• Journal of Microbiology and Biotechnology
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• v.27 no.6
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• pp.1163-1170
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• 2017

Clostridium difficile releases two exotoxins, toxin A and toxin B, which disrupt the epithelial cell barrier in the gut to increase mucosal permeability and trigger inflammation with severe diarrhea. Many studies have suggested that enteric nerves are also directly involved in the progression of this toxin-mediated inflammation and diarrhea. C. difficile toxin A is known to enhance neurotransmitter secretion, increase gut motility, and suppress sympathetic neurotransmission in the guinea pig colitis model. Although previous studies have examined the pathophysiological role of enteric nerves in gut inflammation, the direct effect of toxins on neuronal cells and the molecular mechanisms underlying toxin-induced neuronal stress remained to be unveiled. Here, we examined the toxicity of C. difficile toxin A against neuronal cells (SH-SY5Y). We found that toxin A treatment time- and dose-dependently decreased cell viability and triggered apoptosis accompanied by caspase-3 activation in this cell line. These effects were found to depend on the up-regulation of reactive oxygen species (ROS) and the subsequent activation of p38 MAPK and induction of $p21^{Cip1/Waf1}$. Moreover, the N-acetyl-$\text\tiny L$-cysteine (NAC)-induced down-regulation of ROS could recover the viability loss and apoptosis of toxin A-treated neuronal cells. These results collectively suggest that C. difficile toxin A is toxic for neuronal cells, and that this is associated with rapid ROS generation and subsequent p38 MAPK activation and $p21^{Cip1/Waf1}$ up-regulation. Moreover, our data suggest that NAC could inhibit the toxicity of C. difficile toxin A toward enteric neurons.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.4
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• pp.265-271
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• 2009

Nitric oxide (NO) has both neuroprotective and neurotoxic effects depending on its concentration and the experimental model. We tested the effects of NG-nitro-L-arginine methyl ester (L-NAME), a nonselective nitric oxide synthase (NOS) inhibitor, and aminoguanidine, a selective inducible NOS (iNOS) inhibitor, on kainic acid (KA)-induced seizures and hippocampal CA3 neuronal death. L-NAME (50 mg/kg, i.p.) and/or aminoguanidine (200 mg/kg, i.p.) were administered 1 h prior to the intracerebroventricular (i.c.v.) injection of KA. Pretreatment with L-NAME significantly increased KA-induced CA3 neuronal death, iNOS expression, and activation of microglia. However, pretreatment with aminoguanidine significantly suppressed both the KA-induced and L-NAME-aggravated hippocampal CA3 neuronal death with concomitant decreases in iNOS expression and microglial activation. The protective effect of aminoguanidine was maintained for up to 2 weeks. Furthermore, iNOS knockout mice ($iNOS^{-1-}$) were resistant to KA-induced neuronal death. The present study demonstrates that aminoguanidine attenuates KA-induced neuronal death, whereas L-NAME aggravates neuronal death, in the CA3 region of the hippocampus, suggesting that NOS isoforms play different roles in KA-induced excitotoxicity.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.3
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• pp.279-286
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• 1998

It has been well documented that transient forebrain global ischemia causes selective neuronal degeneration in hippocampal CA1 pyramidal neurons with a delay of a few days. The mechanism of this delayed hippocampal CA1 pyramidal neuronal death (DND) is still controversial. To delineate the mechanisms of the DND, the effects of treatment with MK-801, an NMDA receptor antagonist, kynurenic acid, a NMDA/non-NMDA receptor antagonist, and/or cycloheximide, a protein synthesis inhibitor, on the DND were investigated in male Wistar rats. To examine the participation of apoptotic neuronal death in the DND, TUNEL staining was performed in ischemic brain section. Global ischemia was induced by 4-vessel occlusion for 20 min. All animals in this study showed the DND 3 and 7 days after the ischemic insult. The DND that occured 3 days and 7 days after the ischemia were not affected by pretreatment with MK-801 (1 mg/kg), but markedly attenuated by the pretreatment with kynurenic acid (500 mg/kg). Treatment with cycloheximide (1 mg/kg) also markedly inhibited the DND. The magnitudes of attenuation by the two drugs were similar. The magnitude of attenuation by co-treatments with kynurenic acid and cycloheximide was not greater than that with any single treatment. TUNEL staining was negative in the sections obtained 1 or 2 days after the ischemic insults, but it was positive at hippocampal CA1 pyramidal cells in sections collected 3 days after the ischemia. These results suggested that the DND should be mediated by the activation of non-NMDA receptor, not by the activation of NMDA receptor and that the activation of AMPA receptor should induce the apoptotic process in the DND.

