Ca2+-dependent inhibition of branched-chain α-ketoacid dehydrogenase kinase by thiamine pyrophosphate.

Catabolism of the branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) is regulated by the branched-chain α-ketoacid dehydrogenase (BCKDH) complex, which in turn is regulated by phosphorylation catalyzed by BCKDH kinase (BDK). Thiamine pyrophosphate (TPP) is required as a coenzyme for the E1 component of the BCKDH complex and can also bring about activation of the complex by inhibiting BDK. The present study shows that free Ca2+ in the physiological range greatly increases the sensitivity of BDK to inhibition by TPP (IC50 of 2.5 µM in the presence of 1 µM free Ca2+). This novel mechanism may be responsible for the stimulation of BCAA oxidation by conditions that increase mitochondrial free Ca2+ levels, e.g. in skeletal muscle during exercise.

Glial influence on the blood brain barrier.

The Blood Brain Barrier (BBB) is a specialized vascular structure tightly regulating central nervous system (CNS) homeostasis. Endothelial cells are the central component of the BBB and control of their barrier phenotype resides on astrocytes and pericytes. Interactions between these cells and the endothelium promote and maintain many of the physiological and metabolic characteristics that are unique to the BBB. In this review we describe recent findings related to the involvement of astroglial cells, including radial glial cells, in the induction of barrier properties during embryogenesis and adulthood. In addition, we describe changes that occur in astrocytes and endothelial cells during injury and inflammation with a particular emphasis on alterations of the BBB phenotype.

Cyclopropane-based conformational restriction of GABA by a stereochemical diversity-oriented strategy: identification of an efficient lead for potent inhibitors of GABA transports.

A series of cyclopropane-based conformationally restricted γ-aminobutyric acid (GABA) analogs with stereochemical diversity, that is, the trans- and cis-2,3-methano analogs Ia and Ib and their enantiomers ent-Ia and ent-Ib, and also the trans- and cis-3,4-methano analogs IIa and IIb and their enantiomers ent-IIa and ent-Iib, were synthesized from the chiral cyclopropane units Type-a and Type-b that we developed. These analogs were systematically evaluated with four GABA transporter (GAT) subtypes. The trans-3,4-methano analog IIa had inhibitory effects on GAT3 (IC50=23.9µM) and betaine-GABA transporter1 (5.48µM), indicating its potential as an effective lead compound for the development of potent GAT inhibitors due to its hydrophilic and low molecular weight properties and excellent ligand efficiency.

Neuronal injury induces microglial production of macrophage inflammatory protein-1α in rat corticostriatal slice cultures.

Chemokines are potent chemoattractants for immune and hematopoietic cells. In the central nervous system, chemokines play an important role in inflammatory responses through activation of infiltrating leukocytes and/or resident glial cells. We previously demonstrated that N-methyl-D-aspartate (NMDA)-evoked neuronal injury induced astrocytic production of monocyte chemoattractant protein-1 (MCP-1, CCL2) via sustained activation of extracellular signal-regulated kinase (ERK) in rat organotypic slice cultures. In the present study, we examined mRNA expression and protein production of macrophage inflammatory protein-1α (MIP-1α, CCL3) induced by NMDA-evoked neuronal injury in the slice cultures. MIP-1α mRNA expression was transiently increased by NMDA treatment in a concentration-dependent manner. Double-fluorescence immunohistochemistry revealed that MIP-1α was produced predominantly in microglia. Depletion of microglial cells from the slice cultures by pretreatment with liposome-encapsulated clodronate abrogated the increase in MIP-1α mRNA expression after NMDA treatment. NMDA-induced MIP-1α mRNA expression was partially but significantly inhibited by the c-Jun N-terminal kinase inhibitor SP600125; conversely, the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 enhanced it. U0126, a MAP kinase/ERK kinase inhibitor, did not affect mRNA expression. These results, combined with our previous findings, demonstrate that NMDA-evoked neuronal injury differentially induces MIP-1α and MCP-1 production in microglia and astrocytes, respectively, through activation of different intracellular signaling pathways.

