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A novel repulsive guidance protein, draxin
Abstracts P1-e16 APC2 plays an essential role in the axonal projection through the regulation of microtubule stability Takafumi Shintani 1,2 , Masaru Ihra 1 , Sachiko Tani 1,2 , Juichi Sakuraba 1,2 , Hiraki Sakuta 1,2 , Masaharu Noda 1,2 1 Div. Molecular Nuerobiology, National Institute for Basic Biology, Okazaki, Japan; 2 Sch Life Science, Graduate Univ. for Advanced Studies, Okazaki, Japan
Growth cones at the tips of growing axons are key cellular structures that detect guidance cues and mediate axonal growth. Recently, an increasing number of studies have suggested that dynamic microtubules in the growth cone play an essential role for growth cone stirring. Here, we examined the functional role of adenomatous polyposis coli (APC)-like molecule, APC2, in the development of topographic retinotectal projections. This molecule is distributed along microtubules in growth cones as well as axon shafts of retinal axons. Knockdown of APC2 in chick retina by specific shRNA expression reduces microtubule stability in retinal axons, and resultantly exhibits drastic abnormalities in retinotectal projection. These results suggest that APC2 plays a critical role in the establishment of nervous system through the regulation of microtubule stability. doi:10.1016/j.neures.2009.09.399
P1-e17 Cytoskeletal dynamics during neurite retraction induced by a protein phosphatase inhibitor, Calyculin A Ayumu Inutsuka, Makoto Goda, Yoshinori Fujiyoshi Department Biophys, Kyoto University, Kyoto, Japan Calyculin A (CL-A) is an inhibitor of protein phosphatase 1 (PP1) and PP2A. Previous studies have reported that CL-A induced rapid neurite retraction in developing neurons. Some of them attributed this phenomenon to microtubule (MT) depolymerization, while CL-A is also reported to induce actomyosin activation in various kind of cells. Here, we investigated the cytoskeletal dynamics during the CL-A-induced retraction in cultured hippocampal neurons. Our analyses with the LC-PolScope showed that cytoskeletons in neurites were pulled back, while keeping their polymerization state. We also observed bending of MTs during the retraction by fluorescent microscopy. These observations implicate additional mechanisms other than MT depolymerization. Indeed, the involvement of actomyosin was revealed: CL-A enhanced phosphorylation of myosin light chain (MLC), and furthermore, CL-A-induced neurite retraction was inhibited by myosin inhibitors or actin depolymerizers. Taken together, we suggest that the reorganization of MTs in CL-A-treated neurons is induced by a strong contractile force by actomyosin activation. doi:10.1016/j.neures.2009.09.400
P1-e18 Localization of tissue plasminogen activator and plasminogen in mouse brains Naoko Inoue, Seiji Miyata Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan Tissue plasminogen activator (tPA), a serine protease that activates plasminogen to plasmin, plays an important role in fibrinolysis. tPA is also abundantly expressed in brains and recent studies have indicated that tPA contributes to the synaptic plasticity and neuronal development. However, little attention has been given to the localization of tPA and plasminogen. In the present study, we demonstrated using the confocal microscopy that strong immunoreactivity of tPA was observed chiefly at dendrites of CA3 pyramidal neurons and axons of mossy fibers in the hippocampus and at somata of layers 2/3 in the cerebral cortex. The immunoreactivity of plasminogen was seen mainly at somata of parvalbumin-positive interneurons in the hippocampus and at somata of layer 2/3 and 5 in the cerebral cortex. These results demonstrate specific localization of tPA and plasminogen at a subpopulation of the hippocampus and cerebral cortex in mouse brains. doi:10.1016/j.neures.2009.09.401
P1-e19 Receptor type prtein tyrosine phosptasse control dendritogenesis in hippocampal neuorns Hitomi Asai 1 , Shota Yokoyama 1 , Nobuaki Maeda 2 , Seiji Miyata 1 1 2
Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan; Tokyo Metropolitan Institute of Neuroscience, Japan
