Papers - Yuzaki, Michisuke

The C-terminal juxtamembrane region of the δ2 glutamate receptors controls its export from the endoplasmic reticulum

'MATSUDA SHINJI, HANNEN ROSALIND, MATSUDA KEIKO, YAMADA NOBUAKI, TUBBS THOMAS, YUZAKI MICHISUKE'

European Journal of Neuroscience 19   1683-1690 2004.02

Research paper (scientific journal), Joint Work, Accepted

Specific assembly with NR3B subunit controls surface expression and calcium permeability of NMDA receptors.

'MATSUDA KEIKO, FLETCHER MATT, KAMIYA YOSHINORI, YUZAKI MICHISUKE'

Journal of Neuroscience  ( 23 ) 10064-10073 2003

Research paper (scientific journal), Joint Work

Novel role of δ2-glutamate receptors in AMPA receptor trafficking and cerebellar learning

'HIRAI HIROKAZU, LAUNEY, THOMAS, MIKAWA SUMIKO, TORASHIMA TAKASHI, YANAGIHARA DAY, KASAURA TSUYOSHI, MIYAMOTO AKIHIRO, YUZAKI MICHISUKE'

Nature Neuroscience  ( 6 ) 869-876 2003

Research paper (scientific journal), Joint Work

A hot spot for hotfoot mutations in the gene encoding δ2 glutamate receptor.

'WANG YING, MATSUDA SHINJI, DREWS VALERIE, TORASHIMA TAKASHI, MEISLER MIRIAM H., YUZAKI MICHISUKE'

European Journal of Neuroscience  ( 17 ) 1581-1590 2003

Research paper (scientific journal), Joint Work

New insights into the structure and function of glutamate receptors - The orphan receptor δ2 reveals its family’s secrets.

YUZAKI MICHISUKE

The Keio Journal of Medicine  ( 52 ) 92-99 2003

Research paper (scientific journal), Single Work

記憶素子としてのグルタミン酸受容体

YUZAKI MICHISUKE

Clinical Neuroscience  ( 21 ) 1471 2003

Research paper (scientific journal), Single Work

記憶はどのようにして形成されるか？−最近の話題

YUZAKI MICHISUKE

慶應医学  ( 80 ) 131-139 2003

Research paper (scientific journal), Single Work

小脳の形成と機能における神経伝達物質受容体の役割-d2型グルタミン酸受容体を例として

YUZAKI MICHISUKE

脳の科学  ( 25 ) 563-570 2003

Research paper (scientific journal), Single Work

Abundant extrasynaptic expression of α3β4-containing nicotinic acetylcholine receptors in the medial habenula–interpeduncular nucleus pathway in mice

Tsuzuki A., Yamasaki M., Konno K., Miyazaki T., Takei N., Tomita S., Yuzaki M., Watanabe M.

Scientific Reports 14 ( 1 )  2024.12

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Nicotinic acetylcholine receptors (nAChRs) in the medial habenula (MHb)–interpeduncular nucleus (IPN) pathway play critical roles in nicotine-related behaviors. This pathway is particularly enriched in nAChR α3 and β4 subunits, both of which are genetically linked to nicotine dependence. However, the cellular and subcellular expression of endogenous α3β4-containing nAChRs remains largely unknown because specific antibodies and appropriate detection methods were unavailable. Here, we successfully uncovered the expression of endogenous nAChRs containing α3 and β4 subunits in the MHb–IPN pathway using novel specific antibodies and a fixative glyoxal that enables simultaneous detection of synaptic and extrasynaptic molecules. Immunofluorescence and immunoelectron microscopy revealed that both subunits were predominantly localized to the extrasynaptic cell surface of somatodendritic and axonal compartments of MHb neurons but not at their synaptic junctions. Immunolabeling for α3 and β4 subunits disappeared in α5β4-knockout brains, which we used as negative controls. The enriched and diffuse extrasynaptic expression along the MHb–IPN pathway suggests that α3β4-containing nAChRs may enhance the excitability of MHb neurons and neurotransmitter release from their presynaptic terminals in the IPN. The revealed distribution pattern provides a molecular and anatomical basis for understanding the functional role of α3β4-containing nAChRs in the crucial pathway of nicotine dependence.

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Neuronal DSCAM regulates the peri-synaptic localization of GLAST in Bergmann glia for functional synapse formation

Dewa K.i., Arimura N., Kakegawa W., Itoh M., Adachi T., Miyashita S., Inoue Y.U., Hizawa K., Hori K., Honjoya N., Yagishita H., Taya S., Miyazaki T., Usui C., Tatsumoto S., Tsuzuki A., Uetake H., Sakai K., Yamakawa K., Sasaki T., Nagai J., Kawaguchi Y., Sone M., Inoue T., Go Y., Ichinohe N., Kaibuchi K., Watanabe M., Koizumi S., Yuzaki M., Hoshino M.

Nature Communications 15 ( 1 )  2024.12

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In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.

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Lack of evidence for direct ligand-gated ion channel activity of GluD receptors

Itoh M., Piot L., Mony L., Paoletti P., Yuzaki M.

Proceedings of the National Academy of Sciences of the United States of America 121 ( 31 )  2024.07

ISSN  00278424

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Delta receptors (GluD1 and GluD2), members of the large ionotropic glutamate receptor (iGluR) family, play a central role in numerous neurodevelopmental and psychiatric disorders. The amino-terminal domain (ATD) of GluD orchestrates synapse formation and maturation processes through its interaction with the Cbln family of synaptic organizers and neurexin (Nrxn). The transsynaptic triad of Nrxn–Cbln–GluD also serves as a potent regulator of synaptic plasticity, at both excitatory and inhibitory synapses. Despite these recognized functions, there is still debate as to whether GluD functions as a "canonical" ion channel, similar to other iGluRs. A recent report proposes that the ATD of GluD2 imposes conformational constraints on channel activity; removal of this constraint by binding to Cbln1 and Nrxn, or removal of the ATD, reveals channel activity in GluD2 upon administration of glycine (Gly) and d-serine (d-Ser), two GluD ligands. We were able to reproduce currents when Gly or d-Ser was administered to clusters of heterologous human embryonic kidney 293 (HEK293) cells expressing Cbln1, GluD2 (or GluD1), and Nrxn. However, Gly or d-Ser, but also l-glutamate (l-Glu), evoked similar currents in naive (i.e., untransfected) HEK293 cells and in GluD2-null Purkinje neurons. Furthermore, no current was detected in isolated HEK293 cells expressing GluD2 lacking the ATD upon administration of Gly. Taken together, these results cast doubt on the previously proposed hypothesis that extracellular ligands directly gate wild-type GluD channels.

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Kainate receptors regulate synaptic integrity and plasticity by forming a complex with synaptic organizers in the cerebellum

Kakegawa W., Paternain A.V., Matsuda K., Aller M.I., Iida I., Miura E., Nozawa K., Yamasaki T., Sakimura K., Yuzaki M., Lerma J.

Cell Reports 43 ( 7 )  2024.07

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Kainate (KA)-type glutamate receptors (KARs) are implicated in various neuropsychiatric and neurological disorders through their ionotropic and metabotropic actions. However, compared to AMPA- and NMDA-type receptor functions, many aspects of KAR biology remain incompletely understood. Our study demonstrates an important role of KARs in organizing climbing fiber (CF)-Purkinje cell (PC) synapses and synaptic plasticity in the cerebellum, independently of their ion channel or metabotropic functions. The amino-terminal domain (ATD) of the GluK4 KAR subunit binds to C1ql1, provided by CFs, and associates with Bai3, an adhesion-type G protein-coupled receptor expressed in PC dendrites. Mice lacking GluK4 exhibit no KAR-mediated responses, reduced C1ql1 and Bai3 levels, and fewer CF-PC synapses, along with impaired long-term depression and oculomotor learning. Remarkably, introduction of the ATD of GluK4 significantly improves all these phenotypes. These findings demonstrate that KARs act as synaptic scaffolds, orchestrating synapses by forming a KAR-C1ql1-Bai3 complex in the cerebellum.

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The hidden face of GluD1 at inhibitory synapses

Itoh M., Yuzaki M.

Cell Research 34 ( 6 ) 405 - 406 2024.06

ISSN  10010602

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Activity-driven synaptic translocation of LGI1 controls excitatory neurotransmission

Cuhadar U., Calzado-Reyes L., Pascual-Caro C., Aberra A.S., Ritzau-Jost A., Aggarwal A., Ibata K., Podgorski K., Yuzaki M., Geis C., Hallerman S., Hoppa M.B., de Juan-Sanz J.

Cell Reports 43 ( 5 )  2024.05

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The fine control of synaptic function requires robust trans-synaptic molecular interactions. However, it remains poorly understood how trans-synaptic bridges change to reflect the functional states of the synapse. Here, we develop optical tools to visualize in firing synapses the molecular behavior of two trans-synaptic proteins, LGI1 and ADAM23, and find that neuronal activity acutely rearranges their abundance at the synaptic cleft. Surprisingly, synaptic LGI1 is primarily not secreted, as described elsewhere, but exo- and endocytosed through its interaction with ADAM23. Activity-driven translocation of LGI1 facilitates the formation of trans-synaptic connections proportionally to the history of activity of the synapse, adjusting excitatory transmission to synaptic firing rates. Accordingly, we find that patient-derived autoantibodies against LGI1 reduce its surface fraction and cause increased glutamate release. Our findings suggest that LGI1 abundance at the synaptic cleft can be acutely remodeled and serves as a critical control point for synaptic function.

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Human-induced pluripotent stem cell-derived neural stem/progenitor cell ex vivo gene therapy with synaptic organizer CPTX for spinal cord injury

Saijo Y., Nagoshi N., Kawai M., Kitagawa T., Suematsu Y., Ozaki M., Shinozaki M., Kohyama J., Shibata S., Takeuchi K., Nakamura M., Yuzaki M., Okano H.

Stem Cell Reports 19 ( 3 ) 383 - 398 2024.03

ISSN  22136711

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The transplantation of neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs) has shown promise in spinal cord injury (SCI) model animals. Establishing a functional synaptic connection between the transplanted and host neurons is crucial for motor function recovery. To boost therapeutic outcomes, we developed an ex vivo gene therapy aimed at promoting synapse formation by expressing the synthetic excitatory synapse organizer CPTX in hiPSC-NS/PCs. Using an immunocompromised transgenic rat model of SCI, we evaluated the effects of transplanting CPTX-expressing hiPSC-NS/PCs using histological and functional analyses. Our findings revealed a significant increase in excitatory synapse formation at the transplantation site. Retrograde monosynaptic tracing indicated extensive integration of transplanted neurons into the surrounding neuronal tracts facilitated by CPTX. Consequently, locomotion and spinal cord conduction significantly improved. Thus, ex vivo gene therapy targeting synapse formation holds promise for future clinical applications and offers potential benefits to individuals with SCI.

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Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains

Nonaka H., Sakamoto S., Shiraiwa K., Ishikawa M., Tamura T., Okuno K., Kondo T., Kiyonaka S., Susaki E.A., Shimizu C., Ueda H.R., Kakegawa W., Arai I., Yuzaki M., Hamachi I.

Proceedings of the National Academy of Sciences of the United States of America 121 ( 6 )  2024

ISSN  00278424

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Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse–chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

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Brain-Specific Angiogenesis Inhibitor 3 Is Expressed in the Cochlea and Is Necessary for Hearing Function in Mice

Saegusa C., Kakegawa W., Miura E., Aimi T., Mogi S., Harada T., Yamashita T., Yuzaki M., Fujioka M.

International Journal of Molecular Sciences 24 ( 23 )  2023.12

ISSN  16616596

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Mammalian auditory hair cells transduce sound-evoked traveling waves in the cochlea into nerve stimuli, which are essential for hearing function. Pillar cells located between the inner and outer hair cells are involved in the formation of the tunnel of Corti, which incorporates outer-hair-cell-driven fluid oscillation and basilar membrane movement, leading to the fine-tuned frequency-specific perception of sounds by the inner hair cells. However, the detailed molecular mechanism underlying the development and maintenance of pillar cells remains to be elucidated. In this study, we examined the expression and function of brain-specific angiogenesis inhibitor 3 (Bai3), an adhesion G-protein-coupled receptor, in the cochlea. We found that Bai3 was expressed in hair cells in neonatal mice and pillar cells in adult mice, and, interestingly, Bai3 knockout mice revealed the abnormal formation of pillar cells, with the elevation of the hearing threshold in a frequency-dependent manner. Furthermore, old Bai3 knockout mice showed the degeneration of hair cells and spiral ganglion neurons in the basal turn. The results suggest that Bai3 plays a crucial role in the development and/or maintenance of pillar cells, which, in turn, are necessary for normal hearing function. Our results may contribute to understanding the mechanisms of hearing loss in human patients.

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C1ql1-Bai3 signaling is necessary for climbing fiber synapse formation in mature Purkinje cells in coordination with neuronal activity

Aimi T., Matsuda K., Yuzaki M.

Molecular Brain 16 ( 1 )  2023.12

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Changes in neural activity induced by learning and novel environments have been reported to lead to the formation of new synapses in the adult brain. However, the underlying molecular mechanism is not well understood. Here, we show that Purkinje cells (PCs), which have established adult-type monosynaptic innervation by climbing fibers (CFs) after elimination of weak CFs during development, can be reinnervated by multiple CFs by increased expression of the synaptic organizer C1ql1 in CFs or Bai3, a receptor for C1ql1, in PCs. In the adult cerebellum, CFs are known to have transverse branches that run in a mediolateral direction without forming synapses with PCs. Electrophysiological, Ca2+-imaging and immunohistochemical studies showed that overexpression of C1ql1 or Bai3 caused these CF transverse branches to elongate and synapse on the distal dendrites of mature PCs. Mature PCs were also reinnervated by multiple CFs when the glutamate receptor GluD2, which is essential for the maintenance of synapses between granule cells and PCs, was deleted. Interestingly, the effect of GluD2 knockout was not observed in Bai3 knockout PCs. In addition, C1ql1 levels were significantly upregulated in CFs of GluD2 knockout mice, suggesting that endogenous, not overexpressed, C1ql1-Bai3 signaling could regulate the reinnervation of mature PCs by CFs. Furthermore, the effects of C1ql1 and Bai3 overexpression required neuronal activity in the PC and CF, respectively. C1ql1 immunoreactivity at CF-PC synapses was reduced when the neuronal activity of CFs was suppressed. These results suggest that C1ql1-Bai3 signaling may mediate CF synaptogenesis in mature PCs, potentially in concert with neuronal activity.

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Rab21 regulates caveolin-1-mediated endocytic trafficking to promote immature neurite pruning

Shikanai M., Ito S., Nishimura Y.V., Akagawa R., Fukuda M., Yuzaki M., Nabeshima Y.i., Kawauchi T.

EMBO Reports 24 ( 3 )  2023.03

ISSN  1469221X

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Transmembrane proteins are internalized by clathrin- and caveolin-dependent endocytosis. Both pathways converge on early endosomes and are thought to share the small GTPase Rab5 as common regulator. In contrast to this notion, we show here that the clathrin- and caveolin-mediated endocytic pathways are differentially regulated. Rab5 and Rab21 localize to distinct populations of early endosomes in cortical neurons and preferentially regulate clathrin- and caveolin-mediated pathways, respectively, suggesting heterogeneity in the early endosomes, rather than a converging point. Suppression of Rab21, but not Rab5, results in decreased plasma membrane localization and total protein levels of caveolin-1, which perturbs immature neurite pruning of cortical neurons, an in vivo-specific step of neuronal maturation. Taken together, our data indicate that clathrin- and caveolin-mediated endocytic pathways run in parallel in early endosomes, which show different molecular regulation and physiological function.

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Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution changes

Nonaka H., Mino T., Sakamoto S., Oh J.H., Watanabe Y., Ishikawa M., Tsushima A., Amaike K., Kiyonaka S., Tamura T., Radu Aricescu A., Kakegawa W., Miura E., Yuzaki M., Hamachi I.

Chem 9 ( 2 ) 523 - 540 2023.02

ISSN  24519308

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Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/excretion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein, we report a new method termed “fixation-driven chemical cross-linking of exogenous ligands (FixEL),” which traps and images exogenously administered molecules of interest (MOIs) in complex tissues. This method relies on protein-MOI interactions and chemical cross-linking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOIs such as the scaffold of PET tracer derivatives or drug-like small molecules.

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Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue

Ojima K., Kakegawa W., Yamasaki T., Miura Y., Itoh M., Michibata Y., Kubota R., Doura T., Miura E., Nonaka H., Mizuno S., Takahashi S., Yuzaki M., Hamachi I., Kiyonaka S.

Nature Communications 13 ( 1 )  2022.12

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Direct activation of cell-surface receptors is highly desirable for elucidating their physiological roles. A potential approach for cell-type-specific activation of a receptor subtype is chemogenetics, in which both point mutagenesis of the receptors and designed ligands are used. However, ligand-binding properties are affected in most cases. Here, we developed a chemogenetic method for direct activation of metabotropic glutamate receptor 1 (mGlu1), which plays essential roles in cerebellar functions in the brain. Our screening identified a mGlu1 mutant, mGlu1(N264H), that was activated directly by palladium complexes. A palladium complex showing low cytotoxicity successfully activated mGlu1 in mGlu1(N264H) knock-in mice, revealing that activation of endogenous mGlu1 is sufficient to evoke the critical cellular mechanism of synaptic plasticity, a basis of motor learning in the cerebellum. Moreover, cell-type-specific activation of mGlu1 was demonstrated successfully using adeno-associated viruses in mice, which shows the potential utility of this chemogenetics for clarifying the physiological roles of mGlu1 in a cell-type-specific manner.

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The complement C3-complement factor D-C3a receptor signalling axis regulates cardiac remodelling in right ventricular failure

Ito S., Hashimoto H., Yamakawa H., Kusumoto D., Akiba Y., Nakamura T., Momoi M., Komuro J., Katsuki T., Kimura M., Kishino Y., Kashimura S., Kunitomi A., Lachmann M., Shimojima M., Yozu G., Motoda C., Seki T., Yamamoto T., Shinya Y., Hiraide T., Kataoka M., Kawakami T., Suzuki K., Ito K., Yada H., Abe M., Osaka M., Tsuru H., Yoshida M., Sakimura K., Fukumoto Y., Yuzaki M., Fukuda K., Yuasa S.

Nature Communications 13 ( 1 )  2022.12

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Failure of the right ventricle plays a critical role in any type of heart failure. However, the mechanism remains unclear, and there is no specific therapy. Here, we show that the right ventricle predominantly expresses alternative complement pathway-related genes, including Cfd and C3aR1. Complement 3 (C3)-knockout attenuates right ventricular dysfunction and fibrosis in a mouse model of right ventricular failure. C3a is produced from C3 by the C3 convertase complex, which includes the essential component complement factor D (Cfd). Cfd-knockout mice also show attenuation of right ventricular failure. Moreover, the plasma concentration of CFD correlates with the severity of right ventricular failure in patients with chronic right ventricular failure. A C3a receptor (C3aR) antagonist dramatically improves right ventricular dysfunction in mice. In summary, we demonstrate the crucial role of the C3-Cfd-C3aR axis in right ventricular failure and highlight potential therapeutic targets for right ventricular failure.

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In vivo nanoscopic landscape of neurexin ligands underlying anterograde synapse specification

Nozawa K., Sogabe T., Hayashi A., Motohashi J., Miura E., Arai I., Yuzaki M.

Neuron  2022.10

ISSN  08966273

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Excitatory synapses are formed and matured by the cooperative actions of synaptic organizers, such as neurexins (Nrxns), neuroligins (Nlgns), LRRTMs, and Cbln1. Recent super-resolution nanoscopy developments have revealed that many synaptic organizers, as well as glutamate receptors and glutamate release machinery, exist as nanoclusters within synapses. However, it is unclear how such nanodomains interact with each other to organize excitatory synapses in vivo. By applying X10 expansion microscopy to epitope tag knockin mice, we found that Cbln1, Nlgn1, and LRRTM1, which share Nrxn as a common presynaptic receptor, form overlapping or separate nanodomains depending on Nrxn with or without a sequence encoded by splice site 4. The size and position of glutamate receptor nanodomains of GluD1, NMDA, and AMPA receptors were regulated by Cbln1, Nlgn1, and LRRTM1 nanodomains, respectively. These findings indicate that Nrxns anterogradely regulate the postsynaptic nanoscopic architecture of glutamate receptors through competition and coordination of Nrxn ligands.

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Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking

Ojima K., Shiraiwa K., Soga K., Doura T., Takato M., Komatsu K., Yuzaki M., Hamachi I., Kiyonaka S.

Nature Communications 12 ( 1 )  2021.12

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The regulation of glutamate receptor localization is critical for development and synaptic plasticity in the central nervous system. Conventional biochemical and molecular biological approaches have been widely used to analyze glutamate receptor trafficking, especially for α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-type glutamate receptors (AMPARs). However, conflicting findings have been reported because of a lack of useful tools for analyzing endogenous AMPARs. Here, we develop a method for the rapid and selective labeling of AMPARs with chemical probes, by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological temperature in culture medium. This method allows us to quantify AMPAR distribution and trafficking, which reveals some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method is also successfully expanded to selectively label N-methyl-D-aspartate-type glutamate receptors. Thus, bioorthogonal two-step labeling may be a versatile tool for investigating the physiological and pathophysiological roles of glutamate receptors in neurons.

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Subunit-dependent and subunit-independent rules of AMPA receptor trafficking during chemical long-term depression in hippocampal neurons

Matsuda S., Yuzaki M.

Journal of Biological Chemistry 297 ( 2 )  2021.08

ISSN  00219258

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Long-term potentiation (LTP) and long-term depression (LTD) of excitatory neurotransmission are believed to be the neuronal basis of learning and memory. Both processes are primarily mediated by neuronal activity–induced transport of postsynaptic AMPA-type glutamate receptors (AMPARs). While AMPAR subunits and their specific phosphorylation sites mediate differential AMPAR trafficking, LTP and LTD could also occur in a subunit-independent manner. Thus, it remains unclear whether and how certain AMPAR subunits with phosphorylation sites are preferentially recruited to or removed from synapses during LTP and LTD. Using immunoblot and immunocytochemical analysis, we show that phosphomimetic mutations of the membrane-proximal region (MPR) in GluA1 AMPAR subunits affect the subunit-dependent endosomal transport of AMPARs during chemical LTD. AP-2 and AP-3, adaptor protein complexes necessary for clathrin-mediated endocytosis and late endosomal/lysosomal trafficking, respectively, are reported to be recruited to AMPARs by binding to the AMPAR auxiliary subunit, stargazin (STG), in an AMPAR subunit–independent manner. However, the association of AP-3, but not AP-2, with STG was indirectly inhibited by the phosphomimetic mutation in the MPR of GluA1. Thus, although AMPARs containing the phosphomimetic mutation at the MPR of GluA1 were endocytosed by a chemical LTD-inducing stimulus, they were quickly recycled back to the cell surface in hippocampal neurons. These results could explain how the phosphorylation status of GluA1-MPR plays a dominant role in subunit-independent STG-mediated AMPAR trafficking during LTD.

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Destroy the old to build the new: Activity-dependent lysosomal exocytosis in neurons

Ibata K., Yuzaki M.

