Biomedical subjects

Shun Nakamura

Publications and source records attributed to Shun Nakamura.

At least 19 recordsLinked to original sources

PubMed2006

Ouabain-induced isoform-specific localization change of the Na+, K+-ATPase alpha subunit in the synaptic plasma membrane of rat brain.

Na+, K+-ATPase is one of major membrane proteins that has two subunits, alpha and beta. The alpha subunit has the ATPase activity and the ouabain binding site. Among four isoforms of the alpha subunit, expression of alpha1, alpha2, and alpha3, but not alpha4, is observed in matured rat brain. Ouabain is one of cardiac glycosides, and endogenous ouabain-like compounds have been recognized as a new class of steroid hormone. The alpha subunit is considered as their endogenous receptor. Recent studies envisaged the importance of membrane microdomains (MDs) as signaling platforms, which are recovered as a detergent-resistant membrane microdomain fraction (DRM). Although this ATPase has been considered as a non-DRM protein, some amount of the alpha subunit was found to be a component of the DRM prepared from the synaptic plasma membrane fraction (SPM) of rat brain. Ouabain treatment increased the amount of alpha3 isoform, but not alpha1, in the DRM derived from synaptosome fraction and SPM. These results suggest that the localization of the alpha subunit of Na+, K+-ATPase is regulated with isoform-specific mechanisms and the physiological importance of DRM in the signal transduction of the endogenous ouabain-like steroid hormone in neurons.

Animals↗

PubMed2006

TrkB-T1 regulates the RhoA signaling and actin cytoskeleton in glioma cells.

Recently, the truncated TrkB receptor, T1, has been reported to be involved in the control of cell morphology via the regulation of Rho proteins, through which T1 binds Rho guanine nucleotide dissociation inhibitor (Rho GDI) 1 and dissociates it in a brain-derived neurotrophic factor (BDNF)-dependent manner. However, it is unclear whether T1 signaling regulates the downstream of Rho signaling and the actin cytoskeleton. In this study, we investigated this question using C6 rat glioma cells, which express T1 endogenously. Rho GDI1 was dissociated from T1 in a BDNF-dependent manner, which also causes decreases in the activities of Rho-signaling molecules such as RhoA, Rho-associated kinase, p21-activated kinase, and extracellular-signal regulated kinase1/2. Moreover, BDNF treatment resulted in the disappearance of stress fibers in the cells treated with lysophosphatidic acid, an activator of RhoA, and in morphological changes in cells. Furthermore, a competitive assay with cyan fluorescent protein fusion proteins of T1-specific sequences reduced the effects of BDNF. These results suggest that T1 regulates the Rho-signaling pathways and the actin cytoskeleton.

Actins↗

PubMed2006

[Nano -level imaging for analyzing protein structure and function].

The present manuscript outlines the nano-level imaging project, which is under promotion by the three national research institutes and supported by a research grant from the Ministry of Health, Labor and Welfare (nano-001). This research project targets collecting fundamental information regarding comprehensive understanding of cardiovascular, neurological and the other disorders, developing new diagnostic and therapeutic methods by visualizing protein structure and function in atomic(sub-nano level) or molecular (nano-level) resolution. The results of the current projects will be extended into drug design, clinical diagnostic technology and medical materials in near future.

Nanotechnology↗

PubMed2005

Role of Fabp7, a downstream gene of Pax6, in the maintenance of neuroepithelial cells during early embryonic development of the rat cortex.

Pax6 is a transcription factor with key functional roles in the developing brain. Pax6 promotes neuronal differentiation via transcriptional regulation of the Neurogenin2 (Ngn2) gene, although Pax6 expression appears in proliferating neuroepithelial cells before the onset of neurogenesis. Here, we identified Fabp7 (BLBP/B-FABP), a member of the fatty acid-binding protein (FABP) family, as a downregulated gene in the embryonic brain of Pax6 mutant rat (rSey2/rSey2) by microarray analysis. Marked reduction of Fabp7 expression was confirmed by quantitative PCR. Spatiotemporal expression patterns of Fabp7 in the wild-type rat embryos from embryonic day 10.5 (E10.5) to E14.5 were similar to those of Pax6, and expression of Fabp7 was undetectable in the rSey2/rSey2 cortex. The expression pattern of Fabp7 in the wild-type mouse embryo at E10.5 (corresponding to E12.5 rat) was different from that in the rat embryo, and no change of expression was observed in the Sey/Sey mouse embryo. Overexpression of exogenous Pax6 mainly induced ectopic expression of Fabp7, rather than of Ngn2, in the early cortical primordium. Interestingly, knocking-down FABP7 function by electroporation of Fabp7 small interfering RNA severely curtailed cell proliferation but promoted neuronal differentiation. We conclude that Fabp7 is a downstream gene of Pax6 transcription factor in the developing rat cortex and essential for maintenance of neuroepithelial cells during early cortical development.