Receptor-type protein tyrosine phosphatase (RPTP) has two isoforms, shorter receptor form of RPTP (sRPTP) and full-length receptor form of RPTP (fRPTP). Confocal microscopic observation showed that the immunoreactivity of RPTP was observed throughout cells such as axons, growth cones, and dendrites at the early stages of neuronal culture, while it was seen in dendrites at the late stages. The
S95
overexpression of sRPTP increased the total length of dendritic branches by facilitating the dendritic arborization, whereas that of fRPTP increased the average length of dendritic branches and vice versa decreased the dendritic arborization. The downregulation of RPTP expression using RNA interference decreased the total length of dendritic branches by attenuating the dendritic arborization and growth. Thus, the present study demonstrates that neuronal RPTP is responsible for the dendritogenesis and two RPTP isoforms have different functions. doi:10.1016/j.neures.2009.09.402
P1-e20 Roles of mDia, a Rho effector, in neural development Ryota Shinohara 1 , Hiroshi Kamijo 1 , Hiroyuki Hioki 2 , Takeshi Ishizaki 1 , Tomoyuki Furuyashiki 1 , Shuh Kaneko 2 , Toshimasa 1 Narumiya 1
Department Pharmacol., Kyoto University Grad. Sch. Med., Kyoto, Japan; Department Morphol. Brain Sci., Kyoto University Grad. Sch. Med., Kyoto, Japan 2
Rho GTPases are critical for neural development. However, Rho functions in this process remain poorly understood. mDia is a Rho effector critical for cytoskeletal reorganization, and two isoforms, mDia1 and mDia3, are highly expressed in embryonic CNS. To examine their roles, we generated mDia-deficient mice. Mice deficient in both isoforms (mDia-DKO), but not those lacking either, were partially lethal with growth retardation. Surviving mDia-DKO was impaired in left–right limb coordination during locomotion. Consistently, at the spinal cord of mDia-DKO, dorsal funiculi including corticospinal axons were reduced, and tracer experiments corroborate dysplasia of these axons. Further, axonal growth cones were many fewer in mDia-DKO neurons in vitro than wild-type neurons, whereas axonal elongation remains intact. These data suggest a critical role of mDia in axonal guidance, perhaps through maintaining axonal growth cone. doi:10.1016/j.neures.2009.09.403
P1-e21 Estimated connections that capture population burst-like activities Masaki Nomura 1 , Daisuke Ito 3 , Hiroki Tamate 3 , Kazutoshi Gohara 3 , Toshio Aoyagi 2 1
Grad. Sch. of Lett., Kyoto University, Japan; 2 Grad. Sch. of Informatics, Kyoto University, Japan; 3 Grad. Sch. of Eng., Hokkaido University, Japan
Rat cortical neurons were cultured, and the multi-dimensional activities from the culture were recorded using 8×8 multi-electrode arrays. As the neurons grew, they built rich synaptic connections, and burst-like population activities were observed in the culture. Using the data during burst-like population activities, the connectivity between electrodes was estimated by a dynamic Bayesian analysis. A connection matrix and intrinsic firing rates were obtained from estimation. Then, the binary neuronal network model was simulated with the estimated connection matrix and intrinsic firing rates. The model was found to capture the burst-like population activities. Furthermore, the effect of excitation and inhibition balance on burst-like population activities was explored. doi:10.1016/j.neures.2009.09.404
P1-e22 A novel repulsive guidance protein, draxin Yohei Shinmyo 1 , Md. Shahidul Islam 1 , Tatsuya Okafuji 1 , Yuhong Su 1,2 , Iftekhar Bin Naser 1,2 , Giasuddin Ahmed 1,2 , Sanbing Zhang 1,2 , Kunimasa Ohta 1 , Hideaki Tanaka 1,2 1
Department of Dev. Neurobiol., Kumamoto University, Japan; Kumamoto University, Japan
2
GCOE
The correct selection of axonal path toward to their target is known accomplished by extracellular guidance molecules. We have identified a novel secreted protein, draxin (dorsal repulsive axon guidance protein), which acts as a roof plate-derived chemorepellent for commissural axons in chick spinal cord. Here, we report that draxin is required for the midline crossing of forebrain commissures. draxin deficient mice showed severe defects in axonal projection of all three commissures, the corpus callosum, hippocampal commissure and anterior commissure, in the forebrain. draxin was expressed in the regions that surround the forebrain commissures, such as the midline glial cells. In addition, we showed that draxin repelled neurite outgrowth from cortical explants at E17, when corpus callosal axons are crossing the midline. Thus, we propose that draxin repulsion is essential for proper guidance of forebrain commissural axons, preventing them from misprojecting before reaching the midline. doi:10.1016/j.neures.2009.09.405
