Akama Kuniko, Horikoshi Tomoe, Nakayama Takashi, Otsu Masahiro, Imaizumi Noriaki, Nakamura Megumi, Toda Tosifusa, Inuma Michiko, Hirano Hisashi, Kondo Yasushi, Suzuki Yutaka, Inoue Nobuo
Department of Chemistry, Graduate School of Science, Chiba University, Chiba 263-8522, Japan.
Biochim Biophys Acta. 2013 Feb;1834(2):601-10. doi: 10.1016/j.bbapap.2012.12.002. Epub 2012 Dec 8.
Understanding astrocytogenesis is valuable for the treatment of nervous system disorders, as astrocytes provide structural, metabolic and defense support to neurons, and regulate neurons actively. However, there is limited information about the molecular events associated with the differentiation from primate ES cells to astrocytes. We therefore investigated the differentially expressed proteins in early astrocytogenesis, from cynomolgus monkey ES cells (CMK6 cell line) into astrocyte progenitor (AstP) cells via the formation of primitive neural stem spheres (Day 4), mature neural stem spheres (NSS), and neural stem (NS) cells in vitro, using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). We identified 66 differentially expressed proteins involved in these five differentiation stages. Together with the results of Western blotting, RT-PCR, and a search of metabolic pathways related to the identified proteins, these results indicated that collapsin response mediator protein 2 (CRMP2), its phosphorylated forms, and cellular retinoic acid binding protein 1 (CRABP1) were upregulated from ES cells to Day 4 and NSS cells, to which differentiation stages apoptosis-associated proteins such as caspases were possibly related; Phosphorylated CRMP2s were further upregulated but CRABP1 was downregulated from NSS cells to NS cells, during which differentiation stage considerable axon guidance proteins for development of growth cones, axon attraction, and repulsion were possibly readied; Nonphosphorylated CRMP2 was downregulated but CRABP1 was re-upregulated from NS cells to AstP cells, in which differentiation stage reorganization of actin cytoskeleton linked to focal adhesion was possibly accompanied. These results provide insight into the molecular basis of early astrocytogenesis in monkey.
了解星形胶质细胞生成对于治疗神经系统疾病具有重要价值，因为星形胶质细胞为神经元提供结构、代谢和防御支持，并积极调节神经元。然而，关于从灵长类胚胎干细胞分化为星形胶质细胞所涉及的分子事件的信息有限。因此，我们利用二维凝胶电泳（2-DE）和液相色谱-串联质谱（LC-MS-MS），研究了食蟹猴胚胎干细胞（CMK6细胞系）在体外通过形成原始神经干细胞球（第4天）、成熟神经干细胞球（NSS）和神经干细胞，向星形胶质细胞祖细胞（AstP）分化过程中早期星形胶质细胞生成中差异表达的蛋白质。我们鉴定出了66种在这五个分化阶段差异表达的蛋白质。结合蛋白质免疫印迹、逆转录-聚合酶链反应结果以及对与鉴定出的蛋白质相关的代谢途径的搜索，这些结果表明，从胚胎干细胞到第4天和NSS细胞阶段，塌陷反应介导蛋白2（CRMP2）及其磷酸化形式以及细胞视黄酸结合蛋白1（CRABP1）上调，这两个分化阶段可能与半胱天冬酶等凋亡相关蛋白有关；从NSS细胞到神经干细胞阶段，磷酸化的CRMP2进一步上调，但CRABP1下调，在此分化阶段可能准备了大量用于生长锥发育、轴突吸引和排斥的轴突导向蛋白；从神经干细胞到AstP细胞阶段，非磷酸化的CRMP2下调，但CRABP1再次上调，此分化阶段可能伴随着与粘着斑相关的肌动蛋白细胞骨架的重组。这些结果为猴子早期星形胶质细胞生成的分子基础提供了见解。