Neuroscience Research 167   38 - 46 2021.06

ISSN  01680102

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Lysosomes are organelles that support diverse cellular functions such as terminal degradation of macromolecules and nutrient recycling. Additionally, lysosomes can fuse with the plasma membrane, a phenomenon referred to as lysosomal exocytosis, to release their contents, including hydrolytic enzymes and cargo proteins. Recently, neuronal activity has been shown to induce lysosomal exocytosis in dendrites and axons. Secreted lysosomal enzyme cathepsin B induces and stabilizes synaptic structural changes by degrading the local extracellular matrix. Extracellular matrix reorganization could also enhance the lateral diffusion of the co-released synaptic organizer Cbln1 along the surface of axons to facilitate new synapse formation. Similarly, lateral diffusion of dendritic AMPA-type glutamate receptors could be facilitated to enhance functional synaptic plasticity. Therefore, lysosomal exocytosis is a powerful way of building new cellular structures through the coordinated destruction of the old environment. Understanding the mechanisms by which lysosomal exocytosis is regulated in neurons is expected to lead to the development of new therapeutics for neuronal plasticity following spinal cord injury or neurodegenerative disease.

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The autism-associated protein CHD8 is required for cerebellar development and motor function

Kawamura A., Katayama Y., Kakegawa W., Ino D., Nishiyama M., Yuzaki M., Nakayama K.I.

Cell Reports 35 ( 1 )  2021.04

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Mutations in the gene encoding the chromatin remodeler chromodomain helicase DNA-binding protein 8 (CHD8) are a highly penetrant risk factor for autism spectrum disorder (ASD). Although cerebellar abnormalities have long been thought to be related to ASD pathogenesis, it has remained largely unknown whether dysfunction of CHD8 in the cerebellum contributes to ASD phenotypes. We here show that cerebellar granule neuron progenitor (GNP)-specific deletion of Chd8 in mice impairs the proliferation and differentiation of these cells as well as gives rise to cerebellar hypoplasia and a motor coordination defect, but not to ASD-like behavioral abnormalities. CHD8 is found to regulate the expression of neuronal genes in GNPs. It also binds preferentially to promoter regions and modulates local chromatin accessibility of transcriptionally active genes in these cells. Our results have thus uncovered a key role for CHD8 in cerebellar development, with important implications for understanding the contribution of this brain region to ASD pathogenesis.

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Site-specific covalent labeling of His-tag fused proteins with N-acyl-N-alkyl sulfonamide reagent

Thimaradka V., Hoon Oh J., Heroven C., Radu Aricescu A., Yuzaki M., Tamura T., Hamachi I.

Bioorganic and Medicinal Chemistry 30 2021.01

ISSN  09680896

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The ability to incorporate a desired functionality into proteins of interest in a site-specific manner can provide powerful tools for investigating biological systems and creating therapeutic conjugates. However, there are not any universal methods that can be applied to all proteins, and it is thus important to explore the chemical strategy for protein modification. In this paper, we developed a new reactive peptide tag/probe pair system for site-specific covalent protein labeling. This method relies on the recognition-driven reaction of a peptide tag and a molecular probe, which comprises the lysine-containing short histidine tag (KH6 or H6K) and a binuclear nickel (II)- nitrilotriacetic acid (Ni2+-NTA) complex probe containing a lysine-reactive N-acyl-N-alkyl sulfonamide (NASA) group. The selective interaction of the His-tag and Ni2+–NTA propeles a rapid nucleophilic reaction between a lysine residue of the tag and the electrophilic NASA group of the probe by the proximity effect, resulting in the tag-site-specific functionalization of proteins. We characterized the reactive profile and site-specificity of this method using model peptides and proteins in vitro, and demonstrated the general utility for production of a nanobody-chemical probe conjugate without compromising its binding ability.

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PIP <inf>3</inf> -Phldb2 is crucial for LTP regulating synaptic NMDA and AMPA receptor density and PSD95 turnover

Xie M., Ishikawa Y., Yagi H., Iguchi T., Oka Y., Kuroda K., Iwata K., Kiyonari H., Matsuda S., Matsuzaki H., Yuzaki M., Fukazawa Y., Sato M.

Scientific Reports (Scientific Reports)  9 ( 1 ) 4305 2019.12

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© 2019, The Author(s). The essential involvement of phosphoinositides in synaptic plasticity is well-established, but incomplete knowledge of the downstream molecular entities prevents us from understanding their signalling cascades completely. Here, we determined that Phldb2, of which pleckstrin-homology domain is highly sensitive to PIP 3 , functions as a phosphoinositide-signalling mediator for synaptic plasticity. BDNF application caused Phldb2 recruitment toward postsynaptic membrane in dendritic spines, whereas PI3K inhibition resulted in its reduced accumulation. Phldb2 bound to postsynaptic scaffolding molecule PSD-95 and was crucial for localization and turnover of PSD-95 in the spine. Phldb2 also bound to GluA1 and GluA2. Phldb2 was indispensable for the interaction between NMDA receptors and CaMKII, and the synaptic density of AMPA receptors. Therefore, PIP 3 -responsive Phldb2 is pivotal for induction and maintenance of LTP. Memory formation was impaired in our Phldb2 −/− mice.

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Hyaluronan synthesis supports glutamate transporter activity.

Hayashi MK, Nishioka T, Shimizu H, Takahashi K, Kakegawa W, Mikami T, Hirayama Y, Koizumi S, Yoshida S, Yuzaki M, Tammi M, Sekino Y, Kaibuchi K, Shigemoto-Mogami Y, Yasui M, Sato K

Journal of neurochemistry 150 ( 3 ) 249 - 263 2019.08

ISSN  0022-3042

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Activity-Dependent Secretion of Synaptic Organizer Cbln1 from Lysosomes in Granule Cell Axons.

Ibata K, Kono M, Narumi S, Motohashi J, Kakegawa W, Kohda K, Yuzaki M

Neuron 102 ( 6 ) 1184 - 1198.e10 2019.06

ISSN  0896-6273

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Interneuronal NMDA receptors regulate long-term depression and motor learning in the cerebellum

Kono M., Kakegawa W., Yoshida K., Yuzaki M.

Journal of Physiology (Journal of Physiology)  597 ( 3 ) 903 - 920 2019.02

ISSN  00223751

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© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society Key points: NMDA receptors (NMDARs) are required for long-term depression (LTD) at parallel fibre–Purkinje cell synapses, but their cellular localization and physiological functions in vivo are unclear. NMDARs in molecular-layer interneurons (MLIs), but not granule cells or Purkinje cells, are required for LTD, but not long-term potentiation induced by low-frequency stimulation of parallel fibres. Nitric oxide produced by NMDAR activation in MLIs probably mediates LTD induction. NMDARs in granule cells or Purkinje cells are dispensable for motor learning during adaptation of horizontal optokinetic responses. Abstract: Long-term potentiation (LTP) and depression (LTD), which serve as cellular synaptic plasticity models for learning and memory, are crucially regulated by N-methyl-d-aspartate receptors (NMDARs) in various brain regions. In the cerebellum, LTP and LTD at parallel fibre (PF)–Purkinje cell (PC) synapses are thought to mediate certain forms of motor learning. However, while NMDARs are essential for LTD in vitro, their cellular localization remains controversial. In addition, whether and how NMDARs mediate motor learning in vivo remains unclear. Here, we examined the contribution of NMDARs expressed in granule cells (GCs), PCs and molecular-layer interneurons (MLIs) to LTD/LTP and motor learning by generating GC-, PC- and MLI/PC-specific knockouts of Grin1, a gene encoding an obligatory GluN1 subunit of NMDARs. While robust LTD and LTP were induced at PF–PC synapses in GC- and PC-specific Grin1 (GC-Grin1 and PC-Grin1, respectively) conditional knockout (cKO) mice, only LTD was impaired in MLI/PC-specific Grin1 (MLI/PC-Grin1) cKO mice. Application of diethylamine nitric oxide (NO) sodium, a potent NO donor, to the cerebellar slices restored LTD in MLI/PC-Grin1 cKO mice, suggesting that NO is probably downstream to NMDARs. Furthermore, the adaptation of horizontal optokinetic responses (hOKR), a cerebellar motor learning task, was normally observed in GC-Grin1 cKO and PC-Grin1 cKO mice, but not in MLI/PC-Grin1 cKO mice. These results indicate that it is the NMDARs expressed in MLIs, but not in PCs or GCs, that play important roles in LTD in vitro and motor learning in vivo.

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In vivo Two-Photon Imaging of Anesthesia-Specific Alterations in Microglial Surveillance and Photodamage-Directed Motility in Mouse Cortex.

Sun W, Suzuki K, Toptunov D, Stoyanov S, Yuzaki M, Khiroug L, Dityatev A

Frontiers in neuroscience 13   421 2019

ISSN  1662-4548

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PhotonSABER: new tool shedding light on endocytosis and learning mechanisms in vivo.

Matsuda S, Kakegawa W, Yuzaki M

Communicative & integrative biology 12 ( 1 ) 34 - 37 2019

ISSN  1942-0889

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Mice lacking EFA6C/Psd2, a guanine nucleotide exchange factor for Arf6, exhibit lower Purkinje cell synaptic density but normal cerebellar motor functions.

Saegusa S, Fukaya M, Kakegawa W, Tanaka M, Katsumata O, Sugawara T, Hara Y, Itakura M, Okubo T, Sato T, Yuzaki M, Sakagami H

PloS one 14 ( 5 ) e0216960 2019

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Spatiotemporal regulation of the GPCR activity of BAI3 by C1qL4 and Stabilin-2 controls myoblast fusion

Hamoud N., Tran V., Aimi T., Kakegawa W., Lahaie S., Thibault M., Pelletier A., Wong G., Kim I., Kania A., Yuzaki M., Bouvier M., Côté J.

Nature Communications (Nature Communications)  9 ( 1 ) 4470 2018.12

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© 2018, The Author(s). Myoblast fusion is tightly regulated during development and regeneration of muscle fibers. BAI3 is a receptor that orchestrates myoblast fusion via Elmo/Dock1 signaling, but the mechanisms regulating its activity remain elusive. Here we report that mice lacking BAI3 display small muscle fibers and inefficient muscle regeneration after cardiotoxin-induced injury. We describe two proteins that repress or activate BAI3 in muscle progenitors. We find that the secreted C1q-like1–4 proteins repress fusion by specifically interacting with BAI3. Using a proteomic approach, we identify Stabilin-2 as a protein that interacts with BAI3 and stimulates its fusion promoting activity. We demonstrate that Stabilin-2 activates the GPCR activity of BAI3. The resulting activated heterotrimeric G-proteins contribute to the initial recruitment of Elmo proteins to the membrane, which are then stabilized on BAI3 through a direct interaction. Collectively, our results demonstrate that the activity of BAI3 is spatiotemporally regulated by C1qL4 and Stabilin-2 during myoblast fusion.

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Improvement of cerebellar ataxic gait by injecting Cbln1 into the cerebellum of cbln1-null mice

Takeuchi E., Ito-Ishida A., Yuzaki M., Yanagihara D.

Scientific Reports (Scientific Reports)  8 ( 1 ) 6184 2018.12

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© 2018 The Author(s). Patients and rodents with cerebellar damage display ataxic gaits characterized by impaired coordination of limb movements. Here, gait ataxia in mice with a null mutation of the gene for the cerebellin 1 precursor protein (cbln1-null mice) was investigated by kinematic analysis of hindlimb movements during locomotion. The Cbln1 protein is predominately produced and secreted from cerebellar granule cells. The cerebellum of cbln1-null mice is characterized by an 80% reduction in the number of parallel fiber-Purkinje cell synapses compared with wild-Type mice. Our analyses identified prominent differences in the temporal parameters of locomotion between cbln1-null and wild-Type mice. The cbln1-null mice displayed abnormal hindlimb movements that were characterized by excessive toe elevation during the swing phase, and by severe hyperflexion of the ankles and knees. When recombinant Cbln1 protein was injected into the cerebellum of cbln1-null mice, the step cycle and stance phase durations increased toward those of wild-Type mice, and the angular excursions of the knee during a cycle period showed a much closer agreement with those of wild-Type mice. These findings suggest that dysfunction of the parallel fiber-Purkinje cell synapses might underlie the impairment of hindlimb movements during locomotion in cbln1-null mice.

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Cellular and Subcellular Localization of Endogenous Neuroligin-1 in the Cerebellum

Nozawa K., Hayashi A., Motohashi J., Takeo Y., Matsuda K., Yuzaki M.

Cerebellum (Cerebellum)  17 ( 6 ) 709 - 721 2018.12

ISSN  14734222

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© 2018, Springer Science+Business Media, LLC, part of Springer Nature. Synapses are precisely established, maintained, and modified throughout life by molecules called synaptic organizers, which include neurexins and neuroligins (Nlgn). Despite the importance of synaptic organizers in defining functions of neuronal circuits, the cellular and subcellular localization of many synaptic organizers has remained largely elusive because of the paucity of specific antibodies for immunohistochemical studies. In the present study, rather than raising specific antibodies, we generated knock-in mice in which a hemagglutinin (HA) epitope was inserted in the Nlgn1 gene. We have achieved high-throughput and precise gene editing by delivering the CRISPR/Cas9 system into zygotes. Using HA-Nlgn1 mice, we found that HA-Nlgn1 was enriched at synapses between parallel fibers and molecular layer interneurons (MLIs) and the glomeruli, in which mossy fiber terminals synapse onto granule cell dendrites. HA immunoreactivity was colocalized with postsynaptic density 95 at these synapses, indicating that endogenous Nlgn1 is localized at excitatory postsynaptic sites. In contrast, HA-Nlgn1 signals were very weak in dendrites and somata of Purkinje cells. Interestingly, HA-immunoreactivities were also observed in the pinceau, a specialized structure formed by MLI axons and astrocytes. HA-immunoreactivities in the pinceau were significantly reduced by knockdown of Nlgn1 in MLIs, indicating that in addition to postsynaptic sites, Nlgn1 is also localized at MLI axons. Our results indicate that epitope-tagging by electroporation-based gene editing with CRISPR/Cas9 is a viable and powerful method for mapping endogenous synaptic organizers with subcellular resolution, without the need for specific antibodies for each protein.

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Caveolin-1 Promotes Early Neuronal Maturation via Caveolae-Independent Trafficking of N-Cadherin and L1.

Shikanai M, Nishimura YV, Sakurai M, Nabeshima YI, Yuzaki M, Kawauchi T

iScience 7   53 - 67 2018.09

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La Dolce Vita of Neurexin: Synaptic Partnerships through Glycosaminoglycans.

Suzuki K, Yuzaki M

Cell 174 ( 6 ) 1337 - 1338 2018.09

ISSN  0092-8674

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Optogenetic Control of Synaptic AMPA Receptor Endocytosis Reveals Roles of LTD in Motor Learning

Kakegawa W., Katoh A., Narumi S., Miura E., Motohashi J., Takahashi A., Kohda K., Fukazawa Y., Yuzaki M., Matsuda S.

Neuron (Neuron)  99 ( 5 ) 985 - 998.e6 2018.09

ISSN  08966273

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© 2018 Elsevier Inc. Long-term depression (LTD) of AMPA-type glutamate receptor (AMPA receptor)-mediated synaptic transmission has been proposed as a cellular substrate for learning and memory. Although activity-induced AMPA receptor endocytosis is believed to underlie LTD, it remains largely unclear whether LTD and AMPA receptor endocytosis at specific synapses are causally linked to learning and memory in vivo. Here we developed a new optogenetic tool, termed PhotonSABER, which enabled the temporal, spatial, and cell-type-specific control of AMPA receptor endocytosis at active synapses, while the basal synaptic properties and other forms of synaptic plasticity were unaffected. We found that fiberoptic illumination to Purkinje cells expressing PhotonSABER in vivo inhibited cerebellar motor learning during adaptation of the horizontal optokinetic response and vestibulo-ocular reflex, as well as synaptic AMPA receptor decrease in the flocculus. Our results demonstrate that LTD and AMPA receptor endocytosis at specific neuronal circuits were directly responsible for motor learning in vivo. Video Abstract: Kakegawa et al. show that AMPA receptor endocytosis at parallel fiber-Purkinje cell synapses in the cerebellar flocculus plays a direct role in motor learning in vivo, using a new optogenetic tool, PhotonSABER, which enables the acute inhibition of AMPA receptor endocytosis during LTD.

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Rab family small GTPases-mediated regulation of intracellular logistics in neural development

Shikanai M., Yuzaki M., Kawauchi T.

Histology and Histopathology (Histology and Histopathology)  33 ( 8 ) 765 - 771 2018.08

ISSN  02133911

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© 2018, Histology and Histopathology. All rights reserved. Rab family small GTPases play essential roles in various cellular events via the regulation of intracellular logistics comprising a large number of membrane traffic pathways. Emerging evidence reveals the physiological roles of Rab proteins in several tissues, including developing brains. Many Rab proteins, such as Rab5, Rab6, Rab7, Rab8, Rab10, Rab11, Rab17 and Rab18, are shown to regulate neurite outgrowth in PC12 cells and/or axon and dendrite formation in primary cultured neurons. Recent studies have also revealed in vivo roles of several Rab family small GTPases in brain development and its related neurological disorders. In this review, we introduce the physiological function of Rab family proteins in neural development with particular focus on neurite outgrowth and neuronal migration.

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[Molecular Mechanisms for Learning and Memory: What Happens at the Synapses?].

Kakegawa W, Yuzaki M

Brain and nerve = Shinkei kenkyu no shinpo 70 ( 7 ) 677 - 687 2018.07

ISSN  1881-6096

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Functional interactions between transient receptor potential M8 and transient receptor potential V1 in the trigeminal system: Relevance to migraine pathophysiology

Kayama Y., Shibata M., Takizawa T., Ibata K., Shimizu T., Ebine T., Toriumi H., Yuzaki M., Suzuki N.

Cephalalgia (Cephalalgia)  38 ( 5 ) 833 - 845 2018.04

ISSN  03331024

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© 2017, © International Headache Society 2017. Background: Recent genome-wide association studies have identified transient receptor potential M8 (TRPM8) as a migraine susceptibility gene. TRPM8 is a nonselective cation channel that mediates cool perception. However, its precise role in migraine pathophysiology is elusive. Transient receptor potential V1 (TRPV1) is a nonselective cation channel activated by noxious heat. Both TRPM8 and TRPV1 are expressed in trigeminal ganglion (TG) neurons. Methods: We investigated the functional roles of TRPM8 and TRPV1 in a meningeal inflammation-based migraine model by measuring the effects of facial TRPM8 activation on thermal allodynia and assessing receptor coexpression changes in TG neurons. We performed retrograde tracer labeling to identify TG neurons innervating the face and dura. Results: We found that pharmacological TRPM8 activation reversed the meningeal inflammation-induced lowering of the facial heat pain threshold, an effect abolished by genetic ablation of TRPM8. No significant changes in the heat pain threshold were seen in sham-operated animals. Meningeal inflammation caused dynamic alterations in TRPM8/TRPV1 coexpression patterns in TG neurons, and colocalization was most pronounced when the ameliorating effect of TRPM8 activation on thermal allodynia was maximal. Our tracer assay disclosed the presence of dura-innervating TG neurons sending collaterals to the face. Approximately half of them were TRPV1-positive. We also demonstrated functional inhibition of TRPV1 by TRPM8 in a cell-based assay using c-Jun N-terminal kinase phosphorylation as a surrogate marker. Conclusions: Our findings provide a plausible mechanism to explain how facial TRPM8 activation can relieve migraine by suppressing TRPV1 activity. Facial TRPM8 appears to be a promising therapeutic target for migraine.

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Two Classes of Secreted Synaptic Organizers in the Central Nervous System

Yuzaki M.

Annual Review of Physiology (Annual Review of Physiology)  80   243 - 262 2018.02

ISSN  00664278

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© 2018 by Annual Reviews. All rights reserved. Research in the last two decades has identified many synaptic organizers in the central nervous system that directly regulate the assembly of pre- and/or postsynaptic molecules, such as synaptic vesicles, active zone proteins, and neurotransmitter receptors. They are classified into secreted factors and cell adhesion molecules, such as neurexins and neuroligins. Certain secreted factors are termed extracellular scaffolding proteins (ESPs) because they are components of the synaptic extracellular matrix and serve as a scaffold at the synaptic cleft. These include Lgi1, Cbln1, neuronal pentraxins, Hevin, thrombospondins, and glypicans. Diffusible secreted factors, such as Wnts, fibroblast growth factors, and semaphorins, tend to act from a distance. In contrast, ESPs remain at the synaptic cleft and often help synaptic adhesion and/or accumulation of postsynaptic receptors. Many fundamental questions remain about when, how, and why various synaptic organizers establish and modify the vast numbers of connections during development and throughout life.

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Nav1.2 haplodeficiency in excitatory neurons causes absence-like seizures in mice.

Ogiwara I, Miyamoto H, Tatsukawa T, Yamagata T, Nakayama T, Atapour N, Miura E, Mazaki E, Ernst SJ, Cao D, Ohtani H, Itohara S, Yanagawa Y, Montal M, Yuzaki M, Inoue Y, Hensch TK, Noebels JL, Yamakawa K

Communications biology 1   96 2018

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Signaling Pathways Relevant to Nerve Growth Factor-induced Upregulation of Transient Receptor Potential M8 Expression.

Kayama Y, Shibata M, Takizawa T, Ibata K, Nakahara J, Shimizu T, Toriumi H, Yuzaki M, Suzuki N

Neuroscience 367   178 - 188 2017.12

ISSN  0306-4522

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Dendritic Homeostasis Disruption in a Novel Frontotemporal Dementia Mouse Model Expressing Cytoplasmic Fused in Sarcoma.

Shiihashi G, Ito D, Arai I, Kobayashi Y, Hayashi K, Otsuka S, Nakajima K, Yuzaki M, Itohara S, Suzuki N

EBioMedicine 24   102 - 115 2017.10

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Neural differentiation of human embryonic stem cells induced by the transgene-mediated overexpression of single transcription factors.

Matsushita M, Nakatake Y, Arai I, Ibata K, Kohda K, Goparaju SK, Murakami M, Sakota M, Chikazawa-Nohtomi N, Ko SBH, Kanai T, Yuzaki M, Ko MSH

Biochemical and biophysical research communications 490 ( 2 ) 296 - 301 2017.08

ISSN  0006-291X

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The C1q complement family of synaptic organizers: not just complementary.

Yuzaki M

Current opinion in neurobiology 45   9 - 15 2017.08

ISSN  0959-4388

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Glutamate transporter GLAST controls synaptic wrapping by Bergmann glia and ensures proper wiring of Purkinje cells.

