Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

Choline uptake into the presynaptic terminal of cholinergic neurons is mediated by the high-affinity choline transporter and is essential for acetylcholine synthesis. In a previous study, we reported that P2X(2) purinoceptors are selectively expressed in OFF-cholinergic amacrine cells of the mouse retina. Under specific conditions, P2X(2) purinoceptors acquire permeability to large cations, such as N-methyl-D-glucamine, and therefore potentially could act as a noncanonical pathway for choline entry into neurons. We tested this hypothesis in OFF-cholinergic amacrine cells of the mouse retina. ATP-induced choline currents were observed in OFF-cholinergic amacrine cells, but not in ON-cholinergic amacrine cells, in mouse retinal slice preparations. High-affinity choline transporters are expressed at higher levels in ON-cholinergic amacrine cells than in OFF-cholinergic amacrine cells. In dissociated preparations of cholinergic amacrine cells, ATP-activated cation currents arose from permeation of extracellular choline. We also examined the pharmacological properties of choline currents. Pharmacologically, alpha,beta-methylene ATP did not produce a cation current, whereas ATP gamma S and benzoyl-benzoyl-ATP (BzATP) activated choline currents. However, the amplitude of the choline current activated by BzATP was very small. The choline current activated by ATP was strongly inhibited by pyridoxalphosphate-6-azophenyl- 2', 4'-sulfonic acid. Accordingly, P2X(2) purinoceptors expressed in HEK-293T cells were permeable to choline and similarly functioned as a choline uptake pathway. Our physiological and pharmacological findings support the hypothesis that P2 purinoceptors, including P2X(2) purinoceptors, function as a novel choline transport pathway and may provide a new regulatory mechanism for cholinergic signaling transmission at synapses in OFF-cholinergic amacrine cells of the mouse retina.
NEW & NOTEWORTHY Choline transport across the membrane is exerted by both the high-affinity and low-affinity choline transporters. We found that choline can permeate P2 purinergic receptors, including P2X(2) purinoceptors, in cholinergic neurons of the retina. Our findings show the presence of a novel choline transport pathway in cholinergic neurons. Our findings also indicate that the permeability of P2X(2) purinergic receptors to choline observed in the heterologous expression system may have a physiological relevance in vivo.

DOI： 10.1152/jn.00506.2016

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The aggregation mechanism of phosphorylated tau is an important therapeutic target for tauopathies, including Alzheimer's disease, although the mechanism by which aggregation occurs is still unknown. Because the phosphorylation process of tau is involved in the trafficking of AMPA receptors, which accompanies the long-term depression (LTD) of synapses, we examined the effect of LTD-inducing low-frequency stimulation (LFS) on the formation of pathological tau aggregates in adult and aged wild-type mice. Our biochemical analysis demonstrated that LFS led to the formation of sarkosyl-insoluble (SI) tau oligomers in aged hippocampi but not in adult hippocampi in wild-type mice. In parallel, electrophysiological experiments showed an increased contribution of the autophagy-lysosomal pathway (ALP) to LTD during aging, although the other properties of LFS-induced LTD that we investigated were not altered. Thus, we anticipate that the increased contribution of the ALP to the LTD cascade is involved in the age-dependent formation of tau oligomers that results from LFS. Analysis of the LC3 ratio, an indicator of autophagosome formation, showed that LFS increased cleaved LC3 (type II) in the aged hippocampus relative to type I LC3, suggesting potentiation of the ALP accompanied by LTD. Pharmacological inhibition of autophagosome formation depressed LFS-induced oligomerization of tau. Prevention of lysosomal function in the ALP enhanced the formation of tau oligomers by LFS. These results suggest the importance of the autophagosome for the LFS-induced oligomerization of tau and suggest a reason for its age dependency. Interestingly, the lysosomal disturbance promoted the formation of the fibrillar form of aggregates consisting of hyper-phosphorylated tau. The LTD-ALP cascade potentially acts as one of the suppliers of pathological aggregates of tau in aged neurons.

DOI： 10.1186/s40478-017-0469-x

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Very-KIND/Kndc1/KIAA1768 (v-KIND) is a brain-specific Ras guanine nucleotide exchange factor carrying two sets of the kinase non-catalytic C-lobe domain (KIND), and is predominantly expressed in cerebellar granule cells. Here, we report the impact of v-KIND deficiency on dendritic and synaptic growth in cerebellar granule cells in v-KIND knockout (KO) mice. Furthermore, we evaluate motor function in these animals. The gross anatomy of the cerebellum, including the cerebellar lobules, layered cerebellar cortex and densely-packed granule cell layer, in KO mice appeared normal, and was similar to wild-type (WT) mice. However, KO mice displayed an overgrowth of cerebellar granule cell dendrites, compared with WT mice, resulting in an increased number of dendrites, dendritic branches and terminals. Immunoreactivity for vGluT2 (a marker for excitatory presynapses of mossy fiber terminals) was increased in the cerebellar glomeruli of KO mice, compared with WT mice. The postsynaptic density around the terminals of mossy fibers was also increased in KO mice. Although there were no significant differences in locomotor ability between KO and WT animals in their home cages or in the open field, young adult KO mice had an increased grip strength and a tendency to exhibit better motor performance in balance-related tests compared with WT animals. Taken together, our results suggest that v-KIND is required for compact dendritic growth and proper excitatory synaptic connections in cerebellar granule cells, which are necessary for normal motor coordination and balance.

DOI： 10.1371/journal.pone.0173175

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Opalin, a central nervous system-specific myelin protein phylogenetically unique to mammals, has been suggested to play a role in mammalian-specific myelin. To elucidate the role of Opalin in mammalian myelin, we disrupted the Opalin gene in mice and analyzed the impacts on myelination and behavior. Opalin-knockout (Opalin(-/-)) mice were born at a Mendelian ratio and had a normal body shape and weight. Interestingly, Opalin(-/-) mice had no obvious abnormalities in major myelin protein compositions, expression of oligodendrocyte lineage markers, or domain organization of myelinated axons compared with WT mice (Opalin(+/+)) mice. Electron microscopic observation of the optic nerves did not reveal obvious differences between Opalin(+/+) and Opalin(-/-) mice in terms of fine structures of paranodal loops, transverse bands, and multi-lamellae of myelinated axons. Moreover, sensory reflex, circadian rhythm, and locomotor activity in the home cage, as well as depression-like behavior, in the Opalin(-/-) mice were indistinguishable from the Opalin(+/+) mice. Nevertheless, a subtle but significant impact on exploratory activity became apparent in Opalin(-/-) mice exposed to a novel environment. These results suggest that Opalin is not critical for central nervous system myelination or basic sensory and motor activities under conventional breeding conditions, although it might be required for fine-tuning of exploratory behavior.

DOI： 10.1371/journal.pone.0166732

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Optical clearing methods facilitate deep biological imaging by mitigating light scattering in situ. Multi-scale high-resolution imaging requires preservation of tissue integrity for accurate signal reconstruction. However, existing clearing reagents contain chemical components that could compromise tissue structure, preventing reproducible anatomical and fluorescence signal stability. We developed Sca /eS, a sorbitol-based optical clearing method that provides stable tissue preservation for immunochemical labeling and three-dimensional (3D) signal rendering. Sca /eS permitted optical reconstructions of aged and diseased brain in Alzheimer's disease models, including mapping of 3D networks of amyloid plaques, neurons and microglia, and multi-scale tracking of single plaques by successive fluorescence and electron microscopy. Human clinical samples from Alzheimer's disease patients analyzed via reversible optical re-sectioning illuminated plaque pathogenesis in the z axis. Comparative benchmarking of contemporary clearing agents showed superior signal and structure preservation by Sca /eS. These findings suggest that Sca /eS is a simple and reproducible method for accurate visualization of biological tissue.

