Biomedical subjects

Shogo Endo

Publications and source records attributed to Shogo Endo.

18 recordsLinked to original sources

PubMed2006

Characterization of mRNA species that are associated with postsynaptic density fraction by gene chip microarray analysis.

We previously reported the partial identification by random sequencing of mRNA species that are associated with the postsynaptic density (PSD) fraction prepared from the rat forebrain [Tian et al., 1999. Mol. Brain Res. 72, 147-157]. We report here further characterization by gene chip analysis of the PSD fraction-associated mRNAs, which were prepared in the presence of RNase inhibitor. We found that mRNAs encoding various postsynaptic proteins, such as channels, receptors for neurotransmitters and neuromodulators, proteins involved in signaling, scaffold and adaptor proteins and cytoskeletal proteins, were highly concentrated in the PSD fraction, whereas those encoding housekeeping proteins, such as enzymes in the glycolytic pathway, were not. We extracted approximately 1900 mRNA species that were highly concentrated in the PSD fraction. mRNAs related to certain neuronal diseases were also enriched in the PSD fraction. We also constructed a cDNA library using the PSD fraction-associated mRNAs as templates, and identified 1152 randomly selected clones by sequencing. Our data suggested that the PSD fraction-associated mRNAs are a very useful resource, in which a number of as yet uncharacterized mRNAs are concentrated. Identification and functional characterization of them are essential for complete understanding of synaptic function.

Animals↗

PubMed2006

Mechanisms for association of Ca2+/calmodulin-dependent protein kinase II with lipid rafts.

Localization of CaMKIIalpha in lipid rafts was demonstrated in both cultured neurons and mammalian cells transfected with plasmid with an insert of CaMKIIalpha cDNA by using sucrose gradient centrifugation and the sensitivity to a cholesterol-extractor, methyl-beta-cyclodextrin. CaMKIIalpha was targeted to lipid rafts possibly through protein-protein interactions via at least three domains (a.a. 261-309, 371-420, and 421-478). The multimeric structure of the full-length molecule also appeared to contribute to efficient lipid raft-targeting. Acylation of CaMKIIalpha did not appear to be a mechanism for the targeting.

Animals↗

PubMed2006

A single amino-acid change in ERK1/2 makes the enzyme susceptible to PP1 derivatives.

We generated extracellular signal-regulated kinase 1/2 (ERK1/2) mutants by introducing a single amino-acid substitution in subdomain V of the catalytic domain and then examined the susceptibility of these mutants to PP1 derivatives originally designed as Src inhibitors. Substituting smaller amino acids (alanine [Ala (A)] or glycine [Gly (G)]) for glutamine [Gln (Q)] in subdomain V drastically increased the susceptibility of ERK1/2 to 1-naphthyl PP1 (1NA-PP1). Wild-type ERK1/2 was resistant to 1NA-PP1 inhibition. ERK1(Q122A) and ERK2(Q103A) were inhibited by 1NA-PP1 at IC(50) values of 1.7 +/- 0.13 and 2.1 +/- 0.18 microM, respectively. ERK1(Q122G) and ERK2(Q103G) were inhibited by 1NA-PP1 with IC(50) values of 3.6 +/- 0.26 and 18 +/- 2.2 microM, respectively. Other derivatives of PP1 (1-naphthylmethyl PP1 and 2-naphthylmethyl PP1) did not significantly inhibit ERK1/2 and its various mutants. In addition, these ERK1/2 mutants were activated by TPA when they were expressed in mammalian cells. These results suggest that the Gln residue of subdomain V is important in determining the susceptibility of ERK1/2 to 1NA-PP1 without significant changes in their enzymatic characteristics.

Amino Acid Sequence↗

PubMed2006

Interaction of LDL receptor-related protein 4 (LRP4) with postsynaptic scaffold proteins via its C-terminal PDZ domain-binding motif, and its regulation by Ca/calmodulin-dependent protein kinase II.