Miyazaki T, Yamasaki M, Hashimoto K, Kohda K, Yuzaki M, Shimamoto K, Tanaka K, Kano M, Watanabe M

Proceedings of the National Academy of Sciences of the United States of America 114 ( 28 ) 7438 - 7443 2017.07

ISSN  0027-8424

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MTCL1 plays an essential role in maintaining Purkinje neuron axon initial segment

Satake, Tomoko, Yamashita, Kazunari, Hayashi, Kenji, Miyatake, Satoko, Tamura-Nakano, Miwa, Doi, Hiroshi, Furuta, Yasuhide, Shioi, Go, Miura, Eriko, Takeo, Yukari H., Yoshida, Kunihiro, Yahikozawa, Hiroyuki, Matsumoto, Naomichi, Yuzaki, Michisuke, Suzuki, Atsushi

EMBO JOURNAL 36 ( 9 ) 1227 - 1242 2017.05

Research paper (scientific journal),  ISSN  0261-4189

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Chemical labelling for visualizing native AMPA receptors in live neurons

Wakayama, Sho, Kiyonaka, Shigeki, Arai, Itaru, Kakegawa, Wataru, Matsuda, Shinji, Ibata, Keiji, Nemoto, Yuri L., Kusumi, Akihiro, Yuzaki, Michisuke, Hamachi, Itaru

NATURE COMMUNICATIONS 8   138 - 150 2017.04

Research paper (scientific journal),  ISSN  2041-1723

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A GluD Coming-Of-Age Story

Yuzaki, Michisuke, Aricescu, A. Radu

TRENDS IN NEUROSCIENCES 40 ( 3 ) 138 - 150 2017.03

Research paper (scientific journal),  ISSN  0166-2236

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Rapid differentiation of human pluripotent stem cells into functional neurons by mRNAs encoding transcription factors

Goparaju, Sravan Kumar, Kohda, Kazuhisa, Ibata, Keiji, Soma, Atsumi, Nakatake, Yukhi, Akiyama, Tomohiko, Wakabayashi, Shunichi, Matsushita, Misako, Sakota, Miki, Kimura, Hiromi, Yuzaki, Michisuke, Ko, Shigeru B. H., Ko, Minoru S. H.

SCIENTIFIC REPORTS 7   25577 - 25587 2017.02

Research paper (scientific journal),  ISSN  2045-2322

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Roles of Cbln1 in Non-Motor Functions of Mice.

Otsuka S, Konno K, Abe M, Motohashi J, Kohda K, Sakimura K, Watanabe M, Yuzaki M

The Journal of neuroscience : the official journal of the Society for Neuroscience 36 ( 46 ) 11801 - 11816 2016.11

ISSN  0270-6474

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Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice

Kotajima-Murakami, Hiroko, Narumi, Sakae, Yuzaki, Michisuke, Yanagihara, Dai

PLOS ONE 11 ( 11 ) 958 - 967 2016.11

Research paper (scientific journal),  ISSN  1932-6203

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Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

Kiyonaka, Shigeki, Kubota, Ryou, Michibata, Yukiko, Sakakura, Masayoshi, Takahashi, Hideo, Numata, Tomohiro, Inoue, Ryuji, Yuzaki, Michisuke, Hamachi, Itaru

NATURE CHEMISTRY 8 ( 10 ) 958 - 967 2016.10

Research paper (scientific journal),  ISSN  1755-4330

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Structural basis for integration of GluD receptors within synaptic organizer complexes.

Elegheert J, Kakegawa W, Clay JE, Shanks NF, Behiels E, Matsuda K, Kohda K, Miura E, Rossmann M, Mitakidis N, Motohashi J, Chang VT, Siebold C, Greger IH, Nakagawa T, Yuzaki M, Aricescu AR

Science (New York, N.Y.) 353 ( 6296 ) 295 - 9 2016.07

ISSN  0036-8075

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Transsynaptic Modulation of Kainate Receptor Functions by C1q-like Proteins.

Matsuda K, Budisantoso T, Mitakidis N, Sugaya Y, Miura E, Kakegawa W, Yamasaki M, Konno K, Uchigashima M, Abe M, Watanabe I, Kano M, Watanabe M, Sakimura K, Aricescu AR, Yuzaki M

Neuron 90 ( 4 ) 752 - 67 2016.05

ISSN  0896-6273

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A novel non-canonical Notch signaling regulates expression of synaptic vesicle proteins in excitatory neurons.

Hayashi Y, Nishimune H, Hozumi K, Saga Y, Harada A, Yuzaki M, Iwatsubo T, Kopan R, Tomita T

Scientific reports 6   23969 2016.04

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A Computational Model for the AMPA Receptor Phosphorylation Master Switch Regulating Cerebellar Long-Term Depression.

Gallimore AR, Aricescu AR, Yuzaki M, Calinescu R

PLoS computational biology 12 ( 1 ) e1004664 2016.01

ISSN  1553-734X

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Controlling the Regional Identity of hPSC-Derived Neurons to Uncover Neuronal Subtype Specificity of Neurological Disease Phenotypes

Imaizumi, Kent, Sone, Takefumi, Ibata, Keiji, Fujimori, Koki, Yuzaki, Michisuke, Akamatsu, Wado, Okano, Hideyuki

STEM CELL REPORTS 5 ( 6 ) 1010 - 1022 2015.12

Research paper (scientific journal),  ISSN  2213-6711

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Rorα regulates multiple aspects of dendrite development in cerebellar purkinje cells in vivo

Takeo Yukari H., Kakegawa Wataru, Miura Eriko, Yuzaki Michisuke

Journal of Neuroscience 35 ( 36 ) 12518 - 12534 2015.09

ISSN  0270-6474

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<p>The establishment of cell-type-specific dendritic arbors is fundamental for proper neural circuit formation. Here, using temporal- and cell-specific knock-down, knock-out, and overexpression approaches, we show that multiple aspects of the dendritic organization of cerebellar Purkinje cells (PCs) are controlled by a single transcriptional factor, retinoic acid-related orphan receptor-alpha (RORα), a gene defective in staggerer mutant mice. As reported earlier, RORawas required for regression of primitive dendrites before postnatal day 4 (P4). RORα was also necessary for PCs to form a single Purkinje layer from P0 to P4. The knock-down of RORafrom P4 impaired the elimination of perisomatic dendrites and maturation of single stem dendrites in PCs at P8. Filopodia and spines were also absent in these PCs. The knock-down of RORα from P8 impaired the formation and maintenance of terminal dendritic branches of PCs at P14. Finally, even after dendrite formation was completed at P21,RORα was required for PCs to maintain dendritic complexity and functional synapses, but their mature innervation pattern by single climbing fibers was unaffected. Interestingly, overexpression of RORain PCs at various developmental stages did not facilitate dendrite development, but had specific detrimental effects on PCs. Because RORα deficiency during development is closely related to the severity of spinocerebellar ataxia type 1, delineating the specific roles of RORain PCs in vivo at different time windows during development and throughout adulthood would facilitate our understanding of the pathogenesis of cerebellar disorders.</p>

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Anterograde C1ql1 signaling is required in order to determine and maintain a single-winner climbing fiber in the mouse cerebellum

Kakegawa Wataru, Mitakidis Nikolaos, Miura Eriko, Abe Manabu, Matsuda Keiko, Takeo Yukari H., Kohda Kazuhisa, Motohashi Junko, Takahashi Akiyo, Nagao Soichi, Muramatsu Shin ichi, Watanabe Masahiko, Sakimura Kenji, Aricescu A. Radu, Yuzaki Michisuke

Neuron 85 ( 2 ) 316 - 330 2015.01

ISSN  0896-6273

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<p>Neuronal networks are dynamically modified by selective synapse pruning during development and adulthood. However, how certain connections win the competition with others and are subsequently maintained is not fully understood. Here, we show that C1ql1, a member of the C1q family of proteins, is provided by climbing fibers (CFs) and serves as acrucial anterograde signal to determine and maintain the single-winner CF in the mouse cerebellum throughout development and adulthood. C1ql1 specifically binds to the brain-specific angiogenesis inhibitor 3 (Bai3), which is a member of the cell-adhesionG-protein-coupled receptor family and expressed on postsynaptic Purkinje cells. C1ql1-Bai3 signaling is required for motor learning but not for gross motor performance or coordination. Because related family members of C1ql1 and Bai3 are expressed in various brain regions, the mechanism described here likely applies to synapse formation, maintenance, and function in multiple neuronal circuits essential for important brain functions.</p>

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Optogenetic control of the function of the Golgi apparatus in neurons

Endo, Junnosuke, Takeo, Yukari, Matsuda, Shinji, Yuzaki, Michisuke

GLYCOBIOLOGY 24 ( 11 ) 1201 - 1201 2014.11

Research paper (scientific journal),  ISSN  0959-6658

Axonal localization of Ca²⁺-dependent activator protein for secretion 2 is critical for subcellular locality of brain-derived neurotrophic factor and neurotrophin-3 release affecting proper development of postnatal mouse cerebellum

Sadakata Tetsushi, Kakegawa Wataru, Shinoda Yo, Hosono Mayu, Katoh-Semba Ritsuko, Sekine Yukiko, Sato Yumi, Saruta Chihiro, Ishizaki Yasuki, Yuzaki Michisuke, Kojima Masami, Furuichi Teiichi

PLoS ONE 9 ( 6 ) e99524 2014.06

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<p>Ca²⁺-dependent activator protein for secretion 2 (CAPS2) is a protein that is essential for enhanced release of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) from cerebellar granule cells. We previously identified dex3, a rare alternative splice variant of CAPS2, which is overrepresented in patients with autism and is missing an exon 3 critical for axonal localization. We recently reported that a mouse model CAPS2 ^Δx3/Δex3 expressing dex3 showed autistic-like behavioral phenotypes including impaired social interaction and cognition and increased anxiety in an unfamiliar environment. Here, we verified impairment in axonal, but not somato-dendritic, localization of dex3 protein in cerebellar granule cells and demonstrated cellular and physiological phenotypes in postnatal cerebellum of CAPS2^Δx3/Δx3 mice. Interestingly, both BDNF and NT-3 were markedly reduced in axons of cerebellar granule cells, resulting in a significant decrease in their release. As a result, dex3 mice showed developmental deficits in dendritic arborization of Purkinje cells, vermian lobulation and fissurization, and granule cell precursor proliferation. Paired-pulse facilitation at parallel fiber- Purkinje cell synapses was also impaired. Together, our results indicate that CAPS2 plays an important role in subcellular locality (axonal vs. somato-dendritic) of enhanced BDNF and NT-3 release, which is indispensable for proper development of postnatal cerebellum. © 2014 Sadakata et al.</p>

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The role of Cbln1 on Purkinje cell synapse formation

Ito-Ishida Aya, Okabe Shigeo, Yuzaki Michisuke

Neuroscience Research 83   64 - 68 2014.06

ISSN  0168-0102

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<p>Cbln1 is a glycoprotein which belongs to the C1q family. In the cerebellum, Cbln1 is produced and secreted from granule cells and works as a strong synapse organizer between Purkinje cells and parallel fibers, the axons of the granule cells. In this update article, we will describe the molecular mechanisms by which Cbln1 induces synapse formation and will review our findings on the axonal structural changes which occur specifically during this process. We will also describe our recent finding that Cbln1 has a suppressive role in inhibitory synapse formation between Purkinje cells and molecular layer interneurons. Our results have revealed that Cbln1 plays an essential role to establish parallel fiber-Purkinje cell synapses and to regulate balance between excitatory and inhibitory input on Purkinje cells.</p>

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Reprogramming non-human primate somatic cells into functional neuronal cells by defined factors

Zhou Zhi, Kohda Kazuhisa, Ibata Keiji, Kohyama Jun, Akamatsu Wado, Yuzaki Michisuke, Okano Hirotaka James, Sasaki Erika, Okano Hideyuki

Molecular Brain 7 ( 1 ) 24 2014.04

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<p>Background: The common marmoset (Callithrix jacchus) is a New World primate sharing many similarities with humans. Recently developed technology for generating transgenic marmosets has opened new avenues for faithful recapitulation of human diseases, which could not be achieved in rodent models. However, the longer lifespan of common marmosets compared with rodents may result in an extended period for in vivo analysis of common marmoset disease models. Therefore, establishing rapid and efficient techniques for obtaining neuronal cells from transgenic individuals that enable in vitro analysis of molecular mechanisms underlying diseases are required. Recently, several groups have reported on methods, termed direct reprogramming, to generate neuronal cells by defined factors from somatic cells of various kinds of species, including mouse and human. The aim of the present study was to determine whether direct reprogramming technology was applicable to common marmosets. Results: Common marmoset induced neuronal (cjiN) cells with neuronal morphology were generated from common marmoset embryonic skin fibroblasts (cjF) by overexpressing the neuronal transcription factors: ASCL1, BRN2, MYT1L and NEUROD1. Reverse transcription-polymerase chain reaction of cjiN cells showed upregulation of neuronal genes highly related to neuronal differentiation and function. The presence of neuronal marker proteins was also confirmed by immunocytochemistry. Electrical field stimulation to cjiN cells increased the intracellular calcium level, which was reversibly blocked by the voltage-gated sodium channel blocker, tetrodotoxin, indicating that these cells were functional. The neuronal function of these cells was further confirmed by electrophysiological analyses showing that action potentials could be elicited by membrane depolarization in current-clamp mode while both fast-activating and inactivating sodium currents and outward currents were observed in voltage-clamp mode. The 5-bromodeoxyuridine (BrdU) incorporation assay showed that cjiN cells were directly converted from cjFs without passing a proliferative state. Conclusions: Functional common marmoset neuronal cells can be obtained directly from embryonic fibroblasts by overexpressing four neuronal transcription factors under in vitro conditions. Overall, direct conversion technology on marmoset somatic cells provides the opportunity to analyze and screen phenotypes of genetically-modified common marmosets. © 2014 Zhou et al.; licensee BioMed Central Ltd.</p>

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Rab8a and Rab8b are essential for several apical transport pathways but insufficient for ciliogenesis

Sato Takashi, Iwano Tomohiko, Kunii Masataka, Matsuda Shinji, Mizuguchi Rumiko, Jung Yongwook, Hagiwara Haruo, Yoshihara Yoshihiro, Yuzaki Michisuke, Harada Reiko, Harada Akihiro

Journal of Cell Science 127 ( 2 ) 422 - 431 2014.01

ISSN  0021-9533

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<p>The small GTP-binding protein Rab8 is known to play an essential role in intracellular transport and cilia formation. We have previously demonstrated that Rab8a is required for localising apical markers in various organisms. Rab8a has a closely related isoform, Rab8b. To determine whether Rab8b can compensate for Rab8a, we generated Rab8b-knockout mice. Although the Rab8b-knockout mice did not display an overt phenotype, Rab8a and Rab8b double-knockout mice exhibited mislocalisation of apical markers and died earlier than Rab8a-knockout mice. The apical markers accumulated in three intracellular patterns in the double-knockout mice. However, the localisation of basolateral and/or dendritic markers of the double-knockout mice seemed normal. The morphology and the length of various primary and/or motile cilia, and the frequency of ciliated cells appeared to be identical in control and double-knockout mice. However, an additional knockdown of Rab10 in double-knockout cells greatly reduced the percentage of ciliated cells. Our results highlight the compensatory effect of Rab8a and Rab8b in apical transport, and the complexity of the apical transport process. In addition, neither Rab8a nor Rab8b are required for basolateral and/or dendritic transport. However, simultaneous loss of Rab8a and Rab8b has little effect on ciliogenesis, whereas additional loss of Rab10 greatly affects ciliogenesis. © 2014. Published by The Company of Biologists Ltd.</p>

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Neural ECM and synaptogenesis

Heikkinen Anne, Pihlajaniemi Taina, Faissner Andreas, Yuzaki Michisuke

Progress in Brain Research 214   29 - 51 2014

ISSN  0079-6123

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<p>Chemical synapses allow neurons to perform complex computations and regulate other systems of the body. At a chemical synapse, pre- and postsynaptic sites are separated by a small space (the synaptic cleft) and surrounded by astrocytes. The basement membrane (BM), a sheetlike, specialized extracellular matrix (ECM), is found ubiquitously in the PNS. It has become clear that the ECMs not only play a structural role but also serve as barriers and filters in the PNS and CNS. Moreover, proteoglycans and tenascin family proteins in the ECM regulate synapse formation and synaptic plasticity. Although CNS synapses lack the BMs, recent results indicate that the BM-associated collagens are also present in the CNS synaptic cleft and affect synaptogenesis in both the CNS and the PNS. The C1q domain-containing family proteins are important components of the CNS synaptic cleft in regulating synapse formation, maintenance, and the pruning process. The ECM is regarded as a crucial component of the tetrapartite synapse, consisting of pre- and postsynaptic neurons, astrocyte, and ECM.</p>

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Cbln1 downregulates the formation and function of inhibitory synapses in mouse cerebellar Purkinje cells

Ito-Ishida Aya, Kakegawa Wataru, Kohda Kazuhisa, Miura Eriko, Okabe Shigeo, Yuzaki Michisuke

European Journal of Neuroscience 39 ( 8 ) 1268 - 1280 2014

ISSN  0953-816X

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<p>The formation of excitatory and inhibitory synapses must be tightly coordinated to establish functional neuronal circuitry during development. In the cerebellum, the formation of excitatory synapses between parallel fibers and Purkinje cells is strongly induced by Cbln1, which is released from parallel fibers and binds to the postsynaptic δ2 glutamate receptor (GluD2). However, Cbln1's role, if any, in inhibitory synapse formation has been unknown. Here, we show that Cbln1 downregulates the formation and function of inhibitory synapses between Purkinje cells and interneurons. Immunohistochemical analyses with an anti-vesicular GABA transporter antibody revealed an increased density of interneuron-Purkinje cell synapses in the cbln1-null cerebellum. Whole-cell patch-clamp recordings from Purkinje cells showed that both the amplitude and frequency of miniature inhibitory postsynaptic currents were increased in cbln1-null cerebellar slices. A 3-h incubation with recombinant Cbln1 reversed the increased amplitude of inhibitory currents in Purkinje cells in acutely prepared cbln1-null slices. Furthermore, an 8-day incubation with recombinant Cbln1 reversed the increased interneuron-Purkinje cell synapse density in cultured cbln1-null slices. In contrast, recombinant Cbln1 did not affect cerebellar slices from mice lacking both Cbln1 and GluD2. Finally, we found that tyrosine phosphorylation was upregulated in the cbln1-null cerebellum, and acute inhibition of Src-family kinases suppressed the increased inhibitory postsynaptic currents in cbln1-null Purkinje cells. These findings indicate that Cbln1-GluD2 signaling inhibits the number and function of inhibitory synapses, and shifts the excitatory-inhibitory balance towards excitation in Purkinje cells. Cbln1's effect on inhibitory synaptic transmission is probably mediated by a tyrosine kinase pathway. © 2014 Federation of European Neuroscience Societies and John Wiley &amp; Sons Ltd.</p>

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Enriched expression of GluD1 in higher brain regions and its involvement in parallel fiber-interneuron synapse formation in the cerebellum

Konno Kohtarou, Matsuda Keiko, Nakamoto Chihiro, Uchigashima Motokazu, Miyazaki Taisuke, Yamasaki Miwako, Sakimura Kenji, Yuzaki Michisuke, Watanabe Masahiko

Journal of Neuroscience 34 ( 22 ) 7412 - 7424 2014

ISSN  0270-6474

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<p>Of the two members of the δ subfamily of ionotropic glutamate receptors, GluD2 is exclusively expressed at parallel fiber-Purkinje cell (PF-PC) synapses in the cerebellum and regulates their structural and functional connectivity. However, little is known to date regarding cellular and synaptic expression of GluD1 and its role in synaptic circuit formation. In the present study, we investigated this issue by producing specific and sensitive histochemical probes for GluD1 and analyzing cerebellar synaptic circuits in GluD1-knock-out mice. GluD1 was widely expressed in the adult mouse brain, with high levels in higher brain regions, including the cerebral cortex, striatum, limbic regions (hippocampus, nucleus accumbens, lateral septum, bed nucleus stria terminalis, lateral habenula, and central nucleus of the amygdala), and cerebellar cortex. In the cerebellar cortex, GluD1 mRNA was expressed at the highest level in molecular layer interneurons and its immunoreactivity was concentrated at PF synapses on interneuron somata. In GluD1-knock-out mice, the density of PF synapses on interneuron somata was significantly reduced and the size and number of interneurons were significantly diminished. Therefore, GluD1 is common to GluD2 in expression at PF synapses, but distinct from GluD2 in neuronal expression in the cerebellar cortex; that is, GluD1 in interneurons and GluD2 in PCs. Furthermore, GluD1 regulates the connectivity of PF-interneuron synapses and promotes the differentiation and/or survival of molecular layer interneurons. These results suggest that GluD1 works in concert with GluD2 for the construction of cerebellar synaptic wiring through distinct neuronal and synaptic expressions and also their shared synapse-connecting function. © 2014 the authors.</p>

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Reevaluation of the role of parallel fiber synapses in delay eyeblink conditioning in mice using Cbln1 as a tool

Emi Kyoichi, Kakegawa Wataru, Miura Eriko, Ito-Ishida Aya, Kohda Kazuhisa, Yuzaki Michisuke

Frontiers in Neural Circuits 7 ( NOV ) 180 2013.11

ISSN  1662-5110

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<p>The delay eyeblink conditioning (EBC) is a cerebellum-dependent type of associative motor learning. However, the exact roles played by the various cerebellar synapses, as well as the underlying molecular mechanisms, remain to be determined. It is also unclear whether long-term potentiation (LTP) or long-term depression (LTD) at parallel fiber (PF)-Purkinje cell (PC) synapses is involved in EBC. In this study, to clarify the role of PF synapses in the delay EBC, we used mice in which a gene encoding Cbln1 was disrupted (cbln1^-/- mice), which display severe reduction of PF-PC synapses. We showed that delay EBC was impaired in cbln1^-/- mice. Although PF-LTD was impaired, PF-LTP was normally induced in cbln1^-/- mice. A single recombinant Cbln1 injection to the cerebellar cortex in vivo completely, though transiently, restored the morphology and function of PF-PC synapses and delay EBC in cbln1^-/- mice. Interestingly, the cbln1^-/- mice retained the memory for at least 30 days, after the Cbln1 injection's effect on PF synapses had abated. Furthermore, delay EBC memory could be extinguished even after the Cbln1 injection's effect were lost.These results indicate that intact PF-PC synapses and PF-LTD, not PF-LTP are necessary to acquire delay EBC in mice. In contrast, extracerebellar structures or remaining PF-PC synapses in cbln1^-/- mice may be sufficient for the expression, maintenance, and extinction of its memory trace. © 2013 Emi, Kakegawa, Miura, Ito-Ishida, Kohda and Yuzaki.</p>

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Cerebellar LTD vs. motor learning-Lessons learned from studying GluD2

Yuzaki Michisuke

Neural Networks 47   36 - 41 2013.11

ISSN  0893-6080

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<p>Synaptic plasticity, such as long-term potentiation and long-term depression (LTD), is believed to underlie learning and memory processes in vivo. The cerebellum is an ideal brain region to obtain definitive proof for this hypothesis. The current belief is that the acquisition of motor learning is stored by LTD at the parallel fiber (PF)-Purkinje cell synapse in the cerebellar cortex. Recently, however, several lines of mutant mice that display normal motor learning in the absence of cerebellar LTD have been reported. A similar dichotomy between synaptic plasticity at the circuitry level and learning at the behavioral level has also been reported in the hippocampus. One possible explanation for this dichotomy is that compensatory pathways at the molecular and circuitry levels play an important role in mice that have been genetically modified for their entire lives. Mice that are genetically modified to be deficient in or to express mutant versions of the δ2 glutamate receptor (GluD2) serve as an interesting model due to the predominant expression of GluD2 at PF-Purkinje cell synapses. Furthermore, two major functions of GluD2-PF synapse formation and LTD induction-can be mechanistically dissociated so that the role of LTD in motor learning can be investigated in the absence of morphological abnormalities caused by altered synapse formation. Therefore, genetic manipulations of GluD2 will help to clarify the relationship between LTD and motor learning in the cerebellum. © 2012 Elsevier Ltd.</p>

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Deletions in GRID2 lead to a recessive syndrome of cerebellar ataxia and tonic upgaze in humans

Hills L. Benjamin, Masri Amira, Konno Kotaro, Kakegawa Wataru, Lam Anh Thu N, Lim-Melia Elizabeth, Chandy Nandini, Hill R. Sean, Partlow Jennifer N., Al-Saffar Muna, Nasir Ramzi, Stoler Joan M., Barkovich A. James, Watanabe Masahiko, Yuzaki Michisuke, Mochida Ganeshwaran H.

