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T Shirao

Publications and source records attributed to T Shirao.

At least 19 recordsLinked to original sources
PubMed2006

In vivo, competitive blockade of N-methyl-D-aspartate receptors induces rapid changes in filamentous actin and drebrin A distributions within dendritic spines of adult rat cortex.

In vitro studies have demonstrated that prolonged N-methyl-D-aspartate receptor (NMDAR) blockade triggers a homeostatic up-regulation of NMDARs at synapses. Such upregulation can also be seen within 30 min in vivo in adult rats, implicating trafficking of reserve pools of NMDARs. Here, we evaluated the involvement of filamentous actin (F-actin), the major cytoskeletal component in spines, in this rapid in vivo homeostatic response, using biotinylated phalloidin as its probe. We also immuno-labeled spines for drebrin A, an F-actin-binding protein found at excitatory synapses and with a proposed role of modulating F-actin's cross-linking with one another and interactions with NMDARs. Quantitative 2-D analysis of ultrastructural images revealed that NMDAR blockade increased filamentous actin labeling per spine by 62.5% (P<0.005). The proportion of dendritic spines immuno-labeled for drebrin A also increased significantly, from 67.5% to 85% following NMDAR blockade (P<0.001), especially among larger spines. The frequency distributions of spine widths and postsynaptic density lengths were not affected by the D-(+)-2-amino-5-phosphonopentanoic acid (D-APV) treatment. However, the average postsynaptic density length was reduced by 25 nm among the fewer, drebrin A immuno-negative spines, indicating that drebrin A confers stability to synapse size. We propose that, in a homeostatic in vivo response, increases of drebrin A and F-actin within spines can enhance NMDAR trafficking by reducing cytoskeletal rigidity within the spine cytoplasm without changing the overt morphology of axo-spinous synapses. Alternatively or in addition, the cytoskeletal redistribution within spine cytoplasm may be triggered by the D-APV-induced, homeostatic up-regulation of NMDAR.

Actin Cytoskeleton↗
PubMed2001

Drebrin expression is increased in spinal motoneurons of rats after axotomy.

Drebrin has been known to act on actin filaments at dendritic spines to cause morphological change, and might be related to the plasticity of synaptic transmission. In the present study, changes of drebrin were examined immunohistochemically in the spinal motoneurons of rats following unilateral sciatic nerve transection. Drebrin-immunoreactivity (-ir) in the motoneurons was significantly increased on the lesioned side after 3 days. Confocal laser-scanning microscopic images of the motoneurons revealed conspicuous increase in drebrin in the submembranous region of the cells. After 10 weeks, drebrin-ir on the lesioned side decreased to a level not significantly different from that on the unlesioned side. The results suggested that drebrin played important roles in synaptic restoration in axotomized motoneurons.

Animals↗
PubMed2001

Brain Abeta amyloidosis in APPsw mice induces accumulation of presenilin-1 and tau.

APPsw transgenic mice (Tg2576) overproducing mutant amyloid beta protein precursor (betaAPP) show substantial brain Abeta amyloidosis and behavioural abnormalities. To clarify the subsequent abnormalities, the disappearance of neurons and synapses and dystrophic neurite formation with accumulated proteins including hyperphosphorylated tau were examined. Tg2576 demonstrated substantial giant core plaques and diffuse plaques. The number of neurons was significantly decreased in the areas containing the amyloid cores compared with all other areas and corresponding areas in non-transgenic littermates in sections visualized by Nissl plus Congo red double staining (p<0.001). The presynaptic protein alpha-synuclein and postsynaptic protein drebrin were also absent in the amyloid cores. betaAPP and presenilin-1 were accumulated in dystrophic neurites in and around the core plaques. Tau phosphorylated at five independent sites was detected in the dystrophic neurites in the amyloid cores. Thus, the giant core plaques replaced normal brain tissues and were associated with subsequent pathological features such as dystrophic neurites and the appearance of hyperphosphorylated tau. These findings suggest a potential role for brain Abeta amyloidosis in the induction of secondary pathological steps leading to mental disturbance in Alzheimer's disease.

Alzheimer Disease↗
PubMed2001

Clustering and anchoring mechanisms of molecular constituents of postsynaptic scaffolds in dendritic spines.