Nicotine- and tar-free cigarette smoke induces cell damage through reactive oxygen species newly generated by PKC-dependent activation of NADPH oxidase.

We examined cytotoxic effects of nicotine/tar-free cigarette smoke extract (CSE) on C6 glioma cells. The CSE induced plasma membrane damage (determined by lactate dehydrogenase leakage and propidium iodide uptake) and cell apoptosis {determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction activity and DNA fragmentation}. The cytotoxic activity decayed with a half-life of approximately 2 h at 37°C, and it was abolished by N-acetyl-L-cysteine and reduced glutathione. The membrane damage was prevented by catalase and edaravone (a scavenger of (•)OH) but not by superoxide dismutase, indicating involvement of (•)OH. In contrast, the CSE-induced cell apoptosis was resistant to edaravone and induced by authentic H(2)O(2) or O(2)(-) generated by the xanthine/xanthine oxidase system, indicating involvement of H(2)O(2) or O(2)(-) in cell apoptosis. Diphenyleneiodonium [NADPH oxidase (NOX) inhibitor] and bisindolylmaleimide I [BIS I, protein kinase C (PKC) inhibitor] abolished membrane damage, whereas they partially inhibited apoptosis. These results demonstrate that 1) a stable component(s) in the CSE activates PKC, which stimulates NOX to generate reactive oxygen species (ROS), causing membrane damage and apoptosis; 2) different ROS are responsible for membrane damage and apoptosis; and 3) part of the apoptosis is caused by oxidants independently of PKC and NOX.

Sustained activation of ERK signaling in astrocytes is critical for neuronal injury-induced monocyte chemoattractant protein-1 production in rat corticostriatal slice cultures.

We previously demonstrated that N-methyl-D-aspartate (NMDA) treatment (50 microM, 3 h) induced astrocytic production of monocyte chemoattractant protein-1 (MCP-1, CCL2), a CC chemokine implicated in ischemic and excitotoxic brain injury, in rat corticostriatal slice cultures. In this study, we investigated the signaling mechanisms for NMDA-induced MCP-1 production in slice cultures. The results showed a close correlation between NMDA-induced neuronal injury and MCP-1 production, and an abrogation of NMDA-induced MCP-1 production in NMDA-pretreated slices where neuronal cells had been eliminated. These results collectively indicate that NMDA-induced neuronal injury led to astrocytic MCP-1 production. NMDA-induced MCP-1 production was significantly inhibited by U0126, an inhibitor of extracellular signal-regulated kinase (ERK). Immunostaining for phosphorylated ERK revealed that transient neuronal ERK activation was initially induced and subsided within 30 min, followed by sustained ERK activation in astrocytes. Treatment with U0126 during only the early phase (U0126 was washed out at 15 or 30 min after NMDA administration) suppressed early activation of ERK in neuronal cells, but not later activation of ERK in astrocytes. In this case, MCP-1 production was not suppressed, suggesting that activation of neuronal ERK is not necessary for MCP-1 production. In contrast, delayed application of U0126 at 3 h after the beginning of NMDA treatment inhibited MCP-1 production to the same degree as that observed when U0126 was applied from 3 h before NMDA administration. These findings suggest that sustained activation of the ERK signaling pathway in astrocytes plays a key role in neuronal injury-induced MCP-1 production.

Activation of the beta-adrenoceptor-protein kinase A signaling pathway within the ventral bed nucleus of the stria terminalis mediates the negative affective component of pain in rats.