Neurology 81 ( 16 ) 1378 - 1386 2013.10

ISSN  0028-3878

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<p>Objective: To identify the genetic cause of a syndrome causing cerebellar ataxia and eye movement abnormalities. Methods: Weidentified 2 families with cerebellar ataxia, eyemovement abnormalities, and global developmental delay. We performed genetic analyses including single nucleotide polymorphism genotyping, linkage analysis, array comparative genomic hybridization, quantitative PCR, and Sanger sequencing. We obtained eye movement recordings of mutant mice deficient for the ortholog of the identified candidate gene, and performed immunohistochemistry using human and mouse brain specimens. Results: All affected individuals had ataxia, eye movement abnormalities, most notably tonic upgaze, and delayed speech and cognitive development. Homozygosity mapping identified the disease locus on chromosome 4q. Within this region, a homozygous deletion of GRID2 exon 4 in the index family and compound heterozygous deletions involving GRID2 exon 2 in the second family were identified. Grid2-deficient mice showed larger spontaneous and random eye movements compared to wild-type mice. In developing mouse and human cerebella, GRID2 localized to the Purkinje cell dendritic spines. Brain MRI in 2 affected children showed progressive cerebellar atrophy, which was more severe than that of Grid2-deficient mice. Conclusions: Biallelic deletions of GRID2 lead to a syndrome of cerebellar ataxia and tonic upgaze in humans. The phenotypic resemblance and similarity in protein expression pattern between humans and mice suggest a conserved role for GRID2 in the synapse organization between parallel fibers and Purkinje cells. However, the progressive and severe cerebellar atrophy seen in the affected individuals could indicate an evolutionarily unique role for GRID2 in the human cerebellum. © 2013 American Academy of Neurology.</p>

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The δ2 glutamate receptor gates long-term depression by coordinating interactions between two AMPA receptor phosphorylation sites

Kohda Kazuhisa, Kakegawa Wataru, Matsuda Shinji, Yamamoto Tadashi, Hirano Hisashi, Yuzaki Michisuke

Proceedings of the National Academy of Sciences of the United States of America 110 ( 10 ) E948 - 57 2013.03

ISSN  0027-8424

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<p>Long-term depression (LTD) commonly affects learning andmemoryin various brain regions. Although cerebellar LTD absolutely requires the δ2 glutamate receptor (GluD2) that is expressed in Purkinje cells, LTDin other brain regions does not;why andhowcerebellar LTD is regulated by GluD2 remains unelucidated. Here, we show that the activitydependent phosphorylation of serine 880 (S880) in GluA2 AMPA receptor subunit, which is an essential step for AMPA receptor endocytosis during LTD induction, was impaired in GluD2-null cerebellum. In contrast, the basal phosphorylation levels of tyrosine 876 (Y876) in GluA2were increased in GluD2-null cerebellum. An in vitro phosphorylation assay revealed that Y876 phosphorylation inhibited subsequent S880 phosphorylation. Conversely, Y876 dephosphorylation was sufficient to restore S880 phosphorylation and LTD induction in GluD2-null Purkinje cells. Furthermore, megakaryocyte protein tyrosine phosphatase (PTPMEG), which binds to the C terminus of GluD2, directly dephosphorylated Y876. These data indicate that GluD2 gates LTD by coordinating interactions between the two phosphorylation sites of the GluA2.</p>

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Unlocking the secrets of the δ2 glutamate receptor

Kohda Kazuhisa, Kakegawa Wataru, Yuzaki Michisuke

Communicative and Integrative Biology 6 ( 6 ) e26466 2013

ISSN  1942-0889

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<p>Long-term changes in synaptic transmission in the central nervous system, such as long-term potentiation and long-term depression (LTD), are believed to underlie learning and memory in vivo. Despite intensive research, the precise molecular mechanisms underlying these phenomena have remained unclear. LTD is most commonly caused by the endocytosis of postsynaptic AMPA-type glutamate receptors, triggered by activity- induced serine phosphorylation of the GluA2 subunit. Interestingly, cerebellar LTD, which occurs at synapses between parallel fibers (PFs; axons of granule cells) and Purkinje cells, is unique in requiring an additional type of glutamate receptor, the δ2 receptor (GluD2). Cbln1 was recently identified as a GluD2 ligand that regulates PF synapse formation and maintenance. However, how GluD2 induces downstream signaling in Purkinje cells to regulate LTD induction is unknown. We here present evidence that GluD2 reduces the tyrosine phosphorylation level of the GluA2 subunit via PTPMEG, a protein tyrosine phosphatase that binds to GluD2's C-terminus. We also found that the serine phosphorylation of GluA2, a crucial step for AMPA-receptor endocytosis, requires prior tyrosine dephosphorylation. Thus, GluD2 may serve as a gatekeeper for LTD induction by coordinating interactions between GluA2's 2 phosphorylation sites. © 2013 Landes Bioscience.</p>

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CAPS1 deficiency perturbs dense-core vesicle trafficking and golgi structure and reduces presynaptic release probability in the mouse brain

Sadakata Tetsushi, Kakegawa Wataru, Shinoda Yo, Hosono Mayu, Katoh-Semba Ritsuko, Sekine Yukiko, Sato Yumi, Tanaka Mika, Iwasato Takuji, Itohara Shigeyoshi, Furuyama Kenichiro, Kawaguchi Yoshiya, Ishizaki Yasuki, Yuzaki Michisuke, Furuichi Teiichi

Journal of Neuroscience 33 ( 44 ) 17326 - 17334 2013

ISSN  0270-6474

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<p>Ca ²⁺ -dependent activator protein for secretion 1 (CAPS1) plays a regulatory role in the dense-core vesicle (DCV) exocytosis pathway, but its functions at the cellular and synaptic levels in the brain are essentially unknown because of neonatal death soon after birth in Caps1 knock-out mice. To clarify the functions of the protein in the brain, we generated two conditional knock-out (cKO) mouse lines: 1) one lacking Caps1 in the forebrain; and 2) the other lacking Caps1 in the cerebellum. Both cKO mouse lines were born normally and grew to adulthood, although they showed subcellular and synaptic abnormalities. Forebrain-specific Caps1 cKO mice showed reduced immunoreactivity for the DCV marker secretogranin II (SgII) and the trans-Golgi network (TGN) marker syntaxin 6, a reduced number of presynaptic DCVs, and dilated trans-Golgi cisternae in the CA3 region. Cerebellum-specific Caps1 cKO mice had decreased immunoreactivity for SgII and brain-derived neurotrophic factor (BDNF) along the climbing fibers. At climbing fiber-Purkinje cell synapses, the number of DCVs was markedly lower and the number of synaptic vesicles was also reduced. Correspondingly, the mean amplitude of EPSCs was decreased, whereas paired-pulse depression was significantly increased. Our results suggest that loss of CAPS1 disrupts the TGN-DCV pathway, which possibly impairs synaptic transmission by reducing the presynaptic release probability. © 2013 the authors.</p>

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Stargazin regulates AMPA receptor trafficking through adaptor protein complexes during long-term depression

Matsuda Shinji, Kakegawa Wataru, Budisantoso Timotheus, Nomura Toshihiro, Kohda Kazuhisa, Yuzaki Michisuke

Nature Communications 4   2759 2013

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<p>Long-term depression (LTD) underlies learning and memory in various brain regions. Although postsynaptic AMPA receptor trafficking mediates LTD, its underlying molecular mechanisms remain largely unclear. Here we show that stargazin, a transmembrane AMPA receptor regulatory protein, forms a ternary complex with adaptor proteins AP-2 and AP-3A in hippocampal neurons, depending on its phosphorylation state. Inhibiting the stargazin-AP-2 interaction disrupts NMDA-induced AMPA receptor endocytosis, and inhibiting that of stargazin-AP-3A abrogates the late endosomal/lysosomal trafficking of AMPA receptors, thereby upregulating receptor recycling to the cell surface. Similarly, stargazin's interaction with AP-2 or AP-3A is necessary for low-frequency stimulus-evoked LTD in CA1 hippocampal neurons. Thus, stargazin has a crucial role in NMDA-dependent LTD by regulating two trafficking pathways of AMPA receptors - transport from the cell surface to early endosomes and from early endosomes to late endosomes/lysosomes - through its sequential binding to AP-2 and AP-3A. © 2013 Macmillan Publishers Limited. All rights reserved.</p>

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Efficient Derivation of Multipotent Neural Stem/Progenitor Cells from Non-Human Primate Embryonic Stem Cells

Shimada Hiroko, Okada Yohei, Ibata Keiji, Ebise Hayao, Ota Shin ichi, Tomioka Ikuo, Nomura Toshihiro, Maeda Takuji, Kohda Kazuhisa, Yuzaki Michisuke, Sasaki Erika, Nakamura Masaya, Okano Hideyuki

PLoS ONE 7 ( 11 ) e49469 2012.11

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<p>The common marmoset (Callithrix jacchus) is a small New World primate that has been used as a non-human primate model for various biomedical studies. We previously demonstrated that transplantation of neural stem/progenitor cells (NS/PCs) derived from mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) promote functional locomotor recovery of mouse spinal cord injury models. However, for the clinical application of such a therapeutic approach, we need to evaluate the efficacy and safety of pluripotent stem cell-derived NS/PCs not only by xenotransplantation, but also allotransplantation using non-human primate models to assess immunological rejection and tumorigenicity. In the present study, we established a culture method to efficiently derive NS/PCs as neurospheres from common marmoset ESCs. Marmoset ESC-derived neurospheres could be passaged repeatedly and showed sequential generation of neurons and astrocytes, similar to that of mouse ESC-derived NS/PCs, and gave rise to functional neurons as indicated by calcium imaging. Although marmoset ESC-derived NS/PCs could not differentiate into oligodendrocytes under default culture conditions, these cells could abundantly generate oligodendrocytes by incorporating additional signals that recapitulate in vivo neural development. Moreover, principal component analysis of microarray data demonstrated that marmoset ESC-derived NS/PCs acquired similar gene expression profiles to those of fetal brain-derived NS/PCs by repeated passaging. Therefore, marmoset ESC-derived NS/PCs may be useful not only for accurate evaluation by allotransplantation of NS/PCs into non-human primate models, but are also applicable to analysis of iPSCs established from transgenic disease model marmosets. © 2012 Shimada et al.</p>

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Presynaptically Released Cbln1 Induces Dynamic Axonal Structural Changes by Interacting with GluD2 during Cerebellar Synapse Formation

Ito-Ishida Aya, Miyazaki Taisuke, Miura Eriko, Matsuda Keiko, Watanabe Masahiko, Yuzaki Michisuke, Okabe Shigeo

Neuron 76 ( 3 ) 549 - 564 2012.11

ISSN  0896-6273

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<p>Differentiation of pre- and postsynaptic sites is coordinated by reciprocal interaction across synaptic clefts. At parallel fiber (PF)-Purkinje cell (PC) synapses, dendritic spines are autonomously formed without PF influence. However, little is known about how presynaptic structural changes are induced and how they lead to differentiation of mature synapses. Here, we show that Cbln1 released from PFs induces dynamic structural changes in PFs by a mechanism that depends on postsynaptic glutamate receptor delta2 (GluD2) and presynaptic neurexin (Nrx). Time-lapse imaging in organotypic culture and ultrastructural analyses in vivo revealed that Nrx-Cbln1-GluD2 signaling induces PF protrusions that often formed circular structures and encapsulated PC spines. Such structural changes in PFs were associated with the accumulation of synaptic vesicles and GluD2, leading to formation of mature synapses. Thus, PF protrusions triggered by Nrx-Cbln1-GluD2 signaling may promote bidirectional maturation of PF-PC synapses by a positive feedback mechanism</p>

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Characteristics of Gait Ataxia in δ2 Glutamate Receptor Mutant Mice, ho15J

Takeuchi Eri, Sato Yamato, Miura Eriko, Yamaura Hiroshi, Yuzaki Michisuke, Yanagihara Dai

PLoS ONE 7 ( 10 )  2012.10

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<p>The cerebellum plays a fundamental, but as yet poorly understood, role in the control of locomotion. Recently, mice with gene mutations or knockouts have been used to investigate various aspects of cerebellar function with regard to locomotion. Although many of the mutant mice exhibit severe gait ataxia, kinematic analyses of limb movements have been performed in only a few cases. Here, we investigated locomotion in ho15J mice that have a mutation of the δ2 glutamate receptor. The cerebellum of ho15J mice shows a severe reduction in the number of parallel fiber-Purkinje synapses compared with wild-type mice. Analysis of hindlimb kinematics during treadmill locomotion showed abnormal hindlimb movements characterized by excessive toe elevation during the swing phase, and by severe hyperflexion of the ankles in ho15J mice. The great trochanter heights in ho15J mice were lower than in wild-type mice throughout the step cycle. However, there were no significant differences in various temporal parameters between ho15J and wild-type mice. We suggest that dysfunction of the cerebellar neuronal circuits underlies the observed characteristic kinematic abnormality of hindlimb movements during locomotion of ho15J mice. © 2012 Takeuchi et al.</p>

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Selective and regulated gene expression in murine Purkinje cells by in utero electroporation

Nishiyama Jun, Hayashi Yukari, Nomura Toshihiro, Miura Eriko, Kakegawa Wataru, Yuzaki Michisuke

European Journal of Neuroscience 36 ( 7 ) 2867 - 2876 2012.10

ISSN  0953-816X

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<p>Cerebellar Purkinje cells, which convey the only output from the cerebellar cortex, play an essential role in cerebellar functions, such as motor coordination and motor learning. To understand how Purkinje cells develop and function in the mature cerebellum, an efficient method for molecularly perturbing them is needed. Here we demonstrate that Purkinje cell progenitors at embryonic day (E)11.5 could be efficiently and preferentially transfected by spatially directed in utero electroporation (IUE) with an optimized arrangement of electrodes. Electrophysiological analyses indicated that the electroporated Purkinje cells maintained normal membrane properties, synaptic responses and synaptic plasticity at postnatal days 25-28. By combining the L7 promoter and inducible Cre/loxP system with IUE, transgenes were expressed even more specifically in Purkinje cells and in a temporally controlled manner. We also show that three different fluorescent proteins could be simultaneously expressed, and that Bassoon, a large synaptic protein, could be expressed in the electroporated Purkinje cells. Moreover, phenotypes of staggerer mutant mice, which have a deletion in the gene encoding retinoid-related orphan receptor α (RORα1), were recapitulated by electroporating a dominant-negative form of RORα1 into Purkinje cells at E11.5. Together, these results indicate that this new IUE protocol, which allows the selective, effective and temporally regulated expression of multiple foreign genes transfected into Purkinje cell progenitors in vivo, without changing the cells' physiological characteristics, is a powerful tool for elucidating the molecular mechanisms underlying early Purkinje cell developmental events, such as dendritogenesis and migration, and synaptic plasticity in mature Purkinje cells. A new in utero electroporation (IUE) protocol has been developed to deliver genes preferentially into cerebellar Purkinje cells. IUE did not alter the physiological characteristics or normal synaptic plasticity of Purkinje cells. IUE allowed selective, effective, and temporally regulated expression of multiple foreign genes in Purkinje cells in vivo. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.</p>

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THE INS and OUTS of GluD2-why and how purkinje cells use the special glutamate receptor

Yuzaki Michisuke

Cerebellum 11 ( 2 ) 438 - 439 2012.06

ISSN  1473-4222

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<p>The δ2 glutamate receptor (GluD2) is predominantly expressed in cerebellar Purkinje cells and plays crucial roles in cerebellar functions. Indeed, the number of synapses between parallel fibers (PFs) and Purkinje cells is specifically and severely reduced in GluD2-null cerebellum. In addition, long-term depression (LTD) at PF-Purkinje cell synapses is impaired in these mice. Nevertheless, the mechanism by which GluD2 regulate these two functions-morphological and functional synaptic plasticity at PF synapses-has remained unclear. Recently, we found that Cbln1, a glycoprotein released from granule cells, was bound to the N-terminal domain of GluD2 and regulated formation and maintenance of PF-Purkinje cell synapses. Furthermore, we found that D-Ser released from Bergmann glia bound the ligand-binding domain of GluD2 and mediated LTD in a manner dependent on the C-terminus. These findings indicate how GluD2 is activated and regulates functions at PF-Purkinje cell synapses. A hypothesis about why GluD2 is employed by PF synapses is also discussed. ©Springer Science+Business Media, LLC 2011.</p>

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Cbln1 and the delta2 glutamate receptor - An orphan ligand and an orphan receptor find their partners

Matsuda Keiko, Yuzaki Michisuke

Cerebellum 11 ( 1 ) 78 - 84 2012.03

ISSN  1473-4222

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<p>Cerebellin was originally discovered as a Purkinje cell-specific peptide more than two decades ago. Later, its precursor protein precerebellin (Cbln1) was found to be produced in cerebellar granule cells. It has become increasingly clear that although the cerebellin peptide may have certain functions, Cbln1 is an actual signaling molecule that belongs to the C1q family. However, the precise function of Cbln1 has been unresolved. Cbln1 is released from granule cells, and disruption of the cbln1 gene in mice causes a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs; axons of granule cells) and results in cerebellar ataxia. The glutamate receptor δ2 (GluD2) is highly expressed on Purkinje cells' dendritic spines which make synapses with PFs. Although GluD2 was identified as a member of the ionotropic glutamate receptors more than 15 years ago, it has been referred to as an orphan receptor because its endogenous ligands are unclear. Interestingly, GluD2-null mice phenocopy cbln1-null mice precisely. Cbln1 and GluD2 have therefore been thought to participate in a common signaling pathway that is required for the formation of PF synapses. We recently established a direct ligand-receptor relationship between Cbln1 and GluD2. The Cbln1-GluD2 complex is located at the cleft of PF-Purkinje cell synapses and bidirectionally regulates both presynaptic and postsynaptic differentiation. © 2010 Springer Science+Business Media, LLC.</p>

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Cerebellar long-term depression requires dephosphorylation of TARP in Purkinje cells

Nomura Toshihiro, Kakegawa Wataru, Matsuda Shinji, Kohda Kazuhisa, Nishiyama Jun, Takahashi Takao, Yuzaki Michisuke

European Journal of Neuroscience 35 ( 3 ) 402 - 410 2012.02

ISSN  0953-816X

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<p>Cerebellar long-term depression (LTD) at parallel fiber (PF)-Purkinje cell synapses is thought to play an essential role in certain forms of motor learning. Like hippocampal LTD, cerebellar LTD is mediated by the endocytosis of AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) receptors at postsynaptic sites. However, similar sets of kinases and phosphatases have opposite regulatory effects on hippocampal and cerebellar LTD, although the mechanisms responsible for this difference remain largely unclear. Activity-dependent dephosphorylation of stargazin (an AMPA receptor auxiliary protein) by calcineurin regulates hippocampal LTD, but whether and how stargazin is involved in cerebellar LTD is unknown. In this study, we showed that stargazin is highly phosphorylated at basal states and is dephosphorylated by the application of high KCl plus glutamate (K-glu) or of a metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), both of which chemically induced LTD in cerebellar slices. This chemically induced dephosphorylation of stargazin was specifically blocked by a calcineurin inhibitor. Indeed, inclusion of the calcineurin auto-inhibitory peptide in the patch pipette solution completely inhibited the LTD induced by the conjunctive stimulation of PFs and Purkinje cells. Furthermore, in Purkinje cells expressing stargazin-9D, in which all nine serine residues are mutated to aspartate, neither conjunctive stimulus nor DHPG treatment induced LTD. Finally, immunohistochemical analyses revealed that neither K-glu nor DHPG induced the endocytosis of AMPA receptors in Purkinje cells expressing stargazin-9D. Together, these results indicate that hippocampal and cerebellar LTD share a common pathway, namely dephosphorylation of stargazin by calcineurin. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.</p>

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NMDA Receptor-Mediated PIP5K Activation to Produce PI(4,5)P ₂ Is Essential for AMPA Receptor Endocytosis during LTD

Unoki Takamitsu, Matsuda Shinji, Kakegawa Wataru, Van Ngo Thai Bich, Kohda Kazuhisa, Suzuki Atsushi, Funakoshi Yuji, Hasegawa Hiroshi, Yuzaki Michisuke, Kanaho Yasunori

Neuron 73 ( 1 ) 135 - 148 2012.01

ISSN  0896-6273

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<p>NMDA receptor activation leads to clathrin-dependent endocytosis of postsynaptic AMPA receptors. Although this process controls long-term depression (LTD) induction in the hippocampus, how it is regulated by neuronal activities is not completely clear. Here, we show that Ca ²⁺ influx through the NMDA receptor activates calcineurin and protein phosphatase 1 to dephosphorylate phosphatidylinositol 4-phosphate 5-kinaseγ661 (PIP5Kγ661), the major phosphatidylinositol 4,5-bisphosphate (PI(4,5)P ₂)-producing enzyme in the brain. Bimolecular fluorescence complementation analysis revealed that the dephosphorylated PIP5Kγ661 became associated with the clathrin adaptor protein complex AP-2 atpostsynapses insitu. NMDA-induced AMPA receptor endocytosis and low-frequency stimulation-induced LTD were completely blocked by inhibiting the association between dephosphorylated PIP5Kγ661 and AP-2 and by overexpression ofa kinase-dead PIP5Kγ661 mutant in hippocampal neurons. Furthermore, knockdown of PIP5Kγ661 inhibited the NMDA-induced AMPA receptor endocytosis. Therefore, NMDA receptor activation controls AMPA receptor endocytosis during hippocampal LTD by regulating PIP5Kγ661 activity at postsynapses. AMPA receptor endocytosis is essential for LTD induction, but it is not clear how this process is controlled. Unoki etal. find that NMDA receptor activation controls AMPA receptor endocytosis during hippocampal LTD by regulating the activity of the enzyme PIP5Kγ661. © 2012 Elsevier Inc.</p>

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A new rapid protocol for eyeblink conditioning to assess cerebellar motor learning

Emi Kyoichi, Kohda Kazuhisa, Kakegawa Wataru, Narumi Sakae, Yuzaki Michisuke

Neurochemical Research 36 ( 7 ) 1314 - 1322 2011.07

ISSN  0364-3190

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<p>Mice with spontaneous and induced mutations causing cerebellar phenotypes have provided key insights into how motor-related memories are stored in cerebellar circuits. Delayed eyeblink conditioning is a form of associative motor learning that depends on the cerebellum. However, neurochemical investigation of the underlying mechanisms has been hampered by the long training period (usually several days) required to establish conditioning. Here, we report a new rapid-training protocol that reliably induced delayed eyeblink conditioning within a single day. The associative memory formation depended on the expression of the δ2 glutamate receptor (GluD2) in cerebellar Purkinje cells. It lasted for several weeks, but could be erased by extinction sessions in a single day. In addition, using the rapid protocol, we found that eyeblink conditioning could be induced in juvenile mice at postnatal day 21, and that the Sindbis-virus-mediated expression of GluD2 could rescue the impaired eyeblink conditioning in GluD2-null mice in vivo. © 2011 Springer Science+Business Media, LLC.</p>

