Biomedical subjects

Nobuyuki Takei

Publications and source records attributed to Nobuyuki Takei.

29 records · Page 2Linked to original sources

PubMed2003

Developmental changes of eukaryotic initiation factor 2B subunits in rat hippocampus.

Regulated protein synthesis is critical for neural development, such as the formation of synapses and neural circuits and the modulation of synaptic plasticity. Protein synthesis is controlled by translation factors, including initiation, elongation and release factors. Here we investigated the developmental changes of eukaryotic initiation factor 2B (eIF2B) subunits in rat hippocampus. The eIF2B beta, gamma, delta and epsilon subunit protein levels were maximal at embryonic day 18 and then decreased during development. Aged hippocampus contained only trace amounts of these subunits. The finding that eIF2B subunit levels are high in developing hippocampus suggests that regulated protein synthesis is active in young, highly plastic brain.

Age Factors↗

PubMed2003

Cellular and subcellular distributions of translation initiation, elongation and release factors in rat hippocampus.

Novel protein synthesis in the brain has been suggested to contribute to the formation of synapses and neural circuits during development and the modulation of long-term synaptic plasticity through life. However, cellular and subcellular distribution of neuronal translation machinery and regulator molecules has not yet been extensively characterized in rat brain. In this report, the distribution of translation factors in the developing hippocampus, a region which is highly plastic, was analyzed by immunohistochemistry and Western blotting. Western blot analysis revealed that the hippocampus expresses the factors in all three steps of translation, initiation factors, elongation factors and a release factor. Immunochemical studies of hippocampal slices and culture showed that all translation factors were observed not only in cell bodies but also in dendrites of hippocampal neurons. In addition, the levels of the individual translation factors differed between hippocampal subregions. The differential distribution of translation factors was also confirmed by Western blotting. These results suggest that regulated protein synthesis occurs in the hippocampus, with differences existing between different subregions such as CA1, CA3 and dentate gyrus.

Animals↗

PubMed2003

PACAP and NGF cooperatively enhance choline acetyltransferase activity in postnatal basal forebrain neurons by complementary induction of its different mRNA species.

Both nerve growth factor (NGF) and pituitary adenylate cyclase activating polypeptide (PACAP) have neurotrophic effects on basal forebrain cholinergic neurons. They promote differentiation, maturation, and survival of these cholinergic neurons in vivo and in vitro. Here we report on the cooperative effects of NGF and PACAP on postnatal, but not embryonic, cholinergic neurons cultured from rat basal forebrain. Combined treatment with NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and PACAP induced an additive increase in choline acetyltransferase (ChAT) activity. There were no cooperative effects on the number of cholinergic neurons, suggesting that ChAT mRNA expression had been induced in each cholinergic neuron. Further analysis revealed that NGF and PACAP led to complementary induction of different ChAT mRNA species, thus enhancing total ChAT mRNA expression. These results explain the cooperative neurotrophic action of NGF and PACAP on postnatal cholinergic neurons.

Animals↗

PubMed2003

A novel rat orthologue and homologue for the Drosophila crooked neck gene in neural stem cells and their immediate descendants.

The crooked neck (crn) gene of Drosophila melanogaster encodes a scaffold protein carrying multiple tetratricopeptide repeat (TPR) motifs, and its mutation results in a reduction in the number of neuroblasts and lethality during larval stages. Here, we isolated two structurally related genes from a rat embryonic brain cDNA library. One gene is the rat orthologue of crn, which encodes 690 amino acids including 16 copies of TPR. The other gene, ATH55, encodes an 855 amino acid protein including 21 TPR motifs, which presumably represents a rat crn homologue and an orthologue of human XAB2. Both genes are highly expressed in embryonic brain but their expressions decrease during development. ATH55-like immunoreactivity is present in the ventricular zone and newly formed cortical plate, while CRN-like immunoreactivity is more abundant in a younger ventricular zone. In agreement, both proteins were found to be enriched in cultured neural stem cells and to decrease in response to cell differentiation signals. As indicated for the yeast CRN-like protein, ATH55 and CRN immunoreactivities were both recovered in the nuclear fraction and detected in the splicing complex carrying pre-mRNA. These findings suggest that both TPR-motif-containing proteins are involved in RNA processing of mammalian neural stem cells and their immediate descendants.

Alternative Splicing↗

PubMed2003

BDNF is upregulated by postnatal development and visual experience: quantitative and immunohistochemical analyses of BDNF in the rat retina.

