Details of a Researcher - Yuzaki, Michisuke

Papers - Yuzaki, Michisuke

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The δ2 'ionotropic' glutamate receptor functions as a non-ionotropic receptor to control cerebellar synaptic plasticity

Kakegawa Wataru, Kohda Kazuhisa, Yuzaki Michisuke

Journal of Physiology 584 ( 1 ) 89 - 96 2007.10

ISSN 0022-3751

The δ2 glutamate receptor (GluRδ2) belongs to the ionotropic glutamate receptor (iGluR) family and plays a crucial role in the induction of cerebellar long-term depression (LTD), a form of synaptic plasticity underlying motor learning. Nevertheless, the mechanisms by which GluRδ2 regulates cerebellar LTD have remained elusive. Because a mutation occurring in lurcher mice causes continuous GluRδ2 channel activity that can be abolished by 1-naphtylacetylspermine (NASP), a channel blocker for Ca²⁺- permeable iGluRs, GluRδ2 is thought to function as an ion channel. Here, we introduced a mutant GluR δ 2 transgene, in which the putative channel pore was disrupted, into GluR δ2 -null Purkinje cells using a virus vector. Surprisingly and similar to the effect of the wild-type GluR δ2 transgene, the mutant GluR δ2 completely rescued the abrogated LTD in GluR δ2-null mice. Furthermore, NASP did not block LTD induction in wild-type cerebellar slices. These results indicate that GluRδ2, a member of the iGluR family, does not serve as a channel in the regulation of LTD induction. © 2007 The Authors. Journal compilation © 2007 The Physiological Society.

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The Lurcher mouse

Vogel Michael W., Caston Jean, Yuzaki Michisuke, Mariani Jean

Brain Research 1140 ( 1 ) 4 - 18 2007.04

ISSN 0006-8993

The Lurcher mouse was first discovered in 1954 as a spontaneously occurring autosomal dominant mutation that caused the degeneration of virtually all cerebellar Purkinje cells and most olivary neurons and granule cells. More recent molecular studies revealed that Lurcher is a gain of function mutation in the δ2 glutamate receptor (GluRδ2) that converts an alanine to threonine in the highly conserved third hydrophobic segment of GluRδ2. The mutation converts the receptor into a constitutively leaky cation channel. The GluRδ2 receptor is predominantly expressed in cerebellar Purkinje cells and in the heterozygous Lurcher mutant (+/Lc). Purkinje cells die due to the mutation in the GluRδ2 receptor, while olivary neurons and granule cells degenerate due to the loss of their Purkinje cell targets. The purpose of the review is to provide highlights from 5 decades of research on the Lurcher mutant that have provided insights into the developmental mechanisms that regulate cell number during development, cerebellar pattern formation, cerebellar physiology, and the role of the cerebellum in CNS function. © 2005 Elsevier B.V. All rights reserved.

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Ho15J-A new hotfoot allele in a hot spot in the gene encoding the δ2 glutamate receptor

Motohashi Junko, Kakegawa Wataru, Yuzaki Michisuke

Brain Research 1140 ( 1 ) 153 - 160 2007.04

ISSN 0006-8993

Hotfoot, a recessive mouse mutation characterized by ataxia and jerky movements of the hindlimbs, is caused by various mutations in the gene (Grid2) encoding the δ2 glutamate receptor (GluRδ2). So far, at least 20 alleles, arising either spontaneously or through the random insertion of transgenes, have been described. Interestingly, most hotfoot mutants have deletions of one or more exons coding for portions of the most amino-terminal domain of GluRδ2. However, because live mice colonies are no longer available for most hotfoot mutants, the possibility that the loss of a part of an intron might affect the splicing of other exons or the general efficiency of transcription could not be ruled out. Here, we report that a newly identified hotfoot mutant, ho15J, was caused by an intragenic deletion of the Grid2 gene, which indeed resulted in a new type of 52-amino-acid deletion in the most amino-terminal domain of GluRδ2. Like GluRδ2 proteins in ho4J mutants, GluRδ2 proteins in ho15J mice were retained in the soma of Purkinje cells, where they were degraded. Long-term depression, a form of synaptic plasticity underlying information storage in the cerebellum, was abrogated, and ho15J mice showed severe motor discoordination on rotarod tests. The agreement between the PCR results for genomic DNA and the RT-PCR results for the ho15J allele supports the view that PCR analyses of grid2 genomic DNA can predict alterations in mRNA and protein. In addition, the present findings underscore the importance of the most amino-terminal domain in GluRδ2 signaling and cerebellar functions. © 2006 Elsevier B.V. All rights reserved.

