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Takeshi Yagi

Publications and source records attributed to Takeshi Yagi.

At least 19 recordsLinked to original sources
PubMed2006

Structure of the cadherin-related neuronal receptor/protocadherin-alpha first extracellular cadherin domain reveals diversity across cadherin families.

The recent explosion in genome sequencing has revealed the great diversity of the cadherin superfamily. Within the superfamily, protocadherins, which are expressed mainly in the nervous system, constitute the largest subgroup. Nevertheless, the structures of only the classical cadherins are known. Thus, to broaden our understanding of the adhesion repertoire of the cadherin superfamily, we determined the structure of the N-terminal first extracellular cadherin domain of the cadherin-related neuronal receptor/protocadherin-alpha4. The hydrophobic pocket essential for homophilic adhesiveness in the classical cadherins was not found, and the functional significance of this structural domain was supported by exchanging the first extracellular cadherin domains of protocadherin and classical cadherin. Moreover, potentially crucial variations were observed mainly in the loop regions. These included the protocadherin-specific disulfide-bonded Cys-X(5)-Cys motif, which showed Ca(2+)-induced chemical shifts, and the RGD motif, which has been suggested to be involved in heterophilic cell adhesion via the active form of beta1 integrin. Our findings reveal that the adhesion repertoire of the cadherin superfamily is far more divergent than would be predicted by studying the classical cadherins alone.

Amino Acid Sequence↗
PubMed2006

Allelic gene regulation of Pcdh-alpha and Pcdh-gamma clusters involving both monoallelic and biallelic expression in single Purkinje cells.

The molecular basis for providing the identity and diversity of single neurons is a key for realizing the brain system. Diverse protocadherin isoforms encoded by the Pcdh-alpha and Pcdh-gamma gene clusters are expressed in all of the vertebrates studied. For the Pcdh-alpha isoforms, differential expression patterns have been found in single Purkinje cells by unusual monoallelic and combinatorial types of gene regulation. Here we investigated total allelic gene regulation in the Pcdh-alpha and -gamma clusters, including the C-type variable exons (C1 to C5) and the Pcdh-gammaA and -gammaB variable exons in single Purkinje cells. Using split single-cell reverse transcription-PCR analysis, almost all of the Purkinje cells at postnatal day 21 biallelically expressed all the C-type isoforms, whereas the Pcdh-alpha isoforms showed both monoallelic and combinatorial expression. The Pcdh-gammaA and -gammaB isoforms also showed differential regulation in each cell with both monoallelic and combinatorial gene regulation. These data indicated that different types of allelic gene regulation (monoallelic versus biallelic) occurred in the Pcdh-alpha and -gamma clusters, although they were spliced into the same constant exons. It has been reported that each C-type Pcdh-alpha or -gamma transcript has a different expression pattern during brain development, suggesting that the different C-type variable exons may code temporal diversity, although the Pcdh-alpha, -gammaA, and -gammaB isoforms were differential and combinatorial gene regulation within a single cell. Thus, the multiple gene regulations in the Pcdh-alpha and -gamma clusters had a potential mechanism for increasing the diversity of individual neurons in the brain.

Alleles↗
PubMed2006

Fyn-induced phosphorylation of beta-adducin at tyrosine 489 and its role in their subcellular localization.

Fyn is a Src-family tyrosine kinase involved in neuronal development, transmission, and plasticity in mammalian central nervous system. We have previously reported that Fyn binds to a cytoskeletal protein, beta-adducin, in a phosphorylation-dependent manner. In the present report, we show that Fyn phosphorylates beta-adducin at tyrosine 489 located in its C-terminal tail domain. Phosphorylation of beta-adducin at Y489 was required for its association with the Fyn-SH2 domain. An antibody specific to the phosphorylated form of beta-adducin was raised in rabbits and showed that Y489 of beta-adducin was phosphorylated in wild type, but not in Fyn(-/-) mice, suggesting that Y489 of beta-adducin is phosphorylated downstream of Fyn in vivo. After phosphorylation at Y489, beta-adducin was translocated to the cell periphery, and colocalized with Fyn. These results suggest that Fyn phosphorylates and binds to beta-adducin at Y489, resulting in translocation of beta-adducin to the Fyn-enriched regions in the plasma membrane.

Animals↗
PubMed2006

The role and expression of the protocadherin-alpha clusters in the CNS.

