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Transient forebrain ischemia induces a delayed neuronal death in the CA1 area of the hippocampus. However, the mechanism leading to this phenomenon has yet to be established. The authors used an mRNA differential-display method to isolate genes for which mRNA levels change only in the hippocampus during ischemia/reperfusion. They succeeded in identifying the product of one down-regulated gene as phosphatidylinositol 4-kinase (PI 4-K). Compared with control levels, PI 4-K mRNA expression in the hippocampus, but not the cerebral cortex, was significantly decreased by 30% and about 80% 1 and 7 days after ischemia/reperfusion, respectively. Interestingly, PI 4-K and PI bisphosphate levels were selectively decreased in the CA1 region, but not other regions, whereas TUNEL-positive cells could be detected 3 days after ischemia. Consistent with these results, PI 4-K expression was suppressed by hypoxia in SK-N-MC neuroblastoma cells before loss of cell viability. Overexpression of wild-type PI 4-K, but not the kinase-negative mutant of PI 4-K (K1789A), recovered the loss of viability induced by hypoxia. These findings strongly suggest that a prior decrease in PI 4-K and PI bisphosphate levels caused by brain ischemia/hypoxia is partly involved in delayed neuronal cell death.

Conditions that perturb the function of the endoplasmic reticulum (ER) lead to an accumulation of proteins and subsequent induction of several responses, such as an increased expression of ER-resident chaperones involved in protein folding and activation of c-jun N-terminal kinase (JNK). These responses are mediated by a transmembrane kinase/ribonuclease, IRE1, which transduces the signal from the ER lumen to the cytosol. Although nuclear transcription factor-κB (NF-κB) is also activated by ER stress, whether this response depends on IRE1 is unknown. In this study, we show that IRE1 is involved in the activation of NF-κB induced by ER stress. NF-κB was activated by ER stress-inducing agents, thapsigargin and tunicamycin. The activation was inhibited by a dominant-negative IRE1. In addition, a dominant-negative TRAF2 also suppressed the activation of NF-κB by ER stress. These results suggest that ER stress-induced NF-κB activation is also mediated by the IRE1-TRAF2 pathway, as well as JNK activation., The present study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Leptin is an important circulating signal for the regulation of food intake and body weight. Glucocorticoids were suggested to play a physiological role in the feedback inhibition of immune/inflammatory responses. In the present study, we examined whether these neuroendocrine effects of glucocorticoids are linked to changes in the leptin-induced expression of IL-1beta and STAT3 activation in the brain. Intravenous injection of leptin induced IL-1beta expression in the hypothalamus. Pretreatment with dexamethasone dose dependently inhibited leptin-induced IL-1beta expression in the hypothalamus. Moreover, dexamethasone inhibited leptin-induced IL-1beta expression in the primary cultured glial cells. In contrast, pretreatment with dexamethasone did not inhibit leptin-induced STAT3 phosphorylation in the hypothalamus. These effects of dexamethasone may not be due to the change in the expression level of the leptin receptor Ob-Ra and Ob-Rb isoforms. Therefore, it is suggested that glucocorticoid negatively regulates leptin-induced IL-1beta expression in the brain.

Senescence-accelerated mouse prone 8 (SAMP8) and prone 10 (SAMP10) are useful murine model of accelerated aging. SAMP8 shows marked impairment of learning and memory, whereas SAMP10 shows brain atrophy and aging-associated depressive behavior. This study examined the expression of glial cell line-derived neurotrophic factor (GDNF) in SAMP8 and SAMP10 brains, relative to that in SAM resistant 1 (SAMR1) controls, which age normally. Hippocampal GDNF mRNA expression decreased in an age-dependent manner (10- vs 2-month-old animals) in the SAMR1, but not in the SAMP8 or SAMP10 strains. Furthermore, GDNF mRNA expression in 2-month-old SAMP8 and SAMP10 strains was less than in SAMR1 specimens of the same age. The number of surviving neurons in the CA1 region decreased with age in SAMP8 and SAMP10, and also decreased relative to the number of neurons in 10-month-old SAMR1 controls. Immunohistochemistry revealed that cells that were positive for GDNF-like activity in 10-month-old SAMP8 and SAMP10 were diffusely distributed, in part, around the pyramidal cell layer in the hippocampus. These findings suggest that low GDNF expression in young SAMP8 and SAMP10 may be involved in hippocampal dysfunctions, such as age-related learning impairment and neuronal death.

Numerous studies have shown that endogenous and/or environmental neurotoxins and oxidative stress may participate in the pathogenesis of Parkinson's disease (PD), but the detailed mechanisms are still unclear. While dopamine (DA) replacement therapy with L-DOPA (levodopa) improves PD symptoms, it does not inhibit the degeneration of DA neurons in the substantia nigra. Recently, bromocriptine, pramipexole and several other agonists of the dopamine D2-receptor subfamily (including D2, D3 and D4-subtypes) have been shown to have neuroprotective effects in parkinsonian models in vitro and in vivo. Their neuroprotective effects may be mediated directly and/or indirectly by antioxidant effects, mitochondrial stabilization or induction of the antiapoptotic Bcl-2 family.

Recent findings suggest that unfolded or misfolded proteins participate in the pathology of several neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. Usually, several stress proteins and glial cells act as intracellular molecular chaperones and show chaperoning neuronal function, respectively. In the brains of patients with neurodegenerative disorders, however, stress proteins are expressed and frequently associated with protein aggregates, and glial cells are activated around degenerative regions. In addition, several stress proteins and glial cells may also regulate neuronal cell death and loss. Therefore, some types of stress proteins and glial cells are considered to be neuroprotective targets. We summarize the current findings regarding the neuroprotective effects of stress proteins and glial cells, and discuss the possibility of using this knowledge to develop new therapeutic strategies to treat neurodegeneration.

The endoplasmic reticulum (ER) is an organelle that plays a crucial role in protein quality control such as protein folding. Evidence to indicate the involvement of ER in maintaining cellular homeostasis is increasing. However, when cells are exposed to stressful conditions, which perturb ER function, unfolded proteins accumulate leading to ER stress. Cells then activate the unfolded protein response (UPR) to cope with this stressful condition. In the present review, we will discuss and summarize recent advances in research on the basic mechanisms of the UPR. We also discuss the possible involvement of ER stress in the pathogenesis of Alzheimer’s disease (AD). Potential therapeutic opportunities for diseases targeting ER stress is also described.