Growth cones at the tips of growing axons are key cellular structures that detect guidance cues and mediate axonal growth. Recently, an increasing number of studies have suggested that dynamic microtubules in the growth cone play an essential role for growth cone stirring. Here, we examined the functional role of adenomatous polyposis coli (APC)-like molecule, APC2, in the development of topographic retinotectal projections. This molecule is distributed along microtubules in growth cones as well as axon shafts of retinal axons. Knockdown of APC2 in chick retina by specific shRNA expression reduces microtubule stability in retinal axons, and resultantly exhibits drastic abnormalities in retinotectal projection. These results suggest that APC2 plays a critical role in the establishment of nervous system through the regulation of microtubule stability. doi:10.1016/j.neures.2009.09.399
P1-e17 Cytoskeletal dynamics during neurite retraction induced by a protein phosphatase inhibitor, Calyculin A Ayumu Inutsuka, Makoto Goda, Yoshinori Fujiyoshi Department Biophys, Kyoto University, Kyoto, Japan Calyculin A (CL-A) is an inhibitor of protein phosphatase 1 (PP1) and PP2A. Previous studies have reported that CL-A induced rapid neurite retraction in developing neurons. Some of them attributed this phenomenon to microtubule (MT) depolymerization, while CL-A is also reported to induce actomyosin activation in various kind of cells. Here, we investigated the cytoskeletal dynamics during the CL-A-induced retraction in cultured hippocampal neurons. Our analyses with the LC-PolScope showed that cytoskeletons in neurites were pulled back, while keeping their polymerization state. We also observed bending of MTs during the retraction by fluorescent microscopy. These observations implicate additional mechanisms other than MT depolymerization. Indeed, the involvement of actomyosin was revealed: CL-A enhanced phosphorylation of myosin light chain (MLC), and furthermore, CL-A-induced neurite retraction was inhibited by myosin inhibitors or actin depolymerizers. Taken together, we suggest that the reorganization of MTs in CL-A-treated neurons is induced by a strong contractile force by actomyosin activation. doi:10.1016/j.neures.2009.09.400
P1-e18 Localization of tissue plasminogen activator and plasminogen in mouse brains Naoko Inoue, Seiji Miyata Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan Tissue plasminogen activator (tPA), a serine protease that activates plasminogen to plasmin, plays an important role in fibrinolysis. tPA is also abundantly expressed in brains and recent studies have indicated that tPA contributes to the synaptic plasticity and neuronal development. However, little attention has been given to the localization of tPA and plasminogen. In the present study, we demonstrated using the confocal microscopy that strong immunoreactivity of tPA was observed chiefly at dendrites of CA3 pyramidal neurons and axons of mossy fibers in the hippocampus and at somata of layers 2/3 in the cerebral cortex. The immunoreactivity of plasminogen was seen mainly at somata of parvalbumin-positive interneurons in the hippocampus and at somata of layer 2/3 and 5 in the cerebral cortex. These results demonstrate specific localization of tPA and plasminogen at a subpopulation of the hippocampus and cerebral cortex in mouse brains. doi:10.1016/j.neures.2009.09.401
P1-e19 Receptor type prtein tyrosine phosptasse control dendritogenesis in hippocampal neuorns Hitomi Asai 1 , Shota Yokoyama 1 , Nobuaki Maeda 2 , Seiji Miyata 1 1 2
Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan; Tokyo Metropolitan Institute of Neuroscience, Japan
Receptor-type protein tyrosine phosphatase (RPTP) has two isoforms, shorter receptor form of RPTP (sRPTP) and full-length receptor form of RPTP (fRPTP). Confocal microscopic observation showed that the immunoreactivity of RPTP was observed throughout cells such as axons, growth cones, and dendrites at the early stages of neuronal culture, while it was seen in dendrites at the late stages. The
S95