Recent technological progress has yielded great amounts of information about the molecular constituents of postsynaptic scaffolds in the dendritic spine. Actin filaments are major cytoskeletal elements in the dendritic spine, and they functionally interact with neurotransmitter receptors via regulatory actin-binding proteins. Drebrin A and alpha-actinin-2 are two major actin-binding proteins in dendritic spines. In adult brains, they are characteristically concentrated in spines, but not in dendritic shafts or cell bodies. Thus, they are part of a unique postsynaptic scaffold consisting of actin filaments, PSD protein family, and neurotransmitter receptors. Localization of NMDA receptors, actin filaments, and actin-binding proteins in spines changes in parallel with development, and in response to synaptic activity. This raises the possibility that clustering and anchoring of these characteristic molecular constituents at postsynaptic scaffolds play important roles in spine function. This article focuses on the clustering and anchoring mechanisms of NMDA receptors and actin filaments, and the involvement of actin-binding proteins, in dendritic spines, and the way in which characteristic postsynaptic scaffolds are built up.

Actin Cytoskeleton↗
PubMed2001

The sulphydryl reagent, N-ethylmaleimide, disrupts sleep and blocks A1 adenosine receptor-mediated inhibition of intracellular calcium signaling in the in vitro ventromedial preoptic nucleus.

To explore the neuronal signaling mechanisms underlying sleep regulation in the rat, the present study examined continuous intra-third ventricle infusion of N-ethylmaleimide (NEM), a sulphydryl reagent that inhibits G(i/o) protein-coupled receptor-mediated signaling pathways. The diurnal infusion of NEM (0.01-10 micromol/10 h) dose-dependently inhibited both non-rapid eye movement sleep and rapid eye movement sleep. A maximal dose of NEM (10 micromol/10 h) dramatically inhibited day-time sleep (-57% for non-rapid eye movement sleep and -89% for rapid eye movement sleep) with a compensatory increase of sleep during the subsequent night-time (+33% for non-rapid eye movement sleep and +259% for rapid eye movement sleep). The day-time brain temperature was also increased by NEM, demonstrating effects of NEM on both sleep and body temperature levels. Immunostaining of the rat hypothalamus with a monoclonal antibody against the A1 adenosine receptor (A1R) was used to explore the distribution of a sleep-related G(i/o) protein-coupled receptor. Robust A1R-like immunoreactivity was found in the ventromedial preoptic nucleus and the supraoptic nucleus. Fura-2-based Ca(2+) imaging analysis of acute hypothalamic slices further demonstrated that the A1R agonist N(6)-cyclopentyladenosine (CPA; 200 nM) inhibited spontaneous Ca(2+) oscillations and high potassium (80 mM)-induced Ca(2+) flux in the ventromedial preoptic nucleus, while NEM (100-300 microM) and an A1R antagonist 8-cyclopentyl-dipropylxanthine (300 nM) blocked the CPA actions and increased the high potassium-induced Ca(2+) flux. From these results we suggest that NEM-sensitive G protein-coupled receptor(s) may play an important role in the regulation of sleep and body temperature in the rat and one possible mechanism is an A1R-mediated regulation of intracellular Ca(2+) concentrations in the ventromedial preoptic nucleus.

Adenosine↗
PubMed2001

Molecular cloning and dendritic localization of rat SH3P7.

SH3P7 was originally isolated by cloning SH3 domain ligand targets from a mouse embryo cDNA library. SH3P7 is an actin-binding protein implicated in antigen reception, JNK1 signalling, and Rac activation. It contains a drebrin homology sequence in its N-terminal region and a cortactin homology sequence (SH3 domain) in its C-terminal region. Both drebrin and cortactin are actin-binding proteins, and both have been suggested as possible regulators of the actin cytoskeleton in neurons. In the present study, we performed cDNA cloning of rat SH3P7, performed RT-PCR analysis, generated polyclonal antibodies against the recombinant rat SH3P7 protein, and examined the distribution of SH3P7 in the rat brain using immunohistochemistry. Sequence analysis revealed that there were at least four isoforms of the SH3P7 protein: SH3P7r1-SH3P7r4. RT-PCR analysis revealed that the predominant isoforms expressed in the brain were SH3P7r1 and SH3P7r3. The relative levels of isoform expression were similar among regions. Immunohistochemistry revealed that the most intense immunolabelling for SH3P7 was observed in the hippocampus and cerebellar cortex. Double-labelling studies with anti-SH3P7 antibody and other neuronal marker proteins revealed that SH3P7 was located primarily in dendrites, and in moderate amounts in cell bodies. Immunoreactivity was absent in the presynaptic terminals. In cultured astrocytes, SH3P7 was localized at protrusive structures of the cell periphery and in the cell body. We concluded that SH3P7 is ubiquitous in the rat brain, and occurs as several isoforms. Also, its dendritic localization suggests that SH3P7 is functionally linked to actin cytoskeleton organization in dendrites.

