神経化学教育口演セッション (Neurochemistry Educational Oral Sessions)

神経化学教育口演セッション4 Basal Ganglia
O3-8-6-1 遺伝子コードされた多色カルシウム指示タンパクを用いた、大脳新皮質における明確に区別される2種類の細胞内カルシウム動態とその生物学的意義の解明 Two patterns of Ca²⁺ dynamics associated with distinct cellular populations and behaviors in the neocortical ventricular zone revealed by multi-color imaging with genetically-encoded indicators ○佐合健¹, 松田知己², 永井健治², 宮田卓樹¹ ○Ken Sagou¹, Tomoki Matsuda², Takeharu Nagai², Takaki Miyata¹ 名古屋大学　医学系研究科　細胞生物学¹, 大阪大学　産業科学研究所　生体分子機能科学研究分野² Department of Anatomy and Cell Biology, Nagoya University Graduate School of Medicine¹, The Institute of Scientific and Industrial Research, Osaka University² (Background) Intracellular calcium ion (Ca²⁺) signaling is widely conserved and regulates a variety of cellular functions. Although previous studies reported intracellular Ca²⁺ fluctuation in the ventricular zone (VZ) of embryonic rodent neocortex, analysis at the single cell level to ask developmental meaning of Ca²⁺ fluctuation in VZ has not been done. Therefore, we employed genetically encoded Ca²⁺ indicators (GECIs) to examine the pattern of Ca²⁺ fluctuation in each VZ cell and asked its possible link to the cell's identity and behavior. (Materials and Methods) For Ca²⁺ imaging, green and red fluorescent GECIs were used: G-GECO localizes in cytoplasm, while R-GECO variant in nucleus. Each plasmid was transfected to precursor cells in VZ of mouse embryos by in utero electroporation, followed by slice preparation. Time-lapse images were obtained using confocal microscopy. For cell-type identification, we used the Fucci system, as well as cell-differentiation-sensitive promoters. (Results and discussion) Two patterns of intracellular Ca²⁺ fluctuation were observed in VZ. One pattern was high in frequency and low in magnitude (designated as "HFLM"), and the other was of low frequency and high magnitude ("LFHM"). The number of cells showing LFHM was smaller than that of cells exhibiting HFLM. LFHM occurred in cells that were identified to be in "either G1 or G0" by Fucci system but yet to drive promoters associated with the neuronal lineage. Directional intracellular transmission of Ca²⁺ influx was observed only for LFHM. Treatment with 2-APB or Xestospongin-c inhibited LFHM but not HFLM, and resulted in retarded cell migration to the intermediate zone, suggesting that LFHM regulated in an IP₃-dependent manner may underlie intracellular processes related to differentiation and/or migration. It is also noted that Ca²⁺ fluctuation in VZ is heterogeneously regulated in cell-type- or cell morphology-dependent manners under three-dimensional microenvironment.	O3-8-6-2 Necdinは脳室下帯における神経前駆細胞の増殖を制御する Necdin regulates neural progenitor cell proliferation in the subventricular zone ○橋長秀典¹, 長谷川孝一¹, 藤原一志郎¹, 吉川和明¹ ○Hidenori Hashinaga¹, Koichi Hasegawa¹, Kazushiro Fujiwara¹, Kazuaki Yoshikawa¹ 大阪大・蛋白質研・神経発生制御¹ Lab. of Regulation of Neuronal Development, Inst. for Protein Res., Osaka Univ.¹ In postnatal mammalian forebrain, the subventricular zone (SVZ) contains a number of neural stem cells (NSCs), which generate transit-amplifying cells that proliferate rapidly and differentiate into neuroblasts and neurons. Necdin, which is expressed predominantly in postmitotic neurons, promotes neuronal differentiation and inhibits apoptosis. Our earlier studies have indicated that necdin is expressed in non-neural stem cells residing in white adipose and hematopoietic tissues to suppress their proliferation. However, it remains unclear whether necdin regulates the proliferation of NSCs and transit-amplifying cells during the postnatal development. In this study, we examined whether necdin suppresses the proliferation of NSCs and neural progenitor cells residing in the SVZ of mice at postnatal day 4 and 28 (P4 and P28). Immunohistochemistry revealed that necdin is present in CD133-positive cells, Sox2-positive neural stem/progenitor cells and Dlx2-positive transit-amplifying cells/neuroblasts at P4. On the other hand, necdin was expressed in cells immunopositive for GFAP, CD133 and Dlx2 at P28. In vivo EdU incorporation assay revealed that necdin-null mice treated with EdU for 24 hours had a larger number of EdU-positive cells than wild- type mice at P4 and P28. We characterized the EdU-positive cells in the SVZ by double-staining for EdU and Sox2, Mash1, or Dlx2 at P4 and P28. We found that EdU-positive cell population was significantly increased in SVZ cells expressing high levels of Sox2 and Mash1 in necdin-deficient mice, suggesting that necdin suppresses the proliferation of transit-amplifying cells at P4 and P28. These results suggest that necdin controls the proliferation during the transition from NSCs to transit-amplifying cells in the SVZ during postnatal development.
