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Calcium dependent-regulation of neurite outgrowth mediated by a protein translational factor eEF2 within nerve growth cones
S204
Abstracts / Neuroscience Research 58S (2007) S1–S244
Measurements of changes in [Ca2+ ]i and [Cl− ]i during P3-d20
P3-d24 How does ephrinB3/EphA4 signaling regulate motor-
Tatsuro Kumada, Atsuo Fukuda Department of Physiology, Hamamatsu University School of Medicine, Hamamatsu, Japan
Takuji Iwasato 1 , Hironori Katoh 2 , Hiroshi Nishimaru 3 , Yukio Ishikawa 2 , Haruhisa Inoue 2 , Y.M. Saito 1 , R. Ando 1 , M. Iwama 1 , Ryosuke Takahashi 2 , Manabu Negishi 2 , Shigeyoshi Itohara 1 1 RIKEN-BSI, Wako, Japan; 2 Kyoto University, Japan; 3 AIST, Japan
glioblastoma cell movement
Malignant glioblastoma cells invasively migrate into widespread regions of brain with changing cellular morphology. Alterations in intracellular ion levels such as [Ca2+ ]i or [Cl− ]i are involved in the regulation of cell motility or cellular volume. However, it has well not known about the spatial-temporal changes in these ion levels, especially [Cl− ]i during invasive migration. To assess it, we have attempted to visualize the cell movement with dynamic morphological alterations and changes in [Ca2+ ]i or [Cl− ]i simultaneously. Combination of time-lapse observation and Ca2+ imaging demonstrated that Ca2+ oscillations were observed in the glioblastoma cells which have the structures like leading process during cell movement. Furthermore, FRET imaging with Yellow-camleon and Clomeleon to calculate [Ca2+ ]i and [Cl− ]i levels, respectively, allowed us to detect the changes in FRET efficiencies by pharmacological applications. These results raise the possibility of analysis of the spatio-temporal characteristic of [Ca2+ ]i and [Cl− ]i by FRET imaging during long-time migration. Research funds: KAKENHI 18700313
P3-d22 Calcium dependent-regulation of neurite outgrowth mediated by a protein translational factor eEF2 within nerve growth cones Akira Iizuka 1 , Masumi Iketani 1 , Kumiko Sengoku 1 , Fumio Nakamura 1 , Masayuki Matsushita 2 , Angus Nairn 3 , Yoshio Goshima 1,4 , Kohtaro Takei 1,4 1 Yokohama City University Graduate School of Medicine, Department of Molecular Pharmacology & Neurobiology, Yokohama, Japan; 2 Mitsubishi Institute of Life Science, Tokyo, Japan; 3 Yale University School of Medicine, Department of Psychiatry, New Haven, USA; 4 CREST, JST, Kawaguchi, Japan
circuit formation?
The ephrin/Eph system plays a central role in neural-circuit formation; however, its downstream effectors are poorly understood. Here we show that a-chimerin Rac-GAP mediates ephrinB3/EphA4 forward signaling. We discovered a spontaneous mouse mutation, miffy (mfy), which results in a rabbit-like hopping gait, along with impaired corticospinal axon guidance and spinal central pattern generators. Using positional cloning, transgene rescue and gene targeting, we demonstrated that dysfunction of a-chimerin leads to mfy phenotypes similar to those of EphA4−/− and ephrinB3−/− mice. a-chimerin interacts with EphA4, and, in response to ephrinB3/EphA4 signaling, inactivates Rac, which is a positive regulator of process outgrowth. Moreover, down-regulation of a-chimerin suppresses ephrinB3-induced growth-cone collapse in cultured neurons. Our findings indicate that ephrinB3/EphA4 signaling prevents growth-cone extension in motor-circuit formation via a-chimerin-induced inactivation of Rac. Research funds: PRESTO, KAKENHI (18021041)
P3-d25 CRMP1 regulates spine maturation through mediating Semaphorin3A signaling in vivo Naoya Yamashita 1 , Asa Morita 1 , Yutaka Uchida 1 , Toshio Ohshima 2 , Fumio Nakamura 1 , Jerome Honnorat 3 , Pappachan Kolattukudy 4 , Yoshio Goshima 1 1 Graduate School of Medicine, Yokohama City University, Yokohama, Japan; 2 RIKEN BSI, Wako, Japan; 3 INSERM U433 Hopital Neurologique, Lyon, France; 4 Biomol. Sci., University of Central Florida, Florida, USA
Local protein synthesis within growth cones is known to regulate growth cone behavior. However, the mechanism of the regulation remains unclear. We found that eukaryotic elongation factor-2 (eEF2) in growth cones regulates neurite outgrowth in calcium-dependent manner. Acute localized loss of eEF2 in growth cones by chromophore-assisted laser inactivation (CALI) resulted in growth arrest of neurite. Extracellular ATP-evoked calcium transient activated EF2 kinase (EF2K) and increased eEF2 phosphorylation in growth cones, and also inhibited neurite outgrowth. This ATP-induced growth arrest of neurite was abolished by CALI of EF2K. The data suggest that local mRNA translation regulated by eEF2 in the growth cone may play an important event in the neurite outgrowth.