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Ca²⁺ permeability of the channel pore is not essential for the δ2 glutamate receptor to regulate synaptic plasticity and motor coordination

Kakegawa Wataru, Miyazaki Taisuke, Hirai Hirokazu, Motohashi Junko, Mishina Masayoshi, Watanabe Masahiko, Yuzaki Michisuke

Journal of Physiology 579 ( 3 ) 729 - 735 2007.03

ISSN 0022-3751

The δ2 glutamate receptor (GluRδ2) plays a crucial role in cerebellar functions; mice with a disrupted GluR δ 2 gene (GluR δ 2^-/-) display impaired synapse formation and abrogated long-term depression (LTD). However, the mechanisms by which GluRδ2 functions have remained unclear. Because a GluRδ2 mutation in lurcher mice causes channel activities characterized by Ca²⁺ permeability, GluRδ2 was previously suggested to serve as a Ca²⁺-permeable channel in Purkinje cells. To test this hypothesis, we introduced a GluRδ2 transgene, which had a mutation (Gln618Arg) in the putative channel pore, into GluRδ2^-/- mice. Interestingly, the mutant transgene rescued the major functional and morphological abnormalities of GluRδ2^-/- Purkinje cells, such as enhanced paired-pulse facilitation, impaired LTD at parallel fibre synapses, and sustained innervation by multiple climbing fibres. These results indicate that the conserved glutamine residue in the channel pore, which is crucial for all Ca²⁺-permeable glutamate receptors, is not essential for the function of GluRδ2. © 2007 The Authors. Journal compilation © 2007 The Physiological Society.

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Impaired cerebellar development and function in mice lacking CAPS2, a protein involved in neurotrophin release

Sadakata Tetsushi, Kakegawa Wataru, Mizoguchi Akira, Washida Miwa, Katoh-Semba Ritsuko, Shutoh Fumihiro, Okamoto Takehito, Nakashima Hisako, Kimura Kazushi, Tanaka Mika, Sekine Yukiko, Itohara Shigeyoshi, Yuzaki Michisuke, Nagao Soichi, Furuichi Teiichi

Journal of Neuroscience 27 ( 10 ) 2472 - 2482 2007.03

ISSN 0270-6474

Ca²⁺-dependent activator protein for secretion 2 (CAPS2/CADPS2) is a secretory granule-associated protein that is abundant at the parallel fiber terminals of granule cells in the mouse cerebellum and is involved in the release of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), both of which are required for cerebellar development. The human homolog gene on chromosome 7 is located within susceptibility locus 1 of autism, a disease characterized by several cerebellar morphological abnormalities. Here we report that CAPS2 knock-out mice are deficient in the release of NT-3 and BDNF, and they consequently exhibit suppressed phosphorylation of Trk receptors in the cerebellum; these mice exhibit pronounced impairments in cerebellar development and functions, including neuronal survival, differentiation and migration of postmitotic granule cells, dendritogenesis of Purkinje cells, lobulation between lobules VI and VII, structure and vesicular distribution of parallel fiber-Purkinje cell synapses, paired-pulse facilitation at parallel fiber-Purkinje cell synapses, rotarod motor coordination, and eye movement plasticity in optokinetic training. Increased granule cell death of the external granular layer was noted in lobules VI-VII and IX, in which high BDNF and NT-3 levels are specifically localized during cerebellar development. Therefore, the deficiency of CAPS2 indicates that CAPS2-mediated neurotrophin release is indispensable for normal cerebellar development and functions, including neuronal differentiation and survival, morphogenesis, synaptic function, and motor leaning/control. The possible involvement of the CAPS2 gene in the cerebellar deficits of autistic patients is discussed. Copyright © 2007 Society for Neuroscience.

