Biomedical subjects

S Kohsaka

Publications and source records attributed to S Kohsaka.

At least 19 recordsLinked to original sources

PubMed2002

Widespread activation of the brainstem preceding the recruiting rhythm in human epilepsies.

The excitability change of the brainstem was investigated before and during the conspicuous epileptic discharge in six patients with generalized convulsive seizures. The discharge consisted of a short duration of recruiting rhythm, which was considered equivalent to the seizure discharge on electroencephalogram. The excitability of the brainstem was measured with the parameters (amplitude and area) of component waves (wave-III and -V) of brainstem auditory evoked potentials. The theoretical background of the analysis is that brainstem auditory evoked potentials are 'far-field' potentials, by which they convey the information on the activity change of the brainstem even during the paroxysmal discharge within the cortex. The excitability of both the ventral (parameters of wave-III) and the dorsal brainstem (parameters of wave-V) exhibited a synchronized change (activation-inactivation). They were enhanced from -2.4+/-0.4 s, reaching the maxima before the onset of the seizure discharge, and decayed corresponding to the emergence of the recruiting rhythm. The results suggest the possibility that the widespread (ventral and dorsal) and synchronized activation of the brainstem triggers the seizure discharge in human generalized epilepsy. During the widespread activation of the brainstem, both the thalamus and the cortex probably undergo a suppressed inhibitory state through the cholinergic activation, precipitating the seizure discharge.

Acoustic Stimulation↗

PubMed2001

Induction of CHOP and apoptosis by nitric oxide in p53-deficient microglial cells.

Excessive nitric oxide (NO) has been implicated in neurotoxicity after stresses such as ischemia. NO toxicity is generally thought to be mediated by the DNA damage-p53 pathway or mitochondrial dysfunction. We investigated the mechanism of NO toxicity by using murine microglial MG5 cells established from p53-deficient mice. When MG5 cells were exposed to bacterial lipopolysaccharide plus interferon-gamma, mRNA and protein for inducible NO synthase (iNOS) were markedly induced, and apoptosis occurred. Under these conditions, we found that mRNA and protein for CHOP/GADD153, a C/EBP family transcription factor which is involved in endoplasmic reticulum (ER) stress-induced apoptosis, are induced. iNOS mRNA was induced 2 h after treatment, whereas CHOP mRNA began to increase at 6 h with a time lag. CHOP mRNA was also induced by NO donors S-nitroso-N-acetyl-DL-penicillamine (SNAP) or NOC18, or a peroxynitrite generator 3-(4-morpholinyl)-sydnonimine hydrochloride (SIN-1). Bip/GRP78, an ER chaperone which is known to be induced by ER stress, was also induced by SNAP or SIN-1, indicating that NO causes ER stress. These results suggest that NO-induced apoptosis in MG5 cells occurs through the ER stress pathway involving CHOP, but is independent of p53.

Animals↗

PubMed2001

Establishment of CHO cell lines expressing four N-methyl-D-aspartate receptor subtypes and characterization of a novel antagonist PPDC.

To develop an assay system that allows the N-methyl-D-aspartate (NMDA) receptor subtype-selective antagonistic potency of drugs, we have established Chinese hamster ovary cell lines expressing the four NMDA receptor subtypes (GluRepsilon1/zeta1-GluRepsilon4/zeta1) heat-indelibly. Using these clonal cells, we found that a novel antagonist, (1S,2R)-1-phenyl-2[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide, was less selective for the GluRepsilon1/zeta1: the IC(50) values for the GluRepsilon1/zeta1-GluRepsilon4/zeta1 were 41.7, 13.3, 12.6 and 11.5 microM, respectively, while two well-known antagonists, DL-2-amino-5-phosphonovaleric acid and ifenprodil, showed the known potency and selectivity for each subtype. Thus, the established clonal cells are of use in characterizing the pharmacological properties of drugs that act on NMDA receptors.

Animals↗

PubMed2001

Nitric oxide inhibits the proliferation of murine microglial MG5 cells by a mechanism involving p21 but independent of p53 and cyclic guanosine monophosphate.

