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Chondroitin sulfate proteoglycans dephosphorylate TrkB and repress the synaptic plasticity
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Abstracts / Neuroscience Research 71S (2011) e108–e415
ber of extracellular axon guidance factors, their receptors and intracellular signaling molecules involved in controlling axon morphology, the signaling pathways in axon development remain to be elucidated. We have previously reported that R-Ras, a member of Ras family GTPases, plays an important role in axon specification and guidance. However, direct effectors of R-Ras in neurons have not been identified except PI3K. In this study, we report that lAfadin, an actin filament-binding protein having Ras association (RA) domain and PDZ domain, functions as an effector of R-Ras and regulates axon branching downstream of R-Ras in cultured hippocampal neurons. Pull-down and immunoprecipitaion assays showed that l-Afadin bound to active R-Ras. In Neuro-2a cells, constitutively active R-Ras recruited l-Afadin to the plasma membrane in a RA domain-dependent manner. In cultured neurons, overexpression of l-Afadin promoted axon branching, while knockdown of l-Afadin suppressed the branching activity. Overexpression of constitutively active R-Ras increased axon arborization and it was partially repressed by knockdown of l-Afadin. These results suggest that activated R-Ras induces axon branching in part by recruiting l-Afadin to the plasma membrane. doi:10.1016/j.neures.2011.07.1469
P4-e20 Chondroitin sulfate proteoglycans dephosphorylate TrkB and repress the synaptic plasticity Dai Kurihara , Toshihide Yamashita Dept. Mol. Neurosci., Grad. Sch. of Med., Osaka Univ., Osaka Chondroitin sulfate proteoglycans (CSPGs) are major components of extracellular matrix (ECM) in the central nervous system. CSPGs in the ECM do not play only structural role, but are involved in the control of plasticity. It has been shown that closure of the critical period in the visual cortex is partly due to the inhibitory effect of CSPGs. Indeed, digestion of CSPGs using chondroitinase ABC increases the spine dynamics after the end of the critical period. It has also been shown that CSPGs play a role in long-term potentiation (LTP). However, the molecular mechanisms remained poorly understood. Brain-derived neurotrophic factor (BDNF) regulates synaptic plasticity via its receptor, tropomyosin-related kinase B (TrkB). BDNF increases the density of dendritic filopodia and spine, and induces LTP. In this work, we show that CSPGs treatment decreases tyrosine phosphorylation of TrkB, leading to the decrease of the dendritic filopodia and spine density. We also found that CSPGs receptor, protein tyrosine phosphatase (PTP) binds to TrkB and dephosphorylates it. PTP is necessary for decrease of the dendritic filopodia and spine density induced by CSPGs. We conclude that TrkB signaling is involved in the repression of synapse plasticity mediated by CSPGs. doi:10.1016/j.neures.2011.07.1470
P4-e21 The Rac activator Dock4 regulates dendritic spine formation in hippocampal neurons Shuhei Ueda , Manabu Negishi, Hironori Katoh Lab. of Mol. Neurobiol., Grad. Sch. of Biostudies, Kyoto Univ., Kyoto, Japan The Rho family small GTPases are critical regulators of the actin cytoskeletal reorganization, and thereby contribute to multiple cellular functions. Rac is a member of the Rho family and has been implicated in various aspects of neuronal development including axonal growth and guidance, dendritic maturation and spine formation. Here we show that the Rac specific activator Dock4, a member of the Dock180-related protein family, is highly expressed at late developmental stages in hippocampal neurons and localizes at dendritic spines. In primary cultured rat hippocampal neurons, shRNA-mediated knockdown of endogenous Dock4 significantly reduced dendritic spine density, which was rescued by coexpressing shRNA-resistant Dock4. On the other hand, coexpression of a Dock4 mutant lacking C-terminal prolinerich region (Dock4-C) failed to rescue the defect in spine formation by Dock4 knockdown. Moreover, Dock4-C mutant failed to localize to dendritic spines. These results suggest that Dock4 is necessary for normal spine formation, and that the C-terminal proline-rich region is important for its function. We next screened Dock4 binding proteins by yeast two-hybrid system using the C-terminal region of Dock4 as bait, and identified Cortactin, an actin cytoskeletal regulatory protein, as a novel binding partner. Indeed, Dock4 formed a complex with Cortactin in hippocampal neurons. Thus, our results suggest a new function of the Rac1 activator Dock4 in dendritic spine formation in hippocampal neurons. Research fund: KAKENHI 21770202. doi:10.1016/j.neures.2011.07.1471