Amino Acid Sequence↗
PubMed2000

Non-muscle myosin IIB-like immunoreactivity is present at the drebrin-binding cytoskeleton in neurons.

Dendritic spines are extremely motile, providing a structural mechanism for synaptic plasticity. Actin-myosin interaction is thought to be responsible for the change in the shape of spine. We have already reported that drebrin, an actin-binding protein, inhibits actin-myosin interaction and is enriched in the dendritic spine of mature neurons. In this study, we prepared the actin cytoskeleton of dendritic spines as an immunoprecipitate with anti-drebrin antibody from adult guinea-pig brain, immunized mice with the cytoskeleton, and obtained a monoclonal antibody (MAb) called MAb G650. MAb G650 reacted with non-muscle myosin IIB, but it did not react with muscle myosin II or non-muscle myosin IIA. Immunoblotting with this antibody revealed that drebrin-binding cytoskeleton contains this myosin IIB-like immunreactivity. Immunohistochemistry using MAb G650 demonstrated that this myosin IIB-like immunreactivity can be detected in the neuronal cell bodies and their apical dendrites, where drebrin is hardly detected. These data demonstrate that a myosin subtype associated with drebrin-binding actin filaments in the dendritic spines is myosin IIB, although this myosin is widely distributed in somato-dendritic subdomains of neurons. Furthermore, it is indicated that the cytoskeletons in dendritic spine were uniquely characterized with actin-binding proteins such as drebrin, but not with myosins.

Animals↗
PubMed2000

The effects of neurotrophin-3 and brain-derived neurotrophic factor on cerebellar granule cell movement and neurite extension in vitro.

Migration of the granule cells is a major stage of cerebellar maturation. Granule cells express neurotrophins and their receptors; however, their role in cell migration has not been defined. In this study we investigated the effects of exogenous neurotrophins on the movement and neurite extension of granule cells from glial-free cerebellar cell reaggregates in vitro. Our results provide direct evidence that neurotrophin-3 and brain-derived neurotrophic factor differentially affect the granule cells. Neurotrophin-3 significantly affected granule cell movements by decreasing the migration index (the ratio of the number of cells that moved further than half the neurite length) and the speed of cell soma movement, but did not affect neurite length or growth cone migration. In contrast, brain-derived neurotrophic factor and neurotrophin-4 acted on growing neurites and growth cones by significantly increasing neurite length and the speed of growth cone migration, but had no effect either on the migration index or on the speed of the cell soma movement. The results suggest that neurotrophins differentially affect neurite extension and the movements of cerebellar granule cells.

Animals↗
PubMed1999

Domain analysis of the actin-binding and actin-remodeling activities of drebrin.

Drebrin is an actin-binding protein which is expressed at highly levels in neurons. When introduced into fibroblasts, it has been known to bind to F-actin and to cause remodeling of F-actin. Here, we performed a domain analysis of the actin-binding and actin-remodeling activities of drebrin. Various fragments of drebrin cDNA were fused with green fluorescent protein cDNA and introduced into Chinese hamster ovary cells. Association of the fusion protein with F-actin and remodeling of the F-actin were examined. We found that the central 85-amino-acid sequence (residues 233-317) was sufficient for the binding to and remodeling of F-actin. The binding activity of this fragment was relatively low compared with that of full-length drebrin, but all the types of abnormalities of F-actin that are observed with full-length drebrin were also observed with this fragment. When this sequence was further fragmented, the actin-binding activity was greatly reduced and the actin-remodeling activity disappeared. The actin-binding activity of the central region of drebrin was confirmed by a cosedimentation assay of chymotryptic fragments of drebrin with purified actin. These data indicate that the actin-binding domain and actin-remodeling domain are identical and that this domain is located at the central region of drebrin.