We cloned here a full-length cDNA of Dem26[Tian et al. (1999)Mol. Brain Res., 72, 147-157], a member of the low-density lipoprotein (LDL) receptor gene family from the rat brain. We originally named the corresponding protein synaptic LDL receptor-related protein (synLRP) [Tian et al. (2002) Soc. Neurosci. Abstr., 28, 405] and have renamed it LRP4 to accord it systematic nomenclature (GenBank(TM) accession no. AB073317). LRP4 protein interacted with postsynaptic scaffold proteins such as postsynaptic density (PSD)-95 via its C-terminal tail sequence, and associated with N-methyl-D-aspartate (NMDA)-type glutamate receptor subunit. The mRNA of LRP4 was localized to dendrites, as well as somas, of neuronal cells, and the full-length protein of 250 kDa was highly concentrated in the brain and localized to various subcellular compartments in the brain, including synaptic fractions. Immunocytochemical study using cultured cortical neurons suggested surface localization in the neuronal cells both in somas and dendrites. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) phosphorylated the C-terminal cytoplasmic region of LRP4 at Ser1887 and Ser1900, and the phosphorylation at the latter site suppressed the interaction of the protein with PSD-95 and synapse-associated protein 97 (SAP97). These findings suggest a postsynaptic role for LRP4, a putative endocytic multiligand receptor, and a mechanism in which CaMKII regulates PDZ-dependent protein-protein interactions and receptor dynamics.

Animals↗

PubMed2005

N-Methyl-D-Aspartic acid suppresses Akt activity through protein phosphatase in retinal ganglion cells.

PURPOSE: To investigate the relationship between Akt activity and retinal ganglion cell (RGC) death induced by N-Methyl-D-Aspartic acid (NMDA) in the rat retina. METHODS: Two microlitres of 1, 10, 50, 100, or 200 mM NMDA, or vehicle was injected into the vitreous cavity of Sprague-Dawley (SD) rats (n=125). Retinal damage was estimated by counting ganglion cells labeled with fluorochrome and retinal apoptosis was detected by TUNEL. Akt activity was determined by immunohistochemical analysis with a specific antibody to the activated (phosphorylated) form of Akt. To investigate the mechanism of dephosphorylation of Akt, Okadaic acid, a potent protein phosphatase inhibitor, was injected 1 h before NMDA injury and accessed the number of phosphorylated Akt positive cells 1 h after NMDA injection. To stimulate Akt activity in the retina, brain derived neurotrophic factor (BDNF) was injected into the vitreous 15 min before NMDA injection. RESULTS: Immunohistochemical analysis revealed a reduction in phosphorylated Akt in RGCs and amacrine cells one hour after NMDA injury. The RGCs and amacrine cells showed TUNEL positivity at 6 h and a decrease in cell number at 7 days after NMDA injury. No other cells in the retina stained positive with phosphorylated Akt antibody and TUNEL. Okadaic acid prevented the dephosphorylation of Akt by NMDA. The exogenous administration of BDNF prevented the dephosphorylation of Akt in N-Shc/ShcC-positive RGCs and significantly suppressed the NMDA-induced RGC death. CONCLUSIONS: These observations suggest that Akt is one of the key signaling proteins in RGC death induced by NMDA, and that the presence of N-Shc/ShcC enhances BDNF-mediated neuroprotection via phosphorylated Akt. The regulation of phosphorylated Akt by growth factors and protein phosphatase activity may play an important role in cell fate following NMDA injury. Thus, an increase in phosphorylated Akt may have potential therapeutic implications in the treatment of glutamate-related disease.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Retinal G-substrate, potential downstream component of NO/cGMP/PKG pathway, is located in subtype of retinal ganglion cells and amacrine cells with protein phosphatases.