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The extreme C-terminus of GluRδ2 is essential for induction of long-term depression in cerebellar slices

Kohda Kazuhisa, Kakegawa Wataru, Matsuda Shinji, Nakagami Ryoichi, Kakiya Naomasa, Yuzaki Michisuke

European Journal of Neuroscience 25 ( 5 ) 1357 - 1362 2007.03

ISSN 0953-816X

Long-term depression (LTD) of parallel fibre (PF)-Purkinje cell synapses in the cerebellum is recognized as a cellular substrate of motor learning. Although the δ2 glutamate receptor (GluRδ2) has been shown to be crucial for LTD, the mechanisms by which GluRδ2 functions remain elusive. In this study, we developed a virus vector-based gene transfer approach to rescue impaired LTD in GluRδ2-null Purkinje cells in cerebellar slice preparations. We demonstrated that LTD was restored in GluRδ2-null Purkinje cells transduced with wild-type but not with mutant GluRδ2, which lacked the PDZ-ligand domain in the C-terminus. Immunohistochemical analysis revealed no difference in expression levels or spine localization patterns between virally introduced wild-type and mutant GluRδ2 proteins. Similarly, LTD was abrogated in Purkinje cells that had been acutely perfused with peptides, hampering the interaction of GluRδ2 with PDZ proteins such as PSD-93, PTPMEG and S-SCAM but not with delphilin. Together, these results indicate that PDZ proteins that bind to the C-terminus of GluRδ2 are not essential for localizing GluRδ2 at synapses but are crucial for conveying signals necessary for the induction of LTD. © The Authors (2007).

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Erratum

Ito-Ishida Aya, Kakegawa Wataru, Yuzaki Michisuke

European Journal of Neuroscience 25 ( 5 ) 1617 2007.03

ISSN 0953-816X

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Characterization of a transneuronal cytokine family Cbln - Regulation of secretion by heteromeric assembly

Iijima Takatoshi, Miura Eriko, Matsuda Keiko, Kamekawa Yuichi, Watanabe Masahiko, Yuzaki Michisuke

European Journal of Neuroscience 25 ( 4 ) 1049 - 1057 2007.02

ISSN 0953-816X

Cbln1, a member of the C1q and tumor necrosis factor superfamily, plays crucial roles as a cerebellar granule cell-derived transneuronal regulator of synapse integrity and plasticity in Purkinje cells. Although other Cbln family members, Cbln2-Cbln4, have distinct spatial and temporal patterns of expression throughout the CNS, their biochemical and biological properties have remained largely uncharacterized. Here, we demonstrated that in mammalian heterologous cells, Cbln2 and Cbln4 were secreted as N-linked glycoproteins, like Cbln1. In contrast, despite the presence of a functional signal sequence, Cbln3 was not secreted when expressed alone but was retained in the endoplasmic reticulum (ER) or cis-Golgi because of its N-terminal domain. All members of the Cbln family formed not only homomeric but also heteromeric complexes with each other in vitro. Accordingly, when Cbln1 and Cbln3 were co-expressed in heterologous cells, a proportion of the Cbln1 proteins was retained in the ER or cis-Golgi; conversely, some Cbln3 proteins were secreted together with Cbln1. Similarly, in wild-type granule cells expressing Cbln1 and Cbln3, Cbln3 proteins were partially secreted and reached postsynaptic sites on Purkinje cell dendrites, while Cbln3 was almost completely degraded in cbln1-null granule cells. These results indicate that like Cbln1, Cbln2 and Cbln4 may also serve as transneuronal regulators of synaptic functions in various brain regions. Furthermore, heteromer formation between Cbln1 and Cbln3 in cerebellar granule cells may modulate each other's trafficking and signaling pathways; similarly, heteromerization of other Cbln family proteins may also have biological significance in other neurons. © The Authors (2007).

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[Life of glutamate receptor--new mechanisms controlling number and location in neurons].

