Eun Joo Baik

Cyclooxygenase-2 selective inhibitors aggravate kainic acid induced seizure and neuronal cell death in the hippocampus

Cyclooxygenase-2 (COX-2) in the brain is expressed constitutively and also increased in pathological conditions such as seizure, cerebral ischemia, and inflammation. This study examined the role of COX-2 in kainic acid-induced seizure and in the following neuronal death by using selective inhibitors. Systemic kainate injection (50 mg/kg; i.p.) in mice evoked seizure within 15 min and led to 29% mortality within 2 h. TUNEL-positive neuronal death peaked at 3 days after injection and was prominent in CA(3a) regions of the hippocampus. NS-398 or celecoxib (10 mg/kg, COX-2 selective inhibitor) and indomethacin (5 mg/kg, nonselective inhibitor) exaggerated kainic acid-induced seizure activity and mortality. COX-2 selective inhibitors induced the seizure at earlier onset and more severe mortality within the first hour than indomethacin and aspirin. NS-398 also aggravated kainic acid-induced TUNEL positive neuronal death and decreased Cresyl violet stained viable neurons, and extended lesions to CA(1) and CA(3b). Kainic acid increased the levels of PGD(2), PGF(2a) and PG E(2) in the hippocampus immediately after injection. Indomethacin attenuated the production of basal and kainic acid-induced prostaglandins. In contrast, NS-398 failed to reduce until the first 30 min after kainic acid injection, during which the animals were severely seizured. It has been challenged the endogenous PGs might have anticonvulsant properties. Thus, COX-2 selective inhibitor, including nonselective inhibitor such as indomethacin, aggravated kainic acid-induced seizure activity and the following hippocampal neuronal death even with variable prostaglandin levels.

An animal model of neuropathic pain employing injury to the sciatic nerve branches

The present study was conducted to develop a new animal model of neuropathic pain employing injury to the distal sciatic nerve branches. Under halothane anesthesia, the tibial, sural, and/or common peroneal nerves were injured and neuropathic pain behaviors were compared among different groups of rats. Different types of injury produced different levels of neuropathic pain. Rats with injury to the tibial and sural nerves showed the most vigorous mechanical allodynia, cold allodynia, and spontaneous pain. These neuropathic pain behaviors were not relieved by functional sympathectomy using guanethidine. The results suggested that injury to the tibial and sural nerves, while leaving the common peroneal nerve intact, can be used as a new animal model of neuropathic pain and that this model represents sympathetically independent pain (SIP). The present animal model is very simple to produce injury and can produce profound and reliable pain behaviors. These features enable the new animal model to be a useful tool in elucidating the mechanisms of neuropathic pain, especially SIP.

Effects of peroxisome proliferator-activated receptor agonists on LPS-induced neuronal death in mixed cortical neurons: associated with iNOS and COX-2

In neurodegenerative disease, the use of non-steroidal anti-inflammatory drugs (NSAIDs) has been regarded as beneficial. The NSAID, an inhibitor of cyclooxygenase (COX), has been also suggested as a ligand of the peroxisome proliferator-activated receptor (PPAR). In cortical neuron-glial co-cultures, we examined the effect of PPAR agonists on lipopolysaccharide(LPS)-induced neuronal death, which has been known to be NO-dependent. LPS induced iNOS expression and the release of nitric oxide in microglia, and COX-2 expression in neurons. PPAR-gamma agonists such as 15d-PGJ(2), ciglitazone and troglitazone prevented LPS-induced neuronal death and abolished LPS-induced NO and PGE(2) release, however PPAR-alpha agonists such as clofibrate and WY14,643 did not produce the same results. PPAR-gamma agonists also reduced LPS-induced iNOS and COX-2 expression, which suggested by interfering with the NF-kappaB signal pathway.

Neuroprotective effects of prostaglandin E2 or cAMP against microglial and neuronal free radical mediated toxicity associated with inflammation.

Prostaglandin E2 (PGE2), a product of the cyclooxygenation of arachidonic acid released from membrane phospholipids, plays a critical role in inflammatory neurodegenerative conditions. Despite its classic role as a proinflammatory molecule, exogenous PGE2 was suggested to have protective roles against neuronal death, although the exact protective mechanisms of PGE2 are not yet defined. Thus, the aim of this study was to examine the effect of exogenous PGE2 on inflammatory neurotoxicity. Lipopolysaccharide (LPS) induced neuronal toxicity, which was associated with terminal transferase dUTP nick end labeling (TUNEL)‐positive neuronal death with increased caspase‐3 activity. In neuron‐glial coculture, LPS markedly induced inducible nitric oxide synthase/nitric oxide (iNOS/NO) release from microglial cells, but not from neurons; however, LPS‐induced oxidative stress such as reactive oxygen species (ROS), measured with 2,7‐dichlorofluorescein diacetate oxidation, was increased in neurons, but not in microglial cells. Exogenous PGE2 (1 μg/ml) rescued the neurons, reducing iNOS/NO release from microglial cells and ROS formation from neurons. PGE2 has been known to increase intracelluar cyclic adenosine monophosphate (cAMP) levels. In this study, we found that intracellular cAMP elevating agents, forskolin, and cAMP analogue, dbcAMP and 8‐Br‐cAMP, also prevented LPS‐induced neuronal death. Thus, these results indicate that exogenous PGE2 protects against LPS‐induced neuronal apoptotic cell death through the intracellular cAMP system, and is associated with the modulation of NO from microglial cells and ROS production from neurons.