Animals↗

PubMed2005

Small GTPase proteins Rin and Rit Bind to PAR6 GTP-dependently and regulate cell transformation.

The novel small GTPases Rin and Rit are close relatives of Ras, and recent studies show that they play a role in mediating neuronal differentiation. However, the direct effectors of Rin and Rit have yet to be fully characterized. Here we showed that Rin and Rit directly bind to the PDZ domain of PAR6, a cell polarity-regulating protein, in a GTP-dependent manner both in vivo and in vitro. Moreover, Rin and Rit can form a ternary complex consisting of PAR6 and Rac/Cdc42, members of the Rho family of small GTPases modulating cell growth and polarity. This ternary complex synergistically potentiates cell transformation in NIH3T3 cells, and the interaction between Rin/Rit and the PDZ domain of PAR6 is important for this effect. These results suggest that the Rin/Rit-PAR6-Rac/Cdc42 ternary complex may work physiologically in the cells, such as in tumorigenesis.

Animals↗

PubMed2005

A truncated tropomyosin-related kinase B receptor, T1, regulates glial cell morphology via Rho GDP dissociation inhibitor 1.

Through tropomyosin-related kinase B (TrkB) receptors, brain-derived neurotrophic factor (BDNF) performs many biological functions such as neural survival, differentiation, and plasticity. T1, an isoform of TrkB receptors that lacks a tyrosine kinase, predominates in the adult mammalian CNS, yet its role remains controversial. In this study, to examine whether T1 transduces a signal and to determine its function, we first performed an affinity purification of T1-binding protein with the T1-specific C-terminal peptide and identified Rho GDP dissociation inhibitor 1 (GDI1), a GDP dissociation inhibitor of Rho small G-proteins, as a signaling protein directly associated with T1. The binding of BDNF to T1 caused Rho GDI1 to dissociate from the C-terminal tail of T1. Astrocytes cultured for 30 d expressed only endogenous T1 among the BDNF receptors. In 30 d cultured astrocytes, Rho GDI1, when dissociated in a BDNF-dependent manner, controlled the activities of the Rho GTPases, which resulted in rapid changes in astrocytic morphology. Furthermore, using 2 d cultured astrocytes that were transfected with T1, a T1 deletion mutant, or cyan fluorescent protein fusion protein of the T1-specific C-terminal sequence, we demonstrated that T1-Rho GDI1 signaling was indispensable for regulating the activities of Rho GTPases and for the subsequent morphological changes among astrocytes. Therefore, these findings indicate that the T1 signaling cascade can alter astrocytic morphology via regulation of Rho GTPase activity.

Amino Acid Motifs↗

PubMed2005

Topological relation of chick thalamofugal visual projections with hyper pallium revealed by three color tracers.

In birds, there are two visual projections from retina to higher pallium, i.e., tectofugal and thalamofugal pathways. The latter one is lateralized in chick and suggested to be involved in visually evoked social behavior, like recognition of novelty, predator, and conspecific animals. We wanted to establish functionally relevant topological connection map between thalamic nuclei and hyperpallium apicale (HA) and carried out tracing study with three color fluorescent tracers. The tracers were serially injected in HA either along with the medial-lateral (M-L) or anterior-posterior (A-P) axis. We found that M-L axis and A-P axis in HA were transferred into the dorsal-ventral axis and the medial-lateral axis, respectively within the nucleus geniculatus lateralis pars dorsalis (GLd). In another word, the medial part of nucleus dorsolateralis anterior thalami pars lateralis (DLLv) projected to anterior part of HA and the ventral part of nucleus dorsolateralis anterior thalami pars lateralis pas ventralis (DLLd) projects to lateral HA. This result suggests that thalamus would process information in parallel through each subnuclei and elaborate coordination among them in relation to topological map presented in higher pallium.

Animals↗

PubMed2005

Craniofacial anomalies of the cultured mouse embryo induced by inhibition of sonic hedgehog signaling: an animal model of holoprosencephaly.