DOI： 10.1038/nn.4107

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© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. L-Serine is required to synthesize membrane lipids such as phosphatidylserine and sphingolipids. Nevertheless, it remains largely unknownhowadiminishedcapacityto synthesize L-serine affects lipid homeostasis in cells and tissues. Here, we show that deprivation of external L-serine leads to the generation of 1-deox-ysphingolipids (doxSLs), including 1-deoxysphinganine, in mouse embryonicfibroblasts(KO-MEFs)lackingD-3-phosphoglyceratede-hydrogenase (Phgdh), which catalyzes the first step in the de novo synthesis of L-serine. A novel mass spectrometry-based lipidomic approach demonstrated that 1-deoxydihydroceramide was the most abundant species of doxSLs accumulated in L-serine-deprived KO-MEFs. Among normal sphingolipid species in KO-MEFs, levels of sphinganine, dihydroceramide, ceramide, and hexosylceramide were significantly reduced after deprivation of external L-serine, whereas those of sphingomyelin, sphingosine, and sphingosine 1-phosphate were retained. The synthesis of doxSLs was suppressed by supplementing the culture medium with L-serine but was potentiated by increasing the ratio of L-al-anine to L-serine in the medium. Unlike with L-serine, depriving cells of external L-leucine did not promote the occurrence of doxSLs. Consistent with results obtained from KO-MEFs, brainspecific deletion of Phgdh in mice also resulted in accumulation of doxSLs in the brain. Furthermore, L-serine-deprived KO-MEFs exhibited increased formation of cytosolic lipid bodies containing doxSLs and other sphingolipids. These in vitro and in vivo studies indicate that doxSLs are generated in the presence of a high ratio of L-alanine to L-serine in cells and tissues lackingPhgdh, andde novosynthesis of L-serineisnecessary to maintain normal sphingolipid homeostasis when the external supply of this amino acid is limited.

DOI： 10.1074/jbc.M114.603860

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Introduction: FUS/TLS is an RNA-binding protein whose genetic mutations or pathological inclusions are associated with neurological diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration, and essential tremor (ET). It is unclear whether their pathogenesis is mediated by gain or loss of function of FUS/TLS.
Results: Here, we established outbred FUS/TLS knockout mice to clarify the effects of FUS/TLS dysfunction in vivo. We obtained homozygous knockout mice that grew into adulthood. Importantly, they did not manifest ALS-or ET-like phenotypes until nearly two years. Instead, they showed distinct histological and behavioral alterations including vacuolation in hippocampus, hyperactivity, and reduction in anxiety-like behavior. Knockout mice showed transcriptome alterations including upregulation of Taf15 and Hnrnpa1, while they have normal morphology of RNA-related granules such as Gems.
Conclusions: Collectively, FUS/TLS depletion causes phenotypes possibly related to neuropsychiatric and neurodegenerative conditions, but distinct from ALS and ET, together with specific alterations in RNA metabolisms.

DOI： 10.1186/s40478-015-0202-6

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Alzheimer disease (AD) is biochemically characterized by increased levels of amyloid beta (A beta) peptide, which aggregates into extracellular A beta plagues in AD brains. Before plague formation, A beta accumulates intracellularly in both AD brains and in the brains of AD model mice, which may contribute to disease progression. Autophagy, which is impaired in AD, clears cellular protein aggregates and participates in A beta metabolism. In addition to a degradative role of autophagy in All metabolism we recently showed that A beta secretion is inhibited in mice Lacking autophagy-related gene 7 (Atg7) in excitatory neurons in the mouse forebrain. This inhibition of A beta secretion Leads to intracellular accumulation of A beta. Here, we used fluorescence and immunoelectron microscopy to elucidate the subcellular Localization of the intracellular A beta accumulation which accumulates in All precursor protein mice lacking Atg7. Autophagy deficiency causes accumulation of p62(+) aggregates, but these aggregates do not contain A beta. However, knockdown of Atg7 induced A13 accumulation in the Golgi and a concomitant reduction of A beta in the multivesicular bodies. This indicates that Atg7 influences the transport of All possibly derived from Golgi to multivesicular bodies.

DOI： 10.1016/j.ajpath.2014.10.011

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Language：English   Publisher：(一社)日本認知症学会  

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Recent genetic linkage analysis has shown that LRRTM1 (Leucine rich repeat transmembrane neuronal 1) is associated with schizophrenia. Here, we characterized Lrrtm1 knockout mice behaviorally and morphologically. Systematic behavioral analysis revealed reduced locomotor activity in the early dark phase, altered behavioral responses to novel environments (open-field box, light-dark box, elevated plus maze, and hole board), avoidance of approach to large inanimate objects, social discrimination deficit, and spatial memory deficit. Upon administration of the NMDA receptor antagonist MK-801, Lrrtm1 knockout mice showed both locomotive activities in the open-field box and responses to the inanimate object that were distinct from those of wild-type mice, suggesting that altered glutamatergic transmission underlay the behavioral abnormalities. Furthermore, administration of a selective serotonin reuptake inhibitor (fluoxetine) rescued the abnormality in the elevated plus maze. Morphologically, the brains of Lrrtm1 knockout mice showed reduction in total hippocampus size and reduced synaptic density. The hippocampal synapses were characterized by elongated spines and diffusely distributed synaptic vesicles, indicating the role of Lrrtm1 in maintaining synaptic integrity. Although the pharmacobehavioral phenotype was not entirely characteristic of those of schizophrenia model animals, the impaired cognitive function may warrant the further study of LRRTM1 in relevance to schizophrenia.

DOI： 10.1371/journal.pone.0022716

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Language：English   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2011.07.1240

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

Neuronal activity has an impact on beta cleavage of amyloid precursor protein (APP) by BACE1 to generate amyloid-beta peptide (A beta). However, the molecular mechanisms underlying this effect remain to be elucidated. Cholesterol dependency of beta cleavage prompted us to analyze immunoisolated APP-containing detergent-resistant membranes from rodent brains. We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains. In living cells, APP associates with syntaxin 1-containing microdomains through X11-Munc18, which inhibits the APP-BACE1 interaction and beta cleavage via microdomain segregation. Phosphorylation of Munc18 by cdk5 causes a shift of APP to BACE1-containing microdomains. Neuronal hyperactivity, implicated in A beta overproduction, promotes the switching of APP microdomain association as well as beta cleavage in a partially cdk5-dependent manner. We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons.

DOI： 10.1083/jcb.200804075

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

We evaluated the preservation of ultra-structure and immunoreactivity in cryosections of central nervous system tissue mounted with and stored in a sucrose-gelatin solution for one month at -20 degrees C or -80 degrees C. The ultra-structure of synaptic structure in these sections was well preserved and comparable to that of freshly cut cryosections. Quantitative analysis of mitochondrial ultra-structure demonstrated gradually lower degrees of preservation in sections stored at -20 degrees C and -80 degrees C compared with that in freshly cut sections. We observed distinct metabotropic glutamate receptor 1 (mGluR1)-immunogold labelling at peri-synaptic sites in freshly cut sections and also in those stored at -20 degrees C and -80 degrees C. Quantitative analysis of mGluR1 immunoreactivity revealed that the total number of immunogold particles per synapse and the number of non-specifically bound particles were similar under all three conditions. However, the percentage of gold particles bound to a specific synaptic region was greatest in freshly cut sections (79.0%) and progressively lower in sections stored at -20 degrees C (76.1%), in which sections were not frozen, and in sections stored at -80 degrees C (68.0%). These data indicate that ultra-thin cryosections may be conveniently stored in a sucrose-gelatin solution at -20 degrees C for cryoultramicrotomy-immunolabelling.

DOI： 10.1111/j.1365-2818.2008.02012.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

In contrast to compact myelin, the series of paranodal loops located in the outermost lateral region of myelin is non-compact; the intracellular space is filled by a continuous channel of cytoplasm, the extracellular surfaces between neighboring loops keep a definite distance, but the loop membranes have junctional specializations. Although the proteins that form compact myelin have been well studied, the protein components of paranodal loop membranes are not fully understood. This report describes the biochemical characterization and expression of Opalin as a novel membrane protein in paranodal loops. Mouse Opalin is composed of a short N-terminal extracellular domain (amino acid residues 1-30), a transmembrane domain (residues 31-53), and a long C-terminal intracellular domain (residues 54-143). Opalin is enriched in myelin of the central nervous system, but not that of the peripheral nervous system of mice. Enzymatic deglycosylation showed that myelin Opalin contained N-and O-glycans, and that the O-glycans, at least, had negatively charged sialic acids. We identified two N-glycan sites at Asn-6 and Asn-12 and an O-glycan site at Thr-14 in the extracellular domain. Site-directed mutations at the glycan sites impaired the cell surface localization of Opalin. In addition to the somata and processes of oligodendrocytes, Opalin immunoreactivity was observed in myelinated axons in a spiral fashion, and was concentrated in the paranodal loop region. Immunogold electron microscopy demonstrated that Opalin was localized at particular sites in the paranodal loop membrane. These results suggest a role for highly sialylglycosylated Opalin in an intermembranous function of the myelin paranodal loops in the central nervous system.