PURPOSE: This study sought to elucidate changes in the levels and distribution of brain-derived neurotrophic factor (BDNF) in the retina throughout aging and depending on visual experience. METHODS: Protein and mRNA levels of BDNF were quantified by enzyme-linked immunosorbent assay (ELISA) and semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Levels were assayed in the retinas of rats on postnatal day (P)2, P7, and P14 (approximate time of eye opening) and at 1 month (M), 3M, 8M, and 18M of age. Changes in BDNF expression and localization in the retina were assessed by immunohistochemistry. The effect of monocular deprivation during infancy on retinal BDNF expression was also examined, by ELISA and immunohistochemistry. RESULTS: Both protein and mRNA levels of BDNF in the rat retina increased after P14. Immunohistochemical analyses revealed that the increase in BDNF protein levels occurred in retinal ganglion cells (RGCs) between P14 and 1M. BDNF immunoreactivity in Müller cell processes was observed in the inner nuclear layer at 1M, but not at P14. The levels of BDNF protein in the retinas of visually deprived eyes were lower than those of control eyes, as quantified by ELISA. Immunohistochemistry showed that BDNF immunoreactivity in RGCs was diminished by visual deprivation, whereas Müller cells were unaffected. CONCLUSIONS: These observations indicate that BDNF expression in RGCs is upregulated in an activity-dependent manner, whereas that in Müller cells is regulated only by development.

Aging↗

PubMed2002

Establishment of a novel enzyme-linked immunosorbent assay for Thy-1; quantitative assessment of neuronal degeneration.

In the central nervous system (CNS), Thy-1 is expressed predominantly on neurons and serves as a specific marker for neurons. In the present study, we established a two-site enzyme-linked immunosorbent assay (ELISA) that detects trace amounts of Thy-1 protein. Recombinant Thy-1 protein expressed in Escherichia coli was purified and used as a standard. Of the regions of the nervous system examined, the highest Thy-1 concentration was found in the striatum followed by the hippocampus, neocortex, cerebellum, spinal cord, retina and optic nerve. We found that injection of a neurotoxin, N-methyl-D-aspartate, into the vitreous cavity reduced the Thy-1 level in the retina. Thy-1 ELISA will be useful for quantitative assessment of neurodegeneration in the CNS.

Animals↗

PubMed2002

Nerve growth factor induces systemic hyperalgesia after thoracic burn injury in the rat.

Acute burn injury is usually associated with pain in the injured and nearby areas. However, we have recently reported that a thoracic scald induces hindpaw hyperalgesia during the healing stage in rats. The present study investigated the cause of the remotely occurring hyperalgesia. Behavioral testing using the von Frey test revealed that rats developed hyperalgesia in the neck and flank as well as the hindpaw 2-3 weeks after injury. The concentration of nerve growth factor (NGF) in the skin of the chest increased markedly during the healing stage. Moreover, rats injected daily with anti-NGF serum after burn injury did not develop hyperalgesia, suggesting that increased NGF in the tissue of the healing skin is a key factor causing systemic hyperalgesia during the recovery stage.

Acute Disease↗

PubMed2002

Brain-derived neurotrophic factor regulates surface expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors by enhancing the N-ethylmaleimide-sensitive factor/GluR2 interaction in developing neocortical neurons.

In hippocampal neurons, the exocytotic process of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors is known to depend on activation of N-methyl-d-aspartate channels and its resultant Ca(2+) influx from extracellular spaces. Here we found that brain-derived neurotrophic factor (BDNF) induced a rapid surface translocation of AMPA receptors in an activity-independent manner in developing neocortical neurons. The receptor translocation became evident within hours as monitored by [(3)H]AMPA binding and was resistant against ionotropic glutamate receptor antagonists as evidenced with surface biotinylation assay. This process required intracellular Ca(2+) and was inhibited by the blockers of conventional exocytosis, brefeldin A, botulinum toxin B, and N-ethylmaleimide. To explore the translocation mechanism of individual AMPA receptor subunits, we utilized the human embryonic kidney (HEK) 293 cells carrying the BDNF receptor TrkB. After the single transfection of GluR2 cDNA or GluR1 cDNA into HEK/TrkB cells, BDNF triggered the translocation of GluR2 but not that of GluR1. Subsequent mutation analysis of GluR2 carboxyl-terminal region indicated that the translocation of GluR2 subunit in HEK293 cells involved its N-ethylmaleimide-sensitive factor-binding domain but not its PDZ-interacting site. Following co-transfection of GluR1 and GluR2 cDNAs, solid phase cell sorting revealed that GluR1 subunits were also able to translocate to the cell surface in response to BDNF. An immunoprecipitation assay confirmed that BDNF stimulation can enhance the interaction of GluR2 with N-ethylmaleimide-sensitive factor. These results reveal a novel role of BDNF in regulating the surface expression of AMPA receptors through a GluR2-NSF interaction.