The aim of this study was to determine the distribution and function of G-substrate, a specific substrate of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-cGMP-dependent protein kinase (PKG) signaling pathway, in normal rat retina and in G-substrate knockout mice. The retinas of adult wild-type rats and mice and G-substrate knockout mice were studied immunohistologically to characterize the upstream and downstream components of the NO-cGMP-PKG pathway. Immunoblot analysis showed that the molecular weight of retinal G-substrate was similar to that of cerebellar G-substrate. In adult rats and mice, retinal G-substrate was located in a subpopulation of amacrine cells and in C38-positive retinal ganglion cells (RGCs) but not in alpha RGCs. In addition, retinal G-substrate was co-expressed with other upstream and downstream signaling components of the NO-cGMP-PKG-G-substrate-phosphatase pathway in the adult retina. Electroretinographic (ERG) analysis demonstrated that there was no significant difference between the ERGs of wild-type and G-substrate knockout mice. These results suggest that retinal G-substrate plays a role as a downstream component of the NO-cGMP-PKG pathway. The co-localization of retinal G-substrate with protein Ser/Thr phosphatases suggests that it acts as an endogenous protein phosphatase inhibitor as in the cerebellum.

Amacrine Cells↗

PubMed2005

p55 protein is a member of PSD scaffold proteins in the rat brain and interacts with various PSD proteins.

p55 is a membrane-associated guanylate kinase (MAGuK) family member that consists of a single PDZ followed by SH3, HOOK and guanylate kinase (GuK or GK) domains. We investigated rat p55 (r-p55) in the brain. r-p55 mRNA was expressed widely in various tissues and in various regions of the brain. r-p55 protein was also expressed widely in various rat tissues, including brain and erythrocytes. The protein was enriched in the synaptic plasma membrane and postsynaptic density (PSD) fractions of the forebrain. An immunocytochemical study using cultured cortical neurons suggested postsynaptic localization of r-p55 protein. Pull-down assay showed that r-p55 protein interacted with r-p55 itself and various PSD proteins, such as PSD-95, SAP97, GKAP, CASK, GRIP, neuroligin, cadherin, tubulin, actin, alpha-internexin, neurofilament-L and Ca(2+)/calmodulin-dependent protein kinase II, through its PDZ, SH3, HOOK or GK domains. The interaction with PSD-95 was found to occur between the PDZ domains of PSD-95 and the HOOK and GK domains of r-p55 protein. These findings, together with the presence of r-p55 puncta in a period of early synaptogenesis, suggest that r-p55 protein functions as one of postsynaptic scaffold component in an early stage of synaptogenesis in the brain. r-p55 protein may form a basic structure, which interlinks diverse functional molecules of the PSD necessary for postsynaptic signaling and synaptic adhesion.

Amino Acid Sequence↗

PubMed2005

Neurodegeneration augments the ability of bone marrow-derived mesenchymal stem cells to fuse with Purkinje neurons in Niemann-Pick type C mice.

After transplantation, adult bone marrow-derived mesenchymal stem cells (BM-MSCs) may undergo transdifferentiation and/or cell fusion in response to new environments. However, the mechanism(s) that govern these cell fate switches remain unknown. Here we demonstrate that the pathology associated with murine Niemann-Pick disease type C (NP-C) cerebellum augments the ability of BM-MSCs to fuse with Purkinje neurons. The results suggest that the degenerative microenvironment of Purkinje neurons in the NP-C cerebellum modulates the cell fate switch of BM-MSCs via cell fusion.

Animals↗

PubMed2005

A novel scaffold protein, TANC, possibly a rat homolog of Drosophila rolling pebbles (rols), forms a multiprotein complex with various postsynaptic density proteins.

We cloned from the rat brain a novel gene, tanc (GenBank Accession No. AB098072), which encoded a protein containing three tetratricopeptide repeats (TPRs), ten ankyrin repeats and a coiled-coil region, and is possibly a rat homolog of Drosophila rolling pebbles (rols). The tanc gene was expressed widely in the adult rat brain. Subcellular distribution, immunohistochemical study of the brain and immunocytochemical studies of cultured neuronal cells indicated the postsynaptic localization of TANC protein of 200 kDa. Pull-down experiments showed that TANC protein bound PSD-95, SAP97, and Homer via its C-terminal PDZ-binding motif, -ESNV, and fodrin via both its ankyrin repeats and the TPRs together with the coiled-coil domain. TANC also bound the alpha subunit of Ca2+/calmodulin-dependent protein kinase II. An immunoprecipitation study showed TANC association with various postsynaptic proteins, including guanylate kinase-associated protein (GKAP), alpha-internexin, and N-methyl-D-aspartate (NMDA)-type glutamate receptor 2B and AMPA-type glutamate receptor (GluR1) subunits. These results suggest that TANC protein may work as a postsynaptic scaffold component by forming a multiprotein complex with various postsynaptic density proteins.