DOI： 10.1074/jbc.M801314200

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Streptolysin O (SLO) is a membrane-damaging toxic protein produced by group A streptococci. We performed an ultrastructural analysis of pore formation and the mechanism of hemolysis by SLO, using a mutant form of SLO [SLO(C/A)-SS] and native SLO. SLO(C/A)-SS was unable to penetrate the erythrocyte membrane as a consequence of immobilization that was due to a disulfide bond between domains. The SLO(C/ A)-SS molecules that bound to membranes formed numerous single-layered ring-shaped structures that did not result in pores on the membranes. These structures were similar to the structures formed by native SLO at 0 degrees C. After treatment with dithiothreitol, SLO(C/A)-SS that had bound to membranes formed double-layered rings with pores on the membranes, as does native SLO at room temperature. Our morphological evidence demonstrates that an increase in temperature is necessary for the occurrence of conformational changes and for the formation of double-layered rings after the insertion of domain 3 into the host cell membrane. On the basis of a model of the oligomeric structure of SLO, we propose some new details of the mechanism of hemolysis by SLO. (C) 2007 Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.micinf.2007.06.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Amyloid beta (A beta) toxicity has been hypothesized to initiate the pathogenesis of Alzheimer's disease (AD). The characteristic fibrillar morphology of A beta-aggregates, that constitute the main components of senile plaque, has long been considered to account for the neurotoxicity. But recent reports argue against a primary role for mature fibrils in AD pathogenesis because of the lack of a robust correlation between the severity of neurological impairment and the extent of amyloid deposition. Toxicity from the soluble prefibrillar intermediate entity of aggregates often called oligomer has recently proposed a plausible explanation for this inconsistency. An alternative explanation is based on the observation that certain amyloid fibril morphologies are more toxic than others, indicating that not all amyloid fibrils are equally toxic. Here, we report that it is not only the beta-sheeted fibrillar structure but also the surface physicochemical composition that affects the toxicity of A beta fibrils. For the first time, colloidal gold was used to visualize by electron microscopy positive-charge clusters on A beta fibrils. Chemical modifications as well as point-mutated A beta synthesis techniques were applied to change the surface structures of A beta and to show how local structure affects surface properties that are responsible for electrostatic and hydrophobic interactions with cells. We also report that covering the surface of A beta fibers with myelin basic protein, which has surface properties contrary to those of A beta, suppresses A beta toxicity. On the basis of these results, we propose that the surface structure of A beta fibrils plays an important role in A beta toxicity.

DOI： 10.1021/bi700455c

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Here, we show that cultured Purkinje cells from inositol 1,4,5-trisphosphate receptor type 1 knock-out (IP(3)R1KO) mice exhibited abnormal dendritic morphology. Interestingly, despite the huge amount of IP(3)R1 expression in Purkinje cells, IP(3)R1 in granule cells, not in the Purkinje cells, was responsible for the shape of Purkinje cell dendrites. We also found that BDNF application rescued the dendritic abnormality of IP(3)R1KO Purkinje cells, and that the increase in BDNF expression in response to activation of AMPA receptor (AMPAR) and metabotropic glutamate receptor (mGluR) was impaired in IP(3)R1KO cerebellar granule cells. In addition, we observed abnormalities in the dendritic morphology of Purkinje cells and in the ultrastructure of parallel fiber-Purkinje cell (PF-PC) synapses in IP(3)R1KO mice in vivo. We concluded that activation of AMPAR and mGluR increases BDNF expression through IP(3)R1-mediated signaling in cerebellar granule cells, which contributes to the dendritic outgrowth of Purkinje cells intercellularly, possibly by modifying PF-PC synaptic efficacy.

DOI： 10.1523/JNEUROSCI.3269-06.2006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Trisomy 21 or Down syndrome (DS) is the most common genetic birth defect associated with mental retardation. The over-expression of genes on chromosome 21, including SOD1 (Cu/Zn superoxide dismutase) and APP (amyloid-beta precursor protein) is believed to underlie the increased oxidative stress and neurodegeneration commonly described in DS. However, a segmental trisomy 16 mouse model for DS, Ts1Cje, has a subset of triplicated human chromosome 21 gene orthologs that exclude APP and SOD1. Here, we report that Ts1Cje brain shows decreases of mitochondrial membrane potential and ATP production, increases of reactive oxygen species, hyperphosphorylation of tau without NFT formation, increase of GSK3 beta and JNK/SAPK activities and unaltered A beta PP metabolism. Our findings suggest that genes on the trisomic Ts1Cje segment other than APP and SOD1 can cause oxidative stress, mitochondrial dysfunction and hyperphosphorylation of tau, all of which may play critical roles in the pathogenesis of mental retardation in DS.

DOI： 10.1093/hmg/ddl211

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We examined each step of the protocol for ultracryotomy for central nervous system tissue in order to define and overcome some of the methodological difficulties. The following three steps emerged as critical for the method's success: (1) pretreatment of grids to render them hydrophilic immediately before use; (2) careful collection of ultrathin cryosections during ultracryotomy; (3) removal of the appropriate amount of excess poly(vinyl alcohol)-uranyl acetate (PVA-UA) prior to drying after staining with PVA-UA. By taking account of the three critical steps described above, we succeeded in obtaining ultrathin cryosections, including serial sections, with excellent preservation of ultrastructure, as well as semithin cryosections which are useful for evaluating the quality of the samples and for selecting areas of interest for ultrastructural analysis.
Cytoplasmic organelles in neurons and glial cells, and the fine structure of synapses and myelinated fibers were well preserved. The localization of gold particles after immunostaining for astrocytic glutamate transporter (GLAST), metabotropic glutamate receptor 1 (mGluR1) and neurofilament protein was consistent with previous reports and ultrastructure was well-preserved in all cases.
These findings should be helpful to researchers wishing to carry out ultrastructural and immunogold analyses of cryosections of nervous tissue. (c) 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jneumeth.2005.11.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Mutations in the presenilin 1 ( PS1) gene are responsible for the early onset of familial Alzheimer disease ( FAD). Accumulating evidence shows that PS1 is involved in gamma-secretase activity and that FAD-associated mutations of PS1 commonly accelerate A beta(1-42) production, which causes Alzheimer disease ( AD). Recent studies suggest, however, that PS1 is involved not only in A beta production but also in other processes that lead to neurodegeneration. To better understand the causes of neurodegeneration linked to the PS1 mutation, we analyzed the development of tau pathology, another key feature of AD, in PS1 knock-in mice. Hippocampal samples taken from FAD mutant ( I213T) PS1 knock-in mice contained hyperphosphorylated tau that reacted with various phosphodependent tau antibodies and with Alz50, which recognizes the conformational change of PHF tau. Some neurons exhibited Congo red birefringence and Thioflavin T reactivity, both of which are histological criteria for neurofibrillary tangles ( NFTs). Biochemical analysis of the samples revealed SDS-insoluble tau, which under electron microscopy examination, resembled tau fibrils. These results indicate that our mutant PS1 knock-in mice exhibited NFT-like tau pathology in the absence of A beta deposition, suggesting that PS1 mutations contribute to the onset of AD not only by enhancing A beta(1-42) production but by also accelerating the formation and accumulation of filamentous tau.