• BMB Reports
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• v.35 no.1
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• pp.67-86
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• 2002

Three routes have been identified triggering neuronal death under physiological and pathological conditions. Excess activation of ionotropic glutamate receptors cause influx and accumulation of $Ca^{2+}$ and $Na^+$ that result in rapid swelling and subsequent neuronal death within a few hours. The second route is caused by oxidative stress due to accumulation of reactive oxygen and nitrogen species. Apoptosis or programmed cell death that often occurs during developmental process has been coined as additional route to pathological neuronal death in the mature nervous system. Evidence is being accumulated that excitotoxicity, oxidative stress, and apoptosis propagate through distinctive and mutually exclusive signal transduction pathway and contribute to neuronal loss following hypoxic-ischemic brain injury. Thus, the therapeutic intervention of hypoxic-ischemic neuronal injury should be aimed to prevent excitotoxicity, oxidative stress, and apoptosis in a concerted way.

• Molecules and Cells
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• v.38 no.6
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• pp.535-539
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• 2015

Olfactory stimulation activates multiple signaling cascades in order to mediate activity-driven changes in gene expression that promote neuronal survival. To date, the mechanisms involved in activity-dependent olfactory neuronal survival have yet to be fully elucidated. In the current study, we observed that olfactory sensory stimulation, which caused neuronal activation, promoted activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway and the expression of Bcl-2, which were responsible for olfactory receptor neuron (ORN) survival. We demonstrated that Bcl-2 expression increased after odorant stimulation both in vivo and in vitro. We also showed that odorant stimulation activated Akt, and that Akt activation was completely blocked by incubation with both a PI3K inhibitor (LY294002) and Akt1 small interfering RNA. Moreover, blocking the PI3K/Akt pathway diminished the odorantinduced Bcl-2 expression, as well as the effects on odorant-induced ORN survival. A temporal difference was noted between the activation of Akt1 and the expression of Bcl-2 following odorant stimulation. Blocking the PI3K/Akt pathway did not affect ORN survival in the time range prior to the increase in Bcl-2 expression, implying that these two events, activation of the PI3K pathway and Bcl-2 induction, were tightly connected to promote post-translational ORN survival. Collectively, our results indicated that olfactory activity activated PI3K/Akt, induced Bcl-2, and promoted long term ORN survival as a result.

• Biomolecules & Therapeutics
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• v.25 no.6
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• pp.593-598
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• 2017

The $Na^+/H^+$ exchanger-1 (NHE-1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the brain, heart, and other organs. It is increased by intracellular acidosis through the interaction of intracellular $H^+$ with an allosteric modifier site in the transport domain. In the previous study, we reported that glutamate-induced NHE-1 phosphorylation mediated by activation of protein kinase C-${\beta}$ (PKC-${\beta}$) in cultured neuron cells via extracellular signal-regulated kinases (ERK)/p90 ribosomal s6 kinases (p90RSK) pathway results in NHE-1 activation. However, whether glutamate stimulates NHE-1 activity solely by the allosteric mechanism remains elusive. Cultured primary cortical neuronal cells were subjected to intracellular acidosis by exposure to $100{\mu}M$ glutamate or 20 mM $NH_4Cl$. After the desired duration of intracellular acidosis, the phosphorylation and activation of PKC-${\beta}$, ERK1/2 and p90RSK were determined by Western blotting. We investigated whether the duration of intracellular acidosis is controlled by glutamate exposure time. The NHE-1 activation increased while intracellular acidosis sustained for >3 min. To determine if sustained intracellular acidosis induced NHE-1 phosphorylation, we examined phosphorylation of NHE-1 induced by intracellular acidosis by transient exposure to $NH_4Cl$. Sustained intracellular acidosis led to activation and phosphorylation of NHE-1. In addition, sustained intracellular acidosis also activated the PKC-${\beta}$, ERK1/2, and p90RSK in neuronal cells. We conclude that glutamate stimulates NHE-1 activity through sustained intracellular acidosis, which mediates NHE-1 phosphorylation regulated by PKC-${\beta}$/ERK1/2/p90RSK pathway in neuronal cells.