The pathogenesis of holoprosencephaly is multifactorial, and blockage of Sonic hedgehog signaling is one of the most important causative factors in animal models and human cases. In this study, the authors analyzed facial anomalies of mouse embryos, which were cultured in vitro and exposed to cyclopamine, an alkaloid blocker of Sonic hedgehog signaling. When cultured with cyclopamine for embryonic day 8.5 to 10.5, the whole body size was smaller than normal, and the distance and angle between the nasal placodes were remarkably reduced. Extension of the cranial surface vessels also was noted. No cyclopia was observed. Migration of the cranial neural crest cells seemed to be intact. Expressions of Patched-1 and Gli-1, downstream genes of Sonic hedgehog signaling, also were down-regulated in in situ hybridization and real-time reverse transcriptase-polymerase chain reaction analyses. The authors consider that these facial anomalies represent milder phenotypes of holoprosencephaly.

Animals↗

PubMed2004

The upregulated expression of sonic hedgehog in motor neurons after rat facial nerve axotomy.

Nerve injury leads to the induction of a large number of genes to repair the damage and to restore synaptic transmission. We have attempted to identify molecules whose mRNA expression is altered in response to facial nerve axotomy. Here we report that facial nerve axotomy upregulates Sonic hedgehog (Shh) and its receptor Smoothened (Smo) in facial motor neurons of adult rats, whereas facial nerve axotomy does not upregulate mRNA of Shh or Smo in neonatal rats. We tested whether overexpression of Shh in facial motor neurons of axotomized neonatal rats may promote neuronal survival. Adenovirus-mediated overexpression of Shh, but not that of beta-galactosidase, transiently rescues axotomy-induced neuronal cell death for 3-5 d after axotomy. Finally, the pharmacological inhibitor of Shh signaling, cyclopamine, induces motor neuron death in adult rats after axotomy. These results suggest that Shh plays a regulatory role in nerve injury.

Adenoviridae↗

PubMed2004

Sequence of forebrain activation induced by intraventricular injection of hypertonic NaCl detected by Mn2+ contrasted T1-weighted MRI.

In order to define the sequence of forebrain activation involved in osmoregulation, central activation in response to intracerebroventricular injection of NaCl solution (10 microl of 0.15, 0.5, or 1.5 M) was detected using manganese-contrasted magnetic resonance imaging (MRI) in anesthetized rats. Changes in renal sympathetic nerve activity (RNA) were also measured, and the time courses of forebrain activation and RNA changes compared. NaCl injection resulted in rapid activation of the subfornical organ (SFO), organum vasculosum lamina terminalis (OVLT), and periventricular regions and the lateral hypothalamic area (LHA), then of the paraventricular hypothalamic nucleus (PVN) and supraoptic nucleus (SON). The delay in activation in the PVN and SON showed a wide variation from 0 to 5.78 min, and the average delay in the PVN (2.88+/-0.34 min) and SON (2.90+/-0.39 min) was significantly greater than that in the SFO (0.40+/-0.10 min) and OVLT (0.74+/-0.13 min). NaCl (1.5 M) injection elicited a rapid, large increase in RNA, which consisted of two components, an early rapid increase at 99 s after injection (160+/-27%) and a slower increase at 9 min after injection (209+/-34%). These results suggest that the PVN and SON are activated not only by the afferent input from the SFO and OVLT but also by diffusion of the hypertonic stimulus to these regions and probably by their intrinsic osmosensitivity. The PVN might be responsible for the second slower component of the RNA response, but cannot be responsible for the first component.

Animals↗

PubMed2004

Identification of V-ATPase as a major component in the raft fraction prepared from the synaptic plasma membrane and the synaptic vesicle of rat brain.

Cholesterol is important in the maintenance and remodeling of the synapse. Since membrane cholesterol participates in the formation of the membrane microdomain (raft), the characterization of raft components within membrane structures in the synaptic region could be a good approach to understand the role of cholesterol in the synaptic function. In this study, protein complexes in the raft prepared from synaptic plasma membrane and the synaptic vesicle were analyzed with blue-native polyacrylamide gel electrophoresis and vacuolar H(+)-pump (V-ATPase) was identified as a major raft component using mass spectrometry. The ATPase activity was reduced through cholesterol deprivation with methyl-beta-cyclodextrin. Since the H(+) -gradient is used to transport synaptic transmitters or their precursors into the vesicle, this result suggests the essential role of cholesterol and raft in the synaptic function.