DOI： 10.1074/jbc.MS09145200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Formation of misfolded protein aggregates is a remarkable hallmark of various neurodegenerative diseases including Alzheimer disease, Parkinson disease, Huntington disease, prion encephalopathies, and amyotrophic lateral sclerosis (ALS). Superoxide dismutase 1 (SOD1) immunoreactive inclusions have been found in the spinal cord of ALS animal models and patients, implicating the close involvement of SOD1 aggregates in ALS pathogenesis. Here we examined the molecular mechanism of aggregate formation of ALS-related SOD1 mutants in vitro. We found that long-chain unsaturated fatty acids (FAs) promoted aggregate formation of SOD1 mutants in both dose- and time-dependent manners. Metal-deficient SOD1s, wild-type, and mutants were highly oligomerized compared with holo-SOD1s by incubation in the presence of unsaturated FAs. Oligomerization of SOD1 is closely associated with its structural instability. Heat-treated holo-SOD1 mutants were readily oligomerized by the addition of unsaturated FAs, whereas wild-type SOD1 was not. The monounsaturated FA, oleic acid, directly bound to SOD1 and was characterized by a solid-phase FA binding assay using oleate-Sepharose. The FA binding characteristics were closely correlated with the oligomerization propensity of SOD1 proteins, which indicates that FA binding may change SOD1 conformation in a way that favors the formation of aggregates. High molecular mass aggregates of SOD1 induced by FAs have a granular morphology and show significant cytotoxicity. These findings suggest that SOD1 mutants gain FA binding abilities based on their structural instability and form cytotoxic granular aggregates.

DOI： 10.1074/jbc.M502230200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Zinc is a modulator of glutamatergic inputs in the hippocampus. In the retina, however, we previously reported that endogenous zinc is present in the non-glutamatergic neural processes and earlier electrophysiological studies suggest that zinc is a modulator of inhibitory signaling pathways, which are mediated by glycine and GABA. AII amacrine cells, a subpopulation of glycinergic amacrine cells, are identified by selective immunoreactivity for parvalbumin in the rat retina. In the present study, therefore, we focused on whether zinc is present in AII amacrine cells using silver amplification combined with immunohistochemistry in the rat retina. We also examined whether zinc modulate glycine response in the rat retina by the patch clamp technique. Association of silver precipitates with the parvalbumin-immunoreactive neural processes was observed at the ultrastructural level. We also found that zinc existed in the neural processes which were not parvalbumin-immunoreactive. Glycine-induced responses were augmented when the concentration of Zn2+ was below 10 mu M, but inhibited at Zn2+ concentrations of 50 mu M or more. Our results suggest the notion that zinc in neural processes of retinal neurons modulates the inhibitory signaling pathway, particularly that mediated by glycine receptors in AII amacrine cells.

DOI： 10.1007/s10735-005-1693-4

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Language：English   Publisher：(一社)日本神経化学会  

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It is known that, in the retina, extracellular adenosine triphosphate (ATP) inhibits acetylcholine (ACh) release from cholinergic neurons, but the types of purinoceptors on cholinergic neurons have not been examined. In the present work, we immunohistochemically examined the distribution of the purinoceptors P2X1, P2X2, P2X4, and P2X7 in relation to the cholinergic system of the retina in wild-type mice and transgenic mice expressing green fluorescent protein (GFP). Immunoreactivity for P2X2 was very strong in sublamina a of the inner plexiform layer but very weak in sublamina b of the inner plexiform layer of the retina. Immunoreactivity for P2X2 was colocalized with that for choline acetyltransferase (ChAT). When transgenic mice were treated with the immunotoxin-mediated cell-targeting technology to ablate cholinergic amacrine cells selectively, immunoreactivity for P2X2 and the signals for GFP disappeared in parallel and selectively in the OFF pathway. The distribution of immunoreactivity for P2X1, P2X4, and P2X7 differed from that of ChAT immunoreactivity. The selective distribution of P2X2 purinergic receptors in OFF-type cholinergic amacrine cells indicates that the P2X2 purinergic signaling systems in the ON and OFF pathways of the inner plexiform layer of the mouse retina are functionally different. The distribution of P2X2 purinoceptors may be responsible for the selective regulation of ACh release in the OFF pathway. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/cne.20208

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

This paper describes new cytochemical method for the ultrastructural localization of Co2+ following blockade of synaptic transmission. In the CA1 region of rat hippocampal slices, electrical stimulation of the Schaffer collaterals elicited field excitatory postsynaptic potentials (fEPSPs). The fEPSPs were completely blocked within 2 min after the addition of Co2+ (2 mM). The slice was then fixed and precipitated Co2- was examined by means of a solution containing 2.5% glutaraldehyde, and 10mM K-3[Fe3+(CN)(6)] in 90mM NaCl. Electron spectroscopic imaging confirmed Co in the precipitate. The precipitates were found as clusters on the membranes of the fine apical dendrites and then spine heads of CA1 pyramidal neurons. No clustered precipitate was found when slices were treated: (1) without Co2+: (2) after recovery from the Co2+-induced blockade of fEPSPs, (3) without electrical stimulation of the Schaffer collaterals; and (4) with DL-2-amino-5-phosphonopentanoate and 6-cyano-7-nitroquinoxaline-2,3-dione. After administrating glutamate (5 mM) in the presence of tetrodotoxin (1 muM) and Co2+, precipitates were found on dendritic membranes and spine heads. These results indicate that the Schaffer collaterals stimulation induces the binding of Co2+ on CA1 pyramidal neuron membrane. (C) 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jneumeth.2003.11.008

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Omi/HtrA2 is a mitochondrial serine protease that is released into the cytosol during apoptosis and promotes cytochrome c (Cyt c) dependent caspase activation by neutralizing inhibitor of apoptosis proteins (IAPs) via its IAP-binding motif. The protease activity of Omi/HtrA2 also contributes to the progression of both apoptosis and caspase-independent cell death. In this study, we found that wild-type Omi/HtrA2 is more effective at caspase activation than a catalytically inactive mutant of Omi/HtrA2 in response to apoptotic stimuli, such as UV irradiation or tumor necrosis factor. Although similar levels of Omi/HtrA2 expression, XIAP-binding activity, and Omi/HtrA2 mitochondrial release were observed among cells transfected with catalytically inactive and wild-type Omi/HtrA2 protein, XIAP protein expression after UV irradiation was significantly reduced in cells transfected with wild-type Omi/HtrA2. Recombinant Omi/HtrA2 was observed to catalytically cleave IAPs and to inactivate XIAP in vitro, suggesting that the protease activity of Omi/HtrA2 might be responsible for its IAP-inhibiting activity. Extramitochondrial expression of Omi/HtrA2 indirectly induced permeabilization of the outer mitochondrial membrane and subsequent Cyt c-dependent caspase activation in HeLa cells. These results indicate that protease activity of Omi/HtrA2 promotes caspase activation through multiple pathways.

DOI： 10.1038/sj.cdd.4401343

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：KARGER  

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Publisher：PHYSIOLOGICAL SOCIETY OF JAPAN  

In the retina, while it is known that extracellular ATP inhibits ACh release from cholinergic neurons, the types of purinoceptors on the cholinergic neurons have not been examined. We reported that immunoreactivity for P2X2 localized asymmetrically between the ON and OFF pathways in the cholinergic amacrine cells in the mouse retina. In the present study, we recorded the responses to ATP in the cholinergic amacrine cells of the transgenic mouse retina. Both straburst amacrine cells and displaced amacrine cells responded to 100 μM ATP. ATP induced an inward current. However, part of displaced amacrine cells did not respond to 100 μM ATP. Both types of cells did not respond to 100 μM α,β-methylene ATP. Responses to ATP were inhibited by 100 μM PPADS. When cells received GABAergic-IPSP, 100 μM ATP augmented GABAergic IPSP. These results indicate that inhibitory action of ACh released by ATP is mediated by the presynaptic mechanisms. [Jpn J Physiol 54 Suppl:S162 (2004)]

DOI： 10.14849/psjproc.2004.0_s162_3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Accumulation of insoluble alpha-synuclein aggregates in the brain is characteristic of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Although numerous studies on the aggregation properties of alpha-synuclein have been reported, little is known about its degradation so far. In view of proteolytic degradation, we have found that the serine protease neurosin (kallikrein-6) degrades alpha-synuclein and co-localizes with pathological inclusions such as Lewy bodies and glial cytoplasmic inclusions. In vitro study showed that neurosin prevented alpha-synuclein polymerization by reducing the amount of monomer and also by generating fragmented alpha-synucleins that themselves inhibited the polymerization. Upon cellular stress, neurosin was released from mitochondria to the cytosol, which resulted in the increase of degraded alpha-synuclein species. Down-regulation of neurosin caused accumulation of alpha-synuclein within cultured cells. Thus we concluded that neurosin plays a significant role in physiological alpha-synuclein degradation and also in the pathogenesis of synucleinopathies.