We investigated the effect of nitric oxide (NO) on the proliferation of microglial MG5 cells established from p53-deficient mice. Cells were treated with bacterial lipopolysaccharide and interferon-gamma, and expression of inducible NO synthase (iNOS) and p21/waf1, a cyclin-dependent kinase inhibitor protein which is a critical downstream effector of p53, was investigated by RNA blot and immunoblot analyses. iNOS mRNA was induced 2 h after treatment and increased with time up to 24 h. p21 mRNA was expressed at a low level in untreated cells and increased with a kinetics similar to that for iNOS mRNA. iNOS and p21 proteins were also induced. An NO donor SNAP induced p21 mRNA and protein. SNAP inhibited incorporation of [(3)H]thymidine in MG5 cells in a dose-dependent manner. 8-Bromo-cGMP neither induced p21 mRNA nor inhibited [(3)H]thymidine incorporation. These results suggest that NO inhibits the proliferation of MG5 cells by induction of p21, which occurs independent of p53 and cGMP.

Animals↗

PubMed2001

Iba1 is an actin-cross-linking protein in macrophages/microglia.

Iba1 is a 17-kDa EF hand protein that is specifically expressed in macrophages/microglia and is upregulated during the activation of these cells. When exposed to macrophage colony-stimulating factor (M-CSF), microglia cell line MG5 immediately produces intense membrane ruffles in which Iba1 accumulates together with filamentous actin. In this study, we investigated the physical interaction between Iba1 and actin by centrifugation assay and electron microscopic examination and showed that Iba1 possesses actin-binding and -cross-linking activities. Inhibitory mutant Iba1 that suppresses M-CSF-induced membrane ruffling had lost the actin-cross-linking activity, and it inhibited the cross-linking activity of intact Iba1. These results indicate that Iba1 is a macrophage/microglia-specific actin-cross-linking protein essential for M-CSF-induced membrane ruffling.

Actins↗

PubMed2001

Neurotrophin secretion from cultured microglia.

Because microglia have been suggested to produce neurotrophins, we tested this ability in vitro. Rat primary microglia were found to constitutively secrete a limited amount of brain-derived neurotrophic factor (BDNF), but nerve growth factor (NGF) and neurotrophin-3 (NT-3) were undetectable in the conditioned medium. Stimulation of the cells with lipopolysaccharide (LPS) increased BDNF secretion, and induced NGF secretion. As a first step to examine this regulation system, the association of protein kinase C (PKC) was pharmacologically analyzed. A PKC activator, phorbol-12-myristate-13-acetate, enhanced the secretion of BDNF. Pre-treatment of microglia with a PKC inhibitor, bisindolylmaleimide, suppressed LPS-stimulated BDNF secretion as well as the constitutive one. These results suggest that the PKC signaling cascade is closely associated with BDNF secretion. Among PKC isoforms, PKCalpha probably plays a role in BDNF secretion, based on the results of experiments using a specific PKC activator, 1-oleoyl-2-acetyl-sn-glycerol, and a specific PKC inhibitor, Gö 6976, and by immunoblotting. Taken together, these findings suggest that the secretion of BDNF from microglia is regulated through PKCalpha-associated signal transduction mechanism.

Animals↗

PubMed2001

Impaired microglial activation in the brain of IL-18-gene-disrupted mice after neurovirulent influenza A virus infection.

Knockout of the interleukin-18 (IL-18) gene predisposed mice to impaired clearance of neurovirulent influenza A virus-infected neurons from the brain. In wild-type mice, IL-18 molecule-producing microglia/macrophages emerged in virally attacked regions as early as day 3 after infection. Microglial transformation into macrophages culminated at day 7 to 9, with upregulated expression of Iba1, a novel calcium-binding protein that controls phagocytic functions of microglia/macrophages. In IL-18-/- mice, microglial transformation was interrupted with reduced Iba1 expression. Interferon-gamma (IFN-gamma)-immunopositive neurons appeared in and around virally invaded regions in wild-type mice, peaking in number at day 7, whereas such cells were barely detected in IL-18-/- mice. Stereotaxic microinjection of recombinant IFN-gamma triggered microglial transformation in IL-18-/- mice and upregulated Iba1 expression, leading to effective eradication of virally infected neurons. Collectively, these results suggest that IL-18 plays a key role in activating microglial functions directed against the influenza virus infection by inducing neuronal IFN-gamma in the brain parenchyma.

Animals↗

PubMed2001

Microglia and the early phase of immune surveillance in the axotomized facial motor nucleus: impaired microglial activation and lymphocyte recruitment but no effect on neuronal survival or axonal regeneration in macrophage-colony stimulating factor-deficient mice.

