研究者詳細 - 柚崎　通介

論文 - 柚崎　通介

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Transgenic rescue for characterizing orphan receptors: a review of delta2 glutamate receptor.

Yuzaki M

Transgenic research 14 （ 2 ） 117 - 121 2005年04月

ISSN 0962-8819

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.

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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年

ISSN 1469-221X

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.

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

柚﨑通介

EMBO Report （ 6 ） 90-95 2005年

研究論文（学術雑誌）, 共著, 査読有り

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年04月

ISSN 0953-816X

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.

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The delta2 glutamate receptor—A key molecule controlling synaptic plasticity and structure in Purkinje cells.

柚﨑通介

Cerebellum （ 3 ） 89-93 2004年

研究論文（学術雑誌）, 単著, 査読有り

The δ2 glutamate receptor

Yuzaki Michisuke

Cerebellum 3 （ 2 ） 89 - 93 2004年

ISSN 1473-4222

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.

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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年11月

ISSN 0270-6474

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.

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年08月

ISSN 1097-6256

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.

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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年06月

ISSN 0022-9717

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.

The δ2 glutamate receptor

Yuzaki Michisuke

Neuroscience Research 46 （ 1 ） 11 - 22 2003年05月

ISSN 0168-0102

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.

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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年04月

ISSN 0169-328X

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.

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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年04月

ISSN 0953-816X

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.

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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年01月

ISSN 0169-328X

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.

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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年04月

ISSN 0169-328X

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.

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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年

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年

ISSN 0953-816X

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.

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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年03月

ISSN 0950-9232

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.

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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年

ISSN 0953-816X

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.

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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年04月

ISSN 1097-6256

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.

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Neuronal interleukin-16, (NIL-16)

Kurschner Cornelia, Yuzaki Michisuke

Journal of Neuroscience 19 （ 18 ） 7770 - 7780 1999年09月

ISSN 0270-6474

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.

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