• The Korean Journal of Pain
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• v.22 no.1
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• pp.1-15
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• 2009

Neuropathic pain is often refractory to intervention because of the complex etiology and an incomplete understanding of the mechanisms behind this type of pain. Glial cells, specifically microglia and astrocytes, are powerful modulators of pain and new targets of drug development for neuropathic pain. Glial activation could be the driving force behind chronic pain, maintaining the noxious signal transmission even after the original injury has healed. Glia express chemokine, purinergic, toll-like, glutaminergic and other receptors that enable them to respond to neural signals, and they can modulate neuronal synaptic function and neuronal excitability. Nerve injury upregulates multiple receptors in spinal microglia and astrocytes. Microglia influence neuronal communication by producing inflammatory products at the synapse, as do astrocytes because they completely encapsulate synapses and are in close contact with neuronal somas through gap junctions. Glia are the main source of inflammatory mediators in the central nervous system. New therapeutic strategies for neuropathic pain are emerging such as targeting the glial cells, novel pharmacologic approaches and gene therapy. Drugs targeting microglia and astrocytes, cytokine production, and neural structures including dorsal root ganglion are now under study, as is gene therapy. Isoform-specific inhibition will minimize the side effects produced by blocking all glia with a general inhibitor. Enhancing the anti-inflammatory cytokines could prove more beneficial than administering proinflammatory cytokine antagonists that block glial activation systemically. Research on therapeutic gene transfer to the central nervous system is underway, although obstacles prevent immediate clinical application.

• YAKHAK HOEJI
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• v.45 no.4
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• pp.419-425
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• 2001

The neuronal cell death induced by excess glutamate (Glu) has been implicated in many acute and chronic neurodegenerative diseases including cerebral ischemia. Glu-induced elevation of intra-cellular $Ca^{2+}$ plays a critical role in the excitotoxicity, partly through the activation of a variety of $Ca^{2+}$ dependent enzymes. In the present study, we investigated the Glu-induced modulation of $Ca^{2+}$/calmodulin-dependent protein kinase IV (CaMK IV), a multifunctional enzyme abundantly present in the nuclei of neurons. The exposure of cultured rat cortical neurons to $100{\mu}$M Glu for 3 min dramatically increased CaMK IV activity up to 4.5-fold of the control-treated enzyme activity. The activation was very rapid, reaching peak at 3 min and then declined gradually. Under the same experimental conditions, time-dependent acute and delayed neuronal cell death was observed. Immunoblot analyses using specific antibodies showed that the expressions of CaMK IV and $CaMKK_{\alpha}$ were time-dependently modulated by Glu. Taken together, these results imply that the modulation of CaMK IV activity by Glu may be involved in the cascade of events resulting in neuronal cell death in cortical cultures.

• Journal of Life Science
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• v.25 no.6
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• pp.656-662
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• 2015

Natural products and natural product structures play a general and highly significant role in drug discovery and development process because it has various merits and potentials for new drug source that have extensive clinical experience, development time contraction, excellent stability and safety. In several neurological disorders, neuronal death and excessive activation of microglia (neuro-inflammation) are observed. A number of drug discovery-related neuronal cell death and neuro-inflammation was studied from natural products, respectively. However, until now, it has not been possible to study dual-protection molecules recorded in the Natural Product library. In the present study, using the natural product-derived library of the Institute for Korea Traditional Medical Industry, we investigated dual-protective molecules against glutamate (a classical excitatory neurotransmitter)-induced oxidative stress mediated neuronal cell death and LPS-induced excessive activated microglial cells (immune cells of the brain). Chrysophanol, extracted from Rheum palmatum, had dual-protective effects against both glutamate-induced neuronal cell death and LPS-induced NO production, triggering proinflammatory cytokines and microglia activation and resulting in neuroinflammation. Flow-cytometry analysis revealed that chrysophanol had a scavenger effect, scavenging glutamate- and LPS-induced reactive oxygen species (ROS) produced by neuronal and microglial cells, respectively. Based on the present study, chrysophanol may have an important protective role against neuronal cell death and neuroinflammation in the brain. The results may be helpful for studying drug development candidates for treating central nervous system disorders.

• Molecules and Cells
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• v.40 no.3
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• pp.163-168
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• 2017

Microglia are the primary resident immune cells of the central nervous system (CNS). They are the first line of defense of the brain's innate immune response against infection, injury, and diseases. Microglia respond to extracellular signals and engulf unwanted neuronal debris by phagocytosis, thereby maintaining normal cellular homeostasis in the CNS. Pathological stimuli such as neuronal injury induce transformation and activation of resting microglia with ramified morphology into a motile amoeboid form and activated microglia chemotax toward lesion site. This review outlines the current research on microglial activation and chemotaxis.