P4-f01 Analysis for gene expression program governing specific neural network formation Kenichiro Kuwako , Hideyuki Okano Dep. of Physiol., Keio Sch. of Med., Tokyo, Japan During neural development, axons keep growing toward their final destinations without forming ectopic synapses on their routes. Once axons arrive at their target region, they rapidly quit growing and start to form synapses with target cells, suggesting that totally opposite events will smoothly take place in neurons at the “transition” from axon growth phase to synaptogenesis phase. In hindbrain pontine neurons (PN) whose axons are main afferent networks conveying information to the cerebellar cortex, transcriptional down-regulation of cytoskeletal molecules involved in axon growth and up-regulation of synaptic molecules involved in synaptogenesis occur quite synchronously and rapidly at the transition phase (Diaz et al., Neuron 2002). This suggests that there might be a gene expression program which comprehensively regulates the neuronal expression profiles for prompt transition. In this study, we screened a set of master transcription factors that activates gene expression program governing the transition in PN. DiI-tracing experiments revealed that PN axons arrive at the inner granule layer as early as at postnatal day 4 (P4). From around P10, glomerular structures at the tip of PN axons were observed, suggesting that PN axons started to form synapses with granule cells. Axonal outgrowth of E18.5 PN was strongly inhibited if they were co-cultured with P5 granule cells. Thus, we tested a possibility that target-derived signal activates a gene expression program which is responsible for the transition from axon growth phase to synaptogenesis phase. By DNA microarray analysis, we picked up several transcription factors that were specifically up/down-regulated in PN co-cultured with granule cells. According to the in situ hybridization database, some of them are also significantly up/down-regulated in the pontine nucleus in vivo around the transition phase (P4–P14). We will report additional data of functional analysis for those candidate transcription factors. doi:10.1016/j.neures.2011.07.1472
P4-f02 Single-cell level multi-layered substructures of neocortical layer V Rumi Kurokawa Hosoya
, Hisato Maruoka, Shun Tsuruno, Toshihiko
Hosoya Lab., BSI, RIKEN, Wako, Japan The mammalian neocortex is composed of a large number of neuronal types. We have found that in neocortical layer V specific neuronal types make non-random three-dimensional microorganization. In the radial orientation, pyramidal neurons in layer V have multilayered organization. Pyramidal neurons that have different axonal projection or gene expression make thin sublayers, and multiple sublayers are stacked in layer V. Approximately 5 sublayers can be identified in layer V, and they are often as thin as one to two cells in thickness. These results suggest that layer V is composed of multiple sublayers of functionally different neuronal types, and that this multi-layered structure is built with single-cell precision. Quantitative analysis of gene expression and detailed examination of multiple cell types will be discussed. Research fund: KAKENHI 22115004. doi:10.1016/j.neures.2011.07.1473
P4-f03 Molecular analyses of the development of the spinal nerve in the chick embryo Tomoyuki Masuda 1 , Chie Sakuma 1 , Takayuki Ueno 1 , Yuriko Yamada 1 , Masahiko Taniguchi 2 , Toshiyuki Yamagishi 3 , Takahiro Nagase 4 , Kazuto Kobayashi 5 , Hiroyuki Yaginuma 1 1
Dept of Anat, Fukushima Med Univ Scl Med, Fukushima, Japan 2 Dept of Mol Med Sci, Res Inst Frotier Med, Sapporo Med Univ, Sapporo, Japan 3 Dept of Anat, Grad Sch Med, Osaka City Univ, Osaka, Japan 4 Dept of Human Genome Res, Kazusa DNA Res Inst, Chiba, Japan 5 Dept of Mol Gen, Inst Biomed Sci, Fukushima Med Univ, Fukushima, Japan The neuronal network of the spinal nerve that is composed of dorsal root ganglion (DRG) axons and spinal motor axons is formed during development. Previously, we revealed that several diffusible molecules are involved in the formation of the trajectories of DRG axons. When we perturbed the function of these molecules, the abnormal projections of DRG axons were observed in the mouse or chick embryo. But, the abnormalities that occurred at early stages were corrected later during development. These phenomena suggest the redundancies of axonal guidance molecules to make the precise axonal projection. Using the Kazusa cDNA microarray and its database, now we are