The clustered protocadherins comprise the largest subfamily of the cadherin superfamily and are predominantly expressed in the nervous system. The family of clustered protocadherins (clustered Pcdh family) is substructured into three distinct gene arrays in mammals: Pcdh-alpha, Pcdh-beta, and Pcdh-gamma. These are regulated by multiple promoters and cis-alternative splicing without DNA recombination. Pcdh-alpha proteins interact with beta1-integrin to promote cell adhesion. They also form oligomers with Pcdh-gamma proteins at the same membrane sites. During neuronal maturation, Pcdh-alpha expression is dramatically downregulated by myelination. The clustered Pcdh family has multiple variable exons that differ somewhat in number and sequence across vertebrate species. At the single-cell level, Pcdh-alpha mRNAs are regulated monoallelically, resulting in the combinatorial expression of distinct variable exons from each allele. These findings support the idea that diversified Pcdh molecules contribute to neural circuit development and provide individual cells with their specific identity.

Alternative Splicing↗
PubMed2006

Regulation of dendritic branching and spine maturation by semaphorin3A-Fyn signaling.

A member of semaphorin family, semaphorin3A (Sema3A), acts as a chemorepellent or chemoattractant on a wide variety of axons and dendrites in the development of the nervous systems. We here show that Sema3A induces clustering of both postsynaptic density-95 (PSD-95) and presynaptic synapsin I in cultured cortical neurons without changing the density of spines or filopodia. Neuropilin-1 (NRP-1), a receptor for Sema3A, is present on both axons and dendrites. When the cultured neurons are exposed to Sema3A, the cluster size of PSD-95 is markedly enhanced, and an extensive colocalization of PSD-95 and NRP-1 or actin-rich protrusion is seen. The effects of Sema3A on spine morphology are blocked by PP2, an Src type tyrosine kinase inhibitor, but not by the PP3, the inactive-related compound. In the cultured cortical neurons from fyn(-/-) mice, dendrites bear few spines, and Sema3A does not induce PSD-95 cluster formation on the dendrites. Sema3A and its receptor genes are highly expressed during the synaptogenic period of postnatal days 10 and 15. The cortical neurons in layer V, but not layer III, show a lowered density of synaptic bouton-like structure on dendrites in sema3A- and fyn-deficient mice. The neurons of the double-heterozygous mice show the lowered spine density, whereas those of single heterozygous mice show similar levels of the spine density as the wild type. These findings suggest that the Sema3A signaling pathway plays an important role in the regulation of dendritic spine maturation in the cerebral cortex neurons.

Actins↗
PubMed2006

NMDA-receptor proteins are upregulated in the hippocampus of postnatal heterozygous reeler mice.

Reelin is a large glycoprotein that is secreted into the extracellular matrix. In the embryonic brain, the binding of Reelin to its receptors ApoER2 and VLDLR induces subcellular events that include the activation Fyn tyrosine kinase, and plays a crucial role in cortical formation. Reelin signaling is also involved in postnatal brain functions such as dendrite development and synaptic plasticity. However, the molecular events involved in Reelin signaling in the postnatal brain remain to be elucidated. Here, we evaluated the proteins downstream of Reelin signaling by comparing the tyrosine-phosphorylated proteins in the postnatal hippocampus of heterozygous and homozygous reeler and wild-type mice, by Western blot analyses. We found that the levels of several phosphoproteins were highest in the hippocampus of the heterozygous reeler mice. The most prominent increase was of two 180-kDa phosphoproteins, which were identified as the NR2A and NR2B subunits of NMDA-R. The amounts of these proteins also increased in the hippocampus of heterozygous reeler mice. However, the mRNA levels of the NMDA-R subunits, determined by quantitative RT-PCR, were the same as in wild-type mice. We also found that the increase in NR2A and NR2B proteins in heterozygous reeler was dependent on Fyn, because this change was absent in heterozygous reeler/homozygous Fyn-deficient double-mutant mice. Thus, the NMDA-R protein level is regulated by the Reelin protein level in a Fyn-dependent manner in the mouse brain.

Analysis of Variance↗
PubMed2006

Altered gene expression in the subdivisions of the amygdala of Fyn-deficient mice as revealed by laser capture microdissection and mKIAA cDNA array analysis.