Animals↗

PubMed2002

Lithium induces brain-derived neurotrophic factor and activates TrkB in rodent cortical neurons: an essential step for neuroprotection against glutamate excitotoxicity.

Mechanisms underlying the therapeutic effects of lithium for bipolar mood disorder remain poorly understood. Recent studies demonstrate that lithium has neuroprotective actions against a variety of insults in vitro and in vivo. This study was undertaken to investigate the role of the brain-derived neurotrophic factor (BDNF)/TrkB signaling pathway in mediating neuroprotection of lithium against glutamate excitotoxicity in cortical neurons. Pretreatment with either lithium or BDNF protected rat cerebral cortical neurons from glutamate excitotoxicity. The duration of treatment required to elicit maximal neuroprotection by BDNF (1 day) was much shorter than that by lithium (6 days). K252a, an inhibitor of Trk tyrosine kinases, and a BDNF neutralizing antibody suppressed the neuroprotective effect of lithium. Treatment of cortical neurons with lithium increased the cellular BDNF content in 3 days and the phosphorylation of TrkB at Tyr490 in 5 days, suggesting that long-term lithium administration enhances BDNF expression/secretion, leading to the activation of TrkB receptor. Lithium failed to protect against glutamate excitotoxicity in cortical neurons derived from homozygous and heterozygous BDNF knockout mice, although lithium fully protected cortical neurons prepared from wild type mice littermates. Taken together, these data suggest that the BDNF/TrkB pathway plays an essential role in mediating the neuroprotective effect of lithium.

Animals↗

PubMed2002

Nerve growth factor enhances neurotransmitter release from PC12 cells by increasing Ca(2+)-responsible secretory vesicles through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase.

Neurotrophins play important roles in the differentiation and survival of neurons during development, and in the regulation of synaptic transmission in adult brain. Brief treatment with nerve growth factor (NGF) enhances depolarization and ionomycin-induced dopamine and acetylcholine release from PC12 cells. The enhancing effect appears very quickly and reaches a plateau 10-15 min after application. NGF also enhances hypertonic solution-induced dopamine release, and increases the amount of dopamine released from membrane-permeabilized PC12 cells in the absence of MgATP, suggesting that NGF enhances neurotransmitter release by increasing the number of Ca(2+)-responsive secretory vesicles. The activation of Trk receptors is essential for NGF action, since K252a abolishes the NGF-induced potentiation of dopamine release and brain-derived neurotrophic factor enhanced ionomycin-induced release only in TrkB-expressing cells. NGF-mediated potentiation of dopamine release is completely abolished by wortmannin, a PI 3-kinase inhibitor, and by U0126 and PD98059, MAP kinase kinase inhibitors, indicating that the activation of PI 3-kinase and MAP kinase pathways is essential for NGF action. These findings suggest that NGF regulates neurotransmitter release through the activation of TrkA receptors, possibly by increasing the number of secretory vesicles in a readily releasable pool.

Animals↗

PubMedDate not supplied

Quantitative analyses of mRNA and protein levels of neurotrophin-3 in the rat retina during postnatal development and aging.

PURPOSE: In this study, we sought to elucidate changes in the levels of neurotrophin-3 (NT-3) in the rat retina throughout postnatal development and aging. METHODS: We demonstrated NT-3 localization in the retina by immunohistochemistry. Protein and mRNA levels of NT-3 were quantified by enzyme-linked immunosorbant assay and semiquantitative reverse transcriptase-polymerase chain reaction, respectively. NT-3 protein levels were assayed in the various regions of the central nervous system. Age-associated changes in protein and mRNA levels of NT-3 in the retinas were assessed. RESULTS: NT-3-immunoreactivity localized in the ganglion cell layer, inner nuclear layer, and outer nuclear layer. NT-3 content in the retina was relatively high in the examined regions of the central nervous system. Retinal NT-3 protein levels decreased after eye opening, whereas mRNA levels were constant. Both mRNA and protein levels of NT-3 in the retinas of aged animals remained constant. CONCLUSIONS: Our observations suggest that NT-3 regulation in the retina is independent of increasing visual stimuli after eye opening. Stable expression of NT-3 in the adult retina suggests a possible role in the maintenance of the retinal environment throughout later life.

Aging↗