Abstracts / Neuroscience Research 71S (2011) e108–e415
ber of extracellular axon guidance factors, their receptors and intracellular signaling molecules involved in controlling axon morphology, the signaling pathways in axon development remain to be elucidated. We have previously reported that R-Ras, a member of Ras family GTPases, plays an important role in axon specification and guidance. However, direct effectors of R-Ras in neurons have not been identified except PI3K. In this study, we report that lAfadin, an actin filament-binding protein having Ras association (RA) domain and PDZ domain, functions as an effector of R-Ras and regulates axon branching downstream of R-Ras in cultured hippocampal neurons. Pull-down and immunoprecipitaion assays showed that l-Afadin bound to active R-Ras. In Neuro-2a cells, constitutively active R-Ras recruited l-Afadin to the plasma membrane in a RA domain-dependent manner. In cultured neurons, overexpression of l-Afadin promoted axon branching, while knockdown of l-Afadin suppressed the branching activity. Overexpression of constitutively active R-Ras increased axon arborization and it was partially repressed by knockdown of l-Afadin. These results suggest that activated R-Ras induces axon branching in part by recruiting l-Afadin to the plasma membrane. doi:10.1016/j.neures.2011.07.1469
P4-e20 Chondroitin sulfate proteoglycans dephosphorylate TrkB and repress the synaptic plasticity Dai Kurihara , Toshihide Yamashita Dept. Mol. Neurosci., Grad. Sch. of Med., Osaka Univ., Osaka Chondroitin sulfate proteoglycans (CSPGs) are major components of extracellular matrix (ECM) in the central nervous system. CSPGs in the ECM do not play only structural role, but are involved in the control of plasticity. It has been shown that closure of the critical period in the visual cortex is partly due to the inhibitory effect of CSPGs. Indeed, digestion of CSPGs using chondroitinase ABC increases the spine dynamics after the end of the critical period. It has also been shown that CSPGs play a role in long-term potentiation (LTP). However, the molecular mechanisms remained poorly understood. Brain-derived neurotrophic factor (BDNF) regulates synaptic plasticity via its receptor, tropomyosin-related kinase B (TrkB). BDNF increases the density of dendritic filopodia and spine, and induces LTP. In this work, we show that CSPGs treatment decreases tyrosine phosphorylation of TrkB, leading to the decrease of the dendritic filopodia and spine density. We also found that CSPGs receptor, protein tyrosine phosphatase (PTP) binds to TrkB and dephosphorylates it. PTP is necessary for decrease of the dendritic filopodia and spine density induced by CSPGs. We conclude that TrkB signaling is involved in the repression of synapse plasticity mediated by CSPGs. doi:10.1016/j.neures.2011.07.1470
P4-e21 The Rac activator Dock4 regulates dendritic spine formation in hippocampal neurons Shuhei Ueda , Manabu Negishi, Hironori Katoh Lab. of Mol. Neurobiol., Grad. Sch. of Biostudies, Kyoto Univ., Kyoto, Japan The Rho family small GTPases are critical regulators of the actin cytoskeletal reorganization, and thereby contribute to multiple cellular functions. Rac is a member of the Rho family and has been implicated in various aspects of neuronal development including axonal growth and guidance, dendritic maturation and spine formation. Here we show that the Rac specific activator Dock4, a member of the Dock180-related protein family, is highly expressed at late developmental stages in hippocampal neurons and localizes at dendritic spines. In primary cultured rat hippocampal neurons, shRNA-mediated knockdown of endogenous Dock4 significantly reduced dendritic spine density, which was rescued by coexpressing shRNA-resistant Dock4. On the other hand, coexpression of a Dock4 mutant lacking C-terminal prolinerich region (Dock4-C) failed to rescue the defect in spine formation by Dock4 knockdown. Moreover, Dock4-C mutant failed to localize to dendritic spines. These results suggest that Dock4 is necessary for normal spine formation, and that the C-terminal proline-rich region is important for its function. We next screened Dock4 binding proteins by yeast two-hybrid system using the C-terminal region of Dock4 as bait, and identified Cortactin, an actin cytoskeletal regulatory protein, as a novel binding partner. Indeed, Dock4 formed a complex with Cortactin in hippocampal neurons. Thus, our results suggest a new function of the Rac1 activator Dock4 in dendritic spine formation in hippocampal neurons. Research fund: KAKENHI 21770202. doi:10.1016/j.neures.2011.07.1471
