Chihiro Hisatsune

Characterization of Fyn-mediated tyrosine phosphorylation sites on GluRε2 (NR2B) subunit of the N-methyl-D-aspartate receptor

The N-methyl-d-aspartate (NMDA) receptors play critical roles in synaptic plasticity, neuronal development, and excitotoxicity. Tyrosine phosphorylation of NMDA receptors by Src-family tyrosine kinases such as Fyn is implicated in synaptic plasticity. To precisely address the roles of NMDA receptor tyrosine phosphorylation, we identified Fyn-mediated phosphorylation sites on the GluRε2 (NR2B) subunit of NMDA receptors. Seven out of 25 tyrosine residues in the C-terminal cytoplasmic region of GluRε2 were phosphorylated by Fyn in vitro. Of these 7 residues, Tyr-1252, Tyr-1336, and Tyr-1472 in GluRε2 were phosphorylated in human embryonic kidney fibroblasts when co-expressed with active Fyn, and Tyr-1472 was the major phosphorylation site in this system. We then generated rabbit polyclonal antibodies specific to Tyr-1472-phosphorylated GluRε2 and showed that Tyr-1472 of GluRε2 was indeed phosphorylated in murine brain using the antibodies. Importantly, Tyr-1472 phosphorylation was greatly reduced infyn mutant mice. Moreover, Tyr-1472 phosphorylation became evident when hippocampal long term potentiation started to be observed, and its magnitude became larger in murine brain. Finally, Tyr-1472 phosphorylation was significantly enhanced after induction of long term potentiation in the hippocampal CA1 region. These data suggest that Tyr-1472 phosphorylation of GluRε2 is important for synaptic plasticity.

Abnormal taste perception in mice lacking the type 3 inositol 1,4,5-trisphosphate receptor

Inositol 1,4,5-trisphosphate receptor (IP3R) is one of the important calcium channels expressed in the endoplasmic reticulum and has been shown to play crucial roles in various physiological phenomena. Type 3 IP3R is expressed in taste cells, but the physiological relevance of this receptor in taste perception in vivo is still unknown. Here, we show that mice lacking IP3R3 show abnormal behavioral and electrophysiological responses to sweet, umami, and bitter substances that trigger G-protein-coupled receptor activation. In contrast, responses to salty and acid tastes are largely normal in the mutant mice. We conclude that IP3R3 is a principal mediator of sweet, bitter, and umami taste perception and would be a missing molecule linking phospholipase C β2 to TRPM5 activation.

Osteoblasts induce Ca2+ oscillation-independent NFATc1 activation during osteoclastogenesis

Intercellular cross-talk between osteoblasts and osteoclasts is important for controlling bone remolding and maintenance. However, the precise molecular mechanism by which osteoblasts regulate osteoclastogenesis is still largely unknown. Here, we show that osteoblasts can induce Ca2+ oscillation-independent osteoclastogenesis. We found that bone marrow-derived monocyte/macrophage precursor cells (BMMs) lacking inositol 1,4,5-trisphosphate receptor type2 (IP3R2) did not exhibit Ca2+ oscillation or differentiation into multinuclear osteoclasts in response to recombinant receptor activator of NF-κB ligand/macrophage colony-stimulating factor stimulation. IP3R2 knockout BMMs, however, underwent osteoclastogenesis when they were cocultured with osteoblasts or in vivo in the absence of Ca2+ oscillation. Furthermore, we found that Ca2+ oscillation-independent osteoclastogenesis was insensitive to FK506, a calcineurin inhibitor. Taken together, we conclude that both Ca2+ oscillation/calcineurin-dependent and -independent signaling pathways contribute to NFATc1 activation, leading to efficient osteoclastogenesis in vivo.

Phosphorylation–dependent interaction of the N-methyl- d-aspartate receptor ε2 subunit with phosphatidylinositol 3-kinase

The NMDA receptors (NMDARs) are ion channels through which Ca2+ influx triggers various intracellular responses. Tyrosine phosphorylation of NMDARs regulates NMDA channel activities, which may be important in neuronal plasticity. The biological significance of the tyrosine phosphorylation events, however, differs among NMDAR subunits: tyrosine phosphorylation of NMDARε1 increases NMDA channel activities, but that of NMDARε2 does not. Since signal transductions from various cell surface receptors are mediated by protein–protein interaction through phosphotyrosine and the Src homology 2 (SH2) domain, we examined the possibility that phosphotyrosines in NMDARε2 contribute to the intracellular signalling events.