DOI： 10.1093/hmg/ddg283

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We examined the effects of the expansion of glutamine repeats on the early stage of protein fibrillization. Small-angle x-ray scattering (SAXS) and electron microscopic studies revealed that the elongation of polyglutamine from 35 to 50 repeats in protein induced a large assembly of the protein upon incubation at 37 degreesC and that its formation was completed in similar to 3 h. A bead modeling procedure based on SAXS spectra indicated that the largely assembled species of the protein, quasi-aggregate, is composed of 80 to similar to90 monomers and a bowl-like structure with long and short axes of 400 and 190 Angstrom, respectively. Contrary to fibril, the quasi-aggregate did not show a peak at S = 0.21 Angstrom(-1) corresponding to the 4.8-Angstrom spacing of beta-pleated sheets in SAXS spectra, and reacted with a monoclonal antibody specific to expanded polyglutamine. These results imply that beta-sheets of expanded polyglutamines in the quasi-aggregate are not orderly aligned and are partially exposed, in contrast to regularly oriented and buried beta-pleated sheets in fibril. The formation of non-fibrillary quasi-aggregate in the early phase of fibril formation would be one of the major characteristics of the protein containing an expanded polyglutamine.

DOI： 10.1074/jbc.M209852200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Alzheimer's disease (AD) may be caused by toxic aggregates formed from amyloid-beta (Abeta) peptides. By using Thioflavin T, a dye that specifically binds to beta-sheet structures, we found that highly toxic forms of Abeta-aggregates were formed at the initial stage of fibrillogenesis, which is consistent with recent reports on Abeta oligomers. Formation of such aggregates depends on factors that affect both nucleation and elongation. As reported previously, addition of Abeta42 systematically accelerated the nucleation of Abeta40, most likely because of the extra hydrophobic residues at the C terminus of Abeta42. At Abeta42-increased specific ratio (Abeta40: Abeta42 = 10: 1), on the other hand, not only accelerated nucleation but also induced elongation were observed, suggesting pathogenesis of early-onset AD. Because a larger proportion of Abeta40 than Abeta42 was still required for this phenomenon, we assumed that elongation does not depend only on hydrophobic interactions. Without any change in the C-terminal hydrophobic nature, elongation was effectively induced by mixing wild type Abeta40 with Italian variant Abeta40 (E22K) or Dutch variant (E22Q). We suggest that Abeta peptides in specific compositions that balance hydrophilic and hydrophobic interactions promote the formation of toxic beta-aggregates. These results may introduce a new therapeutic approach through the disruption of this balance.

DOI： 10.1074/jbc.M212785200

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER-VERLAG TOKYO  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Neurofibrillary tangles (NFTs) are found in a wide range of neurodegenerative disorders, including Alzheimer's disease. The major component of NFTs is aberrantly hyperphosphorylated microtubule-associated protein tau. Because appropriate in vivo models have been lacking, the role of tau phosphorylation in NFTs formation has remained elusive. Here, we describe a new model in which adenovirus-mediated gene expression of tau, DeltaMEKK, JNK3, and GSK-3beta in COS-7 cells produces most of the pathological phosphorylation epitopes of tau including AT100. Furthermore, this co-expression resulted in the formation of tau aggregates having short fibrils that were detergent-insoluble and Thioflavin-S-reactive. These results suggest that aberrant tau phosphorylation by the combination of these kinases may be involved in "pretangle," oligomeric tau fibril formation in vivo.

DOI： 10.1074/jbc.M202241200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Aggregates of green fluorescent protein (GFP)-fused truncated N-terminal huntingtin containing abnormally long polyglutamine tracts (150 repeats of glutamine residue) were purified from an ecdysone-inducible mutant neuro2A cell line (HD150Q-28) by using a fluorescence-activated cell sorter. To analyze the aggregate-interacting proteins, we subjected the purified aggregates to SDS-PAGE; prominent protein bands in the gel were digested with Achromobactor lysyl endopeptidase, followed by a HPLC-mass spectrometry (MS) analysis. The resulting data of tandem MS analysis revealed that, in addition to ubiquitin and widely reported chaperone proteins such as heat shock cognate 70 (HSC70), human DNA J-1 (HDJ-1), and HDJ-2, the translational elongation factor-1alpha (EF-1alpha) and heat shock protein 84 (HSP84) also were recognized as aggregate-interacting proteins. Sequestration of these proteins to aggregates was confirmed further by several immunochemical methods. We confirmed that, in addition to the other known proteins, EF-1alpha and HSP84 also colocalized with the intracellular aggregates. An assay of the transient expression of EF-1alpha and HSP84 in HD150Q-28 cells revealed that both proteins improved cell viability. Moreover, the rate of aggregate formation decreased in both transfectants. Our study suggests that both EF-1alpha and HSP84 are involved in the neurodegenerative process of polyglutamine diseases.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

The R406W tau mutation found in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) causes a hereditary tauopathy clinically resembling Alzheimer's disease. Expression of modest levels of the longest human tau isoform with this mutation under the control of the alpha-calcium-calmodulin-dependent kinase-11 promoter in transgenic (Tg) mice resulted in the development of congophilic hyperphosphorylated tau inclusions in forebrain neurons. These inclusions appeared as early as 18 months of age. As with human cases, tau inclusions were composed of both mutant and endogenous wild-type tau, and were associated with microtubule disruption and flame-shaped transformations of the affected neurons. Straight tau filaments were recovered from Sarkosyl-insoluble fractions from only the aged Tg brains. Behaviorally, aged Tg mice had associative memory impairment without obvious sensorimotor deficits. Therefore, these mice that exhibit a phenotype mimicking R406W FTDP-17 provide an animal model for investigating the adverse properties associated with this mutation, which might potentially recapitulate some etiological events in Alzheimer's disease.

DOI： 10.1073/pnas.202205599

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

To examine the effects of aggregation-inducing motifs related to neurodegenerative diseases on amyloid formation of host protein, we prepared several chimera myoglobins, in which various aggregation-inducing motifs were inserted. The focused aggregation-inducing motifs included five (R5) or two (R2) oligopeptide repeats in yeast Sup35p, five octapeptide repeats (OPR) in the human prion protein, a nonamyloid beta component (NAC) in alpha-synuclein, and tandem repeats of 50 glutamines (Q50). Circular dichroism and infrared spectroscopies suggested that the OPR, R5, and Q50 motifs formed an antiparallel beta sheet as well as a random coil, whereas the R2 and NAC motifs mainly formed random coils. The OPR, R5, and Q50 mutants, but not the R2 and NAC mutants, readily formed the SIDS-resistant aggregates under physiological condition, and electron microscopy revealed that the aggregates contained amyloid fibrils. The destabilization and increase in gyration radius of the OPR, R5, and Q50 mutants correlated with the tendency to form amyloid fibrils. A control mutant bearing a nonamyloidgenic sequence was also moderately destabilized but did not form amyloid fibrils. Therefore, we concluded that the OPR, R5, and Q50 motifs, even in a quite stable protein such as myoglobin, led the host protein to formation of amyloid fibrils under physiological condition.