P4-f01 Analysis for gene expression program governing specific neural network formation Kenichiro Kuwako , Hideyuki Okano Dep. of Physiol., Keio Sch. of Med., Tokyo, Japan During neural development, axons keep growing toward their final destinations without forming ectopic synapses on their routes. Once axons arrive at their target region, they rapidly quit growing and start to form synapses with target cells, suggesting that totally opposite events will smoothly take place in neurons at the “transition” from axon growth phase to synaptogenesis phase. In hindbrain pontine neurons (PN) whose axons are main afferent networks conveying information to the cerebellar cortex, transcriptional down-regulation of cytoskeletal molecules involved in axon growth and up-regulation of synaptic molecules involved in synaptogenesis occur quite synchronously and rapidly at the transition phase (Diaz et al., Neuron 2002). This suggests that there might be a gene expression program which comprehensively regulates the neuronal expression profiles for prompt transition. In this study, we screened a set of master transcription factors that activates gene expression program governing the transition in PN. DiI-tracing experiments revealed that PN axons arrive at the inner granule layer as early as at postnatal day 4 (P4). From around P10, glomerular structures at the tip of PN axons were observed, suggesting that PN axons started to form synapses with granule cells. Axonal outgrowth of E18.5 PN was strongly inhibited if they were co-cultured with P5 granule cells. Thus, we tested a possibility that target-derived signal activates a gene expression program which is responsible for the transition from axon growth phase to synaptogenesis phase. By DNA microarray analysis, we picked up several transcription factors that were specifically up/down-regulated in PN co-cultured with granule cells. According to the in situ hybridization database, some of them are also significantly up/down-regulated in the pontine nucleus in vivo around the transition phase (P4–P14). We will report additional data of functional analysis for those candidate transcription factors. doi:10.1016/j.neures.2011.07.1472
P4-f02 Single-cell level multi-layered substructures of neocortical layer V Rumi Kurokawa Hosoya
, Hisato Maruoka, Shun Tsuruno, Toshihiko
Hosoya Lab., BSI, RIKEN, Wako, Japan The mammalian neocortex is composed of a large number of neuronal types. We have found that in neocortical layer V specific neuronal types make non-random three-dimensional microorganization. In the radial orientation, pyramidal neurons in layer V have multilayered organization. Pyramidal neurons that have different axonal projection or gene expression make thin sublayers, and multiple sublayers are stacked in layer V. Approximately 5 sublayers can be identified in layer V, and they are often as thin as one to two cells in thickness. These results suggest that layer V is composed of multiple sublayers of functionally different neuronal types, and that this multi-layered structure is built with single-cell precision. Quantitative analysis of gene expression and detailed examination of multiple cell types will be discussed. Research fund: KAKENHI 22115004. doi:10.1016/j.neures.2011.07.1473
P4-f03 Molecular analyses of the development of the spinal nerve in the chick embryo Tomoyuki Masuda 1 , Chie Sakuma 1 , Takayuki Ueno 1 , Yuriko Yamada 1 , Masahiko Taniguchi 2 , Toshiyuki Yamagishi 3 , Takahiro Nagase 4 , Kazuto Kobayashi 5 , Hiroyuki Yaginuma 1 1
Dept of Anat, Fukushima Med Univ Scl Med, Fukushima, Japan 2 Dept of Mol Med Sci, Res Inst Frotier Med, Sapporo Med Univ, Sapporo, Japan 3 Dept of Anat, Grad Sch Med, Osaka City Univ, Osaka, Japan 4 Dept of Human Genome Res, Kazusa DNA Res Inst, Chiba, Japan 5 Dept of Mol Gen, Inst Biomed Sci, Fukushima Med Univ, Fukushima, Japan The neuronal network of the spinal nerve that is composed of dorsal root ganglion (DRG) axons and spinal motor axons is formed during development. Previously, we revealed that several diffusible molecules are involved in the formation of the trajectories of DRG axons. When we perturbed the function of these molecules, the abnormal projections of DRG axons were observed in the mouse or chick embryo. But, the abnormalities that occurred at early stages were corrected later during development. These phenomena suggest the redundancies of axonal guidance molecules to make the precise axonal projection. Using the Kazusa cDNA microarray and its database, now we are