Yuzaki M

Seikagaku. The Journal of Japanese Biochemical Society 79 ( 1 ) 16 - 27 2007.01

ISSN 0037-1017

A life of glutamate receptor-New mechanisms controlling its number and location in neurons

Yuzaki Michisuke

Seikagaku 79 ( 1 ) 16 - 27 2007

ISSN 0037-1017

Characterization of the δ2 glutamate receptor-binding protein delphilin

Matsuda Keiko, Matsuda Shinji, Gladding Clare M., Yuzaki Michisuke

Journal of Biological Chemistry 281 ( 35 ) 25577 - 25587 2006.09

ISSN 0021-9258

The glutamate receptor δ2 (GluR δ2) is predominantly expressed at parallel fiber-Purkinje cell postsynapses and plays crucial roles in synaptogenesis and synaptic plasticity. Although the mechanism by which GluRδ2 functions remains unclear, its lack of channel activity and its role in controlling the endocytosis of δ-amino-3-hydroxy-5-methyl-4- isoxazolepropionate (AMPA) receptors have suggested that GluRδ2 may convey signals by interacting with intracellular signaling molecules. Among several proteins that interact with GluRδ2, delphilin is unique in that it is selectively expressed at parallel fiber-Purkinje cell synapses and that, in addition to a single PDZ domain, it contains a formin homology domain that is thought to regulate actin dynamics. Here, we report a new isoform of delphilin, designated as L-delphilin, that has alternatively spliced N-terminal exons encoding an additional PDZ domain. Although original delphilin, designated S-delphilin, was palmitoylated at the N terminus, this region was spliced out in Ldelphilin. As a result, S-delphilin was associated with plasma membranes in COS cells and dendritic spines in hippocampal neurons, whereas L-delphilin formed clusters in soma and dendritic shafts. In addition, S-delphilin, but not L-delphilin, facilitated the expression of GluRδ2 on the cell surface. These results indicate that, like PSD-95 and GRIP/ABP, delphilin isoforms with differential palmitoylation and clustering capabilities may provide two separate intracellular and surface GluRδ2 pools and may control GluRδ2 signaling in Purkinje cells. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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ERK1/2 but not p38 MAP kinase is essential for the long-term depression in mouse cerebellar slices

Ito-Ishida Aya, Kakegawa Wataru, Yuzaki Michisuke

European Journal of Neuroscience 24 ( 6 ) 1617 - 1622 2006.09

ISSN 0953-816X

Mitogen-activated protein kinase (MAPK) cascade is essential for synaptic plasticity and learning. In the hippocampus, three different MAPK subfamilies, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK and c-Jun NH ₂-terminal protein kinase (JNK), selectively regulate activity-dependent glutamate receptor trafficking during long-term potentiation (LTP), long-term depression (LTD), and depotentiation after LTP, respectively. Although LTP and LTD at cerebellar parallel fibre (PF)-Purkinje cell synapses are thought to be controlled by glutamate receptor trafficking, the involvement of MAPK subfamilies has not been systemically studied in cerebellar slice preparations. To clarify the role of the MAPK cascade in cerebellar LTD, we performed biochemical and electrophysiological analyses using ICR mouse cerebellar slices. Immunoblot analyses using phosphorylation-specific antibodies for MAPKs revealed that among the three MAPKs, ERK1/2 was specifically activated by phorbol ester, which could induce LTD in cerebellar slices. In addition, U0126, a specific inhibitor of the MAPK kinase-ERK1/2 pathway, abrogated the induction of LTD in cerebellar slices, whereas SB203580 and SP600125, specific inhibitors of p38 MAPK and JNK, respectively, had no effect. Although metabotropic glutamate receptor 1 (mGluR1) has been suggested as a possible downstream target of ERK1/2 in cell-culture preparations, mGluR1-activated slow excitatory postsynaptic currents (EPSCs) were not affected by U0126 treatment in slices. These findings indicate that unlike hippocampal LTD mediated by p38 MAPK, glutamate receptor trafficking during cerebellar LTD was regulated by a distinct mechanism involving ERK1/2 in slice preparations. © The Authors (2006).

