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Functional analysis of Nepro, a gene required for the maintenance of neocortex neural progenitor cells
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Abstracts / Neuroscience Research 71S (2011) e108–e415
to improve clinical symptom of Parkinsonism. Induction of fully functional neural network along with plasticity requires both appropriate differentiation stage of transplanted neural precursor cells and adequate environment of recipient.
P3-d12 Non-cell-autonomous control of the orientation of stem cell polarity and division
doi:10.1016/j.neures.2011.07.983
During the formation and maintenance of tissues, the directional division of stem cells is a major mechanism for the establishment of tissue polarity. Drosophila embryonic neural stem cells, neuroblasts, asymmetrically divide perpendicular to the overlying epithelial layer, budding off daughter ganglion mother cells and their descendant neurons on the opposite side, thereby determining the initial orientation of neural tissue growth. The formation of neuroblast polarity is known to be regulated by Par-complex in a cellautonomous manner. On the other hand, non-cell-autonomous mechanisms, dependent on a cue from the epithelial cells, are believed to regulate the relative orientation of neuroblast polarity, although the mechanisms underlying this process remain unknown. To identify this signaling from the epithelium, we have performed deficiency screens for genes encoding transmembrane proteins, and found that one mutation compromises the orientation of neuroblast polarity without affecting its formation, resulting in defective neural tissue growth. We will discuss about the mechanism of this cell-extrinsic orientation of neuroblast polarity, and as the Par-complex polarizes various types of cells, dictating asymmetric divisions, our finding may help to understand the universal mechanism for the control of tissue polarity, by orienting polarized stem cells and their divisions.
P3-d10 Studying nascent daughter cells neighborship to better understand the mechanism of cell fate determination in the neocortical and retinal neurogenesis Mayumi Okamoto 1 , Ken Sagou 1 , Toshihiko Fujimori 2 , Takaki Miyata 1 1
Dep. Anatomy and Cell Biol., Nagoya Univ. Grad. Sch. of Med 2 NIBB, Div. of Embryology Cell–cell interactions are very important for developmental events that construct the central nervous system. Previous studies done at the cellpopulation level have demonstrated that molecules mediating cell–cell interactions (e.g. Delta and Notch) contribute to the cell fate choices. However, we still cannot tell exactly when and where these interactions occur for nascent daughter cells born at the apical/ventricular surface of the neuroepithelium (NE) or ventricular zone (VZ). NE/VZ is heterogeneous with cells differing in differentiation state and cell cycle phase, allowing cells to “neighbor” (make contacts to) a variety of types of cells. It is possible that such “neighborship” dynamically changes for each cell, because cell movements are an active. To ask whether/how the history of neighborship for each daughter cell might affect its subsequent fate determination, we are comprehensively monitoring the composition of cells that surround a given nascent (<3 h-old) daughter cell. Confocal microscopy to obtain optical slices parallel to the apical/ventricular surface using brain/retinal primordia prepared from ROSA26 Lyn-venus mice allows us to observe outlines of all cells in the periventricular area. Our live observations suggest that daughter cells born almost simultaneously within a limited area can be different as to how (i.e. by which types of cells) they are surrounded. Now we are combining live markers for cell differentiation or cell cycle status to identify the spatiotemporal and cell-type parameters for neighbors that nascent daughter cells encounter, hoping to link the quality/history of such encounters with fate choices. doi:10.1016/j.neures.2011.07.984