O3-8-6-3 リーリンシグナルは受容体を介して神経細胞移動を制御する Reelin signaling is involved in the migratory process of cortical neurons in addition to their layer formation in developing cerebral cortex ○廣田ゆき¹, 久保健一郎¹, 仲嶋一範¹ ○Yuki Hirota¹, Ken-ichiro Kubo¹, Kazunori Nakajima¹ 慶應義塾大学医学部解剖学¹ Dept Anatomy, Keio Univ, Tokyo¹ During development of the mammalian neocortex, excitatory neurons are produced from both apical progenitors in the ventricular zone and basal progenitors in the subventricular zone. They then migrate towards the pial surface. In this process, earlier-born neurons form the deep layers, whereas later-born neurons migrate past the existing layers and form the more superficial layers, in a so-called "inside-out" pattern. This layer formation by neurons is regulated by several signaling cascades, including the Reelin signaling. Reelin is a glycoprotein mainly secreted by Cajal-Retzius neurons in the marginal zone, and known to function via its lipoprotein receptors, apolipoprotein E receptor2 (ApoER2) and very low density lipoprotein receptor (Vldlr), and the cytoplasmic adaptor protein disabled 1 (Dab1). Reeler mice lacking the Reelin protein, ApoER2/Vldlr double knockout mice and Dab1 mutants show an inverted formation of the cortical layers, indicating that Reelin signaling is essential for the correct formation of layered neocortex. However, precise mechanisms by which Reelin signaling controls the neuronal migration process remains unclear. To gain insight into how Reelin signaling controls individual migrating neurons, we performed knockdown experiments for ApoER2 and found that knockdown of ApoER2 resulted in an abnormal migratory pattern in the subventricular zone and the intermediate zone. These results suggest that ApoER2 functions are required for normal migration of neurons in a cell-autonomous manner.	O3-8-6-4 軸索ガイダンス因子FLRT2による視床大脳皮質路の形成制御 Repulsive guidance molecule FLRT2 regulates development of the thalamocortical projections ○山岸覚¹, 佐藤康二¹ ○Satoru Yamagishi¹, Koji Sato¹ 浜松医科大学解剖学講座神経機能学分野¹ Dept Anatomy, Hamamatsu University School of Medicine, Hamamatsu, Japan¹ Recently we reported fibronectin and leucine-rich transmembrane protein-2/3 (FLRT2/3) are novel axon guidance molecules that bind to Unc5s (Yamagishi et al., EMBO J, 2011). During development, FLRT2 and FLRT3 extra cellular domains (ECD) are shed by metalloproteinase from neurons and act as repulsive guidance molecules for axons and somata of Unc5B and Unc5D positive neurons. In the developing murine striatum, FLRT2 is expressed in a low lateral to high medial gradient. In addition, FLRT2 is not expressed at corridor region, where interneurons make a bridge to navigate the thalamic projections to the cortex. These expression patterns suggest that FLRT2 is working as a repellent for thalamic axons. Indeed, in some FLRT2 knockout mice, thalamocortical axons showed abnormal projection at E15.5 by DiI tracing. This result suggests that FLRT2 is involved in making proper thalamocortical projections.

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