The regulatory mechanism of Bcl-2 protein expression was investigated in SH-SY5Y cells, the human neuroblastoma cell line that expresses natively Bcl-2 proteins. WHen the cells were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or retinoic acid, the level of Bcl-2 protein was increased compared with the control. These effects were inhibited by pretreatment with a protein kinase C (PKC) inhibitor, staurosporine or calphostin C. The level of Bcl-2 protein was also increased by treatment with carbachol, a muscarinic acetylcholine receptor (mAChR) agonist, and the effect were also inhibited by pretreatment with staurosporine or calphostin C. An addition, a carbachol-induced increase in Bcl-2 protein levels and a transient elevation of [Ca2+]i were inhibited by pretreatment with 4-DAMP (4-diphenylacetoxy-N-methylpiperidine), an m3 mAChR antagonist. In contrast, the level of Bcl-2 protein was decreased by treatment with dibutyryl cAMP (diBu-cAMP), forskolin, or cholera toxin, and the effects of diBu-cAMP were inhibited by pretreatment with a protein kinase A (PKA) inhibitor, H-89. From these results, we suggest that the expression of Bcl-2 proteins is regulated by PKC and PKA in positive and negative manners, respectively, in SH-SY5Y cells. Furthermore, the nucleosomal DNA fragmentation induced by serum depletion for 4 h was observed in SH-SY5Y cells when the level of Bcl-2 protein was down-regulated by treatment with 1 mM diBu-cAMP for 3 days, although the DNA fragmentation by serum depletion for 4 h was not observed in nontreatment cells, indicating that Bcl-2 proteins whose expression is regulated by PKC and PKA play important roles in serum depletion-induced apoptosis.

Stresses that impair the function of the endoplasmic reticulum (ER) lead to an accumulation of unfolded protein in the ER. Under these conditions, the expression of a variety of genes involved in preventing the accumulation of the unfolded proteins is induced. Yeast Hrd1p is an ER stress-inducible ER membrane protein that acts as a ubiquitin ligase (E3) with a RING finger motif and plays a role in the ubiquitination of proteins in the ER. We report here the identification and characterization of a human homolog to yeast Hrd1p. The predicted structures are highly conserved from yeast to humans. Indeed, human HRD1 was localized to the ER and ubiquitinated its substrates. Furthermore, it was found that human HRD1 was up-regulated by ER stress via IRE1 and ATF6, which are ER stress transducers. Interestingly, 293 cells stably expressing wild-type HRD1, but not the C329S mutant, afforded resistance to ER stress-induced apoptosis. These results suggest that the production of HRD1 is up-regulated to protect against ER stress-induced apoptosis by degrading unfolded proteins accumulated in the ER.

Application of a brief period of ischemia, i.e. preconditioning treatment of the middle cerebral artery territory, has been known to produce ischemic tolerance, reducing cerebral infarction volume in the penumbra region after lethal ischemia. However, little is known about the molecular mechanisms responsible for preconditioning-induced ischemic tolerance. In the present study, we examined the difference in the phosphorylation pattern of cyclic AMP responsive element binding protein (CREB) after 1 h of focal cerebral ischemia between preconditioned and non-preconditioned rats by immunohistochemistry and Western blotting. The phosphorylation of CREB in the penumbra region was more rapidly enhanced in the preconditioned rats than in the non-preconditioned rats after 1 h of ischemia. The result suggested that the immediate enhancement in the phosphorylation of CREB in the penumbra region prevented the spread of infarction in the preconditioned rats.

Procalcitonin (PCT), the precursor protein of calcitonin (CT), has been considered recently as a significant indicator of bacterial infection and sepsis. However, the major source of PCT in sepsis remains unclear. The hypothalamic-pituitary-adrenal axis is activated during sepsis. Moreover, immunoreactive CT (iCT) can be detected in the pituitary. Therefore, we examined the effects of lipopolysaccharide (LPS) administration on CT mRNA expression in the pituitary. After administration of LPS, CT mRNA expression in the pituitary was increased significantly. The increase of CT mRNA was associated with significant elevations of the iCT levels in the serum. These results imply that the pituitary is one of the sources of the serum PCT during sepsis.

Leptin is known to be an important circulating signal for regulation of food intake and body weight. Recent evidence has suggested that leptin is involved in infection and inflammation. The afferent vagus nerve is known to be an important component for transmitting peripheral immune signals to the brain, such as interleukin (IL)-1β expression in the brain, anorexia, and fever responses. In the present study, we investigated whether intravenous leptin-induced IL-1β expression in the hypothalamus is mediated via afferent vagus nerve. IL-1β transcripts in the hypothalamus were significantly increased on RT-PCR assessment 1 h after the administration of leptin (1 mg/kg iv) to mice. Subdiaphragmatic vagotomy did not significantly modify intravenous leptin-induced IL-1β expression in the hypothalamus compared with that in sham-treated mice. These data suggest that circulating leptin directly acts in the brain independently of afferent vagus nerve input originating from the subdiaphragmatic organs.

The Ret receptor tyrosine kinase is part of a functional receptor complex for the glial cell line-derived neurotrophic factor (GDNF) family. We examined the expression of Ret mRNA after transient forebrain ischemia, and explored the effect of local GDNF-pretreatment in rat hippocampus on Ret mRNA expression. Transient forebrain ischemia induced Ret mRNA expression in the hippocampus, with a peak effect at 12 h. Whereas intrahippocampal microinjection of GDNF (1.0 microg) in sham-operated rats induced the expression of Ret mRNA (peak at 6 to 12 h), the expected increase of Ret mRNA induced by ischemia was blunted by local GDNF-pretreatment. Immunohistochemical investigation revealed that ischemia-induced Ret receptor expression in the hippocampal CA1 region was also reduced by local GDNF-pretreatment. These findings suggest that GDNF modulates the expression of Ret, and that GDNF signaling pathways that involve the Ret receptor tyrosine kinase might play an important role in brain injury induced by ischemia.

The chemical chaperone 4-phenylbutyric acid (4-PBA) has potential as an agent for the treatment of neurodegenerative diseases. However, the requirement of high concentrations warrants chemical optimization for clinical use. In this study, novel naphthalene derivatives with a greater chemical chaperone activity than 4-PBA were synthesized with analogy to the benzene ring. All novel compounds showed chemical chaperone activity, and 2 and 5 possessed high activity. In subsequent experiments, the protective effects of the compounds were examined in Parkinson’s disease model cells, and low toxicity of 9 and 11 was related to amphiphilic substitution with naphthalene.