Pain is an unpleasant sensory and emotional experience. The neural systems underlying the sensory component of pain have been studied extensively, but we are only beginning to understand those underlying its affective component. The bed nucleus of the stria terminalis (BNST) has been implicated in stress responses and negative affective states, such as anxiety, fear, and aversion. Recently, we demonstrated the crucial role of the BNST in the negative affective component of pain using the conditioned place aversion (CPA) test. In the present study, we investigated the involvement of the beta-adrenoceptor-protein kinase A (PKA) signaling pathway within the BNST, in particular, within the ventral part of the BNST (vBNST), in pain-induced aversion in male Sprague Dawley rats. In vivo microdialysis showed that extracellular noradrenaline levels within the vBNST were significantly increased by intraplantar formalin injection. Using the CPA test, we found that intra-vBNST injection of timolol, a beta-adrenoceptor antagonist, dose-dependently attenuated the intraplantar-formalin-induced CPA (F-CPA) without reducing nociceptive behaviors. Experiments with subtype-selective antagonists demonstrated the essential role of beta(2)-adrenoceptors in F-CPA. Intra-vBNST injection of isoproterenol, a beta-adrenoceptor agonist, dose-dependently produced CPA even in the absence of noxious stimulation. This isoproterenol-induced CPA was reversed by the coinjection of Rp-cyclic adenosine monophosphorothioate (Rp-cAMPS), a selective PKA inhibitor. Furthermore, intra-vBNST injection of Rp-cAMPS dose-dependently attenuated the F-CPA. Together, these results suggest that PKA activation within the vBNST via the enhancement of beta-adrenergic transmission is important for the negative affective component of pain.

Reciprocal regulation of ATPgammaS-induced monocyte chemoattractant protein-1 production by ERK and p38 MAP kinases in rat corticostriatal slice cultures.

Monocyte chemoattractant protein-1 (MCP-1, CCL2) is a well-defined chemokine implicated in the pathology of various neurodegenerative diseases and brain injuries, such as Alzheimer's disease, multiple sclerosis, stroke, and traumatic injury. We investigated the effect of the activation of P2 purinoceptors on MCP-1 production in rat corticostriatal slice cultures. Treatment with adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), a hydrolysis-resistant adenosine triphosphate (ATP) analog, induced MCP-1 production in astrocytes. The induction was in a concentration-dependent manner and was antagonized by a P2 purinoceptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid. The inhibition of an extracellular signal-regulated kinase (ERK) pathway by PD98059 and U0126 significantly suppressed ATPgammaS-induced MCP-1 mRNA expression and protein production, while inhibition of c-Jun N-terminal kinase by SP600125 resulted in the partial suppression. Conversely, SB203580, a p38 mitogen-activated protein (MAP) kinase inhibitor, significantly enhanced ATPgammaS-induced MCP-1 production. Similar effects of ERK and p38 MAP kinase inhibitors on MCP-1 production were observed in the slices stimulated by ATP and BzATP. These results demonstrate that astrocytic MCP-1 production induced by P2 purinoceptor stimulation is reciprocally regulated by ERK and p38 MAP kinases in the organotypic slice cultures.

Cross-feedback with Partner Contributes to Performance Accuracy in Finger-tapping Rhythm Synchronization between One Leader and Two Followers.

As observed in musical ensembles, people synchronize with a leader together with other people. This study aimed to investigate whether interdependency with a partner improves performance accuracy in rhythm synchronization with the leader. Participants performed a synchronization task via auditory signal by finger tapping in which two followers simultaneously synchronized with a leader: an isochronous metronome or a human leader with or without feedback from the followers. This task was conducted with and without cross-feedback (CFB) between the followers. The followers' weak mutual tempo tracking via the CFB and the followers' strong tempo tracking to the leader improved the tempo stability. Additionally, because the interdependency between the followers was weaker than the followers' dependency on the human leader, the CFB did not enlarge the synchronization error between the human leader and the followers, which occurred in synchronization with the metronome. Thus, the CFB between the followers contributed to accuracy in synchronization with the human leader. The results suggest that in ensembles, players should strongly attend to the leader and should attempt to be less conscious of partners to maintain the appropriate balance between influences from the leader and partners.

Analysis of Sir2E in the cellular slime mold Dictyostelium discoideum: cellular localization, spatial expression and overexpression.