Fyn-tyrosine-kinase-deficient mice exhibit increased fearfulness and display enhanced excitability in the amygdala. To gain insight into the molecular changes associated with the increased excitability of the amygdala, we used a newly developed cDNA array system comprising mouse KIAA cDNA clones to identify novel genes differentially expressed in the amygdala of fyn(-/-) and fyn(+/-) mice following administration of N-methyl-D-aspartate (NMDA). Laser capture microdissection in combination with PCR-based cDNA amplification allowed us to analyze gene expression in each amygdalar subdivision. The statistical significance of the differential expressions was tested by one-way analysis of variance (ANOVA) by the false discovery rate controlling approach. Among the 805 mKIAA cDNA clones tested, only the expression level of mKIAA1577 (Zinc finger SWIM domain containing protein 6; gene name, Zswim6) showed statistically significant change in regard to the genotype and amygdalar subdivision. Namely, only the lowered expression of mKIAA1577 in the central nucleus of fyn(-/-) mice 1 h after NMDA administration (2.1-fold lower relative to fyn(+/-) mice) was statistically significant. In situ hybridization analysis confirmed the downregulation of the mRNA in the central nucleus of the fyn(-/-) mice 1 h after NMDA administration (3.2-fold lower relative to fyn(+/-) mice). The NMDA-induced change in gene expression was partially blocked by the NMDA antagonist D-AP-5. These results suggest that Fyn deficiency was responsible for the NMDA-induced downregulation of a specific gene in the amygdalar central nucleus.

2-Amino-5-phosphonovalerate↗
PubMed2006

Fyn is required for haloperidol-induced catalepsy in mice.

Fyn-mediated tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits has been implicated in various brain functions, including ethanol tolerance, learning, and seizure susceptibility. In this study, we explored the role of Fyn in haloperidol-induced catalepsy, an animal model of the extrapyramidal side effects of antipsychotics. Haloperidol induced catalepsy and muscle rigidity in the control mice, but these responses were significantly reduced in Fyn-deficient mice. Expression of the striatal dopamine D(2) receptor, the main site of haloperidol action, did not differ between the two genotypes. Fyn activation and enhanced tyrosine phosphorylation of the NMDA receptor NR2B subunit, as measured by Western blotting, were induced after haloperidol injection of the control mice, but both responses were significantly reduced in Fyn-deficient mice. Dopamine D(2) receptor blockade was shown to increase both NR2B phosphorylation and the NMDA-induced calcium responses in control cultured striatal neurons but not in Fyn-deficient neurons. Based on these findings, we proposed a new molecular mechanism underlying haloperidol-induced catalepsy, in which the dopamine D(2) receptor antagonist induces striatal Fyn activation and the subsequent tyrosine phosphorylation of NR2B alters striatal neuronal activity, thereby inducing the behavioral changes that are manifested as a cataleptic response.

Animals↗
PubMed2006

Embryonic stem cell-derived neuron models of Parkinson's disease exhibit delayed neuronal death.

Establishment of a Parkinson's disease (PD) neuron model was attempted with mouse embryonic stem (ES) cells. ES cell lines over-expressing mouse nuclear receptor-related 1 (Nurr1), together with human wild-type and alanine 30 --> proline (A30P) and alanine 53 --> threonine (A53T) mutant alpha-synuclein were established and subjected to differentiation into dopaminergic neurons. The ES cell-derived dopaminergic neurons expressing wild-type or mutant alpha-synuclein exhibited the fundamental characteristics consistent with dopaminergic neurons in the substantia nigra. The ES cell-derived PD model neurons exhibited increased susceptibility to oxidative stress, proteasome inhibition, and mitochondrial inhibition. Cell viability of PD model neurons and the control neurons was similar until 28 days after differentiation. Nonetheless, after that time, PD model neurons gradually began to undergo neuronal death over the course of 1 month, showing cytoplasmic aggregate formation and an increase of insoluble alpha-synuclein protein. Such delayed neuronal death was observed in a mutant alpha-synuclein protein level-dependent manner, which was slightly inhibited by a c-jun N-terminal kinase inhibitor and a caspase inhibitor. Such cell death was not observed when the same ES cell lines were differentiated into oligodendrocytes. The ES cell-derived PD model neurons are considered as prospective candidates for a new prototype modelling PD that would allow better investigation of the underlying neurodegenerative pathophysiology.

Analysis of Variance↗
PubMed2006

Predominant expression of rTERTb, an inactive TERT variant, in the adult rat brain.

The full-length and four alternatively spliced variant mRNA transcripts of rat TERT were identified. One of the variant, rTERTb, was predominantly expressed during brain development in accordance with the decrease in telomerase activity. The rTERTb mRNA encoded an inactive truncated protein. Alternative splicing of TERT may be important during brain development.