CRMP1 is one of the CRMP family members that mediate signal transduction of axon guidance molecules. We here show evidence that CRMP1 is involved in Sema3A-Cdk5 signaling to regulate spine maturation in the cerebral cortex. In the cultured cortical neurons from crmp1+/− mice, Sema3A increased the number of clusters of synapsin I and PSD-95, but this increase was attenuated in crmp1−/− mice. This phenotype seen in crmp1−/− mice was rescued by introduction of wild-type CRMP1 but not CRMP1-T509A/S522A, a mutant unphosphorylatable by Cdk5. Golgiimpregnation method showed that the crmp1−/− layer V cortical neurons showed lowered density of synaptic bouton-like structures and this phenotype had genetic interaction with sema3A. These findings suggest that CRMP1 regulates spine maturation through phosphorylation of CRMP1 by Sema3A-Cdk5 cascade.
Research fund: CREST, KAKENHI(13680851)
Research funds: JST CREST
P3-d23 Neuronal calcium sensor-1 in nerve growth cones independently regulates both the neurite outgrowth and growth cone morphology Masumi Iketani 1 , Chihiro Imaizumi 1 , Andreas Jeromin 2 , Fumio Nakamura 1 , Katsuhiko Mikoshiba 3,4 , Yoshio Goshima 1,5 , Kohtaro Takei 1,5 1 Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama, Japan; 2 Center for Learning & Memory, University of Texas, Austin, USA; 3 Institute of Medical Sciences, University of Tokyo, Tokyo, Japan; 4 Brain Science Institute, RIKEN, Wako, Japan; 5 CREST, JST, Kawaguchi, Japan Neuronal calcium sensor-1 (NCS-1) is a calcium binding protein, which is specifically expressed in the nervous system. NCS-1 was distributed in a cluster and co-localized with the type 1 IP3 receptor in the central domain of the growth cone. The acute localized loss of NCS-1 in the growth cone induced by chromophore-assisted laser inactivation (CALI) resulted in the growth arrest of neurites and lamellipodial and filopodial retractions. The CALI-induced growth arrest of neurites was abolished by the pharmacological inhibition of IP3 receptor, while CALI-induced lamellipodial and filopodial retractions were unaffected. These findings suggest that NCS-1 in growth cones may independently regulate both the neurite outgrowth and growth cone morphology. Research funds: CREST, JST
P3-d26 IgLON adhesion molecules participate in synapse formation of hippocampal neurons Takasi Hashimoto 1 , Shohei Maekawa 2 , Seiji Miyata 1 1 Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan; 2 Department of Bioscience, Graduate School of Science and Technology, Kobe University, Nada-ku, Kobe 657-8501, Japan IgLON cell adhesion molecules are known to belong to a subfamily of immunoglobulin superfamily and comprise four members: LAMP, OBCAM, neurotrimin, and kilon. Several in vitro studies showed that members of IgLON subgroup play a role in neurite outgrowth. However, although ultrastructural analysis demonstrated the localization of IgLON at synaptic region in the adult brain of rat, it has not been studied whether IgLONs are involved in the formation of synapses. In this study, we examined the overexpression of IgLONs on synapse formation of hippocampal neurons. Hippocampal neurons were transfected with a vector containing the GFP and each IgLON coding sequence. The quantitative analysis showed that overexpression of LAMP or OBCAM increased the number of synapses. In contrast, overexpression of kilon or neurotrimin surpressed the synapse formation. These results indicate that IgLON are the synaptic cell adhesion molecules concerning the regulation of synapse formation.