Actins↗
PubMed1999

Change in the shape of dendritic spines caused by overexpression of drebrin in cultured cortical neurons.

Dendritic spines are known to be extremely motile, providing a structural mechanism for synaptic plasticity. Actin filaments are thought to be responsible for the changes in the shape of spines. We tested our hypothesis that drebrin, an actin-binding protein, is a regulator of spine shape. In high-density long-term primary cultures of rat cerebral cortex neurons, drebrin was colocalized with actin filaments at spines. We introduced drebrin tagged with green fluorescent protein (GFP) into these neurons to test the ability of exogenous drebrin to localize at spines and the effect of overexpression of drebrin on spine shape. We observed that exogenous drebrin indeed accumulated in spines. But when the actin-binding domain of drebrin was deleted, the protein was distributed in both spines and dendritic shafts, indicating that accumulation of drebrin in the spines required its actin-binding activity. Statistical analysis of the lengths of spines as determined from confocal laser microscopic images revealed that the spines were significantly longer in GFP-drebrin-expressing neurons than in GFP-expressing neurons. The longer spines labeled with GFP-drebrin were demonstrated to be postsynaptic by double labeling of the presynaptic terminals with antibody against synaptophysin. These results directly indicate that drebrin binds to actin filaments at dendritic spines and alters spine shape.

Actins↗
PubMed1999

Suppression of an actin-binding protein, drebrin, by antisense transfection attenuates neurite outgrowth in neuroblastoma B104 cells.

Drebrins, actin-binding proteins, are dominantly expressed during embryogenesis and accumulated in neurite processes of postmigratory neurons. While the cytoskeletal proteins are the important factors for regulating neurite outgrowth, the cellular mechanism in neurons is still unclear. To address the role of drebrins in the neurite outgrowth, we have studied the effect of suppression of drebrin on a rat neuroblastoma B104 cell line, which constitutively expresses drebrin. Deprivation of serum or addition of gangliosides in the culture medium induced remarkable neurite outgrowth of B104 cells. We transfected B104 cells with an antisense construct of human drebrin E cDNA and found that the drebrin expression was significantly reduced in the stable antisense cell lines. In response to serum deprivation and gangliosides treatment, their ability to extend neurite processes was significantly attenuated. In contrast, the cell proliferation of the antisense transfectants was arrested by serum deprivation similar to control B104 cells. These data suggest that the drebrins are required for neurite outgrowth in neuronal cells.

Animals↗
PubMed1999

High level of adenosine A1 receptor-like immunoreactivity in the CA2/CA3a region of the adult rat hippocampus.

We describe the immunocytochemical distribution of adenosine A1 receptors in the rat hippocampus. Adenosine A1 receptor-like immunoreactivity was seen on the cell soma and dendrites of pyramidal cells and the cell soma and proximal part of dendrites of granule cells, but not on glial cells. Developmentally, adenosine A1 receptor-like immunoreactivity was diffuse on postnatal day 7 and increased in intensity in individual cells by day 21. In the CA2/CA3a region, the adult pattern of A1 receptor distribution was established by day 28. In the adult rat hippocampus, rostrocaudal inspection revealed that immunoreactivity in CA2/CA3a was greatest. Confocal microscopy revealed differences in the staining patterns for the adenosine A receptor and synaptophysin, a marker of presynaptic terminals. This result suggests that the adenosine A1 receptor might have postsynaptic physiological functions. Double-labeling of adenosine A1 receptors and anterogradely-labeled fibers from the supramammillary nucleus showed that the fibers from the supramammillary nucleus terminate directly on the cell soma of the A1 receptor-immunopositive neurons in CA2/CA3a and the dentate gyrus. These results indicate that the adenosine A 1 receptor in CA2/CA3a and the dentate gyrus are in a position to regulate hippocampal theta activity and that resultant strong synaptic depression in CA2/CA3a could play a role in regulating the intrinsic signal flow between CA3 and CA1.