P3-d11 Functional analysis of Nepro, a gene required for the maintenance of neocortex neural progenitor cells Tatsuya Sato , Yuko Muroyama, Tetsuichiro Saito Dept. of Dev. Biol., Grad. Sch. of Med., Chiba Univ., Chiba, Japan In the developing neocortex, projection neurons of the six cortical layers are generated from neural progenitor cells (NPCs). Maintenance of NPCs is essential for the generation of appropriate numbers of the various types of cortical neurons. Notch signaling is required for the maintenance of NPCs by inhibiting neuronal differentiation. The intracellular domain of Notch activates transcription of Hairy and Enhancer-of-split (Hes)-type genes, which encode basic helix–loop–helix (bHLH) transcription factors. It has been shown that Hes1 and Hes5 are required for cortical neurogenesis. However, it remains unclear whether other essential effectors are required for Notch signaling in the developing neocortex. We identified Nepro, a gene expressed in the developing mouse neocortex at early stages. Database analyses revealed that each vertebrate species appears to have a single Nepro homolog. No invertebrate homolog was found. We analyzed function of Nepro in the developing neocortex by in vivo electroporation. Misexpression of Nepro inhibited neuronal differentiation only in the early neocortex. Furthermore, knockdown of Nepro by its siRNA caused precocious differentiation of neurons. Misexpression of Nepro decreased mRNA levels of Ngn2 and Mash1, as did Hes and the constitutively active form of Notch (caNotch), suggesting that Nepro inhibits neuronal differentiation, presumably by repressing proneural genes. We then examined the regulation of Nepro expression by Notch signaling. Nepro was activated by caNotch but not by Hes. The combination of Nepro and Hes maintained NPCs even when Notch signaling was blocked. These results indicate that Nepro is necessary for the maintenance of NPCs in the early neocortex downstream of Notch. To further elucidate the function of Nepro in vivo and to detect Nepro-positive cells, we are currently making Nepro knock-in mice expressing Venus in place of Nepro. Research fund: KAKENHI 20240029. doi:10.1016/j.neures.2011.07.985
Shigeki Yoshiura , Nao Ohta, Fumio Matsuzaki RIKEN Center for Developmental Biology, Kobe, Japan
doi:10.1016/j.neures.2011.07.986
P3-d13 Neurotrophic factor-releasing human neural stem/progenitor cells rescue cultured rat septal neurons from amyloid -induced neurotoxicity Narisorn Kitiyanant 1,2 , Clive N. Svendsen 3 , Yindee Kitiyanant 2,4 , Wipawan Thangnipon 1
1 Research Center for Neuroscience, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand 2 Reproductive and Stem Cell Biology Research Group, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand 3 Cedars-Sinai Regenerative Medicine Institute, Los Angeles, CA, USA 4 Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand
A well-known feature of Alzheimer’s disease (AD) is the reduction in choline acetyltransferase (ChAT), an enzyme for acetylcholine synthesis, activity of the basal forebrain cholinergic neurons resulting from deposition of amyloid plaques. Amyloid plaques deposition plays a major role in inducing and regulating reactive oxygen species (ROS) production. Recently, many evidences have suggested that trophic factor systems in AD brain are altered, resulting in ROS production and subsequent neuronal cell death from oxidative stress. Many growth factors including brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF)-1 and glial cell-derived neurotrophic factor (GDNF) are known to protect neuronal cell death in several neurodegenerative models. Therefore, transplantation of those neurotrophic factor-releasing human neural stem/progenitor cells might exert rescuing effects in AD model. In this study, cultured neurons prepared from the septal nucleus of embryonic day 16–17 (E16–17) rat brain were treated with amyloid (1–42) (A1–42 ) at the various forms and different concentrations. To determine neurotoxicity of A1–42 , cultured septal neuron survival was measured using MTT assay. The cultured septal neurons were co-cultured with genetically modified human neural stem/progenitor cells (gmhNPC) to secrete BDNF, IGF-1 and GDNF in tissue culture inserts following exposure to A1–42 . Our results showed that gmhNPC co-cultures significantly rescued cultured septal neurons as well as increase ChAT expression from A1–42 induced neurotoxicity, suggesting that the gmhNPC may be useful for AD treatment. Research fund: Commission on Higher Education, Ministry of Education, Thailand. doi:10.1016/j.neures.2011.07.987
P3-d14 Differential composition of lamin subtypes in the glial cells of adult rat brain Yasuharu Takamori , Tetsuji Mori, Taketoshi Wakabayashi, Yukie Hirahara, Taro Koike, Hisao Yamada Department of Anatomy and Cell Science, Kansai Medical University, Osaka, Japan Lamins are intermediate-filament proteins that form a nuclear lamina, a filamentous meshwork underlying the nuclear envelope. Three lamin subtypes,
Abstracts / Neuroscience Research 71S (2011) e108–e415
to improve clinical symptom of Parkinsonism. Induction of fully functional neural network along with plasticity requires both appropriate differentiation stage of transplanted neural precursor cells and adequate environment of recipient.