The main purposes of our study were to consider the effect of autophagy on auditory cells under oxidative stress, and the function of possible crosstalk among p62, Keap1 and Nrf2 in autophagy-deficient auditory cells. First, we described how cell death was induced in auditory cell line (HEI-OC1) exposed to H2O2. We found that the decision for the cell death of auditory cells under oxidative stress depends on the balance between autophagy and necrosis due to ATP depletion, and autophagy plays a cytoprotective function in oxidative stress-induced necrosis. Our data clearly suggested that autophagy was a cell survival mechanism in H2O2-induced cell death, based on the observation that suppression of autophagy by knockdown of Atg7 sensitized, whereas activation of autophagy by rapamycin protected against H2O2-induced cell death. Next, our results regarding the relationship among p62, Nrf2 and Keap1 by siRNA paradoxically showed that p62 creates a positive feedback loop in the Keap1/Nrf2 pathway. Autophagy impaired by Atg7 knockdown degrades Keap1 in a p62-dependent manner, whereas Nrf2 is activated. As a result, the cell death induced by H2O2 was promoted in auditory cells. Taken together, these results suggested that the autophagy pathway maintained signaling crosstalk with the Keap1-Nrf2 system through p62 in auditory cells under oxidative stress.

Members of the epidermal growth factor family play important roles in the regulation of cell growth, proliferation, and survival. However, the specific roles of each epidermal growth factor family member with respect to brain injury are not well understood. Gene chip assay screens have revealed drastic increases in the expression of the epidermal growth factor family members amphiregulin and epiregulin following lipopolysaccharide stimulation, which activates an immune response. Both immune activity and endoplasmic reticulum stress are activated during cerebral ischemia. We found that the expression levels of amphiregulin and epiregulin were significantly increased under conditions of cerebral ischemia. Because endoplasmic reticulum stress increased the expression of amphiregulin and epiregulin in glial cells, endoplasmic reticulum stress may be a key mediatory factor of pathophysiological activity. Recombinant epiregulin and amphiregulin proteins effectively inhibited endoplasmic reticulum stress and the subsequent induction of neuronal cell death. Therefore, the upregulation of the epidermal growth factor family members epiregulin and amphiregulin may play a critical role in preventing endoplasmic reticulum stress-induced cell death, thus providing a potential therapy for brain injury.

The E3 ubiquitin ligase HRD1 is found in the endoplasmic reticulum membrane of brain neurons and is involved in endoplasmic reticulum-associated degradation. We previously demonstrated that suppression of HRD1 expression in neurons causes accumulation of amyloid precursor protein, resulting in amyloid β production associated with endoplasmic reticulum stress and apoptosis. Furthermore, HRD1 levels are significantly decreased in the cerebral cortex of Alzheimer’s disease patients because of its insolubility. The mechanisms that affect HRD1 solubility are not well understood. We here show that HRD1 protein was insolubilized by oxidative stress but not by other Alzheimer’s disease-related molecules and stressors, such as amyloid β, tau, and endoplasmic reticulum stress. Furthermore, we raise the possibility that modifications of HRD1 by 4-hydroxy-2-nonenal, an oxidative stress marker, decrease HRD1 protein solubility and the oxidative stress led to the accumulation of HRD1 into the aggresome. Thus, oxidative stress-induced HRD1 insolubilization might be involved in a vicious cycle of increased amyloid β production and amyloid β-induced oxidative stress in Alzheimer’s disease pathogenesis.

Studies on the cellular and molecular mechanism of neurotransmitter receptor-signaling and of neuronal and glial cell responses to stresses seem to be important to elucidate the action mechanism of centrally-acting drugs and to develop novel therapeutics against several diseases in the brain. The present review shows our findings with regard to the membrane receptor-signaling mechanism including serotonin, noradrenaline, glutamate receptors, ion channels, G-proteins, protein kinases and drug actions in Xenopus oocytes injected with rat brain mRNA, NG108-15 cells and brain membranes. Regarding the results of studies on the inter- and intra-cellular mechanism of neurons and glial cells against cerebral ischemia/hypoxia, we review the involvement of a transcription factor NF-kappa B in LPS-elicited inducible NO synthase (iNOS) expression in rat astroglial cells. Then we describe possible involvement of: 1) ADP-ribosylation/nitrosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 2) decrease in mitochondrial membrane potential, release of caspase-3 from mitochondria and degradation of the inhibitor of caspase-activated DNase by activated caspase in NO-induced neuronal apoptosis. We observed that hypoxia results in expression of a molecular chaperon such as protein disulfide isomerase (PDI) and HSP70 in astroglial cells. Our recent findings indicate that overexpression of PDI in the rat hippocampus (in vivo) and in neuroblastoma SK-N-MC cells (in vitro) significantly suppress the hypoxia-induced neuronal death. From physiological/pathophysiological and pharmacological aspects, we review the importance of studies on the cellular and molecular mechanism of membrane receptor-signaling and of stress-responses in the brain to identify functional roles of neuro-glial- as well as neuro-neuronal interaction in the brain.

This study examined the expression of glial cell line-derived neurotrophic factor (GDNF) mRNA and the cellular localization of GDNF production in rats subjected to transient forebrain ischemia induced by four-vessel occlusion. Transient forebrain ischemia induced GDNF mRNA expression in the hippocampus from 3 h to 3 days after the ischemic episode, with peak expression at 6 h. The GDNF mRNA increase in the cerebral cortex was similar to that in the hippocampus, whereas no increase in GDNF mRNA was observed in the striatum and brainstem. Western blot analysis showed that GDNF in the hippocampal CA1 region was increased slightly from 3 to 24 h after the ischemia, and then subsequently declined to below the baseline level. In the hippocampus, GDNF was evenly produced in pyramidal neurons of both sham-operated rats and normal rats, as determined by immunohistochemistry. Interestingly, we found that ischemia-induced reactive astrocytes, as well as surviving neurons, produced GDNF in 3-7 days after the ischemia. On the other hand, in other regions, such as the cerebral cortex, striatum, and brainstem, there was no change in GDNF-positive cells secondary to ischemia. These findings suggest that expression of GDNF mRNA is regulated in part via ischemia-induced neuronal degeneration. They also suggest that ischemia-induced reactive astrocytes may produce GDNF to protect against neuronal death. Therefore, GDNF may play an important role in ischemia-induced neuronal death in the brain.