Abstracts / Neuroscience Research 58S (2007) S1–S244
Measurements of changes in [Ca2+ ]i and [Cl− ]i during P3-d20
P3-d24 How does ephrinB3/EphA4 signaling regulate motor-
Tatsuro Kumada, Atsuo Fukuda Department of Physiology, Hamamatsu University School of Medicine, Hamamatsu, Japan
Takuji Iwasato 1 , Hironori Katoh 2 , Hiroshi Nishimaru 3 , Yukio Ishikawa 2 , Haruhisa Inoue 2 , Y.M. Saito 1 , R. Ando 1 , M. Iwama 1 , Ryosuke Takahashi 2 , Manabu Negishi 2 , Shigeyoshi Itohara 1 1 RIKEN-BSI, Wako, Japan; 2 Kyoto University, Japan; 3 AIST, Japan
glioblastoma cell movement
Malignant glioblastoma cells invasively migrate into widespread regions of brain with changing cellular morphology. Alterations in intracellular ion levels such as [Ca2+ ]i or [Cl− ]i are involved in the regulation of cell motility or cellular volume. However, it has well not known about the spatial-temporal changes in these ion levels, especially [Cl− ]i during invasive migration. To assess it, we have attempted to visualize the cell movement with dynamic morphological alterations and changes in [Ca2+ ]i or [Cl− ]i simultaneously. Combination of time-lapse observation and Ca2+ imaging demonstrated that Ca2+ oscillations were observed in the glioblastoma cells which have the structures like leading process during cell movement. Furthermore, FRET imaging with Yellow-camleon and Clomeleon to calculate [Ca2+ ]i and [Cl− ]i levels, respectively, allowed us to detect the changes in FRET efficiencies by pharmacological applications. These results raise the possibility of analysis of the spatio-temporal characteristic of [Ca2+ ]i and [Cl− ]i by FRET imaging during long-time migration. Research funds: KAKENHI 18700313
P3-d22 Calcium dependent-regulation of neurite outgrowth mediated by a protein translational factor eEF2 within nerve growth cones Akira Iizuka 1 , Masumi Iketani 1 , Kumiko Sengoku 1 , Fumio Nakamura 1 , Masayuki Matsushita 2 , Angus Nairn 3 , Yoshio Goshima 1,4 , Kohtaro Takei 1,4 1 Yokohama City University Graduate School of Medicine, Department of Molecular Pharmacology & Neurobiology, Yokohama, Japan; 2 Mitsubishi Institute of Life Science, Tokyo, Japan; 3 Yale University School of Medicine, Department of Psychiatry, New Haven, USA; 4 CREST, JST, Kawaguchi, Japan
circuit formation?
The ephrin/Eph system plays a central role in neural-circuit formation; however, its downstream effectors are poorly understood. Here we show that a-chimerin Rac-GAP mediates ephrinB3/EphA4 forward signaling. We discovered a spontaneous mouse mutation, miffy (mfy), which results in a rabbit-like hopping gait, along with impaired corticospinal axon guidance and spinal central pattern generators. Using positional cloning, transgene rescue and gene targeting, we demonstrated that dysfunction of a-chimerin leads to mfy phenotypes similar to those of EphA4−/− and ephrinB3−/− mice. a-chimerin interacts with EphA4, and, in response to ephrinB3/EphA4 signaling, inactivates Rac, which is a positive regulator of process outgrowth. Moreover, down-regulation of a-chimerin suppresses ephrinB3-induced growth-cone collapse in cultured neurons. Our findings indicate that ephrinB3/EphA4 signaling prevents growth-cone extension in motor-circuit formation via a-chimerin-induced inactivation of Rac. Research funds: PRESTO, KAKENHI (18021041)
P3-d25 CRMP1 regulates spine maturation through mediating Semaphorin3A signaling in vivo Naoya Yamashita 1 , Asa Morita 1 , Yutaka Uchida 1 , Toshio Ohshima 2 , Fumio Nakamura 1 , Jerome Honnorat 3 , Pappachan Kolattukudy 4 , Yoshio Goshima 1 1 Graduate School of Medicine, Yokohama City University, Yokohama, Japan; 2 RIKEN BSI, Wako, Japan; 3 INSERM U433 Hopital Neurologique, Lyon, France; 4 Biomol. Sci., University of Central Florida, Florida, USA
Local protein synthesis within growth cones is known to regulate growth cone behavior. However, the mechanism of the regulation remains unclear. We found that eukaryotic elongation factor-2 (eEF2) in growth cones regulates neurite outgrowth in calcium-dependent manner. Acute localized loss of eEF2 in growth cones by chromophore-assisted laser inactivation (CALI) resulted in growth arrest of neurite. Extracellular ATP-evoked calcium transient activated EF2 kinase (EF2K) and increased eEF2 phosphorylation in growth cones, and also inhibited neurite outgrowth. This ATP-induced growth arrest of neurite was abolished by CALI of EF2K. The data suggest that local mRNA translation regulated by eEF2 in the growth cone may play an important event in the neurite outgrowth.