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D-Serine regulates cerebellar LTD and motor coordination through the δ glutamate receptor

Kakegawa Wataru, Miyoshi Yurika, Hamase Kenji, Matsuda Shinji, Matsuda Keiko, Kohda Kazuhisa, Emi Kyoichi, Motohashi Junko, Konno Ryuichi, Zaitsu Kiyoshi, Yuzaki Michisuke

Nature Neuroscience 14 ( 5 ) 603 - 613 2011.05

ISSN  1097-6256

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<p>D-Serine (D-Ser) is an endogenous co-agonist for NMDA receptors and regulates neurotransmission and synaptic plasticity in the forebrain. D-Ser is also found in the cerebellum during the early postnatal period. Although D-Ser binds to the δ glutamate receptor (GluD2, Grid2) in vitro, its physiological significance has remained unclear. Here we show that D-Ser serves as an endogenous ligand for GluD2 to regulate long-term depression (LTD) at synapses between parallel fibers and Purkinje cells in the immature cerebellum. D-Ser was released mainly from Bergmann glia after the burst stimulation of parallel fibers in immature, but not mature, cerebellum. D-Ser rapidly induced endocytosis of AMPA receptors and mutually occluded LTD in wild-type, but not Grid2-null, Purkinje cells. Moreover, mice expressing mutant GluD2 in which the binding site for D-Ser was disrupted showed impaired LTD and motor dyscoordination during development. These results indicate that glial D-Ser regulates synaptic plasticity and cerebellar functions by interacting with GluD2. © 2011 Nature America, Inc. All rights reserved.</p>

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Cbln family proteins promote synapse formation by regulating distinct neurexin signaling pathways in various brain regions

Matsuda Keiko, Yuzaki Michisuke

European Journal of Neuroscience 33 ( 8 ) 1447 - 1461 2011.04

ISSN  0953-816X

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<p>Cbln1 (a.k.a. precerebellin) is a unique bidirectional synaptic organizer that plays an essential role in the formation and maintenance of excitatory synapses between granule cells and Purkinje cells in the mouse cerebellum. Cbln1 secreted from cerebellar granule cells directly induces presynaptic differentiation and indirectly serves as a postsynaptic organizer by binding to its receptor, the δ2 glutamate receptor. However, it remains unclear how Cbln1 binds to the presynaptic sites and interacts with other synaptic organizers. Furthermore, although Cbln1 and its family members Cbln2 and Cbln4 are expressed in brain regions other than the cerebellum, it is unknown whether they regulate synapse formation in these brain regions. In this study, we showed that Cbln1 and Cbln2, but not Cbln4, specifically bound to its presynaptic receptor -α and β isoforms of neurexin carrying the splice site 4 insert [NRXs(S4+)] - and induced synaptogenesis in cerebellar, hippocampal and cortical neurons in vitro. Cbln1 competed with synaptogenesis mediated by neuroligin 1, which lacks the splice sites A and B, but not leucine-rich repeat transmembrane protein 2, possibly by sharing the presynaptic receptor NRXs(S4+). However, unlike neurexins/neuroligins or neurexins/leucine-rich repeat transmembrane proteins, the interaction between NRX1β(S4+) and Cbln1 was insensitive to extracellular Ca²⁺ concentrations. These findings revealed the unique and general roles of Cbln family proteins in mediating the formation and maintenance of synapses not only in the cerebellum but also in various other brain regions. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.</p>

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Cbln1 and its family proteins in synapse formation and maintenance

Yuzaki Michisuke

Current Opinion in Neurobiology 21 ( 2 ) 215 - 220 2011.04

ISSN  0959-4388

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<p>Cbln1 is a newly identified synaptic organizer belonging to the C1q family. Unlike other synaptic organizers, a deficiency in Cbln1 is sufficient to cause a severe reduction in the number of synapses between cerebellar Purkinje cells and parallel fibers (PFs). Furthermore, Cbln1 can rapidly induce synaptogenesis and is necessary for maintaining normal synapses in the mature cerebellum in vivo. Cbln1 was recently identified as the missing ligand for the orphan glutamate receptor δ2 (GluD2), which is expressed in Purkinje cells. Furthermore, Cbln1 released from PFs binds to neurexin (NRX) expressed on the presynaptic PFs and GluD2 at the postsynaptic site. The NRX/Cbln1/GluD2 tripartite complex is resistant to low extracellular Ca²⁺ levels and serves as a unique bidirectional synaptic organizer. © 2011 Elsevier Ltd.</p>

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Serotonin Mediates Cross-Modal Reorganization of Cortical Circuits

Jitsuki Susumu, Takemoto Kiwamu, Kawasaki Taisuke, Tada Hirobumi, Takahashi Aoi, Becamel Carine, Sano Akane, Yuzaki Michisuke, Zukin R. Suzanne, Ziff Edward B., Kessels Helmut W., Takahashi Takuya

Neuron 69 ( 4 ) 780 - 792 2011.02

ISSN  0896-6273

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<p>Loss of one type of sensory input can cause improved functionality of other sensory systems. Whereas this form of plasticity, cross-modal plasticity, is well established, the molecular and cellular mechanisms underlying it are still unclear. Here, we show that visual deprivation (VD) increases extracellular serotonin in the juvenile rat barrel cortex. This increase in serotonin levels facilitates synaptic strengthening at layer 4 to layer 2/3 synapses within the barrel cortex. Upon VD, whisker experience leads to trafficking of the AMPA-type glutamate receptors (AMPARs) into these synapses through the activation of ERK and increased phosphorylation of AMPAR subunit GluR1 at the juvenile age when natural whisker experience no longer induces synaptic GluR1 delivery. VD thereby leads to sharpening of the functional whisker-barrel map at layer 2/3. Thus, sensory deprivation of one modality leads to serotonin release in remaining modalities, facilitates GluR1-dependent synaptic strengthening, and refines cortical organization. © 2011 Elsevier Inc.</p>

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Snapin snaps into the dynein complex for late endosome-lysosome trafficking and autophagy

Yuzaki Michisuke

Neuron 68 ( 1 ) 4 - 6 2010.10

ISSN  0896-6273

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<p>Late endosome-lysosome trafficking plays a key role in regulating cell surface signaling and degradation of intracellular components by autophagy. New work by Cai and coworkers in this issue of Neuron provides evidence that snapin regulates the recruitment of late endosomes to the dynein motor complex for retrograde trafficking along microtubules and maturation of lysosomes. © 2010 Elsevier Inc.</p>

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Therapeutic potential of appropriately evaluated safe-induced pluripotent stem cells for spinal cord injury

Tsuji Osahiko, Miura Kyoko, Okada Yohei, Fujiyoshi Kanehiro, Mukaino Masahiko, Nagoshi Narihito, Kitamura Kazuya, Kumagai Gentaro, Nishino Makoto, Tomisato Shuta, Higashi Hisanobu, Nagai Toshihiro, Katoh Hiroyuki, Kohda Kazuhisa, Matsuzaki Yumi, Yuzaki Michisuke, Ikeda Eiji, Toyama Yoshiaki, Nakamura Masaya, Yamanaka Shinya, Okano Hideyuki

Proceedings of the National Academy of Sciences of the United States of America 107 ( 28 ) 12704 - 12709 2010.07

ISSN  0027-8424

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<p>Various types of induced pluripotent stem (iPS) cells have been established by different methods, and each type exhibits different biological properties. Before iPS cell-based clinical applications can be initiated, detailed evaluations of the cells, including their differentiation potentials and tumorigenic activities in different contexts, should be investigated to establish their safety and effectiveness for cell transplantation therapies. Here we show the directed neural differentiation of murine iPS cells and examine their therapeutic potential in amouse spinal cord injury (SCI)model. "Safe" iPS-derived neurospheres, which had been pre-evaluated as nontumorigenic by their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse brain, produced electrophysiologically functional neurons, astrocytes, and oligodendrocytes in vitro. Furthermore, when the safe iPS-derived neurospheres were transplanted into the spinal cord 9 d after contusive injury, they differentiated into all three neural lineages without forming teratomas or other tumors. They also participated in remyelination and induced the axonal regrowth of host 5HT⁺ serotonergic fibers, promoting locomotor function recovery. However, the transplantation of iPSderived neurospheres pre-evaluated as "unsafe" showed robust teratoma formation and sudden locomotor functional loss after functional recovery in the SCI model. These findings suggest that preevaluated safe iPS clone-derived neural stem/progenitor cells may be a promising cell source for transplantation therapy for SCI.</p>

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Recent excitements about excitatory synapses

Scheiffele Peter, Yuzaki Michizuke

European Journal of Neuroscience 32 ( 2 ) 179 - 180 2010.07

ISSN  0953-816X

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Synapse formation and maintenance by C1q family proteins

Yuzaki Michisuke

European Journal of Neuroscience 32 ( 2 ) 191 - 197 2010.07

ISSN  0953-816X

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<p>Several C1q family members, especially the Cbln and C1q-like subfamilies, are highly and predominantly expressed in the central nervous system. Cbln1, a member of the Cbln subfamily, plays two unique roles at parallel ber (PF)-Purkinje cell synapses in the cerebellum: the formation and stabilization of synaptic contact, and the control of functional synaptic plasticity by regulating the postsynaptic endocytotic pathway. The delta2 glutamate receptor (GluD2), which is predominantly expressed in Purkinje cells, plays similar critical roles in the cerebellum. In addition, viral expression of GluD2 or the application of recombinant Cbln1 induces PF- Purkinje cell synaptogenesis in vitro and in vivo. Antigen-unmasking methods were necessary to reveal the immunoreactivities for endogenous Cbln1 and GluD2 at the synaptic junction of PF synapses. We propose that Cbln1 and GluD2 are located at the synaptic cleft, where various proteins undergo intricate molecular interactions with each other, and serve as a bidirectional synaptic organizer. © The Author (2010). Journal Compilation © Federation of European Neuroscience Societies and Blackwell Publishing Ltd.</p>

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A response to Dr. Yue's commentary

Yuzaki Michisuke, Nishiyama Jun

Autophagy 6 ( 4 ) 573 2010.05

ISSN  1554-8627

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Excitotoxicity and autophagy

Nishiyama Jun, Yuzaki Michisuke

Autophagy 6 ( 4 ) 568 - 570 2010.05

ISSN  1554-8627

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<p>The role of autophagy in excitotoxic cell death caused by excessive activation of glutamate receptors has been a contentious issue. Lurcher (Lc) mutant mice, in which a mutant glutamate receptor causes continuous ion flow and kills cerebellar Purkinje cells, have been cited as a model of cell death resulting from autophagy, or "autophagic cell death," in vivo. Here, we reinvestigated Lc-mediated cell death in heterologous cells and cultured neurons as well as in Lc mice in vivo. We show that Lc-mediated cell death is likely not caused by autophagy, but rather by necrosis with autophagic features. Constitutive ion flux per se causes reduction of intracellular ATP levels, which activates the autophagic pathways. Therefore, activation of autophagy might have a homeostatic protective role to maintain intracellular ATP in the Lc model of excitotoxicity. © 2010 Landes Bioscience.</p>

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Cbln1 is a ligand for an orphan glutamate receptor δ2, a bidirectional synapse organizer

Matsuda Keiko, Miura Eriko, Miyazaki Taisuke, Kakegawa Wataru, Emi Kyoichi, Narumi Sakae, Fukazawa Yugo, Ito-lshida Aya, Kondo Tetsuro, Shigemoto Ryuichi, Watanabe Masahiko, Yuzaki Michisuke

Science 328 ( 5976 ) 363 - 368 2010.04

ISSN  0036-8075

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<p>Cbln1, secreted from cerebellar granule cells, and the orphan glutamate receptor 52 (GluD2), expressed by Purkinje cells, are essential for synapse integrity between these neurons in adult mice. Nevertheless, no endogenous binding partners for these molecules have been identified. We found that Cblnl binds directly to the N-terminal domain of GluD2. GluD2 expression by postsynaptic cells, combined with exogenously applied Cbln1, was necessary and sufficient to induce new synapses in vitro and in the adult cerebellum in vivo. Further, beads coated with recombinant Cbln1 directly induced presynaptic differentiation and indirectly caused clustering of postsynaptic molecules via GluD2. These results indicate that the Cbln1-GluD2 complex is a unique synapse organizer that acts bidirectionally on both pre- and postsynaptic components.</p>

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Dynein- and activity-dependent retrograde transport of autophagosomes in neuronal axons

Katsumata Kiyoshi, Nishiyama Jun, Inoue Takafumi, Mizushima Noboru, Takeda Junzo, Yuzaki Michisuke

Autophagy 6 ( 3 ) 378 - 385 2010.04

ISSN  1554-8627

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<p>The accumulation of autophagosomes within axons is often observed in axonopathies associated with various neurological disorders, including those following excitotoxic insults. Nevertheless, the life cycle of autophagosomes in axons is not well understood. In the present study, we used microexplant cultures of cerebellar granule cells from GFP-LC3 transgenic mice to perform time-lapse imaging of LC3-positive dots in identified axons. Since these GFP-LC3 dots were never observed in granule cells on an Atg5-null background, they were considered to represent autophagosomes. Under physiological conditions, the autophagosomes showed bidirectional and saltatory movement with a bias towards one direction. Such vectorial movement was largely blocked by the dynein motor inhibitor EHNA (erythro-9-[3-(2-hydroxynonyl)] adenine), suggesting that the autophagosomes moved towards the soma, where most lysosomes are located. Interestingly, the application of the glutamate analog N-methyl-D-aspartic acid (NMDA) as an excitotoxin increased the number of autophagosomes in axons, while it did not significantly change its movement characteristics. These results suggest that autophagosomes play important roles in axons and are dynamically regulated under physiological and pathological conditions. © 2010 Landes Bioscience.</p>

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Reevaluation of neurodegeneration in lurcher mice

Nishiyama Jun, Matsuda Keiko, Kakegawa Wataru, Yamada Nobuaki, Motohashi Junko, Mizushima Noboru, Yuzaki Michisuke

Journal of Neuroscience 30 ( 6 ) 2177 - 2187 2010.02

ISSN  0270-6474

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<p>The lurcher (Lc) mice have served as a valuable model for neurodegeneration for decades. Although the responsible mutation was identified in genes encoding δ2 glutamate receptors (GluD2s), which are predominantly expressed in cerebellar Purkinje cells, how the mutant receptor (GluD2^Lc) triggers cell death has remained elusive. Here, taking advantage of recent knowledge about the domain structure of GluD2, we reinvestigated Lc-mediated cell death, focusing on the "autophagic cell death" hypothesis. Although autophagy and cell death were induced by the expression of GluD2^Lc in heterologous cells and cultured neurons, they were blocked by the introduction of mutations in the channel pore domain of GluD2^Lc or by removal of extracellular Na⁺. In addition, although GluD2^Lc is reported to directly activate autophagy, mutant channels that are not associated with n-PIST (neuronal isoform of protein-interacting specifically with TC10)-Beclin1 still caused autophagy and cell death. Furthermore, cells expressing GluD2^Lc showed decreased ATP levels and increased AMP-activated protein kinase(AMPK)activities in a manner dependent on extracellular Na⁺. Thus, constitutive currents were likely necessary and sufficient to induce autophagy via AMPK activation, regardless of the n-PIST-Beclin1 pathway in vitro. Interestingly, the expression of dominant-negative AMPK suppressed GluD2^Lc-induced autophagy but did not prevent cell death in heterologous cells. Similarly, the disruption of Atg5, a gene crucial for autophagy, did not prevent but rather aggravated Purkinje-cell death in Lc mice. Furthermore, calpains were specifically activated in Lc Purkinje cells. Together, these results suggest that Lc-mediated cell death was not caused by autophagy but necrosis with autophagic features both in vivo and in vitro. Copyright © 2010 the authors.</p>

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Efficient generation of mature cerebellar Purkinje cells from mouse embryonic stem cells

Tao Osamu, Shimazaki Takuya, Okada Yohei, Naka Hayato, Kohda Kazuhisa, Yuzaki Michisuke, Mizusawa Hidehiro, Okano Hideyuki

Journal of Neuroscience Research 88 ( 2 ) 234 - 247 2010.02

ISSN  0360-4012

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<p>Mouse embryonic stem cells (ESCs) can generate cerebellar neurons, including Purkinje cells (PCs) and their precursor cells, in a .oating culture system called serum-free culture of embryoid body-like aggregates (SFEB) treated with BMP4, Fgf8b, and Wnt3a. Here we successfully established a coculture system that induced the maturation of PCs in ESC-derived Purkinje cell (EDPC) precursors in SFEB, using as a feeder layer a cerebellum dissociation culture prepared from mice at postnatal day (P) 6-8. PC maturation was incomplete or abnormal when the adherent culture did not include feeder cells or when the feeder layer was from neonatal cerebellum. In contrast, EDPCs exhibited the morphology of mature PCs and synaptogenesis with other cerebellar neurons when grown for 4 weeks in coculture system with the postnatal cerebellar feeder. Furthermore, the electrophysiological properties of these EDPCs were compatible with those of native mature PCs in vitro, such as Na⁺ or Ca2 ⁺ spikes elicited by current injections and excitatory or inhibitory postsynaptic currents, which were assessed by whole-cell patch-clamp recordings. Thus, EDPC precursors in SFEB can mature into PCs whose properties are comparable with those of native PCs in vitro. © 2009 Wiley-Liss, Inc.</p>

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Ionotropic glutamate receptor AMPA 1 is associated with ovulation rate

Sugimoto Mayumi, Sasaki Shinji, Watanabe Toshio, Nishimura Shota, Ideta Atsushi, Yamazaki Maya, Matsuda Keiko, Yuzaki Michisuke, Sakimura Kenji, Aoyagi Yoshito, Sugimoto Yoshikazu

PLoS ONE 5 ( 11 )  2010

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<p>Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system by opening ion channels upon the binding of glutamate. Despite the essential roles of glutamate in the control of reproduction and anterior pituitary hormone secretion, there is a limited understanding of how glutamate receptors control ovulation. Here we reveal the function of the ionotropic glutamate receptor AMPA-1 (GRIA1) in ovulation. Based on a genome-wide association study in Bos taurus, we found that ovulation rate is influenced by a variation in the N-terminal leucine/ isoleucine/valine-binding protein (LIVBP) domain of GRIA1, in which serine is replaced by asparagine. GRIA1^Asn has a weaker affinity to glutamate than GRIA1^Ser, both in Xenopus oocytes and in the membrane fraction of bovine brain. This single amino acid substitution leads to the decreased release of gonadotropin-releasing hormone (GnRH) in immortalized hypothalamic GT1-7 cells. Cows with GRIA1^Asn have a slower luteinizing hormone (LH) surge than cows with GRIA1^Ser. In addition, cows with GRIA1^Asn possess fewer immature ovarian follicles before superovulation and have a lower response to hormone treatment than cows with GRIA1^Ser. Our work identified that GRIA1 is a critical mediator of ovulation and that GRIA1 might be a useful target for reproductive therapy. © 2010 Sugimoto et al.</p>

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Distinct expression of C1q-like family mRNAs in mouse brain and biochemical characterization of their encoded proteins

Iijima Takatoshi, Miura Eriko, Watanabe Masahiko, Yuzaki Michisuke

European Journal of Neuroscience 31 ( 9 ) 1606 - 1615 2010

ISSN  0953-816X

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<p>Many members of the C1q family, including complement C1q and adiponectin, and the structurally related tumor necrosis factor family are secreted and play crucial roles in intercellular signaling. Among them, the Cbln (precerebellin) and C1q-like (C1ql) subfamilies are highly and predominantly expressed in the central nervous system. Although the Cbln subfamily serve as essential trans-neuronal regulators of synaptic integrity in the cerebellum, the functions of the C1ql subfamily (C1ql1-C1ql4) remain unexplored. Here, we investigated the gene expression of the C1ql subfamily in the adult and developing mouse brain by reverse transcriptase-polymerase chain reaction and high-resolution in-situ hybridization. In the adult brain, C1ql1-C1ql3 mRNAs were mainly expressed in neurons but weak expression was seen in glia-like structures in the adult brain. The C1ql1 mRNA was predominantly expressed in the inferior olive, whereas the C1ql2 and C1ql3 mRNAs were strongly coexpressed in the dentate gyrus. Although the C1ql1 and C1ql3 mRNAs were detectable as early as embryonic day 13, the C1ql2 mRNA was observed at later embryonic stages. The C1ql1 mRNA was also expressed transiently in the external granular layer of the cerebellum. Biochemical characterization in heterologous cells revealed that all of the C1ql subfamily proteins were secreted and they formed both homomeric and heteromeric complexes. They also formed hexameric and higher-order complexes via their N-terminal cysteine residues. These results suggest that, like Cbln, the C1ql subfamily has distinct spatial and temporal expression patterns and may play diverse roles by forming homomeric and heteromeric complexes in the central nervous system. © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.</p>

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New (but old) molecules regulating synapse integrity and plasticity

Yuzaki M.