P3-d12 Non-cell-autonomous control of the orientation of stem cell polarity and division
doi:10.1016/j.neures.2011.07.983
During the formation and maintenance of tissues, the directional division of stem cells is a major mechanism for the establishment of tissue polarity. Drosophila embryonic neural stem cells, neuroblasts, asymmetrically divide perpendicular to the overlying epithelial layer, budding off daughter ganglion mother cells and their descendant neurons on the opposite side, thereby determining the initial orientation of neural tissue growth. The formation of neuroblast polarity is known to be regulated by Par-complex in a cellautonomous manner. On the other hand, non-cell-autonomous mechanisms, dependent on a cue from the epithelial cells, are believed to regulate the relative orientation of neuroblast polarity, although the mechanisms underlying this process remain unknown. To identify this signaling from the epithelium, we have performed deficiency screens for genes encoding transmembrane proteins, and found that one mutation compromises the orientation of neuroblast polarity without affecting its formation, resulting in defective neural tissue growth. We will discuss about the mechanism of this cell-extrinsic orientation of neuroblast polarity, and as the Par-complex polarizes various types of cells, dictating asymmetric divisions, our finding may help to understand the universal mechanism for the control of tissue polarity, by orienting polarized stem cells and their divisions.
P3-d10 Studying nascent daughter cells neighborship to better understand the mechanism of cell fate determination in the neocortical and retinal neurogenesis Mayumi Okamoto 1 , Ken Sagou 1 , Toshihiko Fujimori 2 , Takaki Miyata 1 1
Dep. Anatomy and Cell Biol., Nagoya Univ. Grad. Sch. of Med 2 NIBB, Div. of Embryology Cell–cell interactions are very important for developmental events that construct the central nervous system. Previous studies done at the cellpopulation level have demonstrated that molecules mediating cell–cell interactions (e.g. Delta and Notch) contribute to the cell fate choices. However, we still cannot tell exactly when and where these interactions occur for nascent daughter cells born at the apical/ventricular surface of the neuroepithelium (NE) or ventricular zone (VZ). NE/VZ is heterogeneous with cells differing in differentiation state and cell cycle phase, allowing cells to “neighbor” (make contacts to) a variety of types of cells. It is possible that such “neighborship” dynamically changes for each cell, because cell movements are an active. To ask whether/how the history of neighborship for each daughter cell might affect its subsequent fate determination, we are comprehensively monitoring the composition of cells that surround a given nascent (<3 h-old) daughter cell. Confocal microscopy to obtain optical slices parallel to the apical/ventricular surface using brain/retinal primordia prepared from ROSA26 Lyn-venus mice allows us to observe outlines of all cells in the periventricular area. Our live observations suggest that daughter cells born almost simultaneously within a limited area can be different as to how (i.e. by which types of cells) they are surrounded. Now we are combining live markers for cell differentiation or cell cycle status to identify the spatiotemporal and cell-type parameters for neighbors that nascent daughter cells encounter, hoping to link the quality/history of such encounters with fate choices. doi:10.1016/j.neures.2011.07.984
P3-d11 Functional analysis of Nepro, a gene required for the maintenance of neocortex neural progenitor cells Tatsuya Sato , Yuko Muroyama, Tetsuichiro Saito Dept. of Dev. Biol., Grad. Sch. of Med., Chiba Univ., Chiba, Japan In the developing neocortex, projection neurons of the six cortical layers are generated from neural progenitor cells (NPCs). Maintenance of NPCs is essential for the generation of appropriate numbers of the various types of cortical neurons. Notch signaling is required for the maintenance of NPCs by inhibiting neuronal differentiation. The intracellular domain of Notch activates transcription of Hairy and Enhancer-of-split (Hes)-type genes, which encode basic helix–loop–helix (bHLH) transcription factors. It has been shown