Previously, we proposed that S-nitroso-cysteine (SNC) was incorporated via the l -type-like amino acid transporters in rat brain slices. In PC12 cells (rat neuronal cell line), SNC inhibited [ 3 H]arachidonic acid (AA) release induced by mastoparan (wasp venom peptide). We investigated the involvement of amino acid transporters on SNC-induced inhibition of [ 3 H]AA release in PC12 cells. SNC inhibited mastoparan-stimulated [ 3 H]AA release in a concentration-dependent manner in normal Na + - and low Na + -containing buffer. The inhibitory effect of 0.6 mM SNC in low Na + buffer decreased by 10 mM l -leucine, l -phenylalanine, l -methionine and l -cysteine. In contrast, l -alanine, l -threonine, l -valine or l -isoleucine showed very limited effects. Addition of l -leucine and l -phenylalanine, but not l -alanine or l -valine, also decreased the inhibitory effect of SNC on ionomycin/Na 3 VO 4 -stimulated [ 3 H]AA release in normal Na + buffer. These findings suggest that SNC is incorporated via the amino acid transporters and inhibits AA release in PC12 cells.

The period doubling bifurcation process in the two-dimensional area preserving mapping is investigated on the basis of symmetry structure analysis. In particular a case of the peirod-4 orbits in the standard map has been studied thoroughly to analyze boundary islands formation around the principal period-4 island, and the onset of the hyperbolic bifurcation without reflection. It is illustrated explicity that the hyperbolic bifurcation without reflection gives rise to the birth of twin orbits with the periodicity of the mother orbit.

This review will discuss the recent literature on the molecular mechanism of NF-κB activation, with special focus on IκBα dynamism involved in iNOS- and chemokine-induction in glial cells. NF-κB, a heterotrimer composed of p50, p65 (Rel A) and IκBα, has been shown to be activated by elimination of the regulatory subunit IκBα from the heterotrimer. The elimination of IκBα (formation of active NF-κB, p50·p65) is due to phosplorylation of serines 32 and 36 of IκBα, followed by polyubiquitination and 26S proteasomal degradation of IκBα. Experiments using stable clones of rat C6 glioma cells transfected with dominant negative IκBα (serines 32 and 36 replaced by alanine) suggest that NF-κB activation (phosphorylation of IκBα) is involved in LPS/IFNγ- or IL-1β/IFNγ-induced iNOS expression. Furthermore, the time courses of phosphorylation, ubiquitination of IκBα and proteasome activity after IL-1β treatment also suggest that 26S proteasomal degradation of IκBα is more crucial for chemokine expression in glial cells.

15-Deoxy-Delta(12,14) prostaglandin J(2) and interleukin-4 are endogenous anti-inflammatory substances. In this study, we examined the effects of 15-deoxy-Delta(12,14) prostaglandin J(2) and interleukin-4 in glial cells from the Toll-like receptor-4-mutant (C3H/HeJ) and wild-type (C3H/HeN) mouse brains. The lipopolysaccharide-induced expression of inducible nitric oxide (NO) synthase and cyclooxygenase-2 in the Toll-like receptor-4-mutant glial cells have significantly lower levels (about half and quarter, respectively) than those in the wild-type cells. Treatment with both interleukin-4 (at 10 ng/ml, for 48 h) and 15-deoxy-Delta(12,14) prostaglandin J(2) (at 3 microM, for 30 min) completely inhibited the lipopolysaccharide-induced expression of inducible NO synthase and cyclooxygenase-2. In contrast, heme oxygenase-1 was induced by 15-deoxy-Delta(12,14) prostaglandin J(2) alone, but was not changed by interleukin-4 or lipopolysaccharide. The inhibitory protein of nuclear factor-kappa B was degraded by lipopolysaccharide in both mutant and wild-type glial cells, and this degradation was not inhibited by either 15-deoxy-Delta(12,14) prostaglandin J(2) or interleukin-4. These results suggest that the response to lipopolysaccharide is partially dependent on Toll-like receptor-4 in mouse glial cells, and that 15-deoxy-Delta(12,14) prostaglandin J(2) and interleukin-4 differently regulate the expression of inducible NO synthase and cyclooxygenase-2, and heme oxygenase-1.

Glucocorticoid-attenuated response genes (GARG) belong to a recently described family of genes responsive to the action of dexamethasone. Full-length cDNA of one member of this family, GARG16, has been cloned from rat microglia and regulation of its mRNA expression has been studied. Moreover, regulation of retinoid/retinoic acid activated transcription factor (RXR/RAR) mRNAs in mixed astrocyte and in purified microglia cultures has been investigated. RARbeta mRNA was undetectable in microglia by RT-PCR, whereas clearly present in the mixed cultures. RXRalpha, RARgamma, and GARG16 mRNAs were found in both culture systems. RXRalpha mRNA was strongly expressed in control microglia but rapidly declined upon treatment with LPS. Conversely, GARG16 mRNA was almost untraceable in control microglia but rapidly increased by LPS. Time-course studies revealed an oscillating behavior of expression of both mRNAs during the first 6 hr, which receded to control levels (RXRalpha high, GARG16 low) at 72 hr of LPS-treatment. Additionally, p38 MAPK and SEK phosphorylations peaked at 1 hr followed by steady declines, whereas MEK and c-Jun showed double peaks at 1+4 hr and 1+6 hr, respectively, before subsiding to control levels. This behavior was not observed in comparative studies with TNF-alpha, interleukin-10 (IL-10), or interferon-gamma inducible protein 10 (IP-10). Finally, inhibitors of p38 MAPK, p42/p44 ERK, and PKCalpha as well as the use of dexamethasone revealed major influences of the p38 MAPK-c-Jun-AP-1 signaling pathway on RXRalpha and GARG16 mRNA expressions. The counter regulatory control of GARG16 and RXRalpha mRNA expression is believed to be an example of a fine-tuned cellular mechanism to react to inflammatory stimuli.

Receptors for many neurotransmitters including catecholamines and acetylcholine (ACh) have been detected on the cell surface of lymphocytes. It has been demonstrated that a human T cell line synthesizes ACh and suggested that ACh may be an autacoid modulating T cell-dependent immune responses. However, the biochemical interactions of the ACh system with the immune system have not been elucidated in detail. We have shown that m1 and m2 muscarinic receptor mRNAs are expressed in human peripheral blood lymphocytes and in human T cell line Jurkat cells and that pretreatment of these cells with a muscarinic receptor agonist enhances interleukin-2 (IL-2) production. We also postulated possible intracellular signaling pathways via which muscarinic receptors regulate IL-2 production in Jurkat cells. The findings suggest that M1 muscarinic receptors are involved in muscarinic receptor-mediated enhancement of IL-2 production in Jurkat cells and that the transcription factor AP-1 and pathways via mitogen-activated protein kinase (MAPK)/extracellular signal regulated protein kinase and c-Jun N-terminal kinase, but not via p38 MAPK, may be involved in the muscarinic receptor-mediated enhancement of IL-2 production. Our findings demonstrate a neuro-immune interaction through muscarinic receptor signaling in immune cells.

