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Intracellular trafficking of pathological missense mutations of neural cell adhesion molecule L1 (L1CAM)
Abstracts found to be obviously thinner in the mutants. These results suggest that pcdh9 may be involved in layer formation in the developing cortex. doi:10.1016/j.neures.2009.09.425
P1-f07 Perturbed development of granule cells of the cerebellum in NB-3 knockout mice Kunie Sakurai 1 , Manabu Toyoshima 1 , Yasuo Takeda 2 , Yasushi Shimoda 1 , Domna Karagogeos 3 , Kazutada Watanabe 1 1
Department Bioeng., Nagaoka University Tech., Japan; 2 Department Clinic. Pharm. & Pharmacol., Kagoshima University Grad. Sch. Med. & Dent. Sci., Japan; 3 Department Basic Sci., Crete University Grad. Sch. Med., Japan
S99
Versican is a chondroitin sulfate proteoglycan belonging to the lectican family and has two glycosaminoglycan (GAG) attachment regions named GAG␣ and GAG, which are alternatively spliced. Here, we analyzed the expression and localization of versican isoforms using GAG␣ and GAG-specific antibodies. Western and immunohistochemical analyses revealed GAG␣-reactive isoform was expressed from neonatal periods to adulthood, whereas GAG-reactive isoform was disappeared within 3 weeks of birth. In the adult brain, GAG␣ immunoreactivity was observed on cortical and hippocampal pyramidal neurons and cerebellar Purkinje cells, but not on parvalbumin-positive interneurons and cerebellar stellate cells, suggesting versican is localized on large projection neurons but not on small interneurons. Double labeling of GAG and perineuronal net markers (WFA and phosphacan) revealed that GAG was different localization from typical perineuronal nets. doi:10.1016/j.neures.2009.09.429
NB-3 (contactin6) is expressed in the developing nervous system after birth and its deficiency causes impairment in motor coordination. In this study, we investigated the contribution of NB-3 to cerebellar development. In the developing cerebellum, NB-3 was expressed in radially migrating granule cells and parallel fibers. The double-immunostaining with NB-3 and VGLUT1 or VGLUT2 demonstrated that NB3 is present at the presynaptic terminals of parallel fibers and absent in those of climbing-fibers. Granule cell development was altered and the number of synapses between parallel fibers and Purkinje cells was reduced in NB-3 knockout mouse as compared to wild type mouse. Thus, our results indicate that NB-3 deficiency affects the postnatal development of granule cells in the cerebellum. doi:10.1016/j.neures.2009.09.426
P1-f08 Involvement of Caspr2 encoded by neuropsychiatric developmental disorder-susceptibility gene CNTNAP2 in the synaptogenesis Yasushi Shimoda, Hidehiro Ueda, Kazutada Watanabe Department Bioeng., Nagaoka University of Tech., Japan Caspr2 is a neural recognition molecule belonging to the neurexin family and is encoded by the CNTNAP2 gene that has recently been reported to be associated with neuropsychiatric developmental disorders such as autism schizophrenia, leading us to hypothesize that Caspr2 may be involved in synaptogenesis. In this study, we examined the localization and function of Caspr2. Immunostaining and biochemical analysis revealed that Caspr2 was localized at synapse in vivo and in vitro. Overexpression of Caspr2 induced the formation of protrusions on neurites. The extracellular domain, but not the intracellular domain, of Caspr2 was required for the induction of protrusions. These results suggest that the extracellular interaction of Caspr2 with its unidentified receptor might be involved in the synaptogenesis. doi:10.1016/j.neures.2009.09.427
Tokushima Bunri University, Japan L1CAM plays an important role in the normal brain development. It has become evident that L1CAM gene mutations cause severe neurological diseases including X-linked hydrocephalus. Here we investigated the relation between L1CAM mutations and neurological diseases, focusing on the intracellular trafficking of L1CAM mutants. We constructed L1CAM mutants referring to the its pathological mutants that have already been reported. Then, we examined the intracellular trafficking of L1CAM mutants and morphological change in neuron expressing L1CAM mutants using confocal microscope. Mutations of L1CAM located in the fibronectin type III domains showed downregulation of the cell surface expression levels and accumulations at these mutants on intracellular compartments. This result implicate that inability of L1CAM mutants to express on the cell surface leads to impair the normal brain development. The mechanism on the intracellular trafficking of L1CAM mutants and the identification of intracellular compartments localizing L1CAM mutants are under investigation. doi:10.1016/j.neures.2009.09.428
