibition, disinhibiting CA1 pyramidal neuronal firing and augmenting seizure susceptibility in kainic acid (KA)-induced epileptic mice. Astrocyte-specific Best1 over-expression in KA-injected Best1 knockout mice fully restored the generation of tonic GABA inhibition and effectively suppressed seizure susceptibility. We demonstrate for the first time that tonic GABA from reactive astrocytes strongly contributes to the compensatory shift of E/I balance in epileptic hippocampi, serving as a good therapeutic target against altered E/I balance in epileptic seizures.

Calpain-2 as a Treatment Target in Prenatal Stress-induced Epileptic Spasms in Infant Rats.

Stress can induce a serious epileptic encephalopathy that occurs during early infancy. Recent studies have revealed that prenatal stress exposure is a risk factor for the development of infantile spasms. Our previous work demonstrates that prenatal stress with betamethasone-induced alterations to the expression of the K+/Cl- co-transporter (KCC2) in gamma-aminobutyric acid (GABA) interneurons lowers the seizure threshold in exposed animals. Here, we further investigated the mechanisms involved in this KCC2 dysfunction and explored possible treatment options. We stressed Sprague-Dawley rats prenatally and further treated dams with betamethasone on gestational day 15, which increases seizure susceptibility and NMDA (N-Methyl-D-aspartate)-triggered spasms on postnatal day 15. In this animal model, first, we evaluated baseline calpain activity. Second, we examined the cleavage and dephosphorylation of KCC2. Finally, we checked the effect of a calpain inhibitor on seizure occurrence. The phosphorylated-N-methyl-Daspartate Receptor 2B (NR2B):non-phosphorylated NR2B ratio was found to be higher in the cortex of the prenatally stressed betamethasone model. We further found that the betamethasone model exhibited increased phosphorylation of calpain-2 and decreased phosphorylation of KCC2 and Glutamic acid decarboxylase 67 (GAD67). After using a calpain inhibitor in prenatal-stress rats, the seizure frequency decreased, while latency increased. GABAergic depolarization was further normalized in prenatal-stress rats treated with the calpain inhibitor. Our study suggests that calpain-dependent cleavage and dephosphorylation of KCC2 decreased the seizure threshold of rats under prenatal stress. Calpain-2 functions might, thus, be targeted in the future for the development of treatments for epileptic spasms.

The extracellular role of Ref-1 as anti-inflammatory function in lipopolysaccharide-induced septic mice.

Apurinic/apyrimidinic endonuclease/redox factor-1 (Ref-1), a multifunctional protein secreted from stimulated cells, has been identified as a new serological biomarker. Despite recent reports on the role of Ref-1 in inflammation, the biological function of secreted Ref-1 remains unknown, especially in vivo. This study aimed to evaluate the possible roles of secreted Ref-1 in lipopolysaccharide-induced systemic inflammation in vivo. We generated a secretory Ref-1 adenoviral vector system, AdPPT-LS-Ref-1, by conjugation of preprotrypsin leading sequence (PPT-LS) with full-length Ref-1 sequences. Expression of tumor necrosis factor-α (TNF-α)-induced vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells and lipopolysaccharide (LPS)-induced cyclooxygenase-2 in Raw264.7 cells was inhibited by secretory Ref-1, and this inhibitory effect was abrogated following neutralization of Ref-1 with anti-Ref-1 antibody. Plasma Ref-1 levels following administration of AdPPT-LS-Ref-1 (2 × 109 ifu, i.p.) for 24 h were substantially higher than those recorded following administration of Adßgal (84.6 ±â€¯7.2 ng/ml vs. 4.4 ±â€¯1.5 ng/ml). Treatment with LPS (10 mg/kg, i.v. for 6 h) markedly increased VCAM-1 expression, cathepsin or myeloperoxidase activity, which were significantly suppressed by treatment with AdPPT-LS-Ref-1. Furthermore, LPS-induced cytokines, such as TNF-α, interleukin (IL)-1ß, IL-6, and monocyte chemoattractant protein 1, were significantly inhibited in AdPPT-LS-Ref-1-treated mice. However, LPS-induced myeloperoxidase activities were not suppressed by treatment with the redox mutant of secretory Ref-1, AdPPT-LS-Ref-1(C65A/C93A), or wild-type AdRef-1. Collectively, these results suggest that secreted Ref-1 has anti-inflammatory properties and that its redox cysteine residue is associated with the anti-inflammatory activity in vivo. Furthermore, our findings indicate that secretory Ref-1 may be useful as a therapeutic biomolecule against systemic inflammation.