ERK1/2 is an endogenous negative regulator of the gamma-secretase activity.

As an essential protease in the generation of amyloid β, γ‐secretase is believed to play an important role in the pathogenesis of Alzheimers disease. Although a great deal of progress has been made in identifying the components of γ‐secretase complex, the endogenous regulatory mechanism of γ‐secretase is unknown. Here we show that γ‐secretase is endogenously regulated via extracellular signal regulated MAP kinase (ERK) 1/2‐dependent mitogen‐activated protein kinase (MAPK) pathway. The inhibition of ERK1/2 activity, either by a treatment with a MEK inhibitor or an ERK knockdown transfection, dramatically increased γ‐secretase activity in several different cell types. JNK or p38 kinase inhibitors had little effect, indicating that the effect is specific to ERK1/2‐dependent MAPK pathway. Conversely, increased ERK1/2 activity, by adding purified active ERK1/2 or EGF‐induced activation of ERK1/2, significantly reduced γsecretase activity, demonstrating down‐regulation of γ‐secretase activity by ERK1/2. Whereas γsecretase expression was not affected by ERK1/2, its activity was enhanced by phosphatase treatment, indicating that ERK1/2 regulates γ‐secretase activity by altering the pattern of phophorylation. Among the components of isolated γ‐secretase complex, only nicastrin was phosphorylated by ERK1/2, and it precipitated with ERK1/2 in a co‐immunoprecipitation assay, which suggests binding between ERK1/2 and nicastrin. Our results show that ERK1/2 is an endogenous regulator of γ‐secretase, which raises the possibility that ERK1/2 down‐regulates γsecretase activity by directly phosphorylating nicastrin.

Involvement of endogenous prostaglandin F2α on kainic acid-induced seizure activity through FP receptor: The mechanism of proconvulsant effects of COX-2 inhibitors

COX-2 and prostaglandins (PGs) might play important roles in epilepsy. In kainic acid-induced seizures, the brain largely increases PGD(2), first from COX-1 and later COX-2-induced PGF(2alpha). Pre-treatment with COX-2 inhibitors such as indomethacin, nimesulide, and celecoxib is known to aggravate kainic acid (KA)-induced seizure activity. However it is not known whether the proconvulsant effect of those non-steroidal anti-inflammatory drugs (NSAIDs) is due to changes in endogenous prostaglandins (PGs), or what types of PGs are involved. The purpose of this study was to determine the effect of intracisternally administered PGs on KA-induced seizures aggravated by pre- or post-treatment with COX-2 inhibitors. Systemic KA injection (10 mg/kg i.p.) in mice evoked mild seizure activity within 15 min. PGs were administrated intracisternally 20 min prior to KA administration. COX inhibitors (indomethacin, nimesulide, and ketoprofen, 10 mg/kg i.p.) were injected 1 h before or 15 min after KA. An additional COX-2 inhibitor, celecoxib, was administered orally. Intracisternally administered PGF(2alpha) (700 ng), but not PGD(2) (700 ng) or PGE(2) (700 ng) completely alleviated KA-induced seizures potentiated by COX-2 inhibitors, and also reduced KA-induced hippocampal neuronal death aggravated by indomethacin. PGF(2alpha) alone did not affect KA-induced seizures. However, an FP receptor antagonist, AL 8810 (10 or 50 ng) which is an 11beta-fluoro analogue of PGF(2alpha) potentiated KA-induced seizure activity dose-dependently. In summary, pre- or post-treatment with COX-2 inhibitors aggravates KA-induced seizures, which suggests to change the endogenous PGF(2alpha). Seizure-induced PGF(2alpha) might act as an endogenous anticonvulsant through FP receptors.

β-Catenin Interacts with MyoD and Regulates Its Transcription Activity

ABSTRACT Wnt regulation of muscle development is thought to be mediated by the β-catenin-TCF/LEF-dependent canonical pathway. Here we demonstrate that β-catenin, not TCF/LEF, is required for muscle differentiation. We showed that β-catenin interacts directly with MyoD, a basic helix-loop-helix transcription factor essential for muscle differentiation and enhances its binding to E box elements and transcriptional activity. MyoD-mediated transactivation is inhibited in muscle cells when β-catenin is deficient or the interaction between MyoD and β-catenin is disrupted. These results demonstrate that β-catenin is necessary for MyoD function, identifying MyoD as an effector in the Wnt canonical pathway.