Animals↗

PubMed2004

Gene expression analysis of the late embryonic mouse cerebral cortex using DNA microarray: identification of several region- and layer-specific genes.

The mammalian neocortex develops layer organizations with regional differences represented by expression of multiple genes at embryonic stages. These genes could play important roles in the formation of areal cyto-architecture, yet, the number of genes identified so far is not sufficient to explain such intricate processes. Here we collected five regions--the medial, dorsal, lateral, rostral and occipital--from the dissected E16.5 mouse cerebral cortex and performed extensive gene expression analysis using the Affymetrix U74Av2 array with probes for 12,500 genes. After relative quantitative analysis, 34, 33 and 15 genes were selected as highly expressed genes in the medial, dorsal and lateral regions, respectively. The combination of GeneChip system, real-time quantitative reverse transcription polymerase chain reaction and in situ hybridization analyses allowed the successful identification of seven genes from the dorsal region (Neuropeptide Y, Wnt7b, TGF-beta RI, Nrf3, Bcl-6, MT4-MMP and Rptp kappa), three genes from the medial region (Hop-pending, HtrA and Crystallin), and three genes from the lateral region (Somatostatin, Ngef and Fxyd7). Particularly, all seven genes identified in the dorsal region demarcated the future somatosensory and auditory areas in the cortical plate with high rostrolateral-low caudomedial gradation. Their expression patterns were not uniform, but delineated either the superficial or the deep layer in the cortical plate. Furthermore, the regional expression pattern of Neuropeptide Y was shifted rostrally and the layer specificity was disorganized in the Pax6-deficient mice. Our results provide new information about a subclass of regionally expressed genes in the cortical plate at the late embryonic stage, which may help understand the molecular mechanisms of neocortical arealization.

Animals↗

PubMed2004

Expression of BDNF and TrkB receptor subtypes in the postnatal developing Purkinje cells of monkey cerebellum.

In the previous study, we have shown the complementary expression of TrkB subtypes (TK+ and T1) in the adult monkey cerebellar cortex. In this study, to clarify when that expression pattern appeared, we examined the expressions of TrkB subtypes and its ligand brain-derived neurotrophic factor (BDNF) by immunohistochemistry and Western blot analysis. At the newborn stage, both TK+ and T1 were expressed uniformly in the cerebellar cortex. At postnatal month 3.5, the uneven expression of TrkB subtypes was observed, while the BDNF immunoreactivity was strongly detected in all regions of the cerebellar cortex. The expression patterns of TrkB subtypes and BDNF at both postnatal month 6 and year 7 were the same as those at postnatal month 3.5. Western blot analysis demonstrated that TK+ and T1 were expressed at high levels in the synaptic membrane from newborn to adult stages. Furthermore, the dimerization of TrkB subtypes changed at postnatal month 3, which was similar to the adult pattern: at the newborn stage, the TK+ and TK- homodimers; after postnatal month 3.5, the TK+ and TK- homodimers, and the TK+/TK- heterodimer. These findings suggest that the localization of TrkB subtypes in each Purkinje would be changed at postnatal month 3.5, resulting in the uneven expression of TrkB subtypes and the change of TrkB dimerization.

Animals↗

PubMed2004

Quantum dots targeted to the assigned organelle in living cells.

Fluorescent nanocrystal quantum dots (QDs) have the potential to be applied to bioimaging since QDs emit higher and far longer fluorescence than conventional organic probes. Here we show that QDs conjugated with signal peptide obey the order to transport the assigned organelle in living cells. We designed the supermolecule of luminescent QDs conjugated with nuclear- and mitochondria-targeting ligands. When QDs with nuclear-localizing signal peptides were added to the culture media, we can visualize the movements of the QDs being delivered into the nuclear compartment of the cells with 15 min incubation. In addition, mitochondrial signal peptide can also transport QDs to the mitochondria in living cells. In conclusion, these techniques have the possibility that QDs can reveal the transduction of proteins and peptides into specific subcellular compartments as a powerful tool for studying intracellular analysis in vitro and even in vivo.

Amino Acid Sequence↗

PubMed2003

Small GTPase Rin induces neurite outgrowth through Rac/Cdc42 and calmodulin in PC12 cells.