Alternative Splicing↗
PubMed2006

[Effect of volume reduction surgery by radiofrequency for enlarged adenoid causing recurrent otitis media with effusion].

PURPOSE: We studied the effect of volume reduction surgery by a radiofrequency (ENTec coblator) for enlarged adenoid causing recurrent otitis media with effusion. MATERIALS AND METHODS: The effect of volume reduction surgery by radiofrequency for enlarged adenoid was studied in 50 children with enlarged adenoid causing recurrent otitis media with effusion from June 2002 to September 2004, while a group of 50 children with tympanostomy-tube placement alone from April 2001 to September 2004, was used as the control group. Volume reduction surgery by radiofrequency system for enlarged adenoid was done with tympanostomy-tube placement under general anesthesia by laryngeal mask. We compared two groups in following 5 different aspects: (1) tympanostomy-tube replacement, (2) remyringotomy, (3) total visits to our clinic after surgery, (4) total days with antibiotics, (5) absence of effusion and normal middle ear function as seen on the tympanogram after tympanostomy-tubes loss. We evaluated the reduction of enlarged adenoid by using the pre-and postoperative obstructive rate of the choana through the nasopharyngoscope. RESULTS: The volume of enlarged adenoid was reduced an average of 52.2% by radiofrequency. The pharyngeal opening of the eustachian tube and the choana could be opened widely. No severe intra or postoperative complications occurred. Compared to the control group treated with tympanostomy tubes alone, postoperative tympanostomy-tube replacement, postoperative remyringotomy, total postoperative visits to our clinic, total postoperative days with antibiotics, and tympanogram types C2 and B after tympanostomy-tubes loss decreased notably in cases with volume reduction surgery by radiofrequency for enlarged adenoid and tympanostomy-tube placement. CONCLUSION: Volume reduction surgery by radiofrequency for enlarged adenoid is considered very safe, effective one-day surgery technique for recurrent otitis media with effusion.

Adenoidectomy↗
PubMed2005

Absence of leukotriene B4 receptor 1 confers resistance to airway hyperresponsiveness and Th2-type immune responses.

Bronchial asthma is an increasingly common disorder that remains poorly understood and difficult to manage. The disease is characterized by airway hyperresponsiveness, chronic inflammation, and mucus overproduction. Based on the finding that leukotriene B4 receptor 1 (BLT1) is expressed highly in Th2 lymphocytes, we analyzed the roles of BLT1 using an OVA-induced bronchial asthma model. BLT1-null mice did not develop airway hyperresponsiveness, eosinophilic inflammation, and hyperplasia of goblet cells. Attenuated symptoms were accompanied by reduced IgE production, and accumulation of IL-5 and IL-13 in bronchoalveolar lavage fluid, suggesting attenuated Th2-type immune response in BLT1-null mice. Peribronchial lymph node cells of sensitized BLT1-null mice showed much attenuated proliferation and production of Th2 cytokines upon re-stimulation with Ag in vitro. Thus, LTB4-BLT1 axis is required for the development of Th2-type immune response, and blockade of LTB4 functions through BLT1 would be novel and useful in the effort to ameliorate bronchial asthma and related Th2-biased immune disorders.

Animals↗
PubMed2005

Developmental pluripotency of the nuclei of neurons in the cerebral cortex of juvenile mice.

Nuclei isolated from green fluorescent protein-marked neurons in the cerebral cortex of juvenile mice (14-21 d after birth) were injected into enucleated oocytes that were allowed to develop into blastocysts. Embryonic stem (ES) cell lines were established from the inner cell mass of 76 cloned blastocysts after injecting 2026 neuronal nuclei. Some ES cells were injected individually into enucleated oocytes (nuclear transfer). Other ES cells were transferred into the blastocoeles of tetraploid blastocysts (tetraploid complementation). Two-cell embryos after nuclear transfer were transferred to the oviducts of surrogate mothers. Four (1.5%) of 272 nuclear-transferred two-cell embryos developed to term, and two (0.7%) developed into fertile adults. Nineteen (1.9%) of 992 tetraploid blastocysts receiving ES cells reached term, and 10 (1.0%) developed into adults. These findings demonstrate that some of the nuclei of differentiated neurons in the cerebral cortex of juvenile mice maintain developmental pluripotency.

Animals↗
PubMed2005

Fc{epsilon}RI-mediated mast cell degranulation requires calcium-independent microtubule-dependent translocation of granules to the plasma membrane.