DOI： 10.1021/bi0258905

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Unfolded Pael receptor (Pael-R) is a substrate of the E3 ubiquitin ligase Parkin. Accumulation of Pael-R in the endoplasmic reticulum (ER) of dopaminergic neurons induces ER stress leading to neurodegeneration. Here, we show that CHIP, Hsp70, Parkin, and Pael-R formed a complex in vitro and in vivo. The amount of CHIP in the complex was increased during ER stress. CHIP promoted the dissociation of Hsp70 from Parkin and Pael-R, thus facilitating Parkin-mediated Pael-R ubiquitination. Moreover, CHIP enhanced Parkin-mediated in vitro ubiquitination of Pael-R in the absence of Hsp70. Furthermore, CHIP enhanced the ability of Parkin to inhibit cell death induced by Pael-R. Taken together, these results indicate that CHIP is a mammalian E4-like molecule that positively regulates Parkin E3 activity.

DOI： 10.1016/S1097-2765(02)00583-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Mutations in the EPM2A gene encoding a dual-specificity phosphatase (laforin) cause Lafora disease (LD), a progressive and invariably fatal epilepsy with periodic acid-Schiff-positive (PAS+) cytoplasmic inclusions (Lafora bodies) in the central nervous system. To study the pathology of LD and the functions of laforin, we disrupted the Epm2a gene in mice. At two months of age, homozygous null mutants developed widespread degeneration of neurons, most of which occurred in the absence of Lafora bodies. Dying neurons characteristically exhibit swelling in the endoplasmic reticulum, Golgi networks and mitochondria in the absence of apoptotic bodies or fragmentation of DNA. As Lafora bodies become more prominent at 4-12 months, organelles and nuclei are disrupted. The Lafora bodies, present both in neuronal and non-neural tissues, are positive for ubiquitin and advanced glycation end-products only in neurons, suggesting different pathological consequence for Lafora inclusions in neuronal tissues. Neuronal degeneration and Lafora inclusion bodies predate the onset of impaired behavioral responses, ataxia, spontaneous myoclonic seizures and EEG epileptiform activity. Our results suggest that LD is a primary neurodegenerative disorder that may utilize a non-apoptotic mechanism of cell death.

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Language：Japanese   Publisher：The Japanese Society of Microscopy  

DOI： 10.11410/kenbikyo1950.37.22

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Language：Japanese   Publisher：公益社団法人 日本顕微鏡学会  

DOI： 10.11410/kenbikyo1950.37.22

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Formation of neurofibrillary tangles (NFTs) is a common neuropathological feature found in several neurodegenerative diseases, including Alzheimer's disease. We have developed a transgenic (Tg) mouse expressing mutant human tau (V337M), derived from frontotemporal dementia parkinsonism-17. V337M Tg mice revealed tau aggregations in the hippocampus, which fulfills the histological criteria for NFTs in human neurodegenerative diseases. Concurrent with the accumulation of RNA and phosphorylated tau, neurons exhibited morphological characteristics of degenerating neurons, which include a loss of microtubules, accumulation of ribosomes, plasma and nuclear membrane ruffling, and swelling of the Golgi network. Thus, mutant tau induces neuronal degeneration associated with the accumulation of RNA and phosphorylated tau. The functional consequences of this neuronal degeneration was evidenced by the reduction of hippocampal neural activity and behavioral abnormality in Tg mice.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cell Press  

Unfolded Pael receptor (Pael-R) is a substrate of the E3 ubiquitin ligase Parkin. Accumulation of Pael-R in the endoplasmic reticulum (ER) of dopaminergic neurons induces ER stress leading to neurodegeneration. Here, we show that CHIP, Hsp70, Parkin, and Pael-R formed a complex in vitro and in vivo. The amount of CHIP in the complex was increased during ER stress. CHIP promoted the dissociation of Hsp70 from Parkin and Pael-R, thus facilitating Parkin-mediated Pael-R ubiquitination. Moreover, CHIP enhanced Parkin-mediated in vitro ubiquitination of Pael-R in the absence of Hsp70. Furthermore, CHIP enhanced the ability of Parkin to inhibit cell death induced by Pael-R. Taken together, these results indicate that CHIP is a mammalian E4-like molecule that positively regulates Parkin E3 activity.

DOI： 10.1016/S1097-2765(02)00583-X

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DOI： 10.2302/kjm.51.supplement1_66

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Formation of neurofibrillary tangles (NFTs) is the most common feature in several neurodegenerative diseases, including Alzheimer's disease (AD). Here we report the formation of filamentous tau aggregations having a beta -sheet structure in transgenic mice expressing mutant human tau. These mice contain a tau gene with a mutation of the frontotemporal dementia parkinsonism (FTDP-17) type, in which valine is substituted with methionine residue 337. The aggregation of tau in these transgenic mice satisfies all histological criteria used to identify NFTs common to human neurodegenerative diseases. These mice, therefore, provide a preclinical model for the testing of therapeutic drugs for the treatment of neurodegenerative disorders that exhibit NFTs. (C) 2001 Elsevier Science.

DOI： 10.1006/nbdi.2001.0439

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

An aberrant structure of the expanded polyglutamine might be involved in the formation of aggregates in CAG repeat diseases. To elucidate structural properties of the expanded polyglutamine, we prepared sperm whale myoglobin (Mb) mutants, in which 12, 28, 35, and 50 repeats of glutamine were inserted at the corner between the C and D helices (Gln(12), Gln(28), Gln(35), and Gln(50), respectively). Circular dichroism and IR spectroscopies showed that the expanded polyglutamine, which was recognized by the monoclonal antibody 1C2 in Gln(28), Gln(35), and Gln(50) Mb forms an antiparallel beta -pleated sheet structure. Gln(50) Mb aggregates were found to comprise an intermolecular antiparallel beta -pleated sheet. Fluorescence together with H-1 NMR spectra revealed partial unfolding of the protein surface in Gln(35) and Gln(50) Mb, although the structural changes in the protein core were rather small. The present results indicate that the fluctuating beta -pleated sheet of the expanded polyglutamine exposed on the protein surface facilitates the formation of aggregates through intermolecular interactions. The present study has first established and characterized structural properties of a molecular model for polyglutamine diseases in which various lengths of polyglutamine including a pathologically expanded glutamine repeat were inserted into a structurally known protein.

DOI： 10.1074/jbc.M107502200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Dscam, a novel cell-adhesion molecule belonging to the Ig-superfamily mediates homophilic intercellular adhesion and is expressed abundantly in the nervous system during development. To gain better understanding on the role of Dscam in neuronal differentiation, we raised an antibody and characterized its protein product. Anti-Dscam antibody detected an similar to 200-kDa protein band in human and mouse brain lysates. Immunohistochemical studies showed that during embryonic development of mice, mouse Dscam is expressed throughout the neuronal tissues and also in nonneuronal tissues such as lung, liver, and limb buds. In adult brain Dscam expression is predominant in the cerebellum, hippocampus, and olfactory bulb. Immunofluorescence double labeling of hippocampal and cerebellar primary cultures revealed that Dscam is associated with axonal and dendritic processes. In view of its cellular localization and spatiotemporal expression pattern, we suggest that Dscam is involved in cell-cell interactions during axonal-dendritic development, and maintenance of functional neuronal networks. (C) 2001 Wiley-Liss.

DOI： 10.1002/jnr.1226

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Amyloid-beta protein (A beta) aggregates in the brain to form senile plaques. By using thioflavin T, a dye that specifically binds to fibrillar structures, we found that metals such as Zn(ll) and Cu(II) normally inhibit amyloid beta -aggregation. Another method for detecting A beta, which does not distinguish the types of aggregates, showed that these metals induce a non-beta -sheeted aggregation, as reported previously. Secondary structural analysis and microscopic studies revealed that metals induced A beta to make non-fibrillar aggregates by disrupting beta -sheet formation. These non-fibrillar A beta aggregates displayed much weaker Congo Red birefringence, and in separate cell culture experiments, were less toxic than self beta -aggregates, as demonstrated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The toxicity of soluble A beta was enhanced in the presence of Cu(Il), which suggests the previously hypothesized role of A beta in generating oxidative stress. Finally, under an acidic condition, similar to that in the inflammation associated with senile plaques, beta -aggregation was robustly facilitated at one specific concentration of Zn(ll) in the presence of heparin. However, because a higher concentration of Zn(II) virtually abolished this abnormal phenomenon, and at normal pH any concentrations strongly inhibit beta -aggregation and its associated cytotoxicity, including its anti-oxidative nature we suggest that Zn(II) has an overall protective effect against beta -amyloid toxicity.