Age Factors↗

PubMed2005

BAALC 1-6-8 protein is targeted to postsynaptic lipid rafts by its N-terminal myristoylation and palmitoylation, and interacts with alpha, but not beta, subunit of Ca/calmodulin-dependent protein kinase II.

We cloned a rat BAALC 1-6-8 isoform cDNA (GenBank Accession No. AB073318) that encoded a 22-kDa protein, and identified endogenous BAALC 1-6-8 protein in the brain. The gene was expressed widely in the frontal part of the brain, and the protein was localized to the synaptic sites and was increased in parallel with synaptogenesis. The protein interacted with the alpha, but not beta, subunit of Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIalpha). The interaction occurred between the N-terminal 35-amino-acid region of BAALC 1-6-8 protein and the C-terminal end of the regulatory domain of CaMKIIalpha, which contains alpha isoform-specific sequence. Thus, the interaction may be CaMKIIalpha-specific. We also found that BAALC 1-6-8 protein, as well as CaMKIIalpha, was localized to lipid rafts and that both myristoylation and palmitoylation of BAALC 1-6-8 N-terminal portion were required for targeting of the protein into lipid rafts. These findings suggest that BAALC 1-6-8 protein play a synaptic role at the postsynaptic lipid raft possibly through interaction with CaMKIIalpha.

Alternative Splicing↗

PubMed2004

Expression of Btcl2, a novel member of Btcl gene family, during development of the central nervous system.

Cell-cell interactions are primarily mediated by secreted and transmembrane proteins which play essential roles in the neuronal circuit formation. However, molecular mechanisms underlying neuronal circuit formation, which is mediated by the cell-cell interactions, remain largely elusive. We isolated and characterized a novel gene, Btcl2 (brain-specific transmembrane protein containing CUB [complement subcomponent C1r/C1s, sea urchin protein Uegf, and BMP-1] and LDLa [low-density lipoprotein receptor domain class A] domains 2), using the signal sequence trap (SST) method. The extracellular domain of BTCL2 contains two CUB domains and an LDLa domain. BTCL2 and BTCL1 have similar domain structures, sharing 51% overall identity. The CUB1, CUB2, and LDLa domains of these two proteins share 63%, 72%, and 84% identity, respectively. The CUB domains of BTCL1 and BTCL2 share significant identity with those of neuropilins. Btcl2 mRNA was detected as a single 6-kb transcript in Northern blot analysis. In situ hybridization (ISH) analysis revealed that both Btcl1 and Btcl2 mRNAs were observed restrictively in brain throughout embryonic and postnatal stages. Btcl1 and Btcl2 mRNAs were expressed uniquely in the pontine nucleus and subplate, which are required for establishing the neuronal circuit formation. These results will aid in resolving the mechanisms underlying neuronal circuit formations (e.g., pontocerebellar and thalamocortical axon guidance) and permit more precise studies aimed at understanding the role of BTCL1 and BTCL2 in the central nervous system.

Amino Acid Sequence↗

PubMed2003

Nitric oxide activates extracellular signal-regulated kinase 1/2 and enhances declustering of ionotropic glutamate receptor subunit 2/3 in rat cerebellar Purkinje cells.