Activation of microglia is among the first cellular changes in the injured CNS. However, little is known about their specific contribution to secondary damage or repair processes in neighboring neurons and nonneuronal cells or to the immune surveillance of the damaged tissue. Animal models with defective microglial response such as osteopetrosis provide an approach to explore these effects. Osteopetrosis (op) is an autosomal recessive mutation with a complete deficiency of the macrophage-colony stimulating factor (MCSF; CSF-1), an important mitogen for brain microglia. In the current study we examined the effects of this MCSF deficiency on the microglial reaction and the overall cellular response to nerve injury in the mouse axotomized facial motor nucleus. In the brain, MCSF receptor immunoreactivity was found only on microglia and was strongly up-regulated following injury. MCSF deficiency led to a failure of microglia to show a normal increase in early activation markers (thrombospondin, MCSF receptor, alpha M beta 2- and alpha 5 beta 1-integrins), to spread on the surface of axotomized motoneurons, and to proliferate after injury. Early recruitment of CD3(+) T-lymphocytes to the facial nucleus 24 hours after injury was reduced by 60%. In contrast, the neuronal and astrocyte response was not affected. There was a normal increase in the neuropeptides calcitonin gene-related peptide and galanin, neuronal c-JUN, and NADPH-diaphorase and a decrease in choline acetyltransferase and acetylcholinesterase. Astrocyte glial fibrillary acidic protein immunoreactivity also showed a normal increase. There was a normal influx of macrophages and granulocytes into the injured facial nerve. Synaptic stripping, neuronal survival, and speed of axonal regeneration were also not affected. The current results show a strong, selective effect of MCSF on the early activation of microglia and, indirectly, on lymphocyte recruitment. This early phase of microglial activation appears not to be involved in the process of repair following peripheral nerve injury. However, it is important in the initiation of inflammatory changes in the brain and in the interaction with the immune system.

Animals↗

PubMed2001

Ability of rat microglia to uptake extracellular glutamate.

Since it has been suggested that microglia in vivo act as glutamate scavengers, this possibility was investigated in primary cultured microglia. The microglia showed specific abilities to uptake (14)C-glutamate depending on incubation time and numbers of cells used. The activity was suppressed by a specific inhibitor for a glial cell-type transporter, glutamate transporter-1 (GLT-1) (EAAT2). However, that of cultured astrocytes was not affected. These results suggest that microglia uptake glutamate by means of GLT-1. Supporting these results, immunoblotting revealed the presence of GLT-1 in the microglia, while only glutamate-aspartate transporter (GLAST) (EAAT1: another glial cell-type transporter) was detected in the astrocytes. All together, these results indicate that microglia can act as glutamate scavengers in vivo by expressing the glutamate transporter GLT-1.

ATP-Binding Cassette Transporters↗

PubMed2001

Co-induction of argininosuccinate synthetase, cationic amino acid transporter-2, and nitric oxide synthase in activated murine microglial cells.

Nitric oxide (NO) produced by activated microglia has been implicated in many pathophysiological events in the brain including neurodegenerative diseases. Cellular NO production depends absolutely on the availability of arginine, a substrate of NO synthase (NOS). Murine microglial MG5 cells were treated with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), and expression of inducible NO synthase (iNOS) and arginine-supplying enzymes was investigated by RNA blot analysis. iNOS mRNA was strongly induced after treatment and reached a maximum at 6-12 h. mRNA for argininosuccinate synthetase (AS), a citrulline-arginine recycling enzyme, increased at 6 h and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and AS proteins were also induced. In addition, mRNA encoding the cationic amino acid transporter-2 (CAT-2) was strongly induced shortly after treatment. Induction of mRNAs for iNOS, AS, and CAT-2 by LPS/IFN-gamma was also observed following stimulation of rat primary microglial cells. These results strongly suggest that both arginine transport by CAT-2 and citrulline-arginine recycling are important for high-output production of NO in activated microglial cells.

Amino Acid Transport Systems, Basic↗

PubMed2001

Brainstem activates paroxysmal discharge in human generalized epilepsy.

In nine patients with generalized epilepsy of convulsive seizures, the excitability change of the brainstem was evaluated over the course of the interictal paroxysmal discharge (poly spike-and-wave complex, poly SWC). The evaluation was carried out by a sequential analysis of brainstem auditory evoked potentials (BAEPs) before and during one sequence of poly SWC. The characteristics of BAEPs, i.e. far-field evoked potentials, allowed the evaluation of the excitability change in the brainstem, which was not influenced by the cortical activity. The excitability in the ventral brainstem, measured with the parameters of wave-III, showed a biphasic fluctuation (deceleration--acceleration) before the onset of poly SWC (minima at -0.7+/-0.4 s). On the other hand, the excitability in the dorsal brainstem, measured with the parameters of wave-V, showed no significant difference over the course of poly SWC. The results suggest that the biphasic excitability change in the ventral brainstem is conveyed to the cortex through the ascending activating system. The excitability acceleration preceded by deceleration in the ventral brainstem probably synchronizes the cortical activity profoundly enough to produce poly SWC through the activation of intralaminar thalamic neurons.