Protein 4.1N Is Required for Translocation of Inositol 1,4,5-Trisphosphate Receptor Type 1 to the Basolateral Membrane Domain in Polarized Madin-Darby Canine Kidney Cells ,

Protein 4.1N was identified as a binding molecule for the C-terminal cytoplasmic tail of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) using a yeast two-hybrid system. 4.1N and IP3R1 associate in both subconfluent and confluent Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell line. In subconfluent MDCK cells, 4.1N is distributed in the cytoplasm and the nucleus; IP3R1 is localized in the cytoplasm. In confluent MDCK cells, both 4.1N and IP3R1 are predominantly translocated to the basolateral membrane domain, whereas 4.1R, the prototypical homologue of 4.1N, is localized at the tight junctions (Mattagajasingh, S. N., Huang, S. C., Hartenstein, J. S., and Benz, E. J., Jr. (2000) J. Biol. Chem. 275, 30573–30585), and other endoplasmic reticulum marker proteins are still present in the cytoplasm. Moreover, the 4.1N-binding region of IP3R1 is necessary and sufficient for the localization of IP3R1 at the basolateral membrane domain. A fragment of the IP3R1-binding region of 4.1N blocks the localization of co-expressed IP3R1 at the basolateral membrane domain. These data indicate that 4.1N is required for IP3R1 translocation to the basolateral membrane domain in polarized MDCK cells.

Inositol 1,4,5-Trisphosphate Receptor Type 1 in Granule Cells, Not in Purkinje Cells, Regulates the Dendritic Morphology of Purkinje Cells through Brain-Derived Neurotrophic Factor Production

Here, we show that cultured Purkinje cells from inositol 1,4,5-trisphosphate receptor type 1 knock-out (IP3R1KO) mice exhibited abnormal dendritic morphology. Interestingly, despite the huge amount of IP3R1 expression in Purkinje cells, IP3R1 in granule cells, not in the Purkinje cells, was responsible for the shape of Purkinje cell dendrites. We also found that BDNF application rescued the dendritic abnormality of IP3R1KO Purkinje cells, and that the increase in BDNF expression in response to activation of AMPA receptor (AMPAR) and metabotropic glutamate receptor (mGluR) was impaired in IP3R1KO cerebellar granule cells. In addition, we observed abnormalities in the dendritic morphology of Purkinje cells and in the ultrastructure of parallel fiber–Purkinje cell (PF-PC) synapses in IP3R1KO mice in vivo. We concluded that activation of AMPAR and mGluR increases BDNF expression through IP3R1-mediated signaling in cerebellar granule cells, which contributes to the dendritic outgrowth of Purkinje cells intercellularly, possibly by modifying PF-PC synaptic efficacy.