Exercise training normalizes elevated firing rate of hypothalamic presympathetic neurons in heart failure rats.

Exercise training (ExT) normalizes elevated sympathetic nerve activity in heart failure (HF), but the underlying mechanisms are not well understood. In this study, we examined the effects of 3 wk of ExT on the electrical activity of the hypothalamic presympathetic neurons in the brain slice of HF rats. HF rats were prepared by ligating the left descending coronary artery. The electrophysiological properties of paraventricular nucleus neurons projecting to the rostral ventrolateral medulla (PVN-RVLM) were examined using the slice patch-clamp technique. The neuronal firing rate was elevated in HF rats, and ExT induced a reduction in the firing rate ( P < 0.01). This ExT-induced decrease in the firing rate was associated with an increased frequency of spontaneous and miniature inhibitory postsynaptic current (IPSCs; P < 0.05). There was no significant change in excitatory postsynaptic current. Replacing Ca2+ with Mg2+ in the recording solution reduced the elevated IPSC frequency in HF rats with ExT ( P < 0.01) but not in those without ExT, indicating an increase in the probability of GABA release. In contrast, ExT did not restore the reduced GABAA receptor-mediated tonic inhibitory current in HF rats. A GABAA receptor blocker (bicuculline, 20 µM) increased the firing rate in HF rats with ExT ( P < 0.01) but not in those without ExT. Collectively, these results show that ExT normalized the elevated firing activity by increasing synaptic GABA release in PVN-RVLM neurons in HF rats. Our findings provide a brain mechanism underlying the beneficial effects of ExT in HF, which may shed light on the pathophysiology of other diseases accompanied by sympathetic hyperactivation.

GABAergic signaling linked to autophagy enhances host protection against intracellular bacterial infections.

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain; however, the roles of GABA in antimicrobial host defenses are largely unknown. Here we demonstrate that GABAergic activation enhances antimicrobial responses against intracellular bacterial infection. Intracellular bacterial infection decreases GABA levels in vitro in macrophages and in vivo in sera. Treatment of macrophages with GABA or GABAergic drugs promotes autophagy activation, enhances phagosomal maturation and antimicrobial responses against mycobacterial infection. In macrophages, the GABAergic defense is mediated via macrophage type A GABA receptor (GABAAR), intracellular calcium release, and the GABA type A receptor-associated protein-like 1 (GABARAPL1; an Atg8 homolog). Finally, GABAergic inhibition increases bacterial loads in mice and zebrafish in vivo, suggesting that the GABAergic defense plays an essential function in metazoan host defenses. Our study identified a previously unappreciated role for GABAergic signaling in linking antibacterial autophagy to enhance host innate defense against intracellular bacterial infection.

C-27-carboxylated oleanane triterpenoids up-regulate TRAIL DISC assembly via p38 MAPK and CHOP-mediated DR5 expression in human glioblastoma cells.

Despite recent tremendous progress, targeting of TNF-related apoptosis-inducing ligand (TRAIL) as a cancer therapy has limited success in many clinical trials, in part due to inactivation of death inducing signaling complex (DISC)-mediated caspase-8 signaling cascade in highly malignant tumors such as glioblastoma. In this study, screening of constituents derived from Astilbe rivularis for TRAIL-sensitizing activity identified C-27-carboxylated oleanolic acid derivatives (C27OAs) including 3ß-hydroxyolean-12-en-27-oic acid (C27OA-1), 3ß,6ß,7α-trihydroxyolean-12-en-27-oic acid (C27OA-2), and 3ß-trans-p-coumaroyloxy-olean-12-en-27-oic acid (C27OA-3) as novel TRAIL sensitizers. Interestingly, these C27OAs did not affect apoptotic cell death induced by either ligation of other death receptor (DR) types, such as TNF and Fas or DNA damaging agents, which suggests that C27OAs effectively and selectively sensitize TRAIL-mediated caspase-8 activation. Mechanistically, C27OAs upregulate the expression of cell surface DR5 and DISC formation without affecting downstream intracellular apoptosis-related proteins. The upregulation of DR5 expression by C27OAs strictly depends on transactivation of C/EBP homology protein, which is regulated through the p38 MAPK pathway, rather than p53 and intracellular reactive oxygen species status. Taken together, our results identify the novel C27OAs as TRAIL sensitizers targeting the upstream DISC assembly of DR5, and provide a rationale for further development of C27OAs for facilitating TRAIL-based chemotherapy in glioblastoma patients.