The novel Ras-like small GTPase Rin is expressed prominently in adult neurons, and binds calmodulin (CaM) through its COOH-terminal-binding motif. It might be involved in calcium/CaM-mediated neuronal signaling, but Rin-mediated signal transduction pathways have not yet been elucidated. Here, we show that expression of Rin induces neurite outgrowth without nerve growth factor or mitogen-activated protein kinase activation in rat pheochromocytoma PC12 cells. Rin-induced neurite outgrowth was markedly inhibited by coexpression with dominant negative Rac/Cdc42 protein or CaM inhibitor treatment. We also found that expression of Rin elevated the endogenous Rac/Cdc42 activity. Rin mutant proteins, in which the mutation disrupted association with CaM, failed to induce neurite outgrowth irrespective of Rac/Cdc42 activation. Disruption of endogenous Rin function inhibited the neurite outgrowth stimulated by forskolin and extracellular calcium entry through voltage-dependent calcium channel evoked by KCl. These findings suggest that Rin-mediated neurite outgrowth signaling requires not only endogenous Rac/Cdc42 activation but also Rin-CaM association, and that endogenous Rin is involved in calcium/CaM-mediated neuronal signaling pathways.

Amino Acid Sequence↗

PubMed2003

Brain-derived neurotrophic factor-dependent unmasking of "silent" synapses in the developing mouse barrel cortex.

Brain-derived neurotrophic factor (BDNF) is a critical modulator of central synaptic functions such as long-term potentiation in the hippocampal and visual cortex. Little is known, however, about its role in the development of excitatory glutamatergic synapses in vivo. We investigated the development of N-methyl-D-aspartate (NMDA) receptor (NMDAR)-only synapses (silent synapses) and found that silent synapses were prominent in acute thalamocortical brain slices from BDNF knockout mice even after the critical period. These synapses could be partially converted to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-containing ones by adding back BDNF alone to the slice or fully converted to together with electric stimulation without affecting NMDAR transmission. Electric stimulation alone was ineffective under the BDNF knockout background. Postsynaptically applied TrkB kinase inhibitor or calcium-chelating reagent blocked this conversion. Furthermore, the AMPAR C-terminal peptides essential for interaction with PDZ proteins postsynaptically prevented the unmasking of silent synapses. These results suggest that endogenous BDNF and neuronal activity synergistically activate AMPAR trafficking into synaptic sites.

Animals↗

PubMed2003

Inhibitory but not excitatory cortical neurons require presynaptic brain-derived neurotrophic factor for dendritic development, as revealed by chimera cell culture.

To address questions of whether endogenous BDNF acts differentially on inhibitory and excitatory neurons, and through what routes, we used chimera culture of cerebral cortical neurons derived from BDNF-/- mice and another type of transgenic mice that express green fluorescence protein and BDNF. Presynaptic BDNF transferred to both types of neurons, GABA-synthesizing enzyme-positive and -negative neurons. The latter neurons were confirmed to be glutamatergic with immunocytochemistry. Dendritic development of the former inhibitory neurons was promoted by endogenous BDNF transferred from presynaptic, excitatory neurons. In contrast, dendritic development of excitatory neurons was not related to the presence or absence of presynaptic BDNF, suggesting that BDNF acts on inhibitory neurons through an anterograde, transsynaptic route so as to promote dendritic development, whereas this is not the case in excitatory neurons.

Animals↗

PubMed2003

Light-dependent development of asymmetry in the ipsilateral and contralateral thalamofugal visual projections of the chick.

Light-exposure of the chick embryo induces development of asymmetry in the thalamofugal visual projections to the Wulst regions of the forebrain since the embryo is turned so that it occludes its left and not its right eye. This asymmetry can be reversed by occluding the embryo's right eye and exposing its left eye to light. Here we show that three sub-regions of the thalamus (two in the dorsolateral anterior thalami (DLA) and one more caudal) have differing asymmetries of contralateral and/or ipsilateral projections. Hence the effect of asymmetrical light stimulation is regionally specific within the thalamus. Lateralised light stimulation appears to promote the development of ipsilateral projections from DLA pars dorsolateralis pars anterioris and contralateral projections from the caudal regions (the nucleus superficialis parvocellularis especially) but it may suppress the development of contralateral projections from the nucleus dorsolateralis anterior thalami pars lateralis rostralis. We also show that the light stimulation causes lateralised expression of c-fos and receptors for neurotransmitters.

Animals↗