P1-f11 Localization of chondroitin sulfate proteoglycan versican in the adult brain with special reference to large progection neurons Noriko Horii 1 , Hiroaki Okuda 2 , Kouko Tatsumi 2 , Masahide Yoshikawa 1 , Shigeaki Ishizaka 1 , Akio Wanaka 2 Dept Parasitol, Nara Med Univ, Nara, Japan; Univ, Nara, Japan
1
Div Struct Cell Bio, NAIST, Nara, Japan; 2 Div Endocrionl Metabolism, NIPS, Aichi, Japan; 3 Miami Project, Univ of Miami, USA
L1cam is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. In humans, mutations in the L1 gene cause X-linked hydrocephalus and MASA syndrome. It has been shown that the L1 cytoplasmic domain (L1CD) is important for neurite outgrowth, branching, and interactions with the cytoskeleton. To explore the function of the L1CD, we made three mice lines in which different parts of the L1CD have been altered. While hydrocephalus is present in L1 knockout mice, these three L1CD lines display normal brain morphology, even though one L1CD line is missing almost the entire L1CD. All mutant lines express L1 during development and in all lines L1 is transported into axons. But the expression of L1 protein in the adult becomes very low in the two L1CD lines that lack the ankyrin-binding region. In addition, they showed a defect in motor function. These data show that the L1CD is not essential for brain formation, but is critical for stable L1 expression, and indicate that L1 is important for motor function in the adult. doi:10.1016/j.neures.2009.09.430
P1-f13 Phagocytic synapses on neurons induced by telencephalin–vitronectin interaction Yutaka Furutani 1 , Miwa Kawasaki 1 , Hitomi Matsuno 1 , Kensaku Mori 2 , Yoshihiro Yoshihara 1 1
P1-f09 Intracellular trafficking of pathological missense mutations of neural cell adhesion molecule L1 (L1CAM) Kanako Fujisaki, Yoshikatu Aikawa, Kouichi Itoh
1
P1-f12 The L1 cytoplasmic domain is essential for L1 expression in the adult Yukiko Nakamura 1 , Suni Lee 2 , Candace Haddox 3 , Eli Weaver 3 , Vance Lemmon 3
2
Dept 2nd Anat, Nara Med
RIKEN BSI, Wako, Japan;
2
Dept Physiol, Univ of Tokyo, Tokyo, Japan
Dendritic filopodia are long, thin, and dynamic protrusions that are converted into mature spines during neural development. Telencephalin (TLCN; ICAM-5) is a unique member of the ICAM family specifically expressed by spiny neurons within the telencephalon and abundantly localized to dendritic filopodia. In this study, we report the identification of an extracellular matrix molecule, vitronectin, as a specific ligand for TLCN. The surface plasmon resonance analysis revealed that the extracellular region of TLCN bound to vitronectin with high affinity. Vitronectin-coated microbeads strongly adhered onto dendrites of cultured hippocampal neurons and induced the formation of cup-like membrane protrusions (phagocytic synapses). TLCN, phospho-ERM, F-actin, and PI(4,5)P2 accumulated to the phagocytic synapses and surrounded the vitronectin-coated beads, all of which are contained in the dendritic filopodia. Therefore, molecular constituents of the dendritic filopodia appear identical or similar to those of the phagocytic synapses. doi:10.1016/j.neures.2009.09.431
P1-f14 Dynamic expression pattern of Neuronal leucine-rich repeat 4 in mouse sensory neurons during development Takayoshi Bando 1 , Yoshihiro Morikawa 1 , Atsushi Miyajima 2 , Emiko Senba 1 1 Department Anat. and Neurobiol, Wakayama Med. University, Wakayama, Japan; 2 Lab. of Cell Growth and Differ, IMCB, University of Tokyo, Tokyo, Japan
Previously, we have identified a novel member of NLRR family, NLRR4, and which is expressed in some brain regions and functions in the hippocampus-dependent memory (Bando et al., Mol. Cell. Biol. 25: 4166-4175). Here, we investigated the expression of NLRR4 in the dorsal root ganglia (DRGs) during development. NLRR4 mRNA was expressed in a subset of DRG neurons during development and the expression was most intense from late embryonic to early postnatal stages. To