It has been reported that Dictyostelium discoideum encodes four silent information regulator 2 (Sir2) proteins (Sir2A-D) showing sequence similarity to human homologues of Sir2 (SIRT1-3). Further screening in a database revealed that D. discoideum encodes an additional Sir2 homologue (Sir2E). The amino acid sequence of Sir2E is not similar to those of SIRTs but is similar to those of proteins encoded by Giardia lamblia, Cryptosporidium hominis and Cryptosporidium parvum. Fluorescence of Sir2E-green fluorescent protein fusion protein was detected in the D. discoideum nucleus, indicating that Sir2E is a nuclear localizing protein. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that D. discoideum expressed sir2E in amoebae in the growth phase and in prestalk cells in the developmental phase. D. discoideum overexpressing sir2E grew faster than the wild type. These results indicate that Sir2E plays important roles both in the growth phase and developmental phase of D. discoideum.

Evaluation of steelmaking slag as basal media for coastal primary producers.

The use of granular steelmaking slag as a substitute for natural sand in the construction of tidal flats was investigated. Using an intertidal flat simulator, we evaluated dephosphorization slag mixed with 8% by dry weight of dredged sediment (DPS+DS) as a basal medium for the growth of benthic macro- and microalgae in comparison with silica sand mixed with 8% dredged sediment (SS+DS). Species compositions of macro- and microalgae were distinctly different between DPS+DS and SS+DS. The mean dry weight of macroalgae on DPS+DS was three orders of magnitude higher than that on SS+DS. Sediment shear strength and pH were higher in DPS+DS than in SS+DS or in the sediment of natural tidal flats. These results suggest that DPS contributes to changing the sediment environment, thereby changing the algal composition compared to the composition on natural tidal flats.

TAK-779, a nonpeptide CC chemokine receptor antagonist, protects the brain against focal cerebral ischemia in mice.

The effect of a nonpeptide CC chemokine receptor antagonist, TAK-779, on ischemic brain injury resulting from 1-hour middle cerebral artery occlusion followed by 48-hour reperfusion was examined in ddY mice. On intracerebroventricular injection of vehicle or TAK-779, infarct volume in the vehicle-treated group was 44.2 +/- 13.2% of the contralateral hemispheric volume, and TAK-779 (25 and 250 ng/mouse) dose-dependently reduced the infarct volume to 35.0 +/- 12.2% and 31.1 +/- 12.9%, respectively. On intravenous injection, infarct volume in the vehicle-treated group was 32.0 +/- 16.1%, and TAK-779 (5 microg per 20 g body weight) significantly reduced this to 22.0 +/- 10.5%. The results showed for the first time that a nonpeptide chemokine receptor antagonist is protective against ischemic brain injury.

ATP induces leukemia inhibitory factor mRNA in cultured rat astrocytes.

Leukemia inhibitory factor (LIF) is a cytokine involved in the survival and differentiation of the neural cells in the central and peripheral nervous systems. In the present study, we examined the effects of various neurotransmitter receptor agonists on LIF mRNA expression in cultured rat astrocytes, microglia and neurons to elucidate the cell types producing LIF and to clarify the neurotransmitter(s) regulating the mRNA expression. The results demonstrated that the expression of LIF mRNA was intensely induced by ATP in the cultured astrocytes. Experiments using ATP, UTP and related compounds showed the involvement of P2Y2 and P2Y4 purinoceptors in the expression induced by ATP.

Kainic acid induces leukemia inhibitory factor mRNA expression in the rat brain: differences in the time course of mRNA expression between the dentate gyrus and hippocampal CA1/CA3 subfields.

Leukemia inhibitory factor (LIF) is a pluripotent cytokine which affects the survival and differentiation of various types of cells both in the hematopoietic and nervous systems. In this study, the time course and localization of LIF mRNA expression following kainic acid-induced seizures were examined by northern blot analyses and in situ hybridization. Northern blot analyses demonstrated that intraperitoneal injection of kainic acid at a convulsive dose induced LIF mRNA expression intensely in the hippocampus and moderately to weakly in the cerebral cortex, thalamus and hypothalamus. The expression peaked at 8-24 h after the injection in the hippocampus and cerebral cortex and at 8 h in the thalamus and hypothalamus. In situ hybridization revealed different time courses of LIF mRNA expression depending on the area of the hippocampus; that is, the expression peaked at 10 h in the granule cell layer of the dentate gyrus, then at 12 h in the polymorph and molecular layers of the dentate gyrus, and finally at 12-24 h in the strata oriens and radiatum of the CA1 and CA3 subfields. It is worth noting that the expression of LIF mRNA was intense in the dentate gyrus, the region where neurogenesis and aberrant network reorganization have been shown to be induced by seizures. The upregulation of LIF mRNA expression in the dentate granule cell layer followed by that in the dentate polymorph and molecular layers may be involved in activity-dependent neurogenesis in the granule cell layer and ectopic migration of granule cells to the polymorph and molecular layers in the dentate gyrus.

