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Met modulates cortical circuit development in vivo
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Symposium and Short Talk Abstracts 3 June 2008 / Int. J. Devl Neuroscience 26 (2008) 835–837
dynamics of competition. We suggest that three elements are essential for rapid and robust competition implicit within the neurotrophic factor hypothesis: target initiated sensitization, punishment, and protection from punishment. doi: 10.1016/j.ijdevneu.2008.09.036 [ST6] Met modulates cortical circuit development in vivo M.C. Judson *, M.Y. Bergman, K.L. Eagleson, P. Levitt Vanderbilt University, USA *Corresponding author. The Met receptor tyrosine kinase has been shown in vitro to regulate neurodevelopmental processes including cell migration, neurite outgrowth, and synaptogenesis, which serve to establish appropriate neuronal connectivity. In addition, a functional promoter variant of the human MET gene was recently discovered to be a significant risk factor for autism spectrum disorder (ASD). ASD and other neurodevelopmental disorders have been postulated to share in common disruptions in cortical connectivity. We thus examined Met signaling in the context of cortical circuit development in vivo. First, we developmentally mapped cortical Met expression in wild type mice using complementary immunoblotting and immunohistochemical approaches. We found that: (1) Met expression peaks between postnatal days 7 and 14, coinciding with principle periods of neurite outgrowth and synaptogenesis, and (2) during the peak period of expression, Met protein is localized to the dendrites and axons of projection neurons. We next used an Emx1cre/loxP approach to conditionally ablate Met signaling in all cells arising from the dorsal pallium, including cortical projection neurons. Quantitative morphometric analyses of pyramidal dendrites revealed an approximately 20% increase in dendritic protrusions in conditional null mice as compared to littermate controls, suggesting aberrant synaptic maturation in these mice. Consistent with this, immunoblotting of synapseenriched cortical fractions showed selective alterations in PSD-95 and phosphorylated CaMKIIT286. Collectively, these findings strongly implicate Met signaling in the development of cortical circuitry in vivo and may provide insight into the etiologies of ASD and other neurodevelopmental disorders. Supported by: Marino Autism Research Institute, NIMH Grant MH080759 and NICHD Grant HD15052 doi: 10.1016/j.ijdevneu.2008.09.037 Symposium session 6: Neural stem cells – during development and in the adult Session Chair: Hongjun Song The Chapel, 10.00-12.00 [SY6.0] Extrinsic and intrinsic mechanisms regulating neural stem cells and neurogenesis in the adult brain H. Song Johns Hopkins University School of Medicine, USA New neurons are continuously generated from adult neural stem/progenitor cells (NSCs) residing in the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus in all mammals examined, including humans. Outside
of these two neurogenic regions, proliferating NSCs rarely give rise to functional neurons under physiological conditions. During active adult neurogenesis, NSCs generate functional neurons through orchestrated steps, including cell proliferation, fate specification, neuronal migration, axonal and dendritic growth, and finally synaptic integration into the existing circuitry. As in other somatic stem cell systems, neurogenesis from NSCs in the two neurogenic regions of the adult brain is tightly regulated by the highly specialized microenvironment surrounding the NPCs. These ‘‘neurogenic niches’’ not only anatomically house adult NSCs, but also functionally control their development in vivo. Using multiple approaches for birth-dating, genetic marking and manipulation of proliferating NSCs and their progeny in the dentate gyrus of adult mice, we have characterized the sequential events of adult neurogenesis in vivo. Our studies have identified signaling molecules within the unique neurogenic niche of the dentate gyrus to either positively or negatively regulate various aspects of adult neurogenesis in an activity-dependent fashion. Furthermore, we have also identified essential intrinsic regulators of adult neurogenesis in vivo, such as Disrupted-in-Schizophrenia 1 (DISC1) and NDEL1. We hope a better understanding of cellular and molecular mechanisms regulating adult neural stem cells and neurogenesis may lead to novel strategies for functional neuronal replacement therapy after injury or degenerative neurological diseases. doi: 10.1016/j.ijdevneu.2008.09.038 [SY6.1] Modeling for neurological diseases using human ES cell-derived neurons B. Blanchi 1, J. Xu 2, H. Wu 1, Z. Pang 2, W. Ge 1, T.C. Su¨dhof 2, Y.E. Sun 1,* 1