The aggregation of high affinity IgE receptors (Fcepsilon receptor I [FcepsilonRI]) on mast cells is potent stimulus for the release of inflammatory and allergic mediators from cytoplasmic granules. However, the molecular mechanism of degranulation has not yet been established. It is still unclear how FcepsilonRI-mediated signal transduction ultimately regulates the reorganization of the cytoskeleton and how these events lead to degranulation. Here, we show that FcepsilonRI stimulation triggers the formation of microtubules in a manner independent of calcium. Drugs affecting microtubule dynamics effectively suppressed the FcepsilonRI-mediated translocation of granules to the plasma membrane and degranulation. Furthermore, the translocation of granules to the plasma membrane occurred in a calcium-independent manner, but the release of mediators and granule-plasma membrane fusion were completely dependent on calcium. Thus, the degranulation process can be dissected into two events: the calcium-independent microtubule-dependent translocation of granules to the plasma membrane and calcium-dependent membrane fusion and exocytosis. Finally, we show that the Fyn/Gab2/RhoA (but not Lyn/SLP-76) signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Identification and characterization of coding single-nucleotide polymorphisms within human protocadherin-alpha and -beta gene clusters.

The human protocadherin (Pcdh) gene clusters are located on chromosome 5q31. Single-nucleotide polymorphisms (SNPs) were detected in the Pcdh-alpha and -beta variable exons, and in the Pcdh-alpha constant exon, in samples from 104 individuals. Among coding SNPs (cSNPs), nonsynonymous (amino acid exchange) SNPs were 2.2 times more common than synonymous (silent) changes in the Pcdh-alpha variable exons, but only 1.2 times more common in the Pcdh-beta variable exons. The nonsynonymous SNPs were high in the ectodomain (EC) 1 encoding region of Pcdh-alpha but not of Pcdh-beta. One 48-kb region of extensive linkage disequilibrium (LD) is reported that has two haplotypes extending from the alpha1 to alpha7 genes in the Pcdh-alpha cluster. Here we identified 15 amino acid exchanges in these two major haplotypes; therefore, the two haplotypes encode different sets of Pcdh-alpha proteins in the brain. The distribution of cSNPs was different for each EC region of Pcdh-alpha or -beta. The frequency of cSNPs was negatively correlated with the paralogous sequence diversity. These results suggested that gene conversion events in homologous regions of the Pcdh-alpha and Pcdh-beta clusters generated the cSNPs. Within the cSNPs, gene conversions were found in Pcdh-alpha4 in the major haplotype, and in Pcdh-beta9. These gene conversions were caused by the unequal crossing-over of homologous sequence regions. Thus, nonsynonymous variations in the Pcdh-alpha and -beta genes are possible contributors to the variations in human brain function.

Alleles↗
PubMed2005

Plexin-a4 mediates axon-repulsive activities of both secreted and transmembrane semaphorins and plays roles in nerve fiber guidance.

It has been proposed that four members of the plexin A subfamily (plexin-As; plexin-A1, -A2, -A3, and -A4) and two neuropilins (neuropilin-1 and neuropilin-2) form complexes and serve as receptors for class 3 secreted semaphorins (Semas), potent neural chemorepellents. The roles of given plexin-As in semaphorin signaling and axon guidance, however, are mostly unknown. Here, to elucidate functions of plexin-A4 in semaphorin signaling and axon guidance events in vivo, we generated plexin-A4 null mutant mice by targeted disruption of the plexin-A4 gene. Plexin-A4 mutant mice were defective in the trajectory and projection of peripheral sensory axons and sympathetic ganglion (SG) axons and the formation of the anterior commissure and the barrels. The defects in peripheral sensory and SG axons were fundamentally related to those of neuropilin-1 or Sema3A mutant embryos reported but were more moderate than the phenotype in these mutants. The growth cone collapse assay showed that dorsal root ganglion axons and SG axons of plexin-A4 mutant embryos partially lost their responsiveness to Sema3A. These results suggest that plexin-A4 plays roles in the propagation of Sema3A activities and regulation of axon guidance and that other members of the plexin-A subfamily are also involved in the propagation of Sema3A activities. Plexin-A4-deficient SG axons did not lose their responsiveness to Sema3F, suggesting that plexin-A4 serves as a Sema3A-specific receptor, at least in SG axons. In addition, the present study showed that plexin-A4 bound class 6 transmembrane semaphorins, Sema6A and Sema6B, and mediated their axon-repulsive activities, independently of neuropilin-1. Our results imply that plexin-A4 mediates multiple semaphorin signals and regulates axon guidance in vivo.

Animals↗