Excitotoxic injury induces production of monocyte chemoattractant protein-1 in rat cortico-striatal slice cultures.

The effect of excitotoxic injury on the production of monocyte chemoattractant protein-1 (MCP-1) was examined in rat cortico-striatal slice cultures. Treatment with 50 microM N-methyl-D-aspartate (NMDA) for 4 h, which caused severe damage in neurons, induced the production of MCP-1 in astrocytes. Production levels were markedly elevated immediately after the treatment, peaked at 4-8 h, and had decreased to nearly the basal level by 72 h. Since the treatment promoted the release of MCP-1 in the slice cultures, but not in the enriched astrocyte cultures, it is unlikely that NMDA directly acted on the astrocytes. These results suggest that information on neuronal injury induced by NMDA is transmitted to astrocytes to induce the production of MCP-1. Organotypic slice cultures are useful for investigating the inflammatory responses of astrocytes in the process of neuronal injury.

Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor alpha in mice.

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors which belong to the nuclear receptor family. We examined whether PPARalpha agonists and resveratrol, a polyphenol contained in grapes, protect the brain against ischemia. To investigate whether resveratrol activates PPARs, we performed a cell-based transfection activity assay using luciferase reporter plasmid. PPARalpha and PPARgamma were activated by resveratrol in primary cortical cultures and vascular endothelial cells. Resveratrol (20 mg/kg, 3 days) reduced infarct volume by 36% at 24 h after middle cerebral artery occlusion in wild-type mice. The PPARalpha agonists fenofibrate (30 mg/kg, 3 days) and Wy-14643 (30 mg/kg, days) exerted similar brain protection. However, resveratrol and fenofibrate failed to protect the brain in PPARalpha knockout mice. The data indicate that PPARalpha agonists protect the brain through PPARalpha.

Accumulating microglia phagocytose injured neurons in hippocampal slice cultures: involvement of p38 MAP kinase.

In this study, microglial migration and phagocytosis were examined in mouse organotypic hippocampal slice cultures, which were treated with N-methyl-D-aspartate (NMDA) to selectively injure neuronal cells. Microglial cells were visualized by the expression of enhanced green fluorescent protein. Daily observation revealed microglial accumulation in the pyramidal cell layer, which peaked 5 to 6 days after NMDA treatment. Time-lapse imaging showed that microglia migrated to the pyramidal cell layer from adjacent and/or remote areas. There was no difference in the number of proliferating microglia between control and NMDA-treated slices in both the pyramidal cell layer and stratum radiatum, suggesting that microglial accumulation in the injured areas is mainly due to microglial migration, not to proliferation. Time-lapse imaging also showed that the injured neurons, which were visualized by propidium iodide (PI), disappeared just after being surrounded by microglia. Daily observation revealed that the intensity of PI fluorescence gradually attenuated, and this attenuation was suppressed by pretreatment with clodronate, a microglia toxin. These findings suggest that accumulating microglia phagocytosed injured neurons, and that PI fluorescence could be a useful indicator for microglial phagocytosis. Using this advantage to examine microglial phagocytosis in living slice cultures, we investigated the involvements of mitogen-activated protein (MAP) kinases in microglial accumulation and phagocytosis. p38 MAP kinase inhibitor SB203580, but not MAP kinase/extracellular signal-regulated kinase inhibitor PD98059 or c-Jun N-terminal kinase inhibitor SP600125, suppressed the attenuation of PI fluorescence. On the other hand, microglial accumulation in the injured areas was not inhibited by any of these inhibitors. These data suggest that p38 MAP kinase plays an important role in microglial phagocytosis of injured neurons.