that Hes1 and Hes5 are required for cortical neurogenesis. However, it remains unclear whether other essential effectors are required for Notch signaling in the developing neocortex. We identified Nepro, a gene expressed in the developing mouse neocortex at early stages. Database analyses revealed that each vertebrate species appears to have a single Nepro homolog. No invertebrate homolog was found. We analyzed function of Nepro in the developing neocortex by in vivo electroporation. Misexpression of Nepro inhibited neuronal differentiation only in the early neocortex. Furthermore, knockdown of Nepro by its siRNA caused precocious differentiation of neurons. Misexpression of Nepro decreased mRNA levels of Ngn2 and Mash1, as did Hes and the constitutively active form of Notch (caNotch), suggesting that Nepro inhibits neuronal differentiation, presumably by repressing proneural genes. We then examined the regulation of Nepro expression by Notch signaling. Nepro was activated by caNotch but not by Hes. The combination of Nepro and Hes maintained NPCs even when Notch signaling was blocked. These results indicate that Nepro is necessary for the maintenance of NPCs in the early neocortex downstream of Notch. To further elucidate the function of Nepro in vivo and to detect Nepro-positive cells, we are currently making Nepro knock-in mice expressing Venus in place of Nepro. Research fund: KAKENHI 20240029. doi:10.1016/j.neures.2011.07.985
Shigeki Yoshiura , Nao Ohta, Fumio Matsuzaki RIKEN Center for Developmental Biology, Kobe, Japan
doi:10.1016/j.neures.2011.07.986
P3-d13 Neurotrophic factor-releasing human neural stem/progenitor cells rescue cultured rat septal neurons from amyloid -induced neurotoxicity Narisorn Kitiyanant 1,2 , Clive N. Svendsen 3 , Yindee Kitiyanant 2,4 , Wipawan Thangnipon 1
1 Research Center for Neuroscience, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand 2 Reproductive and Stem Cell Biology Research Group, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand 3 Cedars-Sinai Regenerative Medicine Institute, Los Angeles, CA, USA 4 Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand
A well-known feature of Alzheimer’s disease (AD) is the reduction in choline acetyltransferase (ChAT), an enzyme for acetylcholine synthesis, activity of the basal forebrain cholinergic neurons resulting from deposition of amyloid plaques. Amyloid plaques deposition plays a major role in inducing and regulating reactive oxygen species (ROS) production. Recently, many evidences have suggested that trophic factor systems in AD brain are altered, resulting in ROS production and subsequent neuronal cell death from oxidative stress. Many growth factors including brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF)-1 and glial cell-derived neurotrophic factor (GDNF) are known to protect neuronal cell death in several neurodegenerative models. Therefore, transplantation of those neurotrophic factor-releasing human neural stem/progenitor cells might exert rescuing effects in AD model. In this study, cultured neurons prepared from the septal nucleus of embryonic day 16–17 (E16–17) rat brain were treated with amyloid (1–42) (A1–42 ) at the various forms and different concentrations. To determine neurotoxicity of A1–42 , cultured septal neuron survival was measured using MTT assay. The cultured septal neurons were co-cultured with genetically modified human neural stem/progenitor cells (gmhNPC) to secrete BDNF, IGF-1 and GDNF in tissue culture inserts following exposure to A1–42 . Our results showed that gmhNPC co-cultures significantly rescued cultured septal neurons as well as increase ChAT expression from A1–42 induced neurotoxicity, suggesting that the gmhNPC may be useful for AD treatment. Research fund: Commission on Higher Education, Ministry of Education, Thailand. doi:10.1016/j.neures.2011.07.987
P3-d14 Differential composition of lamin subtypes in the glial cells of adult rat brain Yasuharu Takamori , Tetsuji Mori, Taketoshi Wakabayashi, Yukie Hirahara, Taro Koike, Hisao Yamada Department of Anatomy and Cell Science, Kansai Medical University, Osaka, Japan Lamins are intermediate-filament proteins that form a nuclear lamina, a filamentous meshwork underlying the nuclear envelope. Three lamin subtypes,