Neuroscience 162 ( 3 ) 633 - 643 2009.09

ISSN  0306-4522

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<p>The δ2 glutamate receptor (GluRδ2) is predominantly expressed in cerebellar Purkinje cells and plays crucial roles in cerebellar functions: GluRδ2-null mice display ataxia and impaired motor learning. Interestingly, the contact state of synapses between parallel fibers (PFs) and Purkinje cells is specifically and severely affected, and the number of normal PF synapses is markedly reduced in GluRδ2-null Purkinje cells. Furthermore, long-term depression at PF-Purkinje cell synapses is abrogated. Cbln1, a member of the C1q/tumor necrosis factor (TNF) superfamily, is predominantly expressed and released from cerebellar granule cells. Unexpectedly, the behavioral, physiological and anatomical phenotypes of cbln1-null mice precisely mimic those of GluRδ2-null mice. Thus, we propose that Cbln1, which is released from granule cells, and GluRδ2, which is predominantly expressed in Purkinje cells, are involved in a common signaling pathway crucial for synapse formation/maintenance and plasticity in the cerebellum. Since molecules related to Cbln1 are expressed in various brain regions other than the cerebellum, other C1q/TNF superfamily proteins may also regulate various aspects of synapses in the CNS. Therefore, an understanding of the signaling mechanisms underlying Cbln1 and GluRδ2 in the cerebellum will provide new insights into the roles of C1q/TNF superfamily proteins as new cytokines that regulate normal and abnormal brain functions. © 2009 IBRO.</p>

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Polarized sorting of AMPA receptors to the somatodendritic domain is regulated by adaptor protein AP-4

Matsuda Shinji, Yuzaki Michisuke

Neuroscience Research 65 ( 1 ) 1 - 5 2009.09

ISSN  0168-0102

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<p>Neurons are highly polarized cells comprising somatodendritic and axonal domains. For proper neuronal function, such as neurotransmission and synaptic plasticity, membrane proteins must be transported to precise positions. α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors (AMPA receptors) are membrane proteins localized in the somatodendritic domain. AMPA receptors mediate excitatory synaptic transmission; thus, regulation of the intracellular trafficking of AMPA receptors has a critical role in synaptic plasticity. An understanding of the molecular mechanisms that regulate AMPA receptor trafficking is essential for gaining further insight into neuronal function. Despite its importance, however, how neurons selectively transport AMPA receptors to the somatodendritic domain is largely unknown. In this Update Article, we discuss recent progress in studies of the mechanisms underlying the somatodendritic targeting of AMPA receptors in neurons. © 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society.</p>

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The N-terminal domain of GluD2 (GluRδ2) recruits presynaptic terminals and regulates synaptogenesis in the cerebellum in vivo

Kakegawa Wataru, Miyazaki Taisuke, Kohda Kazuhisa, Matsuda Keiko, Emi Kyoichi, Motohashi Junko, Watanabe Masahiko, Yuzaki Michisuke

Journal of Neuroscience 29 ( 18 ) 5738 - 5748 2009.05

ISSN  0270-6474

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<p>The δ2 glutamate receptor (GluRδ2; GluD2), which is predominantly expressed on postsynaptic sites at parallel fiber (PF)-Purkinje cell synapses in the cerebellum, plays two crucial roles in the cerebellum: the formation of PF synapses and the regulation of long-term depression (LTD), a form of synaptic plasticity underlying motor learning. Although the induction of LTD and motor learning absolutely require signaling via the cytoplasmic C-terminal domain of GluD2, the mechanisms by which GluD2 regulates PF synaptogenesis have remained unclear. Here, we examined the role of the extracellular N-terminal domain (NTD) of GluD2 on PF synaptogenesis by injecting Sindbis virus carrying wild-type (GluD2^wt) or mutant GluD2 into the subarachnoid supracerebellar space of GluD2-null mice. Remarkably, the expression of GluD2^wt, but not of a mutant GluD2 lacking the NTD (GluD2^ΔNTD), rapidly induced PF synapse formation and rescued gross motor dyscoordination in adult GluD2-null mice just 1 d after injection. In addition, although the kainate receptor GluR6 (GluK2) did not induce PF synaptogenesis, a chimeric GluK2 that contained the NTD of GluD2 (GluD2 ^NTD-GluK2) did. Similarly, GluD2^wt and GluD2 ^NTD-GluK2, but not GluD2^ΔNTD, induced synaptogenesis in heterologous cells in vitro. In contrast, LTD was restored in GluD2-null Purkinje cells expressing a mutant GluD2 lacking the NTD. These results indicate that the NTD of GluD2 is necessary and sufficient for the function of GluD2 in the regulation of PF-Purkinje cell synaptogenesis. Furthermore, our results suggest that GluD2 differently regulates PF synaptogenesis and cerebellar LTD through the extracellular NTD and the cytoplasmic C-terminal end, respectively. Copyright © 2009 Society for Neuroscience.</p>

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Activity-dependent repression of Cbln1 expression

Iijima Takatoshi, Emi Kyoichi, Yuzaki Michisuke

Journal of Neuroscience 29 ( 17 ) 5425 - 5434 2009.04

ISSN  0270-6474

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<p>Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is released from cerebellar granule cells and plays a crucial role in forming and maintaining excitatory synapses between parallel fibers (PFs; axons of granule cells) and Purkinje cells not only during development but also in the adult cerebellum. Although neuronal activity is known to cause morphological changes at synapses, how Cbln1 signaling is affected by neuronal activity remains unclear. Here, we show that chronic stimulation of neuronal activity by elevating extracellular K⁺ levels or by adding kainate decreased the expression of cbln1 mRNA within several hours in mature granule cells in a manner dependent on L-type voltage-dependent Ca²⁺ channels and calcineurin. Chronic activity also reduced Cbln1 protein levels within a few days, during which time the number of excitatory synapses on Purkinje cell dendrites was reduced; this activity-induced reduction of synapses was prevented by the addition of exogenous Cbln1 to the culture medium. Therefore, the activity-dependent downregulation of cbln1 may serve as a new presynaptic mechanism by which PF-Purkinje cell synapses adapt to chronically elevated activity, thereby maintaining homeostasis. In addition, the expression of cbln1 mRNA was prevented when immature granule cells were maintained in high-K ⁺ medium. Since immature granule cells are chronically depolarized before migrating to the internal granule layer, this depolarization-dependent regulation of cbln1 mRNA expression may also serve as a developmental switch to facilitate PF synapse formation in mature granule cells in the internal granule layer. Copyright © 2009 Society for Neuroscience.</p>

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Cbln1 accumulates and colocalizes with Cbln3 and GluRδ2 at parallel fiber-Purkinje cell synapses in the mouse cerebellum

Miura Eriko, Matsuda Keiko, Morgan James I., Yuzaki Michisuke, Watanabe Masahiko

European Journal of Neuroscience 29 ( 4 ) 693 - 706 2009.02

ISSN  0953-816X

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<p>Cbln1 (a.k.a. precerebellin) is secreted from cerebellar granule cells as homohexamer or in heteromeric complexes with Cbln3. Cbln1 plays crucial roles in regulating morphological integrity of parallel fiber (PF)-Purkinje cell (PC) synapses and synaptic plasticity. Cbln1-knockout mice display severe cerebellar phenotypes that are essentially indistinguishable from those in glutamate receptor GluRδ2-null mice, and include severe reduction in the number of PF-PC synapses and loss of long-term depression of synaptic transmission. To understand better the relationship between Cbln1, Cbln3 and GluRδ2, we performed light and electron microscopic immunohistochemical analyses using highly specific antibodies and antigen-exposing methods, i.e. pepsin pretreatment for light microscopy and postembedding immunogold for electron microscopy. In conventional immunohistochemistry, Cbln1 was preferentially associated with non-terminal portions of PF axons in the molecular layer but rarely overlapped with Cbln3. In contrast, antigen-exposing methods not only greatly intensified Cbln1 immunoreactivity in the molecular layer, but also revealed its high accumulation in the synaptic cleft of PF-PC synapses. No such synaptic accumulation was evident at other PC synapses. Furthermore, Cbln1 now came to overlap almost completely with Cbln3 and GluRδ2 at PF-PC synapses. Therefore, the convergence of all three molecules provides the anatomical basis for a common signaling pathway regulating circuit development and synaptic plasticity in the cerebellum. © The Authors (2009).</p>

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Cbln1 binds to specific postsynaptic sites at parallel fiber-Purkinje cell synapses in the cerebellum

Matsuda Keiko, Kondo Tetsuro, Iijima Takatoshi, Matsuda Shinji, Watanabe Masahiko, Yuzaki Michisuke

European Journal of Neuroscience 29 ( 4 ) 707 - 717 2009.02

ISSN  0953-816X

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<p>Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is a unique molecule that is not only required for maintaining normal parallel fiber (PF)-Purkinje cell synapses, but is also capable of inducing new PF synapses in adult cerebellum. Although Cbln1 is reportedly released from granule cells, where and how Cbln1 binds in the cerebellum has remained largely unclear, partly because Cbln1 undergoes proteolysis to yield various fragments that are differentially detected by different antibodies. To circumvent this problem, we characterized the Cbln1-binding site using recombinant Cbln1. An immunohistochemical analysis revealed that recombinant Cbln1 preferentially bound to PF-Purkinje cell synapses in primary cultures and acute slice preparations in a saturable and replaceable manner. Specific binding was observed for intact Cbln1 that had formed a hexamer, but not for the N-terminal or C-terminal fragments of Cbln1 fused to other proteins. Similarly, mutant Cbln1 that had formed a trimer did not bind to the Purkinje cells. Immunoreactivity for the recombinant Cbln1 was observed in weaver cerebellum (which lacks granule cells) but was absent in pcd cerebellum (which lacks Purkinje cells), suggesting that the binding site was located on the postsynaptic sites of PF-Purkinje cell synapses. Finally, a subcellular fractionation analysis revealed that recombinant Cbln1 bound to the synaptosomal and postsynaptic density fractions. These results indicate that Cbln1, released from granule cells as hexamers, specifically binds to a putative receptor located at the postsynaptic sites of PF-Purkinje cell synapses, where it induces synaptogenesis. © The Authors (2009).</p>

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[How polarized sorting to neuronal dendrites is achieved

Matsuda Shinji, Yuzaki Michisuke

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 53 ( 16 Suppl ) 2214 - 2219 2008.12

ISSN  0039-9450

AP-4

Matsuda Shinji, Yuzaki Michisuke

Autophagy 4 ( 6 ) 815 - 816 2008.08

ISSN  1554-8627

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<p>Neurons are highly polarized cells composed of two distinct domains, the axon and the somatodendritic domain. Although AMPA-type glutamate receptors, which mediate fast excitatory neurotransmission in the vertebrate CNS, are preferentially expressed in the somatodendritic domain, the molecular mechanisms underlying such polarized distribution have remained elusive. We recently demonstrated that adaptor protein complex-4 (AP-4) binds to transmembrane AMPA receptor regulatory proteins (TARPs), thereby mediating the selective trafficking of AMPA receptors to the somatodendritic domain; generic disruption of AP-4 (AP-4β^-/-), results in the mislocalization of TARPs and AMPA receptors in the axons. Similarly, low-density lipoprotein receptors and δ2 glutamate receptors are mislocalized in axons, while other cargos, such as NMDA receptors and metabotropic glutamate receptors, are properly excluded from AP-4β^-/- axons. These findings indicate that there exist AP-4-dependent and -independent sorting mechanisms. Unexpectedly, mislocalized AMPA receptors do not reach the cell surface and accumulate in autophagosomes in the bulging portions of AP-4β^-/- axons. Several lines of evidence indicate that mislocalized AMPA receptors activate the autophagic pathway. Since increased autophagy and axonal swelling are suggested to occur in various neuronal disorders, further studies using AP-4β^-/- mice are warranted to understand the mechanisms regulating autophagy in axons. ©2008 Landes Bioscience.</p>

AP-4: autophagy-four mislocalized proteins in axons.

Matsuda S, Yuzaki M

Autophagy 4 ( 6 ) 815 - 6 2008.08

ISSN  1554-8627

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Cbln1 regulates rapid formation and maintenance of excitatory synapses in mature cerebellar purkinje cells in vitro and in vivo

Ito-Ishida Aya, Miura Eriko, Emi Kyoichi, Matsuda Keiko, Iijima Takatoshi, Kondo Tetsuro, Kohda Kazuhisa, Watanabe Masahiko, Yuzaki Michisuke

Journal of Neuroscience 28 ( 23 ) 5920 - 5930 2008.06

ISSN  0270-6474

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<p>Although many synapse-organizing molecules have been identified in vitro, their functions in mature neurons in vivo have been mostly unexplored. Cbln1, which belongs to the C1q/tumor necrosis factor superfamily, is the most recently identified protein involved in synapse formation in the mammalian CNS. In the cerebellum, Cbln1 is predominantly produced and secreted from granule cells; cbln1-null mice show ataxia and a severe reduction in the number of synapses between Purkinje cells and parallel fibers (PFs), the axon bundle of granule cells. Here, we show that application of recombinant Cbln1 specifically and reversibly induced PF synapse formation in dissociated cbln1-null Purkinje cells in culture. Cbln1 also rapidly induced electrophysiologically functional and ultrastructurally normal PF synapses in acutely prepared cbln1-null cerebellar slices. Furthermore, a single injection of recombinant Cbln1 rescued severe ataxia in adult cbln1-null mice in vivo by completely, but transiently, restoring PF synapses. Therefore, Cbln1 is a unique synapse organizer that is required not only for the normal development of PF-Purkinje cell synapses but also for their maintenance in the mature cerebellum both in vitro and in vivo. Furthermore, our results indicate that Cbln1 can also rapidly organize new synapses in adult cerebellum, implying its therapeutic potential for cerebellar ataxic disorders. Copyright © 2008 Society for Neuroscience.</p>

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Phosphorylation of delta2 glutamate receptors at serine 945 is not required for cerebellar long-term depression

Nakagami Ryoichi, Kohda Kazuhisa, Kakegawa Wataru, Kondo Tetsuro, Kato Nobumasa, Yuzaki Michisuke

Keio Journal of Medicine 57 ( 2 ) 105 - 110 2008.06

ISSN  0022-9717

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<p>Long-term depression (LTD) of synaptic transmission at parallel fiber (PF)-Purkinje cell synapses is thought to regulate motor learning and memory formation in the cerebellum. Neuronal activity-evoked protein kinase C (PKC) activation is required for the induction of LTD. In addition, the δ2 glutamate receptor (GluRδ2), which is predominantly expressed at PF-Purkinje cell synapses, is indispensable for the induction of LTD; however, the mechanisms by which GluRδ2 regulates LTD and its relationship with PKC activation remain elusive. Interestingly, GluRδ2 is phosphorylated by PKC on serine 945 (Ser945) near its C-terminus and a postsynaptic protein S-SCAM, which could potentially regulate glutamate receptor trafficking and synaptic plasticity, binds to the extreme C-terminus of GluRδ2 in a phosphorylation-dependent manner on Ser945. Here, using a Sindbis-based virus expression approach, we show that a mutant GluRδ2, in which alanine replaced Ser945 and did not undergo PKC phosphorylation, was normally localized at the postsynaptic sites of PF-Purkinje cell synapses. In addition, like wild-type GluRδ2, the phosphorylation-disrupted GluRδ2 successfully rescued abrogated LTD in GluRδ2-null Purkinje cells. These results indicate that Ser945, a major PKC phosphorylation site of of GluRδ2, may not play a crucial role in induction of LTD in the cerebellum.</p>

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Cbln and C1q family proteins - New transneuronal cytokines

Yuzaki M.

Cellular and Molecular Life Sciences 65 ( 11 ) 1698 - 1705 2008.06

ISSN  1420-682X

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<p>The C1q family is characterized by a C-terminal conserved global C1q domain, which is structurally very similar to the tumor necrosis factor homology domain. Although some C1q family members are expressed in the central nervous system, their functions have not been well characterized. Cbln1, a member of the Cbln subfamily of the C1q family, is predominantly expressed in cerebellar granule cells. Interestingly, Cbln1 was recently shown to play two unique roles at excitatory synapses formed between cerebellar granule cells and Purkinje cells: the formation and stabilization of synaptic contact, and the control of functional synaptic plasticity by regulating the postsynaptic endocytosis pathway. Since other Cbln subfamily members, Cbln2-Cbln4, are expressed in various regions of developing and mature brains, Cbln subfamily proteins may generally serve as a new class of transneuronal regulators of synapse development and synaptic plasticity in various brain regions. © 2008 Birkhaueser.</p>

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Accumulation of AMPA Receptors in Autophagosomes in Neuronal Axons Lacking Adaptor Protein AP-4

Matsuda Shinji, Miura Eriko, Matsuda Keiko, Kakegawa Wataru, Kohda Kazuhisa, Watanabe Masahiko, Yuzaki Michisuke

Neuron 57 ( 5 ) 730 - 745 2008.03

ISSN  0896-6273

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<p>AP-4 is a member of the adaptor protein complexes, which control vesicular trafficking of membrane proteins. Although AP-4 has been suggested to contribute to basolateral sorting in epithelial cells, its function in neurons is unknown. Here, we show that disruption of the gene encoding the β subunit of AP-4 resulted in increased accumulation of axonal autophagosomes, which contained AMPA receptors and transmembrane AMPA receptor regulatory proteins (TARPs), in axons of hippocampal neurons and cerebellar Purkinje cells both in vitro and in vivo. AP-4 indirectly associated with the AMPA receptor via TARPs, and the specific disruption of the interaction between AP-4 and TARPs caused the mislocalization of endogenous AMPA receptors in axons of wild-type neurons. These results indicate that AP-4 may regulate proper somatodendritic-specific distribution of its cargo proteins, including AMPA receptor-TARP complexes and the autophagic pathway in neurons. © 2008 Elsevier Inc. All rights reserved.</p>

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Differential regulation of synaptic plasticity and cerebellar motor learning by the C-terminal PDZ-binding motif of GluRδ2

Kakegawa Wataru, Miyazaki Taisuke, Emi Kyoichi, Matsuda Keiko, Kohda Kazuhisa, Motohashi Junko, Mishina Masayoshi, Kawahara Shigenori, Watanabe Masahiko, Yuzaki Michisuke

Journal of Neuroscience 28 ( 6 ) 1460 - 1468 2008.02

ISSN  0270-6474

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<p>The δ2 glutamate receptor (GluRδ2) is predominantly expressed in Purkinje cells and plays crucial roles in cerebellar functions: GluRδ2^-/- mice display ataxia and impaired motor learning. In addition, long-term depression (LTD) at parallel fiber (PF)-Purkinje cell synapses is abrogated, and synapse formation with PFs and climbing fibers (CFs) is severely disturbed in GluRδ2^-/- Purkinje cells. Recently, we demonstrated that abrogated LTD was restored in GluRδ2^-/- Purkinje cells by the virus-mediated expression of the wild-type GluRδ2 transgene (Tg_wt) but not by that of mutant GluRδ2 lacking the C-terminal seven residues to which several PDZ proteins bind (Tg _ΔCT7). These results indicated that the C terminus of GluRδ2 conveys the signal(s) necessary for LTD. In contrast, other phenotypes of GluRδ2^-/- cerebellum, especially morphological abnormalities at PF and CF synapses, could not be rescued by virus-mediated transient expression. Thus, whether these phenotypes are mediated by the same signaling pathway remains unclear. To address these issues and to further delineate the function of GluRδ2 in vivo, we generated transgenic mice that expressed Tg_ΔCT7 on a GluRδ2^-/- background. Interestingly, although Tg_ΔCT7 restored abnormal PF and CF synapse formation almost completely, it could not rescue abrogated LTD in GluRδ2^-/- Purkinje cells. Furthermore, although the gross motor discoordination of GluRδ2^-/- mice was restored, the cerebellar motor learning underlying delayed eyeblink conditioning remained impaired. These results indicate that LTD induction and motor learning are regulated by signaling via the C-terminal end of GluRδ2, whereas other functions may be differentially regulated by other regions of GluRδ2. Copyright © 2008 Society for Neuroscience.</p>

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Aberrant membranes and double-membrane structures accumulate in the axons of Atg5-null Purkinje cells before neuronal death

Nishiyama Jun, Miura Eriko, Mizushima Noboru, Watanabe Masahiko, Yuzaki Michisuke

Autophagy 3 ( 6 ) 591 - 596 2007.11

ISSN  1554-8627

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<p>Autophagy (macroautophagy) is an evolutionally conserved process by which cytoplasmic proteins and organelles are surrounded by unique double membranes and are subsequently degraded upon fusion with lysosomes. Many autophagy-related genes [Atg) have been identified in yeast; a ubiquitin-like Atg12-Atg5 system is also essential for the elongation of the isolation membrane in mammalian cells. Nevertheless, the regulation of autophagy in neurons remains largely unknown. In this study, we crossed conditional knockout mice Atg5 ^flox/flox with pcp2-Cre transgenic mice, which express Cre recombinase through a Purkinje cell-specific promoter, pcp2. In Atg5 ^flox/flox; pcp2-Cre mice, the Atg5 gene was excised as early as postnatal day 6; Purkinje cells started to degenerate after approximately 8 weeks, and the animals showed an ataxic gait from around 10 months. Initially, however, the Purkinje cells showed axonal swelling around its terminals from as early as 4 weeks after birth. An electron microscopic analysis revealed the accumulation of autophagosome-like double-membrane structures in the swollen regions, together with numerous membranous organelles, such as tubular or sheet-like smooth endoplasmic reticulum and vesicles. These results suggest that Atg5 plays important roles in the maintenance of axon morphology and membrane structures, and its loss of function leads to the swelling of axons, followed by progressive neurodegeneration in mammalian neurons. ©2007 Landes Bioscience.</p>

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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

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<p>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.</p>

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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

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<p>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.</p>

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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

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<p>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.</p>

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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

　View Summary

<p>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.</p>

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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

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<p>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.</p>

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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

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<p>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).</p>

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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

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<p>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).</p>

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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

　View Summary

<p>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.</p>

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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

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<p>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).</p>

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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

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<p>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).</p>

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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

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<p>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.</p>

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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

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<p>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.</p>

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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

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<p>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.</p>

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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

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<p>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.</p>

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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

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<p>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.</p>

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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

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<p>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.</p>

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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

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<p>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.</p>

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Transgenic rescue for characterizing orphan receptors

Yuzaki Michisuke

Transgenic Research 14 ( 2 ) 117 - 121 2005.04

ISSN  0962-8819

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<p>Even though the genomes of several major species have been sequenced, many orphan receptors with unknown ligands and mechanisms of action remain in the CNS. The δ2 glutamate receptor (GluRδ2) is one of such receptors expressed predominantly in the cerebellar Purkinje cells. On the basis of amino acid similarity, it belongs to ionotropic glutamate receptor (iGluR) family, which mediates fast excitatory neurotransmission in the mammalian CNS. Although its null-mutant mice show prominent motor discoordination, the mechanisms by which GluRδ2 participates in the cerebellar functions have been unclear. To gain insight into GluRδ2's mechanisms, we recently generated mice that express either a wild-type or a mutant GluRδ2 transgene, in which the conserved arginine in GluRδ2's N-terminal putative ligand-binding motif was disrupted. By breeding these transgenic mice onto a GluRδ2 ^-/- background, we obtained two transgenic 'rescue' lines. Surprisingly, the mutant GluRδ2 transgene was as effective as the wild-type GluRδ2 in rescuing the GluRδ2-null mice. As the disrupted arginine residue is highly conserved from ancestral bacterial periplasmic amino acid-binding proteins to mammalian iGluRs, we propose that GluRδ2 may not require glutamate-like amino acids and may function in an unconventional manner. This 'transgenic rescue' approach to investigating orphan receptors is a relatively easy but powerful method when a knockout mouse with a distinct phenotype is already available. The advantages and limitations of this approach, together with certain cautions in interpreting the resulting data, are discussed in this review. © Springer 2005.</p>

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Rescue of abnormal phenotypes of the δ2 glutamate receptor-null mice by mutant δ2 transgenes

Hirai Hirdkazu, Miyazaki Taisuke, Kakegawa Wataru, Matsuda Shinji, Mishina Masayoshi, Watanabe Masahiko, Yuzaki Michisuke

EMBO Reports 6 ( 1 ) 90 - 95 2005

ISSN  1469-221X

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<p>The δ2 glutamate receptor (GluR52) has a crucial role in cerebellar functions; disruption of GluRδ2 alleles in mice (δ2^-/-) impairs synapse formation and long-term depression, which is thought to underlie motor learning in the cerebellum, and consequently leads to motor discoordination. However, it has been unclear whether GluRδ2 is activated by glutamate analogues. Here we introduced a GluRδ2 transgene, which had a mutation (Arg514Lys) in the putative ligand-binding motif conserved in all mammalian ionotropic glutamate receptors (iGluRs) and their ancestral bacterial periplasmic amino-acid-binding proteins, into δ2^-/- mice. Surprisingly, a mutant GluRδ2 transgene, as well as a wild-type GluRδ2 transgene, rescued all abnormal phenotypes of δ2 ^-/- mice. Therefore, these results indicate that the conserved arginine residue, which is crucial for the binding of iGluRs to glutamate analogues, is not essential for the restoration of GluRδ2 functions in δ2^-/- mice. © 2005 European Molecular Biology Organization.</p>

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Rescue of abnormal phenotypes of the delta2 glutamate receptor-null mice by mutant delta2 transgenes

HIRAI HIROKAZU, MIYAZAKI T., KAKEGAWA WATARU, MATSUDA SHINJI, MISHINA MASAYOSHI, WATANABE MASAHIKO, YUZAKI MICHISUKE

EMBO Report  ( 6 ) 90-95 2005

Research paper (scientific journal), Joint Work, Accepted

The C-terminal juxtamembrane region of the δ2 glutamate receptor controls its export from the endoplasmic reticulum

Matsuda Shinji, Hannen Rosalind, Matsuda Keiko, Yamada Nobuaki, Tubbs Thomas, Yuzaki Michisuke

European Journal of Neuroscience 19 ( 7 ) 1683 - 1690 2004.04

ISSN  0953-816X

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<p>Functions of ionotropic glutamate receptors (iGluRs) are tightly regulated by the intracellular trafficking of receptor proteins. Unlike other iGluRs that are considerably retained in the intracellular component, the δ2 glutamate receptor (GluRδ2) is efficiently expressed on the Purkinje cell surface. To understand the trafficking mechanism of iGluRs, we deleted various portions of the C-terminal intracellular domain of GluRδ2 and analysed the localization of the mutant proteins in heterologous cells and neurons. Biotinylation assays indicated that GluRδ2 lacking the C-terminal juxtamembrane region of 13 amino acids (region A) was not present on the cell surface. This mutant GluRδ2 was sensitive to endoglycosidase H, which digests unprocessed high-mannose oligosaccharides on proteins retained in the endoplasmic reticulum (ER) or cis-Golgi. Therefore, we concluded that region A is crucial for the transport of GluRδ2 beyond the trans-Golgi to the cell surface. Because the immunostaining pattern of GluRδ2 lacking region A in cultured hippocampal neurons completely overlapped the pattern of fluorescence emitted by ER-resident green fluorescent protein, region A is most likely necessary for GluRδ2's exit from the ER. Furthermore, this region is essential for the proper intracellular trafficking of GluRδ2 in Purkinje cells. Region A does not rely on a dihydrophobic motif or positively charged residues to participate in trafficking, but its function is dependent on the juxtamembrane position. Therefore, we propose that GluRδ2's efficient transport to the cell surface utilizes an unknown but general ER exit mechanism, which probably works in close relation to the membrane of heterologous cells and neurons.</p>

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The delta2 glutamate receptor&#8212;A key molecule controlling synaptic plasticity and structure in Purkinje cells.