Mesenchymal stem cells transmigrate across brain microvascular endothelial cell monolayers through transiently formed inter-endothelial gaps.

Mesenchymal stem cells (MSCs) hold much promise for cell therapy for neurological diseases such as cerebral ischemia and Parkinson's disease. Intravenously administered MSCs accumulate in lesions within the brain parenchyma, but little is known of the details of MSC transmigration across the blood-brain barrier (BBB). To study MSC transmigration across the BBB, we developed an in vitro culture system consisting of rat brain microvascular endothelial cells (BMECs) and bone marrow-derived MSCs using Transwell or Millicell culture inserts. Using this system, we first investigated the influence of the number of MSCs added to the upper chamber on BMEC barrier integrity. The addition of MSCs at a density of 1.5 × 105 cells/cm² led to disruption of the BMEC monolayer structure and decreased barrier function as measured by the transendothelial electrical resistance (TEER). When applied at a density of 1.5 × 104 cells/cm², neither remarkable disruption of the BMEC monolayers nor a significant decrease in TEER was observed until at least 12 h. After cultivation for 24 h under this condition, MSCs were found in the subendothelial space or beneath the insert membrane, suggesting that MSCs transmigrate across BMEC monolayers. Time-lapse imaging revealed that MSCs transmigrated across the BMEC monolayers through transiently formed intercellular gaps between the BMECs. These results show that our in vitro culture system consisting of BMECs and MSCs is useful for investigating the molecular and cellular mechanisms underlying MSC transmigration across the BBB.

Neuronal injury induces cytokine-induced neutrophil chemoattractant-1 (CINC-1) production in astrocytes.

Accumulating evidence indicates a pivotal role for neuroinflammation in ischemic and excitotoxic brain injury. Cytokine-induced neutrophil chemoattractant-1 (CINC-1) is a CXC chemokine implicated in the infiltration of inflammatory cells into the brain parenchyma. In this study, we investigated the effect of N-methyl-D-aspartate (NMDA)-induced neuronal injury on CINC-1 production in the organotypic cortico-striatal slice cultures. Treatment with 50 microM NMDA for 3 - 4 h caused devastating neuronal damage and increased CINC-1 production. Immunohistochemical analysis revealed that the CINC-1 immunoreactivity was predominantly detected in astrocytes. NMDA failed to induce CINC-1 production in enriched astrocyte cultures or neuron-depleted slice cultures, suggesting that NMDA acted on neuronal cells to induce astrocytic CINC-1 production. NMDA-induced CINC-1 mRNA expression was significantly inhibited by U0126, a mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor. These results suggest that NMDA-evoked neuronal injury induced astrocytic CINC-1 production via a MEK/ERK signaling pathway. Manipulation of this signaling pathway may serve as a target for suppressing neuroinflammation and, thereby, treating ischemic brain injury.

Role of enhanced noradrenergic transmission within the ventral bed nucleus of the stria terminalis in visceral pain-induced aversion in rats.

Pain is an unpleasant sensory and emotional experience. We demonstrated the crucial role of the bed nucleus of the stria terminalis (BNST) in the negative affective component of somatic and visceral pain induced by intraplantar formalin and intraperitoneal acetic acid injections, respectively, in rats. Recently, we reported the involvement of enhanced noradrenergic transmission via beta-adrenoceptors within the ventral BNST (vBNST) in formalin-induced aversion. Here, we examined the role of intra-vBNST noradrenergic transmission in the negative affective component of visceral pain induced by intraperitoneal acetic acid injection. In vivo microdialysis showed that extracellular noradrenaline levels within the vBNST significantly increased after intraperitoneal acetic acid injection. Using a conditioned place aversion (CPA) test, we found that intra-vBNST injection of timolol, a beta-adrenoceptor antagonist, dose-dependently attenuated the acetic acid-induced CPA without reducing nociceptive behaviors. These results suggest that enhanced noradrenergic transmission via beta-adrenoceptors within the vBNST plays a pivotal role in the negative affective, but not sensory, component of visceral pain.