Adenosine↗
PubMed1999

Loss of proteins regulating synaptic plasticity in normal aging of the human brain and in Alzheimer disease.

Recent studies suggest that the cognitive impairment associated with normal aging is due to neuronal dysfunction rather than to loss of neurons or synapses. To characterize this dysfunction, molecular indices of neuronal function were quantified in autopsy samples of cerebral cortex. During normal aging, the most dramatic decline was found in levels of synaptic proteins involved in structural plasticity (remodeling) of axons and dendrites. Alzheimer disease, the most common cause of dementia in the elderly, was associated with an additional 81% decrease in levels of drebrin, a protein regulating postsynaptic plasticity. Disturbed mechanisms of plasticity may contribute to cognitive dysfunction during aging and in Alzheimer disease.

Aged↗
PubMed1998

Rapid conversion of drebrin isoforms during synapse formation in primary culture of cortical neurons.

Easier methods to evaluate synapse formation in cultured neurons are desirable to investigate the regulatory mechanisms of synaptic development. We focused on drebrin, which changes from embryonic-type to adult-type isoform during postnatal development. The adult-type isoform of drebrin was detected by Western blotting after seventh day in primary cultured cortical neurons and the expression was coincidental with that of synaptophysin. Reverse transcription-polymerase chain reaction could demonstrate reversal of the predominant drebrin mRNA isoform from embryonic type to adult type between 8 and 13 days of culture. This is an easy and quick method to evaluate synapse formation in vitro.

Animals↗
PubMed1998

Differential expression of rat brain synaptic proteins in development and aging.

We have previously reported the differential involvement of synaptic proteins in Alzheimer's disease (AD). As AD is an aging-associated disease, in the present study we examined the developmental and aging-related changes in synaptic proteins such as synaptophysin, synaptobrevin, synaptotagmin, synaptosomal-associated protein 25 (SNAP-25), syntaxin 1/HPC-1 and drebrin in the rat brain. Immunoblot analyses of brain extracts from embryonic day 19 (E19) to postnatal 96-week-old rats indicated that the protein level of synaptophysin and synaptobrevin increased after birth, being highest at 24 weeks, and then decreased with aging. Synaptotagmin was detected at E19, with levels increasing after birth to 96 weeks. SNAP-25 levels were highest at 4 weeks, and then decreased with aging. Syntaxin 1/HPC-1 levels were high at E19 and 1 week, decreasing rapidly from 2 weeks onwards, and drebrin levels were highest at E19 and 1 week, and decreased during aging. The present results suggest that the expression of each synaptic protein is differentially regulated in development and aging.

Aging↗
PubMed1998

Interactions of drebrin and gephyrin with profilin.

Profilin is an actin monomer-binding protein which stimulates actin polymerization. Recent studies have revealed that profilin interacts with VASP, Mena, Bnilp, Bnrlp, and mDia, all of which have the proline-rich domain. Here, we isolated three profilin-binding proteins from rat brain cytosol by glutathione S-transferase-profilin affinity column chromatography and identified them as Mena, drebrin, and gephyrin. These proteins had a proline-rich domain and directly interacted with profilin.

Amino Acid Sequence↗
PubMed1997

Neurite formation induced in neuroblastoma cells and genetically altered non-neuronal cells.

Ultrastructural changes associated with neurite formation were examined in suitably cultured neuroblastoma cells (Neuro-2a) and drebrin (developmentally regulated brain protein) gene-transfected fibroblasts (L cells) in culture. Both neuroblastoma cells and fibroblasts initially were flattened and epitheloid, with many microspikes or microvilli diffusely distributed over their surfaces. Intracellular organelles were abundant and diffusely arranged. In the transformed state, both cell types were round to oval with long processes where microspikes were concentrated. A constant arrangement of surface and intracellular structures was apparent, though drebrin gene-transfected fibroblasts retained some of their original characteristics. Neurite formation programmed by genes may be initiated by environmental factors in neuroblastoma cells. Neurite-like processes in fibroblasts may be formed due to changes in microfilaments resulting from transfection of the drebrin (actin-binding protein) gene.

Animals↗