P1-f07 Perturbed development of granule cells of the cerebellum in NB-3 knockout mice Kunie Sakurai 1 , Manabu Toyoshima 1 , Yasuo Takeda 2 , Yasushi Shimoda 1 , Domna Karagogeos 3 , Kazutada Watanabe 1 1
Department Bioeng., Nagaoka University Tech., Japan; 2 Department Clinic. Pharm. & Pharmacol., Kagoshima University Grad. Sch. Med. & Dent. Sci., Japan; 3 Department Basic Sci., Crete University Grad. Sch. Med., Japan
S99
Versican is a chondroitin sulfate proteoglycan belonging to the lectican family and has two glycosaminoglycan (GAG) attachment regions named GAG␣ and GAG, which are alternatively spliced. Here, we analyzed the expression and localization of versican isoforms using GAG␣ and GAG-specific antibodies. Western and immunohistochemical analyses revealed GAG␣-reactive isoform was expressed from neonatal periods to adulthood, whereas GAG-reactive isoform was disappeared within 3 weeks of birth. In the adult brain, GAG␣ immunoreactivity was observed on cortical and hippocampal pyramidal neurons and cerebellar Purkinje cells, but not on parvalbumin-positive interneurons and cerebellar stellate cells, suggesting versican is localized on large projection neurons but not on small interneurons. Double labeling of GAG and perineuronal net markers (WFA and phosphacan) revealed that GAG was different localization from typical perineuronal nets. doi:10.1016/j.neures.2009.09.429
NB-3 (contactin6) is expressed in the developing nervous system after birth and its deficiency causes impairment in motor coordination. In this study, we investigated the contribution of NB-3 to cerebellar development. In the developing cerebellum, NB-3 was expressed in radially migrating granule cells and parallel fibers. The double-immunostaining with NB-3 and VGLUT1 or VGLUT2 demonstrated that NB3 is present at the presynaptic terminals of parallel fibers and absent in those of climbing-fibers. Granule cell development was altered and the number of synapses between parallel fibers and Purkinje cells was reduced in NB-3 knockout mouse as compared to wild type mouse. Thus, our results indicate that NB-3 deficiency affects the postnatal development of granule cells in the cerebellum. doi:10.1016/j.neures.2009.09.426
P1-f08 Involvement of Caspr2 encoded by neuropsychiatric developmental disorder-susceptibility gene CNTNAP2 in the synaptogenesis Yasushi Shimoda, Hidehiro Ueda, Kazutada Watanabe Department Bioeng., Nagaoka University of Tech., Japan Caspr2 is a neural recognition molecule belonging to the neurexin family and is encoded by the CNTNAP2 gene that has recently been reported to be associated with neuropsychiatric developmental disorders such as autism schizophrenia, leading us to hypothesize that Caspr2 may be involved in synaptogenesis. In this study, we examined the localization and function of Caspr2. Immunostaining and biochemical analysis revealed that Caspr2 was localized at synapse in vivo and in vitro. Overexpression of Caspr2 induced the formation of protrusions on neurites. The extracellular domain, but not the intracellular domain, of Caspr2 was required for the induction of protrusions. These results suggest that the extracellular interaction of Caspr2 with its unidentified receptor might be involved in the synaptogenesis. doi:10.1016/j.neures.2009.09.427
Tokushima Bunri University, Japan L1CAM plays an important role in the normal brain development. It has become evident that L1CAM gene mutations cause severe neurological diseases including X-linked hydrocephalus. Here we investigated the relation between L1CAM mutations and neurological diseases, focusing on the intracellular trafficking of L1CAM mutants. We constructed L1CAM mutants referring to the its pathological mutants that have already been reported. Then, we examined the intracellular trafficking of L1CAM mutants and morphological change in neuron expressing L1CAM mutants using confocal microscope. Mutations of L1CAM located in the fibronectin type III domains showed downregulation of the cell surface expression levels and accumulations at these mutants on intracellular compartments. This result implicate that inability of L1CAM mutants to express on the cell surface leads to impair the normal brain development. The mechanism on the intracellular trafficking of L1CAM mutants and the identification of intracellular compartments localizing L1CAM mutants are under investigation. doi:10.1016/j.neures.2009.09.428