Exogenous nitric oxide (NO) donors (S-nitroso-N-acetyl-DL-penicillamine and (E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide) and simultaneous application of high potassium and glutamate led to the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in cerebellar Purkinje cells. NO-induced ERK1/2 phosphorylation was abolished by the addition of MEK (MAPK ERK kinase) inhibitor (PD98059) or oxyhemoglobin, and was significantly reduced by the PKG (cGMP-dependent protein kinase) inhibitor, KT5823. KT5823 also reduced the peak levels of ERK1/2 activation induced by high K-glutamate or NO in Purkinje cell somata. NO-induced ERK1/2 phosphorylation remained above basal levels for at least 1 h after NO-donor removal. To address the physiological role of NO-induced ERK activation in Purkinje cells, we examined the effects of NO and 8-bromoadenosine 3', 5'-cyclic monophosphate (8-Br-cGMP) on PKC-dependent receptor declustering, which underlies cerebellar long-term depression. TPA (12-Ø-tetradecanoylphorbol 13-acetate), a potent activator of PKC, induced GluR2/3 receptor declustering. The 8-Br-cGMP or NO-donors enhanced GluR2/3 declustering, but did not induce it. These results suggest an important role for the NO-cGMP-PKG pathway in the activation of ERK1/2 and GluR2/3 (ionotropic glutomate receptor subunit 2/3) declustering in Purkinje cells.

Animals↗

PubMed2003

A novel gene, Btcl1, encoding CUB and LDLa domains is expressed in restricted areas of mouse brain.

A variety of secreted and membrane proteins play key roles in the formation of neuronal circuits in the central nervous system. Using the signal sequence trap method, we isolated and characterized a novel gene, Btcl1 (brain-specific transmembrane protein containing two CUB and an LDLa domains). BTCL1 has significant homology with neuropilin-1 and -2 in their CUB domains. Domain structure of BTCL1 indicates that BTCL1 belongs to a new class of brain-specific CUB domain-containing protein. On Northern blot analysis, Btcl1 mRNA was observed as a single transcript of 3.7 kb specifically in the brain. In situ hybridization analysis revealed that Btcl1 mRNA was highly expressed in the hippocampal CA3 region, olfactory bulb, and neocortex in the adult brain. Expression pattern of mRNA and structural similarity with neuropilin suggest that BTCL1 plays a role in the development and/or maintenance of neuronal circuitry.

Amino Acid Sequence↗

PubMed2003

ERKs regulate PKC-dependent synaptic depression and declustering of glutamate receptors in cerebellar Purkinje cells.

Phorbol esters, such as tetradecanoylphorbol 13-acetate (TPA), have been used extensively in studies of cerebellar long-term depression (LTD), based on the hypothesis that activated protein kinase C (PKC) directly mediates alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor phosphorylation. Here, we show that TPA-induced depression of synaptic transmission between granule cells and Purkinje cells in culture is mediated through activation of the MEK1/2-ERK1/2 pathway. Phosphorylation of ERK1/2 induced by TPA and co-application of high potassium and glutamate was greatly attenuated by preincubating Purkinje cells with the MEK1/2 (MAPK ERK kinase 1/2) inhibitor PD98059. TPA-induced depression of synaptic transmission between granule cells and Purkinje cells was attenuated by PD98059. The MEK1/2 inhibitor also suppressed declustering of the ionotropic glutamate receptor subunit 2/3 (GluR2/3) induced by TPA and co-application of high potassium and glutamate, even though phosphorylation of Ser880 of GluR2/3 was not inhibited significantly in the presence of PD98059. These results suggest that ERK1/2 plays an essential role in TPA-induced depression via regulation of GluR2/3 declustering at the synapse.

Animals↗

PubMed2003

Thr123 of rat G-substrate contributes to its action as a protein phosphatase inhibitor.