YUZAKI MICHISUKE

Cerebellum  ( 3 ) 89-93 2004

Research paper (scientific journal), Single Work, Accepted

The δ2 glutamate receptor

Yuzaki Michisuke

Cerebellum 3 ( 2 ) 89 - 93 2004

ISSN  1473-4222

　View Summary

<p>The orphan glutamate receptor δ2 (GluRδ2) is predominantly expressed in cerebellar Purkinje cells and plays a crucial role in cerebellar functions; mice that lack the GluRδ2 gene display ataxia and impaired motor-related learning tasks. However, when expressed alone or with other glutamate receptors, GluRδ2 does not form functional glutamate-gated ion channels nor does it bind to glutamate analogs. Therefore, the mechanisms by which GluRδ2 participates in cerebellar functions have been elusive. Studies of mutant mice, such as lurcher, hotfoot, and GluRδ2 knockout mice, have provided clues to the structure and function of GluRδ2. Particularly, morphological and electrophysiological analyses of hotfoot and GluRδ2 knockout mice have indicated a unique role of GluRδ2 in aligning and maintaining the postsynaptic element with the presynaptic one at parallel fiber (PF)-Purkinje cell synapses. In addition, GluRδ2 was expressed in newly formed ectopic PF-Purkinje cell synapses found after blockade of electrical activity in adult cerebellum. Moreover, application of an antibody specific for GluRδ2's extracellular N-terminal region abrogated synaptic plasticity. These results indicate that GluRδ2 plays a direct role in synapse formation and synaptic plasticity in adult mice. Based on these results, two hypotheses about mechanisms by which GluRδ2 functions are proposed in this article.</p>

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Specific Assembly with the NMDA Receptor 3B Subunit Controls Surface Expression and Calcium Permeability of NMDA Receptors

Matsuda Keiko, Fletcher Matt, Kamiya Yoshinori, Yuzaki Michisuke

Journal of Neuroscience 23 ( 31 ) 10064 - 10073 2003.11

ISSN  0270-6474

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<p>The NMDA receptor 3B (NR3B) subunit is the most recently identified member of the NMDA receptor family. In heterologous cells, it has been shown to reduce the Ca²⁺ permeability of glutamatergic receptor complexes formed together with NR1 and NR2 subunits and to form the unique excitatory glycine receptor complex with the NR1 subunit. However, it is unclear whether NR3B protein is expressed in and exerts similar functions in neurons. In addition, it is not understood how NR3B interacts with NR1 and NR2 and how such an interaction may regulate the membrane trafficking of the NMDA receptor complex. Here we report that our analysis using an antibody specific for NR3B showed that the NR3B protein is selectively expressed in somatic motor neurons in the brainstem of adult mice. Coimmunoprecipitation and electrophysiological analyses demonstrated that NR3B, when exogenously introduced into hippocampal neurons, can coassemble with endogenous NR1 and NR2A and can reduce the Ca ²⁺ permeability of NMDA currents. In contrast, NR3B was not involved in the excitatory glycine response in neurons under our test conditions. Although NR1 or NR3B alone cannot be transported to the cell surface, coexpression of these subunits mutually supported transport of the NMDA receptor complex by interaction involving the specific regions of the C terminus of NR3B. These results indicate that NR3B may modulate the function of NMDA receptors in somatic motor neurons during adulthood by controlling membrane trafficking and by reducing Ca²⁺ permeability.</p>

New role of δ2-glutamate receptors in AMPA receptor trafficking and cerebellar function

Hirai Hirokazu, Launey Thomas, Mikawa Sumiko, Torashima Takashi, Yanagihara Dai, Kasaura Tsuyoshi, Miyamoto Akihiro, Yuzaki Michisuke

Nature Neuroscience 6 ( 8 ) 869 - 876 2003.08

ISSN  1097-6256

　View Summary

<p>Previous gene knockout studies have shown that the orphan glutamate receptor δ2 (GluRδ2) is critically involved in synaptogenesis between parallel fibers and Purkinje cells during development. However, the precise function of GluRδ2 and whether it is functional in the mature cerebellum remain unclear. To address these issues, we developed an antibody specific for the putative ligand-binding region of GluRδ2, and application of this antibody to cultured Purkinje cells induced AMPA receptor endocytosis, attenuated synaptic transmission and abrogated long-term depression. Moreover, injection of this antibody into the subarachnoidal supracerebellar space of adult mice caused transient cerebellar dysfunction, such as ataxic gait and poor performance in the rotorod test. These results indicate that GluRδ2 is involved in AMPA receptor trafficking and cerebellar function in adult mice.</p>

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New insights into the structure and function of glutamate receptors

Yuzaki Michisuke

Keio Journal of Medicine 52 ( 2 ) 92 - 99 2003.06

ISSN  0022-9717

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<p>The ionotropic glutamate receptors (iGluRs) form ligand-gated ion channels that mediate the vast majority of excitatory neurotransmission in the mammalian brain. These receptors play central roles not only in normal neurodevelopmental and neurophysiological processes but also in certain neuropathological processes. Molecular cloning of genes for iGluRs in the past decade has advanced our understanding of the basic properties of iGluRs, such as ion selectivity, ligand binding, and anchoring at synapses. Although the gene for the δ2 glutamate receptor (GluRδ2) was cloned on the basis of homology screening, GluRδ2 has been referred to as an "orphan" receptor because it does not form functional glutamate-gated ion channels. However, ataxia in many types of mice is caused by spontaneous mutation of GluRδ2. Analysis of two such mutants, lurcher and hotfoot, has provided key insights into the GluRδ2 signaling in neurons. Furthermore, characterization of mutant GluRδ2 has revealed unexpected clues to two fundamental features regarding the structure and function of iGluRs - gating and assembly. Studies have recently shown that the transmembrane region where the lurcher mutation is located probably plays a crucial role in channel gating. The mechanism that controls iGluR subunit assembly seems to involve the extracellular N-terminal domain where the hotfoot mutation is located. An understanding of mechanisms responsible for gating and assembly is essential for the comprehension of neuronal function and dysfunction. Although reverse genetics is useful in deciphering glutamate signaling, these findings demonstrate the power of classic approaches to forward genetics on mutant mice.</p>

The δ2 glutamate receptor

Yuzaki Michisuke

Neuroscience Research 46 ( 1 ) 11 - 22 2003.05

ISSN  0168-0102

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<p>The orphan glutamate receptor δ2 (GluRδ2) is predominantly expressed in Purkinje cells and plays a crucial role in cerebellar functions: mice that lack the GluRδ2 gene display ataxia and impaired synaptic plasticity. However, when expressed alone or with other glutamate receptors, GluRδ2 does not form functional glutamate-gated ion channels nor does it bind to glutamate analogs. Therefore, the mechanisms by which GluRδ2 participates in cerebellar functions have been elusive. Studies of mutant mice such as lurcher, hotfoot, and GluRδ2 knockout mice have provided clues to the structure and function of GluRδ2. GluRδ2 has a channel pore similar to that of other glutamate receptors; the channel is functional at least when the lurcher mutation is present. GluRδ2 must be transported to the Purkinje cell surface to function; the absence of surface GluRδ2 causes the ataxic phenotype of hotfoot mice. In GluRδ2-null mice, the presence of naked spines not innervated by parallel fibers may influence the sustained innervation of mutant Purkinje cells by multiple climbing fibers. From these results, several hypotheses about mechanisms by which GluRδ2 functions are proposed in this article. Further characterization of GluRδ2's functions will provide key insights into normal and abnormal cerebellar functions. © 2003 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved.</p>

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Differential expression and function of apoptosis-associated tyrosine kinase (AATYK) in the developing mouse brain

Tomomura Mineko, Hasegawa Yuki, Hashikawa Tsutomu, Tomomura Akito, Yuzaki Michisuke, Furuichi Teiichi, Yano Ryoji

Molecular Brain Research 112 ( 1-2 ) 103 - 112 2003.04

ISSN  0169-328X

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<p>Apoptosis-associated tyrosine kinase (AATYK) is a non-receptor type tyrosine kinase that is predominantly expressed in adult mouse brain. Although it is also expressed in developing brains, its expression pattern and physiological functions are unclear. In the present study, we analyzed expression profiles of AATYK in developing mouse brains and its functional role and subcellular localization in cultured cerebellar granule cells. Expression of AATYK mRNA and protein increased during postnatal brain development. Immunohistochemical analysis indicated that the protein was differentially expressed in postmitotic neurons within various brain areas including the olfactory bulb, cerebral cortex, hippocampus, thalamus, colliculus, cerebellum, and brain stem. Developmental increases in its expression were also observed in cultured cerebellar granule cells. AATYK protein was largely fractionated into the microsomal fraction and was immunocytochemically distributed in an ER-like meshwork of the granule cell soma, suggesting a possible association with the ER membrane. AATYK protein was also present in neurites. In immature granule cells, overexpression of wild-type AATYK promoted neurite outgrowth, whereas that of tyrosine kinase-defective mutant significantly inhibited it. These results suggest that, in addition to its role in cell death in mature neurons, AATYK has a unique role in promoting neurite extension through its tyrosine kinase activity in developing neurons. © 2003 Elsevier Science B.V. All rights reserved.</p>

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A hot spot for hotfoot mutations in the gene encoding the δ2 glutamate receptor

Wang Ying, Matsuda Shinji, Drews Valerie, Torashima Takashi, Meisler Miriam H., Yuzaki Michisuke

European Journal of Neuroscience 17 ( 8 ) 1581 - 1590 2003.04

ISSN  0953-816X

　View Summary

<p>The orphan glutamate receptor δ2 is selectively expressed in Purkinje cells and plays a crucial role in cerebellar functions. Recently, ataxia in the hotfoot mouse ho4J was demonstrated to be caused by a deletion in the δ2 receptor gene (Grid2) removing the N-terminal 170 arnino acids of the δ2 receptor. To understand how δ2 receptors function, we characterized mutations in eight additional spontaneously occurring hotfoot alleles of Grid2. The mouse Grid2 gene consists of 16 exons, spanning approximately 1.4 Mb. Genomic DNA analysis showed that seven hotfoot mutants had a deletion of one or more exons encoding the N-terminal domain of δ2 receptors. The exception is ho5J, which has a point mutation in exon 12. Deletions in ho7J, ho9J, ho11J and ho12J mice result in the in-frame deletion of between 40 and 95 amino acids. Expression of constructs containing these deletions in HEK293 cells resulted in protein retention in the endoplasmic reticulum or cis-Golgi without transport to the cell surface. Coimmunoprecipitation assays indicated that these deletions also reduce the intermolecular interaction between individual δ2 receptors. These results indicate that the deleted N-terminal regions are crucial for oligomerization of δ2 receptors and their subsequent transport to the cell surface of Purkinje cells. The relatively large size of the Grid2 gene may be one of the reasons why many spontaneous mutations occur in this gene. In addition, the frequent occurrence of in-frame deletions within the N-terminal domain in hotfoot mutants suggests the importance of this domain in the function of δ2 receptors.</p>

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Heteromer formation of δ2 glutamate receptors with AMPA or kainate receptors

Kohda Kazuhisa, Kamiya Yoshinori, Matsuda Shinji, Kato Kunio, Umemori Hisashi, Yuzaki Michisuke

Molecular Brain Research 110 ( 1 ) 27 - 37 2003.01

ISSN  0169-328X

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<p>The δ2 glutamate receptor (GluRδ2) is predominantly expressed in the postsynaptic densities of parallel fiber-Purkinje cell synapses and plays a crucial role in cerebellar function. However, the mechanisms by which GluRδ2 participates in cerebellar functions are largely unknown because GluRδ2 does not bind glutamate analogs. We investigated the possibility that GluRδ2 may be involved in channel formation together with other glutamate receptor families. We transiently expressed lurcher mutant AMPA receptor GluR1^Lc and kainate receptor GluR6^Lc in HEK293 cells. Cells expressing these constitutively active channels displayed a rectifying current-voltage (I-V) relationship. However, when cells were co-transfected with GluRδ2^Lc, which had the arginine residue in the channel pore region, cells displayed a linear I-V relationship, a result that indicates GluRδ2^Lc formed functional heteromeric channels with GluR1^Lc or GluR6^Lc. Assembly of GluRδ2 with GluR1 or GluR6 was further confirmed by co-immunoprecipitation assays in HEK293 cells. In addition, GluRδ2 receptors were partially co-immunoprecipitated from cerebellar synaptosomal fractions by antibodies against GluR2 or KA2. In contrast to lurcher channels, expression of wild-type GluRδ2 significantly reduced the glutamate-induced current of the wild-type GluR1 receptors without affecting channel properties, such as current kinetics, dose-response relationship, and single-channel conductance. Thus, the heteromeric channel created by the association of wild-type GluR1 and GluRδ2 may not be gated by glutamate and does not participate in glutamate-induced currents. These results suggest that GluRδ2 and AMPA or kainate receptors can assemble to form heteromeric receptors in vitro and could modify glutamate signaling in vivo. These findings may help explain the role of GluRδ2. © 2002 Elsevier Science B.V. All rights reserved.</p>

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Cloning and characterization of a novel NMDA receptor subunit NR3B

Matsuda Keiko, Kamiya Yoshinori, Matsuda Shinji, Yuzaki Michisuke

Molecular Brain Research 100 ( 1-2 ) 43 - 52 2002.04

ISSN  0169-328X

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<p>We report the cloning and characterization of a novel NMDA receptor subunit cDNA, which encodes a predicted polypeptide of 1003 amino acids. Phylogenic analysis indicates that this new subunit is most closely related to NR3A. Therefore, we term it NR3B. Important functional domains of glutamate receptors, such as the ligand-binding domain, the channel pore, and the channel gate, are conserved in NR3B. NR3B mRNA was expressed highly in pons, midbrain, medulla, and the spinal cord, but at low levels in the forebrain and the cerebellum. Although NR3A mRNA expression decreases sharply after the second postnatal weeks, NR3B mRNA expression levels in whole brain were constant during postnatal development and into adult. Coimmunoprecipitation analysis showed that NR3B could form NMDA receptor complex with NR1a and NR2A subunits in heterologous cells. Although expression of NR3B alone did not reconstitute functional NMDA receptors, coexpression of NR3B reduced the Ca²⁺ permeability of glutamate-induced currents in cells expressing NR1a and NR2A. These results indicate that NR3B is a dominant modulatory subunit that can modify the function of NMDA receptors. Since high Ca²⁺ permeability of NMDA receptors is thought to be a key feature for NMDA receptors to play critical roles in neurodevelopment, synaptic plasticity, and neuronal death, NR3B may contribute to the regulation of these physiological and pathological processes. © 2002 Elsevier Science B.V. All rights reserved.</p>

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Antibody against a putative ligand-binding site reveals delta2 glutamate receptor function

Hirai Hirokazu, Launey Thomas, Mikawa Sumiko, Yanagihara Dai, Kasaura Tsuyoshi, Miyamoto Akihiro, Yuzaki Michisuke

Annals of the New York Academy of Sciences 978   519 2002

ISSN  0077-8923

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Mutation in hotfoot-4J mice results in retention of δ2 glutamate receptors in ER

Matsuda Shinji, Yuzaki Michisuke

European Journal of Neuroscience 16 ( 8 ) 1507 - 1516 2002

ISSN  0953-816X

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<p>The orphan glutamate receptor δ2 is selectively expressed in Purkinje cells and plays a critical role in cerebellar function. Recently, the ataxia of hotfoot-4J (ho-4J) mice was shown to be caused by a 170-amino acid deletion in the N-terminal region of δ2 receptors. To understand δ2 receptor function, we characterized these mutant receptors (δ2^ho) in Purkinje cells. Immunohistochemical staining showed that δ2^ho receptors of the ho-4J homozygotes were abundantly expressed but localized to the Purkinje cell soma; in wild-type mice, δ2 receptors were predominantly present at distal dendrites of Purkinje cells. In addition, δ2^ho receptors of the ho-4J mice were sensitive to endoglycosidase H, a finding suggesting that δ2^ho receptors were not transported beyond the endoplasmic reticulum (ER) or cis-Golgi apparatus. To gain further insights into the mechanisms of this phenomenon, we characterized δ2^ho receptors in transfected HEK293 cells. δ2^ho receptors expressed in HEK293 cells were also sensitive to endoglycosidase H. Immunohistochemical staining showed that δ2^ho receptors colocalized with proteins retained in the ER. Furthermore, δ2^ho receptors were not labelled by membrane-impermeable biotinylation reagents. Coimmunoprecipitation assays showed that the intermolecular interaction of δ2^ho receptors was significantly weaker than those of wild-type δ2 receptors, a finding suggesting that the ho-4J region is involved in oligomerization of δ2 receptors. Thus, δ2^ho receptors were retained in the ER, probably by the quality control mechanism that detects unstable oligomers. We conclude that the absence of δ2 receptors on the cell surface by failed transport from the ER of Purkinje cells causes ataxia.</p>

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Characterization of the apoptosis-associated tyrosine kinase (AATYK) expressed in the CNS

Tomomura M., Fernandez-Gonzales A., Yano R., Yuzaki M.

Oncogene 20 ( 9 ) 1022 - 1032 2001.03

ISSN  0950-9232

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<p>We isolated three related cDNA clones from a mouse cerebellar library; the type I cDNA was identical to the gene encoding the apoptosis-associated tyrosine kinase (AATYK), whose expression in myeloid precursor cells is increased during growth arrest or apoptosis. Low levels of AATYK mRNA expression were seen in adult mouse brains but not in embryos. In situ hybridization confirmed the widespread expression of AATYK mRNA in neurons throughout the adult brain. AATYK possessed tyrosine kinase activity and was autophosphorylated when expressed in 293 cells. AATYK mRNA expression was rapidly induced in cultured cerebellar granule cells during apoptosis induced by a low concentration of KCl (5 mM). Levels of endogenous AATYK protein were increased only slightly, but they were accompanied by an increase in molecular weight during apoptosis. Results of the tyrosine phosphatase treatments indicated that the increase in molecular weight was partly caused by tyrosine phosphorylation. The number of apoptotic granule cells overexpressing wild-type AATYK protein was significantly greater than the number of apoptotic granule cells overexpressing a mutant AATYK that lacked tyrosine kinase activity in low concentrations of KCl. These findings suggest that through its tyrosine kinase activity, AATYK is involved in the apoptosis of mature neurons.</p>

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Purification of Purkinje cells by fluorescence-activated cell sorting from transgenic mice that express green fluorescent protein

Tomomura M., Rice D. S., Morgan J. I., Yuzaki M.

European Journal of Neuroscience 14 ( 1 ) 57 - 63 2001

ISSN  0953-816X

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<p>The cerebellar Purkinje cell has been the focus of numerous studies involving the analysis of development and information processing in the nervous system. Purkinje cells represent less than 0.1% of the total cell content of the cerebellum. To facilitate studies of molecules that are expressed in such a small proportion of neurons, we have established procedures for the purification of these cells. Transgenic mice were developed in which the expression of green fluorescent protein (GFP) was controlled by the L7 promoter. In adult cerebellum, GFP fluorescence was only detected in Purkinje cells, where it filled dendrites, soma and axons. GFP fluorescence was detected in Purkinje cells as early as embryonic day 17 and increased during development in vivo and in dissociated cerebellar culture. Mirroring endogenous L7 expression, high levels of GFP were observed in retinal rod bipolar cells. Lower levels of GFP were seen in olfactory periglomerular cells, neurons in the interpeduncular nucleus, and superior colliculus neurons. Cerebella from transgenic mice were dissociated by mild enzymatic treatment and Purkinje cells were isolated by fluorescence-activated cell sorting (FACS). By selecting optimal parameters, a fraction of viable Purkinje cells that was 94% pure was obtained. These results indicate that FACS is a powerful tool for isolating Purkinje cells from postnatal L7-GFP transgenic mice. GFP-positive neurons will also be useful in the real-time observation of dendritic morphogenesis and axonal outgrowth during development, or after neuronal activity in vitro.</p>

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Mutation of a glutamate receptor motif reveals its role in gating and δ2 receptor channel properties

Kohda Kazuhisa, Wang Ying, Yuzaki Michisuke

Nature Neuroscience 3 ( 4 ) 315 - 322 2000.04

ISSN  1097-6256

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<p>Despite its importance in the cerebellum, the functions of the orphan glutamate receptor δ2 are unknown. We examined a mutant δ2 receptor channel in lurcher mice that was constitutively active in the absence of ligand. Because this mutation was within a highly conserved motif (YTANLAAF), we tested its effect on several glutamate receptors. Mutant δ2 receptors showed distinct channel properties, including double rectification of the current- voltage relationship, sensitivity to a polyamine antagonist and moderate Ca²⁺ permeability, whereas other constitutively active mutant glutamate channels resembled wildtype channels in these respects. Moreover, the kinetics of ligand-activated currents were strikingly altered. We conclude that the δ2 receptor has a functional ion channel pore similar to that of glutamate receptors. The motif may have a role in the channel gating of glutamate receptors.</p>

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Neuronal interleukin-16, (NIL-16)

Kurschner Cornelia, Yuzaki Michisuke

Journal of Neuroscience 19 ( 18 ) 7770 - 7780 1999.09

ISSN  0270-6474

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<p>Interleukin (IL)-16 is a proinflammatory cytokine that has attracted widespread attention because of its ability to block HIV replication. We describe the identification and characterization of a large neuronal IL-16 precursor, NIL-16. The N-terminal half of NIL-16 constitutes a novel PDZ domain protein sequence, whereas the C terminus is identical with splenocyte- derived mouse pro-IL-16. IL-16 has been characterized only in the immune system, and the identification of NIL-16 marks a previously unsuspected connection between the immune and the nervous systems. NIL-16 is a cytosolic protein that is detected only in neurons of the cerebellum and the hippocampus. The N-terminal portion of NIL-16 interacts selectively with a variety of neuronal ion channels, which is similar to the function of many other PDZ domain proteins that serve as intracellular scaffolding proteins. Among the NIL-16-interacting proteins is the class C α1 subunit of a mouse brain calcium channel (mbC α1). The C terminus of NIL-16 can be processed by caspase-3, resulting in the release of secreted IL-16. Furthermore, in cultured cerebellar granule neurons undergoing apoptosis, NIL-16 proteolysis parallels caspase-3 activation. Cerebellar granule neurons express the IL-16 receptor CD4. Exposure of these cells to IL-16 induces expression of the immediate-early gene, c-fos, via a signaling pathway that involves tyrosine phosphorylation. This suggests that IL-16 provides an autocrine function in the brain. Therefore, we hypothesize that NIL-16 is a dual function protein in the nervous system that serves as a secreted signaling molecule as well as a scaffolding protein.</p>

Characterization of L-homocysteate-induced currents in Purkinje cells from wild-type and NMDA receptor knockout mice

Yuzaki Michisuke, Connor John A.