The senescence-accelerated mouse (SAM) is known to be a murine model for accelerated aging. The SAMP8 strain shows age-related deterioration of learning and memory at an earlier age than control mice (SAMR1). In the present study, we investigated the changes in expressions of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in the brain of SAMP8. In the hippocampus of 10 months old SAMP8, the expression of IL-1 mRNA was significantly elevated in comparison with that of SAMR1. In both strains of SAMs, increases in IL-1beta protein in the brain were observed at 10 months of age compared with 2 and 5 months. The only differences found between the strain in protein levels were at 10 months and were elevations in IL-1beta in the hippocampus and hypothalamus, and in TNF-alpha and IL-6 in the cerebral cortex and the hippocampus in SAMP8 as compared with SAMR1. However, lipopolysaccharide-induced increases in the expression of these cytokines in brain did not differ between SAMP8 and SAMR1. Increases in expression of proinflammatory cytokines in the brain may be involved in the age-related neural dysfunction and/or learning deficiency in SAMP8.

It has been demonstrated from studies using NF-kappaB inhibitors that NF-kappaB may be involved in the iNOS induction stimulated by cytokines and/or lipopolysaccharide (LPS) in various cell types and tissues. However, the actions of the inhibitors are less selective and highly cytotoxic. We constructed stable clones of C6 cells transfected with two types of IkappaBalpha mutant genes (IkappaBalpha(SS --AA); Ser-32/36 to Ala-32/36, IkappaBalpha(KK --RR); Lys-21/22 to Arg-21/22). IkappaBalpha(SS --AA) strongly inhibited (1) LPS-, IL-1beta-, and TNF-alpha-induced nuclear translocation and DNA binding of NF-kappaB to the kappaB site; and (2) iNOS induction stimulated by LPS or IL-1beta plus IFN-gamma. These results indicate that NF-kappaB plays a critical role in cytokines and/or LPS-induced iNOS induction. Surprisingly, similar to the endogenous IkappaBalpha, IkappaBalpha(KK --RR) was degraded by various stimuli, and proteasome inhibitors blocked this event. These results suggest that another Lys residue(s), other than Lys-21/22, may be required for the ligand-induced IkappaBalpha degradation by the ubiquitin-proteasome pathway.

Interleukin-4 (IL-4) likely is one of the key players in the concert of immunosuppressive factors in brain. Therefore, influences of the cytokine on mRNA expression of endogenous mediators of inflammation, such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha), interferon-inducible protein 10 (IP-10), interleukin-3 receptor-beta (IL-3R-beta), and of another antiinflammatory cytokine, IL-10, have been evaluated in the present study by semi-quantitative RT-PCR. Primary rat mixed glial cultures and isolated microglial cells, the resident immunocytes of the brain, have been used as rich sources of these mRNAs in response to the bacterial cell wall component lipopolysaccharide (LPS). Time-course studies showed peak levels of LPS-increased mRNAs at approximately 4 h. Interestingly, IL-10 mRNA was elevated also upon the LPS-stimulus. IL-4, given 30 min before LPS, inhibited increases of all mRNAs significantly, including IL-10 mRNA. IL-4, however, induced peroxisome proliferator-activated receptor (PPAR)-gamma in cultured microglia. This induction was completely inhibited by simultaneous administration of LPS. The data confirms IL-4 as an important antiinflammatory cytokine and gives some idea of cross-talk between intracellular signaling evoked by pro- and antiinflammatory substances.

Recently, we have reported that glial cell line-derived neurotrophic factor (GDNF), which supports the survival of dopaminergic neurons, prevents delayed neuronal death in the hippocampal CA1 region induced by transient forebrain ischemia. In the present study, we examined the role of GDNF in the expression of tyrosine hydroxylase (TH) mRNA induced by transient forebrain ischemia in rats. The expression of TH mRNA was increased in a time-dependent manner, with a significant increase in 24 h to 7 days, in the hippocampus after induction of transient forebrain ischemia, as determined using the reverse transcription and polymerase chain reaction method. Although it has been suggested that the increase of dopamine beta-hydroxylase mRNA expression correlates with the activation of noradrenergic neurons, no increase of dopamine beta-hydroxylase mRNA in the hippocampus was observed in our system. Western blot analysis revealed that TH protein, but not dopamine beta-hydroxylase protein, was produced in a time-dependent manner in the hippocampus during the ischemia. Interestingly, the induction level of TH mRNA was reduced by intrahippocampal microinjection of GDNF (1.0 microg), and this local GDNF treatment also reduced the increase of TH-like immunohistochemistry-positive terminals in the hippocampus. In contrast, local GDNF treatment of normal rats increased the TH mRNA expression at 6-12 h. These findings suggest that GDNF protects against neuronal degeneration including delayed neuronal death in the hippocampal CA1 region by modulating the expression levels of TH mRNA and protein.

To examine the role of leptin in the immune function of the brain, we examined the effect of leptin on interleukin-1beta (IL-1beta) expression in mouse primary cultured glial cells. The expression of leptin receptor isoforms Ob-Ra and Ob-Rb mRNA was detected by RT-PCR analysis of total RNA from primary cultured glial cells. Protein of leptin receptor was also expressed in mouse primary cultured glial cells as evaluated by Western blotting analysis. Leptin increased the expression of IL-1beta mRNA evaluated by RT-PCR. The expression of IL-1beta transcript peaked 2 to 6 h after leptin application. These results indicate that leptin could induce IL-1beta transcript in the brain and that one of the target cells of the leptin-induced IL-1beta transcript may be a glial cell.

We isolated and identified a stress protein that is up-regulated in response to hypoxia in primary-cultured glial cells. Protein-disulfide isomerase (PDI) was up-regulated not only by hypoxia in glia in vitro, but also by transient forebrain ischemia in rats in vivo. To determine whether newly synthesized PDI is involved in tolerance to ischemic stress, we carried out two procedures to induce PDI gene expression in human neuroblastoma SK-N-MC cells, as well as intrahippocampal injection following electroporation of an expression vector capable of overexpressing PDI in rats. Overexpression of this gene resulted in attenuation of the loss of cell viability induced by hypoxia in neuroblastoma SK-N-MC cells and a reduction in the number of DNA-fragmented cells in the CA1 area of the hippocampus in brain ischemic rats, respectively. These findings suggest that up-regulated PDI may play a critical role in resistance to ischemic damage, and that the elevation of levels of this protein in the brain may have beneficial effects against brain stroke.