Adolescent↗

PubMed2001

Molecular characterization of mammalian homologues of class C Vps proteins that interact with syntaxin-7.

Vesicle-mediated protein sorting plays an important role in segregation of intracellular molecules into distinct organelles. Extensive genetic studies using yeast have identified more than 40 vacuolar protein sorting (VPS) genes involved in vesicle transport to vacuoles. However, their mammalian counterparts are not fully elucidated. In this study, we identified two human homologues of yeast Class C VPS genes, human VPS11 (hVPS11) and human VPS18 (hVPS18). We also characterized the subcellular localization and interactions of the protein products not only from these genes but also from the other mammalian Class C VPS homologue genes, hVPS16 and rVPS33a. The protein products of hVPS11 (hVps11) and hVPS18 (hVps18) were ubiquitously expressed in peripheral tissues, suggesting that they have a fundamental role in cellular function. Indirect immunofluorescence microscopy revealed that the mammalian Class C Vps proteins are predominantly associated with late endosomes/lysosomes. Immunoprecipitation and gel filtration studies showed that the mammalian Class C Vps proteins constitute a large hetero-oligomeric complex that interacts with syntaxin-7. These results indicate that like their yeast counterparts, mammalian Class C Vps proteins mediate vesicle trafficking steps in the endosome/lysosome pathway.

Amino Acid Sequence↗

PubMed2001

Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors.

The initial microglial responses that occur after brain injury and in various neurological diseases are characterized by microglial accumulation in the affected sites of brain that results from the migration and proliferation of these cells. The early-phase signal responsible for this accumulation is likely to be transduced by rapidly diffusible factors. In this study, the possibility of ATP released from injured neurons and nerve terminals affecting cell motility was determined in rat primary cultured microglia. Extracellular ATP and ADP induced membrane ruffling and markedly enhanced chemokinesis in Boyden chamber assay. Further analyses using the Dunn chemotaxis chamber assay, which allows direct observation of cell movement, revealed that both ATP and ADP induced chemotaxis of microglia. The elimination of extracellular calcium or treatment with pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, suramin, or adenosine-3'-phosphate-5'-phosphosulfate did not inhibit ATP- or ADP-induced membrane ruffling, whereas AR-C69931MX or pertussis toxin treatments clearly did so. As an intracellular signaling molecule underlying these phenomena, the small G-protein Rac was activated by ATP and ADP stimulation, and its activation was also inhibited by pretreatment with pertussis toxin. These results strongly suggest that membrane ruffling and chemotaxis of microglia induced by ATP or ADP are mediated by G(i/o)-coupled P2Y receptors.

Adenosine Diphosphate↗

PubMed2001

Intact microglia are cultured and non-invasively harvested without pathological activation using a novel cultured cell recovery method.

Because spontaneous host regeneration of damaged tissues is limited, novel therapeutics utilizing cultured cells with the aid of tissue engineering methods are promising alternatives for tissue replacement. One critical shortcoming is current requirement for invasive cell harvest from culture to fabricate cell-based devices. Although microglia that secrete neurotrophic factors are attractive candidates for novel cell transplantation therapy for damaged central nervous system tissue, the intact harvest of cultured microglia is presently not achievable. Therefore, primary microglia were plated onto culture surfaces grafted with the temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). This surface undergoes rapid, reversible temperature-dependent changes in its hydration state and surface hydrophilicity. Microglia attached and proliferated on PIPAAm-grafted dishes at 37 degrees C. By reducing culture temperature, more than 90% of the cells spontaneously detached from the dishes within several minutes without trypsin or EDTA treatment. Recovered and replated microglia exhibited phenotypic properties comparable to those of primary microglia freshly isolated from brain. By contrast, less than 60% of the cells were harvested by trypsin digestion, and exhibited significant alteration of characteristic cellular properties as monitored by pathological states in vivo. This new technology exhibits utility for the preparation of cell sources required for cell transplantation as well as microglial function analysis.

Animals↗

PubMed2001

Effects of bright light exposure on heart rate variability during sleep in young women.