Journal of Neurophysiology 82 ( 5 ) 2820 - 2826 1999

ISSN  0022-3077

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<p>L-Homocysteic acid (HCA), an endogenous excitatory amino acid in the mammalian CNS, potently activates N-methyl-D-aspartate (NMDA) receptors in hippocampal neurons. However, the responses to HCA in Purkinje cells, which lack functional NMDA receptors, have been largely unexplored: HCA may activate conventional non-NMDA receptors by its mixed agonistic action on both NMDA and non-NMDA receptors, or it may activate a novel non-NMDA receptor that has high affinity for HCA. To test these possibilities, we compared the responses to HCA in cultured Purkinje cells with those in hippocampal neurons by using the whole cell patch-clamp technique. To clearly isolate HCA responses mediated by non-NMDA receptors, we complemented pharmacological methods by using neurons from mutant mice (NR(-/-)) that lack functional NMDA receptors. A moderate dose of HCA (100 μM) induced substantial responses in Purkinje cells. These responses were blocked by non- NMDA receptor antagonists but were insensitive to NMDA receptor antagonists. HCA also activated responses mediated by non-NMDA receptors in both wild-type and NR1(-/-) hippocampal neurons. HCA responses in Purkinje cells had a pharmacological profile (EC₅₀ and Hill coefficient) very similar to that of non-NMDA receptor components of HCA responses in hippocampal neurons. Moreover, the amplitude of the non-NMDA receptor component of HCA responses was directly correlated with that of α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA)- and kainate-induced responses in both types of neurons. Finally, in both types of neurons, HCA currents mediated by non- NMDA receptors were potently blocked by the AMPA receptor antagonist GYKI52466. These findings indicate that HCA-activated AMPA receptors in Purkinje cells are similar to those in hippocampal neurons and that there is no distinct HCA-preferring receptor in Purkinje cells. We also found that in hippocampal neurons, the EC₅₀s of HCA for non-NMDA receptors and for NMDA receptors were more similar than originally reported; this finding indicates that HCA is similar to other mixed agonists, such as glutamate. HCA responses may appear to be selective at NMDA receptors in cells that express these receptors, such as hippocampal neurons; in cells that express few functional NMDA receptors, such as Purkinje cells, HCA may appear to be selective at non-NMDA receptors.</p>

Selective activation of calcium permeability by aspartate in Purkinje cells

Yuzaki Michisuke, Forrest Douglas, Curran Tom, Connor John A.

Science 273 ( 5278 ) 1112 - 1114 1996.08

ISSN  0036-8075

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<p>Glutamate and aspartate are endogenous excitatory amino acid neurotransmitters widely distributed in the mammalian central nervous system. Aspartate was shown to induce a large membrane current sensitive to N- methyI-D-aspartate (NMDA) and non-NMDA receptor antagonists in Purkinje cells from mice lacking functional NMDA receptors (NR1(-/-)). This response was accompanied by high permeability to calcium. In contrast, no current was induced by aspartate in hippocampal neurons and cerebellar granule cells from NR1-/- mice. Several other glutamate receptor agonists failed to evoke this response. Thus, in Purkinje cells, aspartate activates a distinct response capable of contributing to synaptic plasticity through calcium permeability.</p>

Functional NMDA receptors are transiently active and support the survival of Purkinje cells in culture

Yuzaki Michisuke, Forrest Douglas, Verselis Lynne M., Sun Shaiu C., Curran Tom, Connor John A.

Journal of Neuroscience 16 ( 15 ) 4651 - 4661 1996.08

ISSN  0270-6474

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<p>Conflicting evidence exists concerning the activity of NMDA receptors (NMDARs) in cerebellar Purkinje cells and their possible functions. To investigate the activity of NMDARs, we used whole-cell recording on immunocytochemically identified Purkinje cells in primary culture. In addition, we used mice with a disrupted NMDAR1 gene that lack functional NMDARs (NR1-/-) to assess the physiological role of NMDARs. In cultures from normal mice, NMDA-mediated currents were detected in all identified Purkinje cells at 4 d in vitro (div). After 14 d, however, NMDA responses were reduced in amplitude, whereas the responses to kainate and glutamate increased steadily in amplitude. In addition, the NMDA-induced current displayed a pronounced desensitization at these later stages; peak current declined to zero during steady application of NMDA. At 7 div, the number of surviving Purkinje cells was less in cultures treated with NMDA antagonists, and their survival was dose-dependent. Purkinje cell survival was correspondingly poorer in cultures from the NR1-/- mice than in wild-type controls, suggesting that NMDAR activity enhances the survival of Purkinje cells in vitro. The addition of moderate doses of NMDA promoted the survival of wild- type Purkinje cells in the presence of tetrodotoxin. Feeder layers of cerebellar granule cells derived from wild-type or NR1-/- mice promoted survival of Purkinje cells to a similar degree, suggesting that the NMDAR in Purkinje cells, but not in other cells, is directly involved in Purkinje cell viability. The results demonstrate that NMDARs transiently produce membrane current in Purkinje cells and may serve as one of the epigenetic factors that support the survival of Purkinje cells in vitro.</p>

A stimulus paradigm inducing long-term desensitization of AMPA receptors evokes a specific increase in BDNF mRNA in cerebellar slices.

Yuzaki M., Furuichi T., Mikoshiba K., Kagawa Y.

Learning & memory (Cold Spring Harbor, N.Y.) 1 ( 4 ) 230 - 242 1994.11

ISSN  1072-0502

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<p>Long-term desensitization of AMPA receptors (LTDA) is a core mechanism of long-term depression, a model of motor learning in the cerebellum. In this study we investigated the expression of neurotrophic factor genes after induction of LTDA in cultured cerebellar slices. LTDA was induced by application of quisqualate and monitored as a population response with a wedge recording technique. The levels of mRNA were quantified by reverse transcription followed by polymerase chain reaction. Quisqualate, at a dose and duration that reliably induced LTDA, elicited a significant and specific increase in BDNF mRNA with a peak at four hours after the application. By cell fractionation, the major source of BDNF mRNA increase was found to be in granule cells. In addition, a small but significant increase of transcripts with specific exon usage was observed in a Purkinje cell fraction. These results indicate that BDNF may be coinduced with LTDA and suggest that the slow and sustained increase of BDNF mRNA might play a role in later phases of synaptic plasticity in the cerebellum.</p>

Targeted disruption of NMDA receptor 1 gene abolishes NMDA response and results in neonatal death

Forrest Douglas, Yuzaki Michisuke, Soares Holly D., Ng Lily, Luk Daniel C., Sheng Morgan, Stewart Colin L., Morgan James I., Connor John A., Curran Tom

Neuron 13 ( 2 ) 325 - 338 1994

ISSN  0896-6273

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<p>In vitro studies have suggested that the NMDA receptor consists of an essential subunit, NR1, and various modulatory NR2 subunits. To test this hypothesis directly in vivo, we generated mice carrying a disrupted NR I allele. NMDA-inducible increases in intracellular calcium and membrane currents were abolished in neurons from homozygous null mutants (NR1^-/-). Thus, NR1 has a unique role, which cannot be substituted by any other subunit, in determining the activity of the endogenous NMDA receptor. A concomitant reduction in levels of NR2B but not NR2A occurred in NR1^-/- mice, demonstrating that there is an interdependence of subunit expression. NR1^-/- mice died 8-15 hr after birth, indicating a vital neonatal function for the NMDA receptor. Although the NMDA receptor has been implicated in several aspects of neurodevelopment, overall neuroanatomy of NR1^-/- mice appeared normal. Pathological evidence suggested that respiratory failure was the ultimate cause of death. © 1994.</p>

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Cerebellar Astrocytes Specifically Support the Survival of Purkinje Cells in Culture

Yuzaki M., Mikoshiba K., Kagawa Y.

Biochemical and Biophysical Research Communications 197 ( 1 ) 123 - 129 1993.11

ISSN  0006-291X

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<p>As a first step toward identifying the factor(s) that is/are produced by astrocytes and support(s) the survival of cerebellar Purkinje cells in dissociated culture, we compared the effect of astrocytes of cerebellar, hippocampal, and cerebral origin. A feeder coverslip of cerebellar astrocytes, which did not have cell-to-cell contact to neuronal culture, increased the percentage of Purkinje cells to about 8-9 fold with no change in the percentage of astrocytes. On the other hand, astrocytes of hippocampal or cerebral origin did not increase the percentage of Purkinje cells at low plating density, whereas they increased the number of astrocytes in neuronal culture. These results indicate that the factor(s), tentatively named as Purkinje-cell suvival factor was/were specifically produced by cerebellar astrocytes and affected directly on Purkinje cells. © 1993 Academic Press. All rights reserved.</p>

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Characterization of metabotropic glutamate receptors in cultured Purkinje cells

Yuzaki M., Mikoshiba K., Kagawa Y.

Annals of the New York Academy of Sciences 707   505 - 508 1993

ISSN  0077-8923

Antibody to the inositol trisphosphate receptor blocks thimerosalenhanced Ca²⁺-induced Ca²⁺ release and Ca²⁺ oscillations in hamster eggs

Miyazaki Shun ichi, Shirakawa Hideki, Nakada Ken, Honda Yoshiko, Yuzaki Michisuke, Nakade Shinji, Mikoshiba Katsuhiko

FEBS Letters 309 ( 2 ) 180 - 184 1992.09

ISSN  0014-5793

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<p>The sulfhydryl reagent thimerosal enhanced the sensitivity of hamster eggs to injected inositol 1,4,5-trisphosphate (InsP₃) or Ca²⁺ to generate regenerative Ca²⁺ release from intracellular pools. A monoclonal antibody (mAb) to the InsP₃ receptor blocked both the InsP₃-induced Ca²⁺ release (IICR) and Ca²⁺-induced Ca²⁺ release (CICR). The mAb also blocked Ca²⁺ oscillations induced by thimerosal. The results indicate that thimerosal enhances IICR sensitized by cytosolic Ca²⁺, but not CICR from InsP₃-insensitive pools, and causes repetitive Ca²⁺ releases from InsP₃-sensitive pools. © 1992.</p>

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Molecular cloning and characterization of the inositol 1,4,5-trisphosphate receptor in Drosophila melanogaster

Yoshikawa Shingo, Tanimura Teiichi, Miyawaki Atsushi, Nakamura Makoto, Yuzaki Michisuke, Furuichi Teiichi, Mikoshiba Katsuhiko

Journal of Biological Chemistry 267 ( 23 ) 16613 - 16619 1992.08

ISSN  0021-9258

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<p>We isolated a cDNA encoding an inositol 1,4,5-trisphosphate receptor (InsP₃R) of Drosophila melanogaster. The predicted Drosophila InsP₃R (2,833 amino acids) has extensive sequence similarity to the mouse InsP₃R. The polypeptide encoded by the cDNA was functionally expressed and showed characteristic InsP₃-binding activity. The Drosophila InsP₃R gene is located at the region 83A5-9 on the third chromosome and expresses throughout development but predominantly in the adult. Localization of the InsP₃R mRNA in adult tissues suggests strong expression in the retina and antenna, indicating the involvement of the InsP₃R in visual and olfactory transduction. In addition, the InsP₃R mRNA is abundant in the legs and thorax, which are enriched with a muscular system. Such localization is apparently consistent with the quantitatively predominant sites for [³H]InsP₃ binding in Drosophila and the fleshfly (Boettcherisca peregrina). The present study points to the likely functional importance of the InsP₃/Ca²⁺ signaling system in Drosophila.</p>

Block of Ca²⁺ wave and Ca²⁺ oscillation by antibody to the inositol 1,4,5-trisphosphate receptor in fertilized hamster eggs

Miyazaki Shun Ichi, Yuzaki Michisuke, Nakada Ken, Shirakawa Hideki, Nakanishi Setsuko, Nakade Shinji, Mikoshiba Katsuhiko

Science 257 ( 5067 ) 251 - 255 1992.07

ISSN  0036-8075

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<p>The concentration of cytoplasmic free calcium (Ca²⁺) increases in various stimulated cells in a wave (Ca²⁺ wave) and in periodic transients (Ca²⁺ oscillations). These phenomena are explained by inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release (IICR) and Ca²⁺-induced Ca²⁺ release (CICR) from separate intracellular stores, but decisive evidence is lacking. A monoclonal antibody to the IP₃ receptor inhibited both IICR and CICR upon injection of IP₃ and Ca²⁺ into hamster eggs, respectively. The antibody completely blocked sperm-induced Ca²⁺ waves and Ca²⁺ oscillations. The results indicate that Ca² release in fertilized hamster eggs is mediated solely by the IP₃ receptor, and Ca²⁺-sensitized IICR, but not CICR, generates Ca²⁺ waves and Ca²⁺ oscillations.</p>

Pharmacological and immunocytochemical characterization of metabotropic glutamate receptors in cultured Purkinje cells

Yuzaki M., Mikoshiba K.

Journal of Neuroscience 12 ( 11 ) 4253 - 4263 1992

ISSN  0270-6474

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<p>Metabotropic glutamate receptor (mGluR) is highly expressed in cerebellar Purkinje cells. The purpose of this study was pharmacological and immunocytochemical characterization of the mGluR in single cerebellar neurons, especially Purkinje cells. Ca²⁺ imaging with fura-2 in cultured cerebellar neurons, identified immunocytochemically, was used to record the direct effects of drugs in stable conditions. In addition, the expression of mGluR was examined, and expression of the intracellular receptor for inositol trisphosphate (IP₃) produced by mGluR activation was studied immunocytochemically with specific antibodies. Purkinje neurons and some other neurons showed Ca²⁺-mobilizing responses to mGluR agonists. These responses were mediated by mGluR because they were not blocked by ionotropic GluR antagonists, were independent of the caffeine-sensitive Ca²⁺ pool, and were blocked by inhibitors of IP₃-induced Ca²⁺ release. This is the first pharmacological characterization of mGluR at single Purkinje cells. The results differed as follows from those in earlier studies in which phosphoinositide turnover of the entire population of cerebellar cells was monitored: (1) the mGluR responses were not blocked by pertussis toxin or D,L-2-amino-3-phosphonopropionic acid; (2) glutamate was a potent agonist, whereas L-aspartate was ineffective; and (3) the dose-response relationship showed an all-or-none tendency. The metabotropic response of Purkinje cells changed markedly during development, with a sharp peak after day 4 of culture, whereas mGluR and IP₃ receptor proteins increased steadily during maturation. This apparent desensitization of mGluR was not blocked by inhibitors of protein kinase C (PKC) or ADP-ribosyltransferase. The metabotropic responses were mainly localized to the center of the somata of Purkinje cells even on day 4, whereas both receptor proteins were expressed throughout the cell. These results suggest that the function of mGluR is spatially and developmentally controlled by a posttranslational mechanism involving a mechanism other than phosphorylation by PKC or ADP-ribosylation.</p>

MK-801 blocked the functional NMDA receptors in identified cerebellar neurons

Yuzaki Michisuke, Kudo Yoshihisa, Akita Kyoko, Miyawaki Atsushi, Mikoshiba Katsuhiko

Neuroscience Letters 119 ( 1 ) 19 - 22 1990.10

ISSN  0304-3940

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<p>In order to clarify the nature of N-methyl-D-aspartate (NMDA) receptors in cerebellum, where heterogeneity of the NMDA receptor has been suggested, we investigated the action of MK-801 on the NMDA-induced [Ca²⁺]_i rise in cultured cerebellar neurons using video-assisted microfluorometry. MK-801 caused a potent and selective blockade of the NMDA-activated [Ca²⁺]_i elevation. The blockade caused by MK-801 was dependent on the presence of NMDA, i.e., use-dependent. There was no difference in the mode of blockade between immunocytochemically identified Purkinje and non-Purkinje cells, although the relative size of the NMDA-induced [Ca²⁺]_i rise was significantly less in Purkinje cells. These results indicate that the NMDA receptors in cultured cerebellar neurons are coupled with the same channels as those in other brain regions. © 1990.</p>

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Mode of blockade by MK-801 of N-methyl-d-aspartate-induced increase in intracellular Ca²⁺ in cultured mouse hippocampal neurons

Yuzaki Michisuke, Miyawaki Atsushi, Akita Kyoko, Kudo Yoshihisa, Ogura Akihiko, Ino Hidetoshi, Mikoshiba Katsuhiko

Brain Research 517 ( 1-2 ) 51 - 56 1990.05

ISSN  0006-8993

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<p>Microfluorometry with fura-2 was applied to study the action of the anticonvulsant (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) on N-methyl-d-aspartate (NMDA)-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]₁) in cultured mouse hippocampal neurons. MK-801 caused a potent and long-lasting blockade of the NMDA-activated [Ca²⁺]_i elevation in a selective manner, not affecting the [Ca²⁺]_i rise induced by quisqualate or kainate. Blockade and recovery from the blockade by MK-801 showed use dependency; the degree of blockade was dependent on the presence of NMDA. The use-dependent onset of antagonism was, however, highly sensitive to the bath temperature. MK-801 applied in the absence of NMDA had no effect on the response to subsequent application of NMDA at 22°C, whereas it reduced the subsequent response to NMDA significantly at 37°C. MK-801 interacted with the receptor-ion channel complex even when Mg²⁺, which is considered to block the open channel, had already blocked the NMDA-induced [Ca²⁺]_i. The recovery from blockade by MK-801 was not accelerated by the application of 10 mM Mg²⁺ for 5 min. These results suggest that MK-801 can gain access to its binding site in the absence of NMDA at physiological temperature, and that this binding site is distinct from that for Mg²⁺. © 1990.</p>

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Multiple deletions in mitochondrial DNA at direct repeats of non-D-loop regions in cases of familial mitochondrial myopathy

Yuzaki Michisuke, Ohkoshi Norio, Kanazawa Ichiro, Kagawa Yasuo, Ohta Shigeo

Biochemical and Biophysical Research Communications 164 ( 3 ) 1352 - 1357 1989.11

ISSN  0006-291X

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<p>Muscle mitochondrial DNAs from two brothers with mitochondrial myopathy associated with peripheral neuropathy had multiple deletions, most of which started in non-D-loop regions, unlike in an autosomal dominant mitochondrial myopathy (Zeviani, M. et al., Nature 339, 309 (1989)). The non-D-loop regions with deletions were amplified by the polymerase chain reaction and the resulting fragments were subcloned and then sequenced. At least 12 deletions of different lengths in different sites were found. However, all the deletions were flanked by short direct repeats (4-12 base pairs). © 1989.</p>

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Autoradiographic visualization of a calcium channel antagonist, [¹²⁵I]ω-conotoxin GVIA, binding site in the brains of normal and cerebellar mutant mice (pcd and weaver)

Maeda Nobuaki, Wada Kentaro, Yuzaki Michisuke, Mikoshiba Katsuhiko

Brain Research 489 ( 1 ) 21 - 30 1989.06

ISSN  0006-8993

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<p>An in vitro autoradiographic technique has been used to localize [¹²⁵I]ω-conotoxin GVIA binding sites in the brains of normal and cerebellar mutant mice. In the brains of normal mice, the highest densities of binding sites were observed at glomeruli of the olfactory bulb, cerebral cortex, caudate nucleus-putamen, hippocampus, and the nucleus of the solitary tract. Moderate densities of the silver grains occured on the granular layer of the olfactory bulb, the molecular layer of the dentate gyrus, the molecular layer of the cerebellum, and the cochlear nucleus. No specific binding appeared in the white matter or the deep nucleus of the cerebellum, the corpus callosum, the internal capsule and the external plexiform layer of the olfactory bulb. Autoradiographic studies of the cerebella of Purkinje cell degeneration (pcd) mice showed that the distribution of binding sites on the molecular layer of the cerebellum are not affected by the degeneration of Purkinje cells. However, only background levels of the silver grains occured on the cerebella of agranular weaver mutant mice, suggesting that the receptor for ω-conotoxin GVIA in the cerebellum are predominantly distributed on the parellel fibers of granule cells. © 1989.</p>

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Cation permeability change caused by l-glutamate in cultured rat hippocampal neurons

Ozawa Seiji, Nakamura Tomoko, Yuzaki Michisuke

Brain Research 443 ( 1-2 ) 85 - 94 1988.03

ISSN  0006-8993

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<p>The ionic mechanism of the membrane permeability changes caused by l-glutamate in hippocampal neurons prepared from 17- to 19-day-old fetal rat in dispersed cell cultures was studied with the whole-cell variation of the patch electrode voltage-clamp technique. The cultured hippocampal neurons became sensitive to glutamate 7 days after plating, and thereafter the sensitivity gradually increased. The conductance increase caused by glutamate was voltage-sensitive, decreasing with membrane hyperpolarization at potentials more negative than -40 mV. The relative permeability of glutamate-activated channels to alkali metal and alkaline earth cations was estimated by reversal potential measurements. The alkali metal cations, Li+, Na+, K+, Rb+ and Cs+ were permeant to the glutamate channels, and the selectively among them was weak. The alkaline earth cations, Ca²⁺, Sr²⁺ and Ba²⁺ were more permeant than the alkali metals. The permeability ratios of these divalent cations relative to Na+ were 2.4 (Ca²⁺), 2.4 (Sr²⁺) and 2.8 (Ba²⁺), respectively. Mg²⁺ was much less permeant and the permeability ratio (P_Mg/P_Na) was only 0.1. Anion conductance made no contribution to the glutamate-induced current. Functional implications of the glutamate-induced increase in Ca²⁺-influx were discussed. © 1988.</p>

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