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Distinct expression of Cbln family mRNAs in developing and adult mouse brains

Miura Eriko, Iijima Takatoshi, Yuzaki Michisuke, Watanabe Masahiko

European Journal of Neuroscience 24 ( 3 ) 750 - 760 2006.08

ISSN 0953-816X

Cbln1 belongs to the C1q and tumour necrosis factor superfamily, and plays crucial roles as a cerebellar granule cell-derived transneuronal regulator for synapse integrity and plasticity in Purkinje cells. Although Cbln2-Cbln4 are also expressed in the brain and could form heteromeric complexes with Cbln1, their precise expressions remain unclear. Here, we investigated gene expression of the Cbln family in developing and adult C57BL mouse brains by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and high-resolution in situ hybridization (ISH) analyses. In the adult brain, spatial patterns of mRNA expression were highly differential depending on Cbln subtypes. Notably, particularly high levels of Cbln mRNAs were expressed in some nuclei and neurons, whereas their postsynaptic targets often lacked or were low for any Cbln mRNAs, as seen for cerebellar granule cells/Purkinje cells, entorhinal cortex/hippocampus, intralaminar group of thalamic nuclei/caudate-putamen, and dorsal nucleus of the lateral lemniscus/central nucleus of the inferior colliculus. In the developing brain, Cbln1, 2, and 4 mRNAs appeared as early as embryonic day 10-13, and exhibited transient up-regulation during the late embryonic and neonatal periods. For example, Cbln2 mRNA was expressed in the cortical plate of the developing neocortex, displaying a high rostromedial to low caudolateral gradient. In contrast, Cbln3 mRNA was selective to cerebellar granule cells throughout development, and its onset was as late as postnatal day 7-10. These results will provide a molecular-anatomical basis for future studies that characterize roles played by the Cbln family. © The Authors (2006).

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A new motif necessary and sufficient for stable localization of the δ2 glutamate receptors at postsynaptic spines

Matsuda Shinji, Matsuda Keiko, Yuzaki Michisuke

Journal of Biological Chemistry 281 ( 25 ) 17501 - 17509 2006.06

ISSN 0021-9258

The number of each subclass of ionotropic glutamate receptors (iGluRs) at the spines is differentially regulated either constitutively or in a neuronal activity-dependent manner. The δ2 glutamate receptor (GluRδ2) is abundantly expressed at the spines of Purkinje cell dendrites and controls synaptic plasticity in the cerebellum. To obtain clues to the trafficking mechanism of the iGluRs, we expressed wild-type or mutant GluRδ2 in cultured hippocampal and Purkinje neurons and analyzed their intracellular localization using immunocytochemical techniques. Quantitative analysis revealed that deletion of the 20 amino acids at the center of the C terminus (region E) significantly reduced the amount of GluRδ2 protein at the spines in both types of neurons. This effect was partially antagonized by the inhibition of endocytosis by high dose sucrose treatment or coexpression of dominant negative dynamin. In addition, mutant GluRδ2 lacking the E region (GluRδ2^ΔE), but not wild-type GluRδ2, was found to colocalize with the endosomal markers Rab4 and Rab7. Moreover, the antibody-feeding assay revealed that GluRδ2^ΔE was internalized more rapidly than GluRδ2^wt. These results indicate that the E region (more specifically, a 12-amino-acid-long segment of the E2 region) is necessary for rendering GluRδ2 resistant to endocytosis from the cell surface at the spines. Furthermore, insertion of the E2 region alone into the C terminus of the GluR1 subtype of iGluRs was sufficient to increase the amount of GluR1 proteins in the spines. Therefore, we propose that the E2 region of GluRδ2 is necessary, and also sufficient, to inhibit endocytosis of the receptor from postsynaptic membranes. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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A mechanism underlying AMPA receptor trafficking during cerebellar long-term potentiation

Kakegawa Wataru, Yuzaki Michisuke

Proceedings of the National Academy of Sciences of the United States of America 102 ( 49 ) 17846 - 17851 2005.12

ISSN 0027-8424

Long-term potentiation (LTP) is mediated by the activity-driven delivery of GluR1 glutamate receptors via Ca²⁺/calmodulin-dependent protein kinase II activity in various brain regions. Recently, postsynaptic LTP was shown to be induced at parallel fiber-Purkinje cell synapses by stimulating the parallel fibers at 1 Hz or applying a NO donor. Here, we demonstrate that NO-evoked postsynaptic LTP in mice cerebellum was blocked by botulinum toxin and enhanced by prior treatment with phorbol ester, which is known to induce GluR2 endocytosis. Interestingly, such LTP was not affected by a Ca ²⁺/calmodulin-dependent protein kinase II inhibitor or a peptide binding to a protein interacting with C kinase 1, but was blocked by a peptide binding to N-ethylmaleimide-sensitive factor, which specifically binds to GluR2. Therefore, although the synaptic incorporation of GluR2 has been reported to be a constitutive pathway, NO-induced postsynaptic LTP in Purkinje cells is likely mediated by a pathway involving N-ethylmaleimide-sensitive factor-dependent GluR2 trafficking. © 2005 by The National Academy of Sciences of the USA.