A role of Disable-1 as a transcription factor in Reelin signaling

s / Neuroscience Research 58S (2007) S1–S244 S51 O2P-AØ4 A role of Disable-1 as a transcription factor in Reelin signaling Toshifumi Morimura1, Chihiro Hisatsune2,3, Mitsuharu Hattori4, Katsuhiko Mikoshiba2,3,5, Masaharu Ogawa1 1 Laboratory for Cell Culture Development, BSI, RIKEN, Saitama, Japan; 2 Division of Molecular Neurobiology, IMS, The University of Tokyo, Tokyo, Japan; 3 Laboratory for Developmental Neuroscience, BSI, RIKEN, Saitama, Japan; 4 Department of Biomedical Science, Graduate School of Pharmaceutical Science, Nagoya City University, Aichi, Japan; 5 ICORP-SORST, JST, Tokyo, Japan Reelin controls neuronal migration and positioning in the developing brain by inducing tyrosine phosphorylation of an intracellular adaptor protein, Disabled-1 (Dab1). We clarified that Dab1 tyrosine phosphorylation initiated the internalization of Reelin, and that Dab1 dissociated from Reelin receptors after their internalization. Furthermore, we found that Reelin induced the nuclear import of tyrosine-phosphorylated Dab1. In the nucleus, Dab1 bound to DNA and regulated gene expression in cooperation with a transcriptional co-activator to control neuronal migration. These results suggest that Reelin regulates correct neuronal migration and positioning by inducing the nuclear import of tyrosine-phosphorylated Dab1. Research funds: KAKENNHI (18780222) O2P-AØ5 The somatodendritic targeting of DNER requires transcytosis Tetsuko Fukuda, Seisuke Yokoyama, Mineko Kengaku Laboratory for Neural Cell Polarity, BSI, RIKEN, Wako, Japan Establishment of neuronal polarity depends on targeting of proteins into different plasma membrane domains, axons and dendrites. It has been shown that many dendritic proteins reach the somatodendritic membrane based on selective sorting and transport of carrier vesicles. Using cultured hippocampal neurons, we examined the trafficking pathways of DNER, a transmembrane protein specifically expressed in CNS dendrites. Mutations of DNER that increased surface expression disrupted its somatodendritic localization. Inhibition of endocytosis resulted in disruption of polarized distribution of DNER, indicating that somatodendritic targeting of DNER was dependent on endocytosis. DNER binds to a clathrin adaptor AP-2 via multiple sites. However, clathrin-dependent endocytosis only partly mediated the somatodendritic targeting of DNER. Instead, a clathrin independent endocytosis was also responsible for its somatodendritic distribution. Thus, selective retention mediated by clathrin-dependentand independent-endocytosis is critical for polarized targeting of a somatodendritic protein. O2P-AØ6 Microtubule dynamics regulating migration of cerebellar granule cells Hiroki Umeshima1, Tomoo Hirano2, Mineko Kengaku1 1 Laboratory for Neural Cell Polarity, RIKEN, BSI, Wako, Japan; 2 Department of Biophysics, Kyoto University, Kyoto, Japan During neuronal migration in the developing brain, it is thought that the centrosome precedes the nucleus and provides a cue for nuclear migration along the microtubules. In time-lapse imaging studies of radially migrating granule cells in mouse cerebellar slices, we observed that the movements of the nucleus and centrosome appeared to occur independently of each other. The nucleus often migrated ahead of the centrosome during its saltatory movement, negating the supposed role of the centrosome in pulling the nucleus. We observed the perinuclear structure consisting of dynamic and stable microtubules, neither of which was converged at the centrosome. In addition, stable microtubules showed the polarized distribution in contrast with the overall distribution of dynamic microtubules. Disruption or excess formation of stable microtubules attenuated nuclear migration, suggesting that polarized arrangement of microtubules is prerequisite for nuclear migration. O2P-AØ7 Functional analysis of mouse Mahya genes Naoya Ichikawa, Tatsuhiko Kadowaki Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan We identified one of the Urbilaterian genes, Mahya, which is specifically conserved between Hymenoptera and Deuterostome. It has been implicated in the higher order of brain functions enabling social behavior and cognitive ability. Mahya encodes a secretory protein with a follistatin-like domain, two immunoglobulin domains, and a C-terminal novel domain. Mouse Mahya genes (mMahya-1 and mMahya-2) are expressed in the olfactory bulb, hippocampus (CA3 region and dentate gyrus), and cerebellum (granule cells and Purkinje cells) of the adult brain. To understand the functions of mMahya genes in the brain functions we have generated KO mice for mMahya-1 and mMahya-2 in collaboration with the RIKEN CDB group. Both of homozygous KO mice are viable, and thus we are analyzing the structural changes in the brain areas and behaviors of these KO mice. O2P-BØ1 The signaling pathways involved in dendritic spine formation via Shc, phospho-tyrosine adaptor molecule Yoko Shiraisi-Yamaguchi1,2, Nozomu Mori2 1 JSPS, Tokyo, Japan; 2 Department of Anatomy and Neurobiology, Nagasaki University School of Medicine, Nagasaki, Japan Shc binds to phospho-tyrosine residues of the activated receptors. In central nervous system, N-Shc (neural Shc) is predominantly expressed and transmits neurotrophin signals from the TrkB receptor to Ras/MAPK pathway. Studies on the N-Shc knock out mice reveals a novel function of N-shc; N-Shc is also involved in the regulation of NMDA receptor functions and hippocampal synaptic plasticity. Since N-Shc also interacts with key regulators for actin-cytoskeletal rearrangements such as Crk and Grit (RICS), we then tested whether N-Shc is involved in the formation of synaptic architecture, dendritic spines. Following exogenously overexpression of N-Shc in primary cultured hippocampal neurons, the spine number was significantly reduced, and the effect was more severe when N-Shc/Crk cascade was disturbed. However, additive inhibition of N-Shc/Ras/MAPK cascade rescued the spine formation. Those results indicate that both N-Shc/Crk and N-Shc/Ras/MAPK pathways cooperatively regulate dendritic spine formation. Research funds: Grant-in-Aid for JSPS Fellows O2P-BØ2 Roles of monoacylglycerol lipase in the regulation of endocannabinoid signaling Yuki Hashimotodani1, Takako Ohno-Shosaku2, Masanobu Kano3 1 Department of Neurophysiology, Graduate School of Medicine, Osaka University, Osaka, Japan; 2 Department of Impair. Study, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan; 3 Department of Cellular Neuroscience, Graduate School of Medicine, Osaka University, Osaka, Japan Endogenous cannabinoids (eCBs) are released from postsynaptic neurons and retrogradely suppress synaptic transmission by acting on presynaptic CB1 cannabinoid receptors. 2-Arachidonoylglycerol (2-AG), a major molecule of eCBs, is degraded by monoacylglycerol lipase (MGL), which is expressed at presynaptic terminals. In this study, we investigated how MGL regulates eCB signaling at hippocampal inhibitory synapses. Inhibition of MGL resulted in a gradual suppression of basal IPSCs. This suppression was blocked by a CB1 antagonist and attenuated by inhibiting 2-AG synthesis, suggesting that 2-AG is constitutively released and degraded by MGL. MGL inhibition also prolonged the recovery of IPSCs from the suppression by exogenous 2-AG application or by eCB released by depolarization. These results indicate that presynaptic MGL plays an important role in both regulating basal eCB tone and terminating the retrograde eCB signal. Research funds: KAKENHI 17023021 17100004