University of California, Los Angeles, USA University of Texas, USA *Corresponding author. 2
Human embryonic stem cells (hESCs) are characterized as being capable of self-renewal and pluripotent. Up on differentiation, hESCs are prone to generating neurons, making them perhaps the only stable and genetically tractable source for production of large quantities of human neurons, which are invaluable materials for studying functions of genes in human neurons, especially those genes closely linked to neurological diseases. Here we report the establishment of a step-wise differentiation protocol that allows hESCs to be effectively converted into almost pure cultures of Nestin-Sox2 double positive human neural stem/progenitor cells (hNPCs), which in turn generate cultures of 70–80% pure human neurons (hNus) that are Map2 and TuJ1 positive. Importantly, these hNus, when co-cultured with astrocytes, form functional neural networks. Using this system we developed a human neuron-based Rett syndrome (RTT) model. RTT is a severe neurodevelopmental disorder caused by mutations in Mecp2, a transcriptional repressor that binds to methylated DNA. It is unclear how Mecp2 mutations lead to dysfunction of the nervous system, and no effective treatments for RTT are available. Using lenti-viral Mecp2-specific shRNA knock-down approaches, we have created Mecp2-deficient hNus either derived from hESC clones with stable lenti-viral integration sites, or from lenti-virally infected hNPCs with variable viral integration sites. We have discovered that knockdown of Mecp2 causes a dramatic shift of spontaneous synaptic responses from excitatory to inhibitory. This observation of increased synaptic GABAergic activity in Mecp2 knockdown hNus is in general consistent with that observed using
Symposium and Short Talk Abstracts 3 June 2008 / Int. J. Devl Neuroscience 26 (2008) 835–837
dynamics of competition. We suggest that three elements are essential for rapid and robust competition implicit within the neurotrophic factor hypothesis: target initiated sensitization, punishment, and protection from punishment. doi: 10.1016/j.ijdevneu.2008.09.036 [ST6] Met modulates cortical circuit development in vivo M.C. Judson *, M.Y. Bergman, K.L. Eagleson, P. Levitt Vanderbilt University, USA *Corresponding author. The Met receptor tyrosine kinase has been shown in vitro to regulate neurodevelopmental processes including cell migration, neurite outgrowth, and synaptogenesis, which serve to establish appropriate neuronal connectivity. In addition, a functional promoter variant of the human MET gene was recently discovered to be a significant risk factor for autism spectrum disorder (ASD). ASD and other neurodevelopmental disorders have been postulated to share in common disruptions in cortical connectivity. We thus examined Met signaling in the context of cortical circuit development in vivo. First, we developmentally mapped cortical Met expression in wild type mice using complementary immunoblotting and immunohistochemical approaches. We found that: (1) Met expression peaks between postnatal days 7 and 14, coinciding with principle periods of neurite outgrowth and synaptogenesis, and (2) during the peak period of expression, Met protein is localized to the dendrites and axons of projection neurons. We next used an Emx1cre/loxP approach to conditionally ablate Met signaling in all cells arising from the dorsal pallium, including cortical projection neurons. Quantitative morphometric analyses of pyramidal dendrites revealed an approximately 20% increase in dendritic protrusions in conditional null mice as compared to littermate controls, suggesting aberrant synaptic maturation in these mice. Consistent with this, immunoblotting of synapseenriched cortical fractions showed selective alterations in PSD-95 and phosphorylated CaMKIIT286. Collectively, these findings