Rat G-substrate cDNA was isolated from a cerebellar library and characterized. The deduced amino acid sequence of rat G-substrate contained two putative phosphorylation sites for PKG at Thr72 and Thr123; the amino acid sequences (KPRRKDT(p)PA) around these sites are conserved in human, mouse and rabbit. G-substrate phosphorylated by PKG inhibited the catalytic subunits of both protein phosphatase-1 (IC(50) 14.1 nM) and -2A (IC(50) 5.9 nM). Mutation of Thr123 (site 2) to Ala significantly reduced the inhibition of both PP-1 and PP-2A, while mutation of Thr72 (site 1) to Ala had little effect on inhibitory activity. In situ hybridization analysis revealed that G-substrate mRNA was localized exclusively in cerebellar Purkinje cells. Immunoperoxidase staining showed that in Purkinje cells, G-substrate was present in somata, dendrites and axons. In rat cerebellar slices, activation of PKG with a nitric oxide (NO) donor, NOR3, or 8-Br-cGMP, increased phosphorylation of G-substrate, as demonstrated with a phosphorylation-specific antibody. These results characterize further the inhibition of PP-1 and PP-2A by phospho-G-substrate, and demonstrate its physiological phosphorylation in rat Purkinje cells.

Amino Acid Sequence↗

PubMed2003

A novel ubiquitin-specific protease, synUSP, is localized at the post-synaptic density and post-synaptic lipid raft.

Recent reports suggest an important role for protein ubiquitination in synaptic plasticity. We cloned, from the rat brain, a novel gene that encoded an ubiquitin-specific protease (USP), and termed this protein synaptic ubiquitin-specific protease (synUSP, GenBankTM Accession no. AB073880). The homologous human gene was mapped to a locus on chromosome 1p36.12. The deduced synUSP protein consisted of 1036 amino acids, and possessed an ubiquitin-like domain at the C-terminus, Cys- and His-boxes, leucine zipper motifs, and six amino acid-repeats of L/ILCPHG. The protein possessed de-ubiquitinating activity toward a model substrate, as expected from its sequence. The protein of 125 kDa was present in the rat brain; in particular, it was enriched in the post-synaptic density and the dendritic lipid raft fractions. The immunostaining of cortical neurons confirmed the post-synaptic localization. The mRNA for synUSP was localized to dendrites, as well as somas, of neuronal cells. Thus, both the mRNA and the protein were localized in the post-synaptic compartments. These results suggest a regulatory mechanism for the ubiquitin-related system at the post-synaptic sites.

Amino Acid Sequence↗

PubMed2002

Glial protein S100B modulates long-term neuronal synaptic plasticity.

Glial cells are traditionally regarded as elements for structural support and ionic homeostasis, but have recently attracted attention as putative integral elements of the machinery involved in synaptic transmission and plasticity. Here, we demonstrate that calcium-binding protein S100B, which is synthesized in considerable amounts in astrocytes (a major glial cell subtype), modulates long-term synaptic plasticity. Mutant mice devoid of S100B developed normally and had no detectable abnormalities in the cytoarchitecture of the brain. These mutant mice, however, had strengthened synaptic plasticity as identified by enhanced long-term potentiation (LTP) in the hippocampal CA1 region. Perfusion of hippocampal slices with recombinant S100B proteins reversed the levels of LTP in the mutant slices to those of the wild-type slices, indicating that S100B might act extracellularly. In addition to enhanced LTP, mutant mice had enhanced spatial memory in the Morris water maze test and enhanced fear memory in the contextual fear conditioning. The results indicate that S100B is a glial modulator of neuronal synaptic plasticity and strengthen the notion that glial-neuronal interaction is important for information processing in the brain.

Animals↗

PubMedDate not supplied

Targeted DNA transfection into the mouse central nervous system using laser-induced stress waves.

We investigated the feasibility of gene transfer into the mouse central nervous system (CNS) by applying nanosecond pulsed laser-induced stress waves (LISWs). Intraventricular or hippocampal injection of a reporter gene [enhanced green fluorescent protein (EGFP)] followed by application of LISWs showed this method to be efficient in the CNS of newborn and adult mice. Cells expressing EGFP reside at least 3.5 mm from the surface of the tissue, while no apparent damage was detected. Additionally, expression of EGFP was limited to the area that was exposed to LISWs. Using this method, the formulation of plasmid DNA by cationic transfer reagent polyethylenimine proved to be effective for improving transfer efficiency into the CNS.

Animals↗