DOI： 10.1074/jbc.M010706200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS LTD  

In the retina, zinc is believed to be a modulator of synaptic transmission and a constituent: of metalloenzymes. To determine whether the intracellular localization of zinc correlates with function, we examined the localization of endogenous zinc in the rat retina using the silver amplification method. By light microscopy, reaction products were detected in the pigment epithelial cells (PE), the inner segment of photoreceptors (IS), the outer nuclear layer (ONL) and the inner nuclear layer (INL), the outer plexiform layer (OPL) and the inner plexiform layer (IPL), and the ganglion cell layer (CC). The heaviest accumulation of precipitate was observed in PE and IS, whereas only a little precipitate was found in GC. When the intracellular zinc was chelated with diethyldithiocarbamate, a small amount of precipitate was observed only in ONL. By electron microscopy, zinc was associated with three compartments. In OPL and IPL, zinc was associated with neural processes, while in PE, IS, INL and GC it was associated with the Golgi apparatus. In ONL, zinc was associated with the nucleus. Zinc in the neural processes is believed to act as a modulator of synaptic transmission, and zinc associated with the Golgi apparatus is assumed to catalyze metalloenzyme reactions.

DOI： 10.1177/002215540104900109

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To clarify toxic effects of long-term oral administration of low dose cadmium (Cd) on the liver and kidney, six groups of female Sprague-Dawley rats were fed a diet containing Cd-polluted rice or CDCl2 at concentrations up to 40 ppm, and killed after 12, 18, and 22 months. With toxicological parameters, including histopathology, there was no evidence of Cd-related hepato-renal toxicity, despite a slight decrease of mean corpuscular volume and mean corpuscular hemoglobin of red blood cells with 40 ppm CDCl2. Dose-dependent accumulation of Cd was observed in the liver and kidneys with peak levels of 130±42 μg/g and 120±20 μg/g, respectively, at 18 months in animals treated with 40 ppm CDCl2. A dose-dependent increase in urinary Cd levels became evident with time. Induction of metallothionein (MT) was also observed in the liver and kidney with a high correlation to the corresponding Cd levels. In the proximal renal tubular epithelia of 40 ppm CdCl2-treated rats at 22 months, prominent accumulation of Cd was observed in secondary lysosomes associated with MT deposits in their exocytotic residual bodies. The results demonstrated that, in contrast to the case with high-dose Cd-administration, renal toxicity is not induced by long-term oral administration of low amounts of Cd, although tissue accumulation does occur. Possible protective mechanisms may be operating.

DOI： 10.2131/jts.26.337

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The progressive myoclonus epilepsy of Lafora type is an autosomal recessive disorder caused by mutations in the EPM2A gene. EPM2A is predicted to encode a putative tyrosine phosphatase protein, named laforin, whose full sequence has not yet been reported. In order to understand the function of the EPM2A gene, we isolated a full-length cDNA, raised an antibody and characterized its protein product. The full-length clone predicts a 38 kDa laforin that was very close to the size detected in transfected cells, Recombinant laforin was able to hydrolyze phosphotyrosine as well as phosphoserine/threonine substrates, demonstrating that laforin is an active dual-specificity phosphatase. Biochemical, immunofluorescence and electron microscopic studies on the full-length laforin expressed in HeLa cells revealed that laforin is a cytoplasmic protein associated with polyribosomes, possibly through a conformation-dependent protein-protein interaction. We analyzed the intracellular targeting of two laforin mutants with missense mutations. Expression of both mutants resulted in ubiquitin-positive perinuclear aggregates suggesting that they were misfolded proteins targeted for degradation. Our results suggest that laforin is involved in translational regulation and that protein misfolding may be one of the molecular bases of the Lafora disease phenotype caused by missense mutations in the EPM2A gene.

DOI： 10.1093/oxfordjournals.hmg.a018916

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Language：Japanese  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN ACAD  

During electrical stimulation of the frog neuromuscular preparation in Ringer solution, CaCl2 (1.8 mM) was replaced by equimolar CoCl2 (Co-Ringer solution). Muscular contraction due to the motor nerve stimulation was blocked completely within 2 min, while muscular contraction due to direct muscle stimulation was reduced but not completely blocked. The tissue was fixed with 2.5% glutaraldehyde prepared in Go-Ringer solution. After rapid wash in Ringer solution, the tissue was transfered into an aqueous solution containing 10 mM K3Fe(CN)(6), (precipitating agent of Co2+) and 90 mM NaCl. In conventional transmission electron microscopy, large precipitates were observed close to both sides of the presynaptic membrane, outside of the sarcolemma in subsynaptic and extrasynaptic area, on the myofibrils and close to both sides of the membrane of the sarcoplasmic reticulum in the vicinity of the T-tubules. It is presumed that the sites where the precipitates were localized correspond to the voltage dependent calcium channels of the presynaptic membrane, sarcoplasmic membrane and membrane of sarcoplasmic reticulum, The precipitates found on the myofibrils are considered as Co2+ which penetrated the endplate channels and directed towards the sarcoplasmic reticulum. To our knowledge, the present electron microscopic observation of Co2+ precipitated with potassium ferricyanide is the first attempt in the cobalt cytochemistry.

DOI： 10.2183/pjab.76.7

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Partially purified bovine bone morphogenetic proteiun (BMP) and type collagen as carrier were combined and lyophilized. The freeze-dried pellets were implanted into the dorsal stubcutaneous tissues of 3 week old Wistar strain rats. The specimens were removed at variotus periods and embedded in Epon812. The none-decalcified specimens were observed by electron microscopy for differentiation of mesenchymal cells into osteogenic cells in the tissues surrounding the implanted pellets.

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First lower molar dental papillae, isolated from day-17 mouse embryos, were embedded in 10 E l of BGJ-b medium including fetal calf serum and 0.5% agar. Two E1 of BMP solution at the different dose of BMP were added to the medium before getting. The papillae embedded in the semi-solidmedium were deposited on Millipore filters, and cultured for 6 days. This BMP fraction promoted functional differentiatioun of odontoblast-like cells and stimtulated the matrix secretion, dependent on the concentration of BMP.

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Language：English   Publisher：Japan Society of Histochemistry and Cytochemistry  

We have seen reporting on the calcium ion distribution in cells and tissues with X-ray microanalysis (EDX) since 1978 [9] and with electron energy-loss spectroscopy (ESS) imaging since 1983 [10, 17], under various fixing conditions [12-16, 18-24]. Almost ten years ago, we tried a fixative containing NHA (N,N-naphthaloylhydroxamine) as a chemical precipitant for Ca<2+> ions under microwaved conditions [12, 19]. However, the data did not seem to be as sensitive or useful, under conventional electron microscopy and computerized X-ray microanalysis, compared to the two-step replacement method combined with microwave fixation using potassium (K) oxalate followed by potassium (K) antimonate, for the detection of Ca<2+> ions [12, 19-21]. Recently, however, we used ESS to analyze same tissue sections which were fixed with NHAcontaining fixative with microwave fixation ten years previously. We discovered with ESS analysis that NHA is a sensitive and ideal chemical reagent for calcium ion deteclion in a cell giving the same view as that of frozen dried or freeze-substituted tissue sections. Calcium was clearly seen and widely distributed in developing chondroblast endoplasmic ultrastructures: ribosomes, cell and endoplasmic membranes, mitochondria, Golgi, and nucleus. However, free mesen-chymal cells which were not involved in calcifying functions had only a very small amount of calcium. Calcium was widely distributed in the lacunae of the surrounding space of the chondroblasts and inmature matrix, but the exoplasm of the chondroblasts was almost calcium-free. Other elemental images in the young chondrocytes or osteoblasts, i.e.. P・L, S・L, O・K, and N・K, were seen clearly superimposed on the cell ultrastructures, i.e.. the cell membranes, ribosomes, endoplasmic reticula, mitochondria, Golgi, and nucleus [24].