Differential roles of cyclooxygenase isoforms after kainic acid-induced prostaglandin E2 production and neurodegeneration in cortical and hippocampal cell cultures

Prostaglandins, which are cyclooxygenase (COX) products, are pathologically up-regulated, and have been proven to be closely associated with neuronal death. In this study, we investigated a role of COX isoforms (COX-1 and COX-2) in kainic acid-induced neuronal death in cultured murine cortical or hippocampal neurons. In primary cortical neurons, both indomethacin (COX-1/-2 nonselective inhibitor) and aspirin (COX-1 preferential inhibitor) reduced basal and kainic acid-induced PGE(2) production significantly and prevented neuronal cell death after kainic acid treatment. In contrast, NS398 (COX-2 selective inhibitor) had no effect on kainic acid-induced neuronal cell death. In hippocampal neurons, however, COX-2 inhibitors prevented both kainic acid-induced neuronal death and PGE(2) production. COX-2 expression was remarkably up-regulated by kainic acid in hippocampal neurons; whereas in cortical neurons, COX-2 expression was comparatively less significant. Astrocytes were unresponsive to kainic acid in terms of PGE(2) production and cell death. In conclusion, we suggest that the release of PGE(2) induced by kainic acid occurred through COX-1 activity rather than COX-2 in cortical neurons. The inhibition of PGE(2) release by COX-1 inhibitors prevented kainic acid-induced cortical neuronal death, while in the hippocampal neurons, COX-2 inhibitors prevented kainic acid-induced PGE(2) release and hippocampal neuronal death.

Arachidonic acid induces neuronal death through lipoxygenase and cytochrome P450 rather than cyclooxygenase

Arachidonic acid (AA) is released from membrane phospholipids during normal and pathologic processes such as neurodegeneration. AA is metabolized via lipoxygenase (LOX)‐, cyclooxygenase (COX)‐, and cytochrome P450 (CYP450)‐catalyzed pathways. We investigated the relative contributions of these pathways in AA‐induced neuronal death. Exposure of cultured cortical neurons to AA (50 μM) yielded significantly apoptotic neuronal death, which was attenuated greatly by LOX inhibitors (nordihydroguaiaretic acid, AA861, and baicalein), or CYP450 inhibitors (SKF525A and metyrapone), rather than COX inhibitors (indomethacin and NS398). AA (10 μM)‐induced neurotoxicity was prevented by all kinds of inhibitors. Compared, the neurotoxic effects of three pathway metabolites, 12‐hydroxyeicosatetraenoic acid (12‐HETE), a major LOX metabolite, induced a significant neurotoxicity. AA also produced reactive oxygen species within 30 min, which was reduced by all inhibitors tested, including COX inhibitors, and AA neurotoxicity was abolished by the antioxidant Trolox. AA treatment also depleted glutathione levels; this depletion was reduced by the LOX or CYP450 inhibitors rather than by the COX inhibitors. Taken together, our data suggested that the LOX pathway likely plays a major role in AA‐induced neuronal death with the modification of intracellular free radical levels.

Calpain-Mediated N-Cadherin Proteolytic Processing in Brain Injury

Neural-cadherin (N-cadherin), a member of the classical cadherin family of transmembrane glycoproteins, mediates cellular recognition and cell–cell adhesion through calcium-dependent homophilic interactions and plays important roles in the development and maintenance of the nervous system. Metalloproteinase is known to cleave N-cadherin, which is further cleaved by γ-secretase. The intracellular domain of N-cadherin interacts with β-catenin, and β-catenin stability is critical for cell–cell adhesion and cell survival. In the present study, we showed that N-cadherin is cleaved specifically by calpain, resulting in the generation of a novel 110 kDa fragment. The cleavage occurred in ischemic brain lesions and in vitro neural cells in the presence of NMDA and ionomycin, and was restored by calpain inhibitors but not matrix metalloproteinase or γ-secretase inhibitors. Calpain directly cleaved N-cadherin in in vitro calpain assays, and calpain inhibitors prevented its cleavage in a dose-dependent manner. Using N-cadherin deletion mutants, we found that calpain cleavage sites exist in at least four regions of the cytoplasmic domain. Treatment with NMDA induced neuronal death, and it suppressed the expression of surface N-cadherin and the N-cadherin/β-catenin interaction, effects that were prevented by calpain inhibitor. Furthermore, calpain-mediated N-cadherin cleavage significantly affected cell–cell adhesion, AKT signaling, the N-cadherin/β-catenin interaction and the Wnt target gene expressions through the accumulation of nuclear β-catenin.