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Hzf protein regulates dendritic localization and BDNF-induced translation of type 1 inositol 1,4,5-trisphosphate receptor mRNA

Iijima Takatoshi, Imai Takao, Kimura Yuki, Bernstein Alan, Okano Hirotaka James, Yuzaki Michisuke, Okano Hideyuki

Proceedings of the National Academy of Sciences of the United States of America 102 ( 47 ) 17190 - 17195 2005.11

ISSN 0027-8424

The localization of certain mRNAs to dendrites and their local translation in synaptic regions are proposed to be involved in certain aspects of synaptic plasticity. A cis-acting element within the 3′ untranslated region (3′ UTR) of the targeted mRNAs, which is bound by a trans-acting RNA-binding protein, controls the dendritic mRNA localization. Here, we identified hematopoietic zinc finger (Hzf) as a trans-acting factor that regulates the dendritic mRNA localization of the type 1 inositol 1,4,5-trisphosphate receptor (IP₃RI), a dendritically localized mRNA in cerebellar Purkinje cells, via binding to the 3′ UTR. In Hzf-deficient mice, the dendritic localization of IP₃RI mRNA and brain-derived neurotrophic factor-induced IP₃RI protein synthesis in the cerebellum were impaired. These findings suggest that Hzf is an RNA-binding protein that controls the dendritic mRNA localization and activity-dependent translation of IP₃RI, and may be involved in some aspects of synaptic plasticity. © 2005 by The National Academy of Sciences of the USA.

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Cbln1 is essential for synaptic integrity and plasticity in the cerebellum

Hirai Hirokazu, Pang Zhen, Bao Dashi, Miyazaki Taisuke, Li Leyi, Miura Eriko, Parris Jennifer, Rong Yongqi, Watanabe Masahiko, Yuzaki Michisuke, Morgan James I.

Nature Neuroscience 8 ( 11 ) 1534 - 1541 2005.11

ISSN 1097-6256

Cbln1 is a cerebellum-specific protein of previously unknown function that is structurally related to the C1q and tumor necrosis factor families of proteins. We show that Cbln1 is a glycoprotein secreted from cerebellar granule cells that is essential for three processes in cerebellar Purkinje cells: the matching and maintenance of pre- and postsynaptic elements at parallel fiber-Purkinje cell synapses, the establishment of the proper pattern of climbing fiber-Purkinje cell innervation, and induction of long-term depression at parallel fiber-Purkinje cell synapses. Notably, the phenotype of cbln1-null mice mimics loss-of-function mutations in the orphan glutamate receptor, GluRδ2, a gene selectively expressed in Purkinje neurons. Therefore, Cbln1 secreted from presynaptic granule cells may be a component of a transneuronal signaling pathway that controls synaptic structure and plasticity. © 2005 Nature Publishing Group.

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Induction of long-term depression and phosphorylation of the δ2 glutamate receptor by protein kinase C in cerebellar slices

Kondo Tetsuro, Kakegawa Wataru, Yuzaki Michisuke

European Journal of Neuroscience 22 ( 7 ) 1817 - 1820 2005.10

ISSN 0953-816X

The phosphorylation of ionotropic glutamate receptors (iGluRs) by protein kinases plays a crucial role in synaptic plasticity. In the cerebellum, protein kinase C (PKC) activation is required for the induction of long-term depression (LTD) at parallel fibre-Purkinje cell synapses. Although δ2 glutamate receptors (GluRδ2), expressed predominantly in Purkinje cells, are essential for cerebellar LTD, little is known about the mechanism by which GluRδ2 participates in LTD or its relationship with PKC activation pathways. We found that a PKC activator, phorbol ester, induced postsynaptic LTD in Purkinje cells in mouse cerebellar slice preparations without significantly changing the presynaptic properties. Under this condition, the GluRδ2 prepared from the cerebellar slices was significantly phosphorylated. Indeed, the C-terminus of the GluRδ2 fused with glutathione-S-transferase (GST) was directly phosphorylated by purified PKC at a specific serine residue. In addition, two-dimensional phosphopeptide mapping analysis indicated that the major phosphorylation site of the GST-fusion protein containing the C-terminus of GluRδ2 was identical to that of GluRδ2 prepared from cerebellar slices. Therefore, GluRδ2 is phosphorylated by PKC in vitro and by an LTD-inducing stimulus in slice preparations. Because this region of GluRδ2 is known to associate with certain intracellular molecules, the PKC phosphorylation status of the C-terminus of GluRδ2 may be involved in new signaling pathways during LTD. © Federation of European Neuroscience Societies.