To investigate the effects of evening bright light on the autonomic nervous system, heart rate variability (HRV) during sleep was analyzed in dim light (DL) and bright light (BL) conditions. We recorded polysomnography in nine healthy young women aged 20-21 years. Time series of % delta power was calculated in the 0.49-2.20 Hz band. Heart rate variability was analyzed from a 10-min segment of slow wave sleep. The low- to high-frequency ratio and the low-frequency component decreased significantly in the BL conditions compared with the DL conditions. However, the power of the high-frequency component did not change in the two conditions. These results indicate that evening BL affects the autonomic nervous system during slow wave sleep.

Adult↗

PubMed2001

Real-time, two-dimensional visualization of ischaemia-induced glutamate release from hippocampal slices.

The involvement of excitatory amino acid (EAA) toxicity in ischaemia-induced neuronal cell death has long been suggested. However, in the hippocampus, the brain site most vulnerable to ischaemia, the detailed spatial and temporal patterns of EAA release are not yet known. To address this issue, we have developed a novel strategy for the continuous, real-time, two-dimensional monitoring of EAA release from brain slices. As EAA detector, we used a cell line transformed with the N-methyl-D-aspartate (NMDA) receptor, which is exclusively activated by EAAs, leading to an increase in the intracellular Ca(2+) level. Combined with a calcium imaging technique, the use of this cell line allowed the temporal and regional analysis of EAA release from a brain slice placed directly on top of the clonal cells in a culture dish. Using this strategy, we demonstrated ischaemia-induced EAA release in rat hippocampal slices. Increased EAA release was seen initially in the CA1 region, about 3 min after the beginning of ischaemia, then in the CA3 region and dentate gyrus, and, finally, throughout the hippocampal slice. Regional differences in extracellular EAA levels were also seen, with more EAA being released from the CA1 region than from the middle dentate gyrus. The present results are especially interesting as neurons in the CA1 region are more vulnerable to ischaemia than those in the CA3 region and dentate gyrus.

Alanine↗

PubMed2001

Fibulin-1 binds the amino-terminal head of beta-amyloid precursor protein and modulates its physiological function.

Genetic studies have implicated amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease. While accumulating lines of evidence indicate that APP has various functions in cells, little is known about the proteins that modulate its biological activity. Toward this end, we employed a two-hybrid system to identify potential interacting factors. We now report that fibulin-1, which contains repetitive Ca(2+)-binding EGF-like elements, binds to APP at its amino-terminal growth factor-like domain, the region that is responsible for its neurotrophic activities. Fibulin-1 expression in the brain is confined to neurons, and is not expressed significantly by astrocytes or microglia. Direct binding of fibulin-1 to the secreted form of APP (sAPP) was demonstrated with a pull-down assay using fragments of both fibulin-1 fused with glutathione-S transferase and sAPP, produced in bacteria and yeast, respectively. The fibulin-1/sAPP heteromer was shown to form in the conditioned medium of transfected COS-7 cells. Furthermore, fibulin-1 blocks sAPP-mediated proliferation of primary cultured rat neural stem cells. These results suggest that fibulin-1 may play a significant role in modulating the neurotrophic activities of APP.

Amino Acid Sequence↗

PubMed2001

Mechanisms underlying extracellular ATP-evoked interleukin-6 release in mouse microglial cell line, MG-5.

Microglia play various important roles in the CNS via the synthesis of cytokines. The ATP-evoked production of interleukin-6 (IL-6) and its intracellular signals were examined using a mouse microglial cell line, MG-5. ATP, but not its metabolites, produced IL-6 in a concentration-dependent manner. Although ATP activated two mitogen-activated protein kinases, i.e. p38 and extracellular signal-regulated protein kinase, only p38 was involved in the IL-6 induction. However, the activation of p38 was not sufficient for the IL-6 induction because 2'- and 3'-O-(4-benzoylbenzoyl) ATP, an agonist to P2X7 receptors, failed to produce IL-6 despite the fact that it activated p38. Unlike in other cytokines in microglial cells, P2Y rather than P2X7 receptors seem to have a major role in the IL-6 production by the cells. The ATP-evoked IL-6 production was attenuated by Gö6976, an inhibitor of Ca(2+)-dependent protein kinase C (PKC). The P2Y receptor responsible for these responses was insensitive to pertussis toxin (PTX) and was linked to phospholipase C. Taken together, ATP acting on PTX-insensitive P2Y receptors activates p38 and Ca(2+)-dependent PKC, thereby resulting in the mRNA expression and release of IL-6 in MG-5. This is a novel pathway for the induction of cytokines in microglia.

Adenosine Triphosphate↗