Nitric oxide has been shown to regulate neurotransmitter release. Previously, we reported that S-nitrosothiols such as S-nitrosocysteine (SNC) stimulate noradrenaline (NA) release in rat hippocampus in vivo and in vitro. To examine the role of sulfhydryl groups in SNC-induced NA release, the effects of metal ions such as Hg2+ and N-ethylmaleimide (NEM, a sulfhydryl alkylating agent) on [3H]NA release from labeled rat brain slices (hippocampus and cerebral cortex) were studied and compared with the effects of SNC. The addition of 200 microM HgCl2, but not Pb2+, Zn2+, or Cd2+, stimulated [3H]NA release from both types of slices in the presence of extracellular CaCl2. p-Chloromercuribenzoic acid (p-CMBA) also stimulated [3H]NA release. NEM stimulated [3H]NA release from both types of slices in the presence and absence of extracellular CaCl2. The effect of 200 microM NEM was enhanced, but the effect of 200 microM SNC was inhibited by co-addition of 200 microM p-CMBA in the absence of extracellular CaCl2. The concentration-response curve of SNC shifted to the right after co-addition of 200 microM p-CMBA or 100 microM HgCl2, although the effect of 200 microM NEM was additive to the effect of SNC. These findings demonstrate that SNC acts as a sulfhydryl agent on proteins that regulate NA release, and that SNC may share the same sulfhydryl groups with Hg compounds. The effect of T-588 ¿(R)-(-)-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino)ethoxy]eth anol hydrochloride¿, a novel cognitive enhancer and a stimulator of NA release, was compared with the effects of sulfhydryl reagents.

The senescence-accelerated mouse (SAM) is known to be a murine model for accelerated aging. A novel inbred SAMP10 has shown age-related brain atrophy and learning deficiency. In the present study, we investigated the changes in learning ability and in ligand binding with muscarinic acetylcholine (mACh) receptors, alpha adrenoceptors and protein kinase C in SAMP10. In Morris's water maze task, in a control strain of SAMR1 at 9 months, the escape latency and path length decreased with increasing trial days, in contrast, escape latency and path length did not decrease in SAMP10. These results indicate that SAMP10 exhibits learning deficiency. The ligand binding activity of mACh receptors decreased in the hippocampus of SAMP10 and the protein kinase C level in the hippocampus of SAMP10 was lower than that of SAMR1. On the other hand, there was no significant difference between SAMR1 and SAMP10 regarding ligand binding activity of alpha(1) and alpha(2) adrenoceptors. Thus, a reduction of mACh receptors and protein kinase C in the brain seems to underlie dysfunction of learning and memory in SAMP10.

Previously we reported that 1-methyl-4-phenylpyridinium ion (MPP(+)), a dopaminergic neurotoxin, induced apoptosis of GH3 cells established from rat anterior pituitary. In the present study, the role of MPP(+) along with that of other apoptotic factors such as Ca(2+) and H(2)O(2) in cell death was examined. Ionomycin induced DNA fragmentation and lactate dehydrogenase (LDH) leakage in GH3 cells. H(2)O(2) also induced LDH leakage. Co-addition of MPP(+), in conditions where MPP(+) had no effect by itself, enhanced ionomycin- and H(2)O(2)-induced cell death. Because the stimulation of phospholipase A(2) (PLA(2)) causing arachidonic acid (AA) release has been proposed to be involved in neuronal cell death, the effect of MPP(+) on AA release in GH3 cells was investigated. MPP(+) treatment for 8 h enhanced ionomycin- and H(2)O(2)-stimulated AA release mediated by activation of cytosolic PLA(2) in a concentration-dependent manner, although MPP(+) by itself had no effect on AA release. An inhibitor of cytosolic PLA(2) inhibited MPP(+)-induced cell death. These findings suggest a synergistic effect of MPP(+) on Ca(2+)- and H(2)O(2)-induced cell death, and the involvement of cytosolic PLA(2) activation in MPP(+)-induced cell death in GH3 cells. Pretreatment with a caspase inhibitor or EGF did not modify the ionomycin- or H(2)O(2)-induced AA release, or enhancement by MPP(+), but the pretreatment inhibited the cell death in the presence and absence of MPP(+). The involvement of caspase(s) on activation of PLA(2) by MPP(+) was excluded, and EGF inhibited MPP(+)-induced cell death downstream of the AA release.

S-Nitroso-cysteine (SNC) inhibits Ca2+-induced noradrenaline (NA) release from PC12 cells. Since SNC stimulated Ca2+ mobilization from intracellular Ca2+ pools and SNC-induced inhibition of NA release was not washed-out, SNC may modify exocytosis-related proteins that overcome Ca2+ mobilization. In the present study, we investigated the effects of SNC on exocytosis-related proteins in PC12 cells. Ionomycin stimulated NA release and increased the immunoreactivity of synaptophysin in the cytosol fraction. A 25-kDa synaptosome-associated protein (SNAP-25), which localizes to plasma membranes and vesicles, increased in the cytosol fraction after stimulation. The increases in these proteins by ionomycin were inhibited in PC12 cells treated with 0.6 mM SNC. Synaptobrevin and synapsin-1 in the cytosol fraction, and syntaxin and 43 kDa growth-associated protein in the membrane fraction were not affected by ionomycin or SNC. Incubation of each protein with SNC did not affect antibody immunoreactivity. [32P]ADP-ribosylation of GTP-binding proteins (Gi/Go) by pertussis toxin, but not Gs by cholera toxin, was inhibited in SNC-treated PC12 cells and by co-addition of SNC to the assay mixture. These findings suggest that 1) SNC inhibits translocation of vesicles containing synaptophysin and SNAP-25, and 2) SNC reacts with cysteine residues in Gi/Go, causing inhibition of ADP-ribosylation by pertussis toxin.

We have previously shown that m1 and m2 muscarinic receptors were expressed on human peripheral blood lymphocytes (hPBL) and that pre-stimulation of these receptors enhanced phytohemagglutinin (PHA)-induced interleukin-2 (IL-2) production. Possible intracellular signal pathways of muscarinic receptors to regulate IL-2 production were examined in human T cell line Jurkat cells. Pretreatment of the cells with muscarinic receptor agonist, oxotremorine M (Oxo-M), enhanced IL-2 production induced by phorbol 12-myristate 13-acetate (PMA)/A23187, while Oxo-M by itself did not affect IL-2 production. The enhancement of IL-2 production by Oxo-M was inhibited by 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) an m1/m3 receptor antagonist. When the cells were pretreated with AF-DX116, an m2 antagonist, the IL-2 production enhanced by Oxo-M was further stimulated. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that m1 and m2 muscarinic receptors exist on Jurkat cells.The stimulation of m1 receptors enhanced the PMA/A23187-induced binding activity to AP-1 consensus sequences in IL-2 promoter and production of c-Fos and c-Jun protein. The stimulation of m1 receptors did not modify the DNA binding of NF-κB, NF-AT or Oct-1. When m1 receptors were stimulated, activities of mitogen-activated protein kinase (MAPK)/extracellular signal regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) were increased, while p38 MAPK was not affected. Incubation with Oxo-M induced a transient increase in [Ca2+]i, which was abolished by pretreatment with 4-DAMP. Treatment with cyclosporin A markedly decreased the PMA/A23187-induced IL-2 promoter activity. This treatment, however, did not affect the enhancement of the promoter activity induced by m1 receptor stimulation.The results suggest that transcription factor AP-1 is involved in the m1 receptor-mediated enhancement of IL-2 transcript in Jurkat cells, and that pathways via MAPK/ERK and JNK, but not via p38 MAPK, are involved in the m1 receptor-mediated enhancement of IL-2 promoter activity.