overexpression of sRPTP increased the total length of dendritic branches by facilitating the dendritic arborization, whereas that of fRPTP increased the average length of dendritic branches and vice versa decreased the dendritic arborization. The downregulation of RPTP expression using RNA interference decreased the total length of dendritic branches by attenuating the dendritic arborization and growth. Thus, the present study demonstrates that neuronal RPTP is responsible for the dendritogenesis and two RPTP isoforms have different functions. doi:10.1016/j.neures.2009.09.402
P1-e20 Roles of mDia, a Rho effector, in neural development Ryota Shinohara 1 , Hiroshi Kamijo 1 , Hiroyuki Hioki 2 , Takeshi Ishizaki 1 , Tomoyuki Furuyashiki 1 , Shuh Kaneko 2 , Toshimasa 1 Narumiya 1
Department Pharmacol., Kyoto University Grad. Sch. Med., Kyoto, Japan; Department Morphol. Brain Sci., Kyoto University Grad. Sch. Med., Kyoto, Japan 2
Rho GTPases are critical for neural development. However, Rho functions in this process remain poorly understood. mDia is a Rho effector critical for cytoskeletal reorganization, and two isoforms, mDia1 and mDia3, are highly expressed in embryonic CNS. To examine their roles, we generated mDia-deficient mice. Mice deficient in both isoforms (mDia-DKO), but not those lacking either, were partially lethal with growth retardation. Surviving mDia-DKO was impaired in left–right limb coordination during locomotion. Consistently, at the spinal cord of mDia-DKO, dorsal funiculi including corticospinal axons were reduced, and tracer experiments corroborate dysplasia of these axons. Further, axonal growth cones were many fewer in mDia-DKO neurons in vitro than wild-type neurons, whereas axonal elongation remains intact. These data suggest a critical role of mDia in axonal guidance, perhaps through maintaining axonal growth cone. doi:10.1016/j.neures.2009.09.403
P1-e21 Estimated connections that capture population burst-like activities Masaki Nomura 1 , Daisuke Ito 3 , Hiroki Tamate 3 , Kazutoshi Gohara 3 , Toshio Aoyagi 2 1
Grad. Sch. of Lett., Kyoto University, Japan; 2 Grad. Sch. of Informatics, Kyoto University, Japan; 3 Grad. Sch. of Eng., Hokkaido University, Japan
Rat cortical neurons were cultured, and the multi-dimensional activities from the culture were recorded using 8×8 multi-electrode arrays. As the neurons grew, they built rich synaptic connections, and burst-like population activities were observed in the culture. Using the data during burst-like population activities, the connectivity between electrodes was estimated by a dynamic Bayesian analysis. A connection matrix and intrinsic firing rates were obtained from estimation. Then, the binary neuronal network model was simulated with the estimated connection matrix and intrinsic firing rates. The model was found to capture the burst-like population activities. Furthermore, the effect of excitation and inhibition balance on burst-like population activities was explored. doi:10.1016/j.neures.2009.09.404
P1-e22 A novel repulsive guidance protein, draxin Yohei Shinmyo 1 , Md. Shahidul Islam 1 , Tatsuya Okafuji 1 , Yuhong Su 1,2 , Iftekhar Bin Naser 1,2 , Giasuddin Ahmed 1,2 , Sanbing Zhang 1,2 , Kunimasa Ohta 1 , Hideaki Tanaka 1,2 1
Department of Dev. Neurobiol., Kumamoto University, Japan; Kumamoto University, Japan
2
GCOE
The correct selection of axonal path toward to their target is known accomplished by extracellular guidance molecules. We have identified a novel secreted protein, draxin (dorsal repulsive axon guidance protein), which acts as a roof plate-derived chemorepellent for commissural axons in chick spinal cord. Here, we report that draxin is required for the midline crossing of forebrain commissures. draxin deficient mice showed severe defects in axonal projection of all three commissures, the corpus callosum, hippocampal commissure and anterior commissure, in the forebrain. draxin was expressed in the regions that surround the forebrain commissures, such as the midline glial cells. In addition, we showed that draxin repelled neurite outgrowth from cortical explants at E17, when corpus callosal axons are crossing the midline. Thus, we propose that draxin repulsion is essential for proper guidance of forebrain commissural axons, preventing them from misprojecting before reaching the midline. doi:10.1016/j.neures.2009.09.405