DOI： 10.1267/ahc.31.217

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Collagen was examined by electron microscopy. The BMP-Colagen induced new bone formation by intramembranous ossification without cartilage. The initial calcification was observed not only on matrix vesicle derived from osteoblasts induced by the BMP but also on the BMP-collagen complex. Thus the BMP-collagen complex may play a role in a storagesite for minerals of initial calcification.

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Other Link： http://search.jamas.or.jp/link/ui/1998047081

Language：Japanese   Publisher：日本硬組織研究技術学会  

A modofied method was established to extracted BMP from bovine bone. SDS-PAGE and Westen blotting were apllied to analize the components of this partially purified BMP. In addition the amino acid sequence was studied using protein sequencer to analyze the unknown bands. Type I collagen derived from bone was used as a carrier whose properties and advantages were studied. The following conclusions are made from this study. 1. The limitation of molecluar weight in tbe early step of BMP purification procedure improves purification efficiency and stability, which makes the applicadon of BMP in big animalos possible. 2. In this method the purified BMP product also contains histone H3, histone 2B and other unknown proteins as well as BMP-2. 3. The atelocollagen derived from bone has proved to be an useful carrier for BMP. This carrier shows characteristics of self absorption and recalcification when embedded in tissues. 4. The minimun bone induction dose of BMP at each step (G-Ext:500μg, Hep:30μg, S200:50μg) and the optimal bone induction dose(G-Ext:5.0mg, Hep:2.0mg, S200:0.3mg) are determined, which makes the applicalion of BMP in cellular biology possible.

CiNii Books

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CiNii Books

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Language：Japanese   Publisher：日本硬組織研究技術学会  

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Other Link： http://search.jamas.or.jp/link/ui/1997001332

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CiNii Books

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=306524

Language：Japanese   Publisher：日本硬組織研究技術学会  

Osteopontin(Osp), osteonectin(Osn),osteocalcin(Osc) gene in new formational bone induced by bone morphogenetic protein(BMP) and tooth germ were examined in this study. BMP-carrier(collagen I) composites were implanted in subcutaneous tissue of the back of rats. At 1,2,3 weeks after implanted, all specimens were immersed in 4% paraformaldehyde and processed for routine light microscopy with or without decalcification. For in situ hybridization, we use single strand RNA probes by DIG RNA Labeling Kit, and stained with Nucleic Acid Detection Kit(Boehringer Mannheim). Osp and Osn signals both appears on the osteoblastic or fibroblastic cells at 1 week after implantation. Osc signal appears on the osteoblasts at 2 weeks after implantation. Osc signal increase on the osteoblasts around mineralized trabecular bone at 3 weeks after implantation. These results suggest that distribution of these bone matrix protein gene was associated with growth and mineralization of bone formation.

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=306533

Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

Multinucleated giant cells (MGCs) that responded to synthetic hydroxyapatite (HAP) implanted in rat mandibles were studied with electron microscopy. HAP used in this study sintered at 200°C (HAP200) and at 125°C (HAP1250) after the synthesis by a wet method. One to three weeks after the intraosseous implantation of HAP, MGCs responding to HAP200 had not only well-developed ruffled border and the clear zone but well-developed perinuclear Golgi complex, many mitochondria and vesicles in their cytoplasms. MGCs responding to HAP1250 had the clear zone, but not the ruffled border although they showed similar cytoplasmic features to those of MGCs responding to HAP200. They merely extended short slender cytoplasmic processes to HAP1250. These results suggest that although osteoclast-like MGCs respond to HAP implanted in the bone, the development of the ruffled border-clear zone system depends on physicochemical properties of HAP. © 1992 Oxford University Press.

Scopus

PubMed

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Language：Japanese   Publisher：特定非営利活動法人 日本口腔科学会  

Immunohistochemical expressions of cytokeratin, amelogenin, and enamelin were studied in odontogenic tumors: ameloblastoma, adenomatoid odontogenic tumor and ameloblastic carcinoma. Four anti-cytokeratin monoclonal antibodies were used in this study: CK-1 to total keratin, SE-K to 56, 56.5, 58 and 68 Kd, NSE-K to 52.5 Kd, and 19 K to 40 Kd. Cytokeratin expression in follicular ameloblastoma was similar to that in tooth germ epithelia, and in plexiform ameloblastoma similar to that in fetal oral mucosa. Both types of ameloblastoma also showed slight immunoreactivity for amelogenin. In adenomatoid odontogenic tumor there was a difference between cytokeratin expression in central cells and in peripheral cells in the tumor nests. Moreover, cytokeratin expression in adenomatoid odontogenic tumor was completely different from those in ameloblastoma and tooth germ. Immunoreactivity for amelogenin and enamelin in adenomatoid odontogenic tumor was found in colloidal drops, calcified masses and lining cell membrane of ductlike structure. Therefore, it is possible that these tumor cells differentiate into enamel protein-producing cells, more than those of ameloblastoma do. Immunohistochemical results in ameloblastic carcinoma showed that this carcinoma is composed of undifferentiated odontogenic tumor cells.

DOI： 10.11277/stomatology1952.40.746

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Language：Japanese   Publisher：歯科基礎医学会  

Activities of acid phosphatase (ACPase) and tartrate-reristant acid phosphatase (TRAP) of multinucleated giant cells (MGCs) responding to hydroxyapatite (HA) implanted in rat jaw bone and subcutaneous tissue were studied by enzyme histochemistry.In the jaw bone, MGCs had ruffled border-like structures on the surface of HA.Both ACPase and TRAP activity were also detected in the cytoplasm of MGCs.Morphological and histochemical features of these MGCs closely resembled those of osteoclasts in the jaw bone.Moreover, both enzyme activities were observed in the mononuclear cells around the MGCs.On the other hand, MGCs in subcutaneous tissue had elongated the flattened cytoplasm along HA surface, and both enzyme activities were basely detected.Also many mononuclear cells that did not show TRAP activity were observed around these MGCs.The present results raise the possibility that the histological environment of the site for HA implantation may play a role in the regulation of the biological characteristics of MGCs responding to HA.

DOI： 10.2330/joralbiosci1965.33.275

J-GLOBAL

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J-GLOBAL

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-LISS  

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Language：Japanese   Publisher：(一社)日本神経学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE INC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE INC  

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Language：Japanese   Publisher：日本基礎老化学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：(公社)日本獣医学会  

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Language：Japanese   Publisher：(一社)日本認知症学会  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：BLACKWELL SCIENCE LTD  

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Event date： 2020.7

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Event date： 2020.3

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Event date： 2019.7

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灌流固定したラットとマウスの脳組織を用いて、Tokuyasu法に準じて凍結超薄切片を作製しレクチン染色による組織化学的検討を行った。大脳皮質内にWGA、ConAの特異的な結合部位の局在を認めた。WGAは一部のシナプス周囲の領域、特にシナプスを形成する神経突起の膜上に局在しており、錐体細胞内の模様構造と毛細血管内皮細胞の内腔側に見られた。ConAは一部の神経突起内に局在した。レクチンはそれぞれいくつかの糖残基と結合することから、レクチン局在部位にはこうした糖残基を含む糖鎖が存在する可能性が高いと考えられる。レクチン組織化学は糖、或いは糖鎖の超微細構造における検索には、有望な手法と思われた。

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In the retina, while it is known that extracellular ATP inhibits ACh release from cholinergic neurons, the types of purinoceptors on the cholinergic neurons have not been examined. We reported that immunoreactivity for P2X2 localized asymmetrically between the ON and OFF pathways in the cholinergic amacrine cells in the mouse retina. In the present study, we recorded the responses to ATP in the cholinergic amacrine cells of the transgenic mouse retina. Both straburst amacrine cells and displaced amacrine cells responded to 100 &mu;M ATP. ATP induced an inward current. However, part of displaced amacrine cells did not respond to 100 &mu;M ATP. Both types of cells did not respond to 100 &mu;M &alpha;,&beta;-methylene ATP. Responses to ATP were inhibited by 100 &mu;M PPADS. When cells received GABAergic-IPSP, 100 &mu;M ATP augmented GABAergic IPSP. These results indicate that inhibitory action of ACh released by ATP is mediated by the presynaptic mechanisms. [Jpn J Physiol 54 Suppl:S162 (2004)]

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