Previously, we reported that (R)-(-)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N- diethylamino)ethoxy] ethanol hydrochloride (T-588), a novel putative cognitive enhancer, stimulated noradrenaline (NA) release from rat cerebral cortical slices. In this study, we investigated the effects of T-588 compared to other secretagogues on NA release from PC12 cells. Addition of as little as 10 microM T-588 stimulated [3H]NA release in a dose-dependent and an extracellular Ca(2+)-independent manner from PC12 cells. Ten micromolar ionomycin-, 300 microM adenosine-5'-O-(gamma-thiotriphosphate)- and 10 microM forskolin-induced extracellular Ca(2+)-dependent [3H]-NA release was further enhanced by 30 microM T-588. Cytosolic synaptophysin and 25-kDa synaptosome-associated protein immunoreactivity was increased by addition of T-588 in a dose-dependent manner. Interestingly, increases in synaptic vesicle-related proteins triggered by T-588 had a 4-min lag time and were completely dependent on extracellular CaCl2. These findings suggest that T-588 stimulates NA release from PC12 cells in a Ca(2+)-independent manner. T-588 also induced the translocation of synaptic vesicles in a Ca(2+)-dependent manner.

Tyrosine phosphorylation has been shown to participate in the signal cascade after receptor stimulation with neurotransmitters and neurotrophins. However, the role of tyrosine phosphorylation in the process(es) of neurotransmitter release has not been well established. The effects of orthovanadate (Na3VO4), an inhibitor of protein-tyrosine phosphatases, on cytosolic free Ca2+ concentrations ([Ca2+]i), phosphotyrosine accumulation and noradrenaline (NA) release in neurosecretory PC12 cells were investigated. Addition of Na3VO4 enhanced ionomycin-stimulated [3H]NA release in a concentration-dependent manner, although Na3VO4 alone had no effect. Na3VO4 also enhanced [3H]NA release induced by P2 receptor stimulation with adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) or by depolarization with 50 mM KCl, which stimulated a [Ca2+]i increase. A cell permeable inhibitor of protein-tyrosine phosphatases, L-p-bromotetramisole oxalate, at 0.3 mM enhanced ionomycin-stimulated [3H]NA release, although pervanadate had no effect. Addition of 5 mM Na3VO4 stimulated phosphotyrosine accumulation in several protein bands such as p130cas, but did not increase [Ca2+]i in PC12 cells. These findings suggest that the tyrosine phosphorylation pathway regulates Ca2+-stimulated NA release without changes of [Ca2+]i in PC12 cells.

It has long been known that pregnancy stimulates a variety of physiological mucosal changes, including pigmentary, elastic, and vascular changes. Pyogenic granuloma represents a vascular lesion observed during gravidism. We present three case reports of pregnancy tumor.The patients ranged in age from 26 to 29 years old, and had lesions located on the tongue. In all cases, surgical excision was performed after delivery.Histopathologically, pregnancy tumor is identical to pyogenic granuloma of unrelated etiology. The majority of lesions exhibit nodular inflammation hyperplasia and numerous small dilatated vascular channels and proliferating endothelial cells. The surface with a thin atrophic epithelium is almost completely ulcerated and coated with a thick, fibrinopurulent exudate. This “epidermal collarette” surrounds the deeper portion. There are three main pathological features: granulomatous, hemangiomatous, and intermediate types. One case was the hemangiomatous type and two cases were the intermediate type.This article also discusses the clinical findings, pathology, therapy and prognosis of pyogenic granuloma.

We previously reported that several stresses can induce cytokine-induced neutrophil chemoattractant expression in a nuclear factor κB (NF-κB)-dependent manner. In this study, we focused further on the regulation of NF-κB. The activation of NF-κB and the subsequent cytokine-induced neutrophil chemoattractant induction in response to interleukin-1β (IL-1β) were inhibited by proteasome inhibitors, MG132 and proteasome inhibitor I. Translocation of NF-κB into nuclei occurs by the phosphorylation, multi-ubiquitination, and degradation of IκBα, a regulatory protein of NF-κB. Nascent IκBα began to degrade 5 min after treatment with IL-1β and disappeared completely after 15 min. However, IκBα returned to basal levels after 45–60 min. Interestingly, resynthesized IκBα was already phosphorylated at Ser-32. These results suggest that 1) the upstream signals are still activated, although the translocation of NF-κB peaks at 15 min; and 2) the regulated protein(s) acts downstream of IκBα phosphorylation. Western blotting showed that the resynthesized and phosphorylated IκB molecules were also upward-shifted by multi-ubiquitination in response to IL-1β treatment. On the other hand, ATP-dependent Leu-Leu-Val-Tyr cleaving activity transiently increased, peaked at 15 min, and then decreased to basal levels at 60 min. Furthermore, the cytosolic fraction that was stimulated by IL-1β for 15 min, but not for 0 and 60 min, could degrade phosphorylated and multi-ubiquitinated IκBα. These results indicate that the transient translocation of NF-κB in response to IL-1β may be partly dependent on transient proteasome activation.