CRMP1 is one of the CRMP family members that mediate signal transduction of axon guidance molecules. We here show evidence that CRMP1 is involved in Sema3A-Cdk5 signaling to regulate spine maturation in the cerebral cortex. In the cultured cortical neurons from crmp1+/− mice, Sema3A increased the number of clusters of synapsin I and PSD-95, but this increase was attenuated in crmp1−/− mice. This phenotype seen in crmp1−/− mice was rescued by introduction of wild-type CRMP1 but not CRMP1-T509A/S522A, a mutant unphosphorylatable by Cdk5. Golgiimpregnation method showed that the crmp1−/− layer V cortical neurons showed lowered density of synaptic bouton-like structures and this phenotype had genetic interaction with sema3A. These findings suggest that CRMP1 regulates spine maturation through phosphorylation of CRMP1 by Sema3A-Cdk5 cascade.
Research fund: CREST, KAKENHI(13680851)
Research funds: JST CREST
P3-d23 Neuronal calcium sensor-1 in nerve growth cones independently regulates both the neurite outgrowth and growth cone morphology Masumi Iketani 1 , Chihiro Imaizumi 1 , Andreas Jeromin 2 , Fumio Nakamura 1 , Katsuhiko Mikoshiba 3,4 , Yoshio Goshima 1,5 , Kohtaro Takei 1,5 1 Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama, Japan; 2 Center for Learning & Memory, University of Texas, Austin, USA; 3 Institute of Medical Sciences, University of Tokyo, Tokyo, Japan; 4 Brain Science Institute, RIKEN, Wako, Japan; 5 CREST, JST, Kawaguchi, Japan Neuronal calcium sensor-1 (NCS-1) is a calcium binding protein, which is specifically expressed in the nervous system. NCS-1 was distributed in a cluster and co-localized with the type 1 IP3 receptor in the central domain of the growth cone. The acute localized loss of NCS-1 in the growth cone induced by chromophore-assisted laser inactivation (CALI) resulted in the growth arrest of neurites and lamellipodial and filopodial retractions. The CALI-induced growth arrest of neurites was abolished by the pharmacological inhibition of IP3 receptor, while CALI-induced lamellipodial and filopodial retractions were unaffected. These findings suggest that NCS-1 in growth cones may independently regulate both the neurite outgrowth and growth cone morphology. Research funds: CREST, JST
P3-d26 IgLON adhesion molecules participate in synapse formation of hippocampal neurons Takasi Hashimoto 1 , Shohei Maekawa 2 , Seiji Miyata 1 1 Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan; 2 Department of Bioscience, Graduate School of Science and Technology, Kobe University, Nada-ku, Kobe 657-8501, Japan IgLON cell adhesion molecules are known to belong to a subfamily of immunoglobulin superfamily and comprise four members: LAMP, OBCAM, neurotrimin, and kilon. Several in vitro studies showed that members of IgLON subgroup play a role in neurite outgrowth. However, although ultrastructural analysis demonstrated the localization of IgLON at synaptic region in the adult brain of rat, it has not been studied whether IgLONs are involved in the formation of synapses. In this study, we examined the overexpression of IgLONs on synapse formation of hippocampal neurons. Hippocampal neurons were transfected with a vector containing the GFP and each IgLON coding sequence. The quantitative analysis showed that overexpression of LAMP or OBCAM increased the number of synapses. In contrast, overexpression of kilon or neurotrimin surpressed the synapse formation. These results indicate that IgLON are the synaptic cell adhesion molecules concerning the regulation of synapse formation.