strongly implicate Met signaling in the development of cortical circuitry in vivo and may provide insight into the etiologies of ASD and other neurodevelopmental disorders. Supported by: Marino Autism Research Institute, NIMH Grant MH080759 and NICHD Grant HD15052 doi: 10.1016/j.ijdevneu.2008.09.037 Symposium session 6: Neural stem cells – during development and in the adult Session Chair: Hongjun Song The Chapel, 10.00-12.00 [SY6.0] Extrinsic and intrinsic mechanisms regulating neural stem cells and neurogenesis in the adult brain H. Song Johns Hopkins University School of Medicine, USA New neurons are continuously generated from adult neural stem/progenitor cells (NSCs) residing in the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus in all mammals examined, including humans. Outside
of these two neurogenic regions, proliferating NSCs rarely give rise to functional neurons under physiological conditions. During active adult neurogenesis, NSCs generate functional neurons through orchestrated steps, including cell proliferation, fate specification, neuronal migration, axonal and dendritic growth, and finally synaptic integration into the existing circuitry. As in other somatic stem cell systems, neurogenesis from NSCs in the two neurogenic regions of the adult brain is tightly regulated by the highly specialized microenvironment surrounding the NPCs. These ‘‘neurogenic niches’’ not only anatomically house adult NSCs, but also functionally control their development in vivo. Using multiple approaches for birth-dating, genetic marking and manipulation of proliferating NSCs and their progeny in the dentate gyrus of adult mice, we have characterized the sequential events of adult neurogenesis in vivo. Our studies have identified signaling molecules within the unique neurogenic niche of the dentate gyrus to either positively or negatively regulate various aspects of adult neurogenesis in an activity-dependent fashion. Furthermore, we have also identified essential intrinsic regulators of adult neurogenesis in vivo, such as Disrupted-in-Schizophrenia 1 (DISC1) and NDEL1. We hope a better understanding of cellular and molecular mechanisms regulating adult neural stem cells and neurogenesis may lead to novel strategies for functional neuronal replacement therapy after injury or degenerative neurological diseases. doi: 10.1016/j.ijdevneu.2008.09.038 [SY6.1] Modeling for neurological diseases using human ES cell-derived neurons B. Blanchi 1, J. Xu 2, H. Wu 1, Z. Pang 2, W. Ge 1, T.C. Su¨dhof 2, Y.E. Sun 1,* 1
University of California, Los Angeles, USA University of Texas, USA *Corresponding author. 2
Human embryonic stem cells (hESCs) are characterized as being capable of self-renewal and pluripotent. Up on differentiation, hESCs are prone to generating neurons, making them perhaps the only stable and genetically tractable source for production of large quantities of human neurons, which are invaluable materials for studying functions of genes in human neurons, especially those genes closely linked to neurological diseases. Here we report the establishment of a step-wise differentiation protocol that allows hESCs to be effectively converted into almost pure cultures of Nestin-Sox2 double positive human neural stem/progenitor cells (hNPCs), which in turn generate cultures of 70–80% pure human neurons (hNus) that are Map2 and TuJ1 positive. Importantly, these hNus, when co-cultured with astrocytes, form functional neural networks. Using this system we developed a human neuron-based Rett syndrome (RTT) model. RTT is a severe neurodevelopmental disorder caused by mutations in Mecp2, a transcriptional repressor that binds to methylated DNA. It is unclear how Mecp2 mutations lead to dysfunction of the nervous system, and no effective treatments for RTT are available. Using lenti-viral Mecp2-specific shRNA knock-down approaches, we have created Mecp2-deficient hNus either derived from hESC clones with stable lenti-viral integration sites, or from lenti-virally infected hNPCs with variable viral integration sites. We have discovered that knockdown of Mecp2 causes a dramatic shift of spontaneous synaptic responses from excitatory to inhibitory. This observation of increased synaptic GABAergic activity in Mecp2 knockdown hNus is in general consistent with that observed using