The effects of nitrogen monoxide (NO)-related compounds on cytosolic free Ca2+ concentrations ([Ca2+]i) and noradrenaline (NA) release in neurosecretory PC12 cells were investigated. The addition of S-nitroso-cysteine (SNC) stimulated [Ca2+]i increases from an intracellular Ca2+ pool continuously in a concentration-dependent manner. Other NO donors, which stimulate cyclic GMP accumulation, did not cause [Ca2+]i increases. After treatment with 0.2 mM SNC, transient increases in [Ca2+]i from the Ca2+ pool induced by caffeine were completely abolished. The addition of N-ethylmaleimide (NEM) caused sustained [Ca2+]i increases from the intracellular Ca2+ pool. Furthermore, caffeine did not stimulate further [Ca2+]i increases in PC12 cells pretreated with NEM. These findings suggest that SNC and NEM predominantly interact with a caffeine-sensitive Ca2+ pool. The addition of dithiothreitol (DTT) to 0.4 mM SNC-stimulated cells reduced [Ca2+]i to basal levels, and the addition of DTT to NEM-stimulated cells locked [Ca2+]i at high levels. The stimulatory effects of SNC but not NEM were not abolished by pretreatment with DTT. These findings suggest that modification of the oxidation status of the sulfhydryl groups on the caffeine-sensitive receptors by SNC or NEM regulates Ca2+ channel activity in a reversible manner. SNC did not stimulate NA release by itself but did inhibit ionomycin-stimulated NA release. In contrast, NEM stimulated NA release in the absence of extracellular CaCl2 and further enhanced ionomycin-stimulated NA release. Ca2+ mobilization by SNC from the caffeine-sensitive pool was not a sufficient factor, and other factors stimulating NA release may be negatively regulated by SNC.

The aim of this study was to elucidate the possible mechanism of HSP induction in response to hypoxia in rat primary astrocytes. Treatment with SB203580, a selective p38 MAP kinase (p38 MAPK) inhibitor, attenuated the increase in HSP70 in a concentration-dependent manner. p38 MAPK was activated in response to hypoxic treatment. These results suggest that p38 MAPK positively regulates hypoxia-induced HSP70 expression in astrocytes.

Pertinent animal models of age-related learning deficiencies are required to elucidate the mechanism of age-related learning deficiencies and to develop novel therapeutic drugs for age-related diseases such as learning defects. Among many strains of accelerated senescence prone, senescence-accelerated mouse (SAM), SAMP8 mice have age-related defects in learning and cognitive abilities. We review recent findings on alterations in SAMP8 brain in neurochemical parameters related to neurotransmission and synaptic plasticity compared to those in SAMR1 brain as the control. In addition, we report the preventive effects of drugs on learning deficiencies in SAMP8 and discuss the usefulness of SAMP8 as an animal model of age-related learning deficiencies.

Glial cell line-derived neurotrophic factor, a member of the transforming growth factor-β superfamily, is a potent neurotrophic factor, which has a variety of biological activities that affect several types of neurons in both the central and peripheral nervous systems. In this study, we examined the effects of glial cell line-derived neurotrophic factor on delayed neuronal death in the hippocampal CA1 region of rats after transient forebrain ischemia. In the control rats pretreated with the vehicle, transient forebrain ischemia-induced delayed neuronal death in the hippocampal CA1 region was observed seven days after reperfusion. Pretreatment with glial cell line-derived neurotrophic factor (1.0 μg), which was directly microinjected into the right hippocampal CA1 region, gave significant protection against the delayed hippocampal neuronal death. On the contralateral side of the hippocampus, which was not injected with glial cell line-derived neurotrophic factor, delayed neuronal death similar to that seen in vehicle-treated control animals was observed. Intracerebroventricular glial cell line-derived neurotrophic factor (2.5 μg) injection also protected against delayed neuronal death. In addition, pretreatment with glial cell line-derived neurotrophic factor gave significant protection against apoptotic cell death induced by brain ischemia in the hippocampal CA1 region, as determined by in situ staining for DNA fragmentation. These findings suggest that glial cell line-derived neurotrophic factor plays an important role in delayed neuronal death induced by brain ischemia.

The peroxisome proliferator-activated receptor-gamma (PPARgamma) is activated by 15-deoxy-delta(12,14) prostaglandin J2 (15d-PGJ2), anti-diabetic thiazolidinediones and several non-steroidal anti-inflammatory drugs (NSAIDs). In rat glial cells, lipopolysaccharide and interferon-gamma (LPS/IFN-gamma) induced expression of both inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1). PPARgamma activators inhibited iNOS expression by LPS and IFN-gamma. However, PPARgamma activator alone induced HO-1 expression and further enhanced LPS/IFN-gamma-induced HO-1 expression. These results suggest that activation of PPARgamma negatively regulate iNOS expression and positively regulates HO-1 expression in glial cells.

To elucidate the intracellular mechanism of NF-kappa B activation, we performed the involvement of I kappa B alpha of NF-kappa B in the expression of inducible NO synthase (iNOS) and chemokine (CINC) following pretreatment with bacterial endotoxin (LPS) or IL-1 beta, respectively, using rat C6 glioma cells. We found that herbimycin A, a tyrosine protein kinase inhibitor, blocked: 1) LPS/IFN gamma-induced iNOS expression, 2) LPS-induced intranuclear translocation of activated NF-kappa B (p50. p65) and 3) IFN gamma-induced autophosphorylation and activation of Jak 2 and Stat 1 as well as intranuclear translocation of phosphorylated Stat 1. Furthermore, transfection of a dominant negative form of I kappa B alpha (SS-->AA) suppressed LPS/IFN gamma-induced iNOS expression, suggesting that NF-kappa B, in particular, I kappa B alpha molecules could play important roles in the iNOS expression. We also found in IL-1 beta-induced CINC expression using cultured C6 glioma cells, the transient translocation of NF-kappa B in response to IL-1 beta is partly dependent on transient proteasome activation. Thus we suggest that the formation of heterodimer p50.p65 from inactive trimer p50.p65.I kappa B alpha, particularly, proteolytic degradation and dissociation of I kappa B alpha from p50.p65 are a critical phase in NF-kappa B activation during LPS-induced iNOS and IL-1 beta-induced CINC expression in astroglial cells.

Recent studies suggest that inflammatory events are associated with plaque formation in the brains of patients with Alzheimer's disease (AD). Treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) of these patients appears to slow the progression of disease. We assessed the occurrence of cyclooxygenases (COX-1 and -2) and peroxisome proliferator-activated receptor-gamma (PPARgamma) in temporal cortex from normal and AD brains using specific antibodies. In AD brains, protein levels of COX-1 were increased in both cytosolic and particulate fractions, and COX-2 protein was also increased in the particulate fraction. On the other hand, PPARgamma level was increased in the cytosolic fraction but not in the particulate fraction. Thus, expression levels of COX-1, COX-2, and PPARgamma may change in AD brains. In addition, several NSAIDs which are also PPARgamma activators, such as indomethacin, inhibited COX-2 expression in glial cells. These results suggest that PPARgamma activators have inhibitory effects on inflammatory events in AD brains.

Geldanamycin induces CHOP expression through a 4-(2-aminoethyl)-benzenesulfonyl fluoride-responsive serine protease