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Potential functional neural repair with grafted neural stem cells of early embryonic neuroepithelial origin

Uchida Koichi, Momiyama Toshihiko, Okano Hideyuki, Yuzaki Michisuke, Koizumi Amane, Mine Yutaka, Kawase Takeshi

Neuroscience Research 52 ( 3 ) 276 - 286 2005.07

ISSN 0168-0102

The fate of grafted neuroepithelial stem cells in the normal mature brain environment was assessed both morphologically and electrophysiologically to confirm their feasibility in the functional repair of damaged neural circuitry. The neuroepithelial stem cells were harvested from the mesencephalic neural plate of transgenic green fluorescence protein-carrying rat embryos, and implanted into the normal adult rat striatum. The short- and long-term differentiation pattern of donor-derived cells was precisely monitored immunohistochemically. The functional abilities of the donor-derived cells and communication between them and the host were investigated using host-rat brain slices incorporating the graft with whole-cell patch-clamp recording. Vigorous differentiation of the neuroepithelial stem cells into mostly neurons was noted in the short-term with positive staining for tyrosine hydroxylase, suggesting that the donor-derived cells were exclusively following their genetically programmed fate, together with gamma-aminobutyric acid (GABA) and glutamate expression. In the long-term, the large number of donor-derived neurons was sustained, but the staining pattern showed expression of dopamine- and adenosine 3′:5′-monophosphate-regulated phosphoprotein 32, suggesting that some neurons were following environmental cues, together with the appearance of some cholinergic neurons. Some donor-derived astrocytes were also seen in the graft. Many action potentials indicating the presence of both dopaminergic and non-dopaminergic patterns could be elicited and recorded in the donor-derived neurons in addition to spontaneous glutamatergic and GABAergic post-synaptic currents which were strongly shown to be of host origin. Neuroepithelial stem cells are therefore an attractive candidate as a source of donor material for intracerebral grafting in functional repair. © 2005 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

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Roles of the N-terminal domain on the function and quaternary structure of the ionotropic glutamate receptor

Matsuda Shinji, Kamiya Yoshinori, Yuzaki Michisuke

Journal of Biological Chemistry 280 ( 20 ) 20021 - 20029 2005.05

ISSN 0021-9258

The α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptors (iGluRs) mediates fast excitatory neurotransmission in the mammalian brain. Although the most N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain is suggested to play a role in the initial assembly of iGluR subunits, it is unclear how this domain is arranged and functions in intact iGluRs. Similarly, although recent crystallographic analyses indicate that the isolated ligand-binding lysine/arginine/ornithine-binding protein domain forms a 2-fold symmetric dimer, the subunit stoichiometry of intact iGluRs remains elusive. Here, we developed a new approach to address these issues. The LIVBP domain of the GIuR1 subunit of AMPA receptors was replaced by leucine-zipper peptides designed to form stable symmetric dimers, trimers, tetramers, or pentamers. All these mutant GluR1s were expressed in human embryonic kidney 293 cells and were transported to the cell surface as well as wild type GluR1. Functional and biochemical analyses indicated that these oligomerizing peptides specifically controlled the formation of the expected number of subunits in a channel complex. However, the channel function was only restored by the tetramer-forming peptide. Although the purified LIVBP domain of GluR1 formed a dimmer in solution, a dimer-forming peptide could not restore the function of GluR1. Moreover, a cross-linking assay indicated that four LIVBP domains are located in proximity to each other. These results suggest that the function of the LIVBP domain is not simply to form initial dimers but to adopt a conformation compatible with the overall tetrameric arrangement of subunits in intact AMPA receptors. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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