P1-f11 Localization of chondroitin sulfate proteoglycan versican in the adult brain with special reference to large progection neurons Noriko Horii 1 , Hiroaki Okuda 2 , Kouko Tatsumi 2 , Masahide Yoshikawa 1 , Shigeaki Ishizaka 1 , Akio Wanaka 2 Dept Parasitol, Nara Med Univ, Nara, Japan; Univ, Nara, Japan
1
Div Struct Cell Bio, NAIST, Nara, Japan; 2 Div Endocrionl Metabolism, NIPS, Aichi, Japan; 3 Miami Project, Univ of Miami, USA
L1cam is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. In humans, mutations in the L1 gene cause X-linked hydrocephalus and MASA syndrome. It has been shown that the L1 cytoplasmic domain (L1CD) is important for neurite outgrowth, branching, and interactions with the cytoskeleton. To explore the function of the L1CD, we made three mice lines in which different parts of the L1CD have been altered. While hydrocephalus is present in L1 knockout mice, these three L1CD lines display normal brain morphology, even though one L1CD line is missing almost the entire L1CD. All mutant lines express L1 during development and in all lines L1 is transported into axons. But the expression of L1 protein in the adult becomes very low in the two L1CD lines that lack the ankyrin-binding region. In addition, they showed a defect in motor function. These data show that the L1CD is not essential for brain formation, but is critical for stable L1 expression, and indicate that L1 is important for motor function in the adult. doi:10.1016/j.neures.2009.09.430
P1-f13 Phagocytic synapses on neurons induced by telencephalin–vitronectin interaction Yutaka Furutani 1 , Miwa Kawasaki 1 , Hitomi Matsuno 1 , Kensaku Mori 2 , Yoshihiro Yoshihara 1 1
P1-f09 Intracellular trafficking of pathological missense mutations of neural cell adhesion molecule L1 (L1CAM) Kanako Fujisaki, Yoshikatu Aikawa, Kouichi Itoh
1
P1-f12 The L1 cytoplasmic domain is essential for L1 expression in the adult Yukiko Nakamura 1 , Suni Lee 2 , Candace Haddox 3 , Eli Weaver 3 , Vance Lemmon 3
2
Dept 2nd Anat, Nara Med
RIKEN BSI, Wako, Japan;
2
Dept Physiol, Univ of Tokyo, Tokyo, Japan
Dendritic filopodia are long, thin, and dynamic protrusions that are converted into mature spines during neural development. Telencephalin (TLCN; ICAM-5) is a unique member of the ICAM family specifically expressed by spiny neurons within the telencephalon and abundantly localized to dendritic filopodia. In this study, we report the identification of an extracellular matrix molecule, vitronectin, as a specific ligand for TLCN. The surface plasmon resonance analysis revealed that the extracellular region of TLCN bound to vitronectin with high affinity. Vitronectin-coated microbeads strongly adhered onto dendrites of cultured hippocampal neurons and induced the formation of cup-like membrane protrusions (phagocytic synapses). TLCN, phospho-ERM, F-actin, and PI(4,5)P2 accumulated to the phagocytic synapses and surrounded the vitronectin-coated beads, all of which are contained in the dendritic filopodia. Therefore, molecular constituents of the dendritic filopodia appear identical or similar to those of the phagocytic synapses. doi:10.1016/j.neures.2009.09.431
P1-f14 Dynamic expression pattern of Neuronal leucine-rich repeat 4 in mouse sensory neurons during development Takayoshi Bando 1 , Yoshihiro Morikawa 1 , Atsushi Miyajima 2 , Emiko Senba 1 1 Department Anat. and Neurobiol, Wakayama Med. University, Wakayama, Japan; 2 Lab. of Cell Growth and Differ, IMCB, University of Tokyo, Tokyo, Japan
Previously, we have identified a novel member of NLRR family, NLRR4, and which is expressed in some brain regions and functions in the hippocampus-dependent memory (Bando et al., Mol. Cell. Biol. 25: 4166-4175). Here, we investigated the expression of NLRR4 in the dorsal root ganglia (DRGs) during development. NLRR4 mRNA was expressed in a subset of DRG neurons during development and the expression was most intense from late embryonic to early postnatal stages. To