Neurotransmitter Roles During Neurogenesis

神经递质在神经发生过程中的作用

• 批准号: 6675658
• 负责人: JEFFERY BARKER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6675658
• 关键词: GABA receptor; acetylcholine; astrocytes; axon; biological signal transduction; calcium channel; cell migration; cell proliferation; central nervous system; dendrites; developmental neurobiology; early embryonic stage; embryo /fetus tissue /cell culture; flow cytometry; growth factor; immunocytochemistry; ion transport; laboratory rat; mammalian embryology; muscarinic receptor; neurogenesis; neurons; neurotransmitters; oligodendroglia; phenotype; stem cells

Summary of Work: The complex and poorly understood process of lineage progression from neural stem cells to neuronal and glial phenotypes has come under increasing study using a variety of in vitro strategies. An important issue to be resolved is how neural stem cells are regulated to either self-renew or differentiate. In vivo these cells line the ventricles of the developing central nervous system (CNS) with variable numbers radiating processes to the pial surface (radial glial form of neural stem cell). The cells integrate signals derived from both carrier proteins in the cerebrospinal fluid filling the ventricles and other cells in the neuroepithelium lining the ventricles. Signals conveyed by carrier proteins and via cell-cell interactions, which could serve to regulate cell lineage progression, have not been eluciated. We have developed a novel strategy to isolate identified subpopulations of neural stem cells and differentiaing progenitors from the CNS during neurogenesis and gliogenesis. The strategy involves labeling of live cells with reagents identifying surface gangliosides, which are conserved throughout vertebrate evolution, and epitopes characteristic of cells undergoing apoptosis in conjunction with fluorescence-activated cell sorting (FACS). This FACS strategy permits prospective cellular and molecular studies of neural stem cells for the first time. During FY 2002 we focused primarily on eluciating the role of basic fibroblast growth factor (bFGF) in neural stem cell biology. Subpopulations of neural stem cells express bFGF and one of its principal receptors and bFGF is necessary and sufficient to sustain the self-renewal of neural stem cells without differentiation. Withdrawal of bFGF from defined, serum-free medium halted self-renewal and resulted in lineage progression primarily into an astrocyte lineage. Inclusion of EGF with bFGF led to self-renewal and differentiation of neurons and glial phenotypes. Imaging of Ca2+ levels in self-renewing and differentiating cells revealed bFGF-mediated regulation via pathways triggering release from intracellular stores and entry from extracellular sources. The characteristics of bFGF-mediated Ca2+ signaling were developmentally regulated and in the most differentiated phenotypes could no longer be detected with the well-established protocol. Thus, bFGF-mediated Ca2+ signaling is widespread and readily detectable among neural stem cells in self-renewal and undergoing lineage progression. We have used pharmacology to identify two lipid pathways transducing bFGF interactions with its receptor into Ca2+ signals. Pharmacological block of these pathways attenuates proliferation of neural stem cells and induces apoptosis. Similar results were obtained with a specific inhibitor of the tyrosine auto-phosphorylation reaction initiated by bFGF following interaction with its receptor. The same inhibitor affected baseline Ca2+ levels, indicating that bFGF-mediated autophosphorylation of neural stem cells regulates Ca2+ levels, which are critical for self-renewal and differentiation. Finally, we have carried out large-scale sorting of the neural stem cell and neuronal and glial populations emerging during the embryonic development of the cortex. cDNA microarray analysis of these populations in collaboration with Dr. Lynn Hudson's lab will be carried out to reveal gene clusters whose abundance correlates with different stages of cell lineage progression.

工作摘要：使用各种体外策略，研究了从神经干细胞到神经元和神经胶质表型的谱系进展过程的复杂且知之甚少。要解决的重要问题是如何调节神经干细胞以自我更新或分化。在体内，这些细胞将发育中心神经系统（CNS）的心室划分，数量可变，将过程辐射到伴侣表面（神经干细胞的径向神经胶质形式）。这些细胞整合了源自脑脊液中的两个载体蛋白，这些信号填充了心室衬里的神经上皮中的其他细胞。尚未提出由载体蛋白和通过细胞 - 细胞相互作用传达的信号（可以用于调节细胞谱系进展）的信号。我们已经开发了一种新的策略，可以在神经发生和神经胶发生过程中分离出鉴定出神经干细胞和与中枢神经系统的差异的亚群。该策略涉及将活细胞标记的试剂鉴定表面神经节苷脂，这些神经节苷脂在整个脊椎动物演化过程中都保守，并且在荧光激活的细胞分析（FACS）结合下，细胞的表达特征。该FACS策略允许首次对神经干细胞的前瞻性细胞和分子研究。在2002财年期间，我们主要集中于阐明基本成纤维细胞生长因子（BFGF）在神经干细胞生物学中的作用。神经干细胞的亚群表达BFGF及其主要受体和BFGF之一是必要的，足以维持没有分化的神经干细胞的自我更新。从定义的无血清培养基中撤出BFGF停止自我更新，并导致谱系进展主要为星形胶质细胞谱系。将EGF纳入BFGF导致神经元和神经胶质表型的自我更新和分化。自我更新和分化细胞中Ca2+水平的成像揭示了BFGF介导的调节通过途径触发细胞内存储并从细胞外来源进入的调节。 BFGF介导的Ca2+信号传导的特征受发展调节，在最分化的表型中，无法通过完善的方案检测到。因此，BFGF介导的Ca2+信号传导在自我更新和正在进行谱系进展的神经干细胞中很普遍，并且很容易检测到。我们已经使用药理学来识别两种脂质途径，将其与受体相互作用转化为Ca2+信号。这些途径的药理阻滞减弱了神经干细胞的增殖并诱导凋亡。用BFGF与其受体相互作用后，用BFGF引发的酪氨酸自磷酸化反应的特异性抑制剂获得了相似的结果。相同的抑制剂影响基线Ca2+水平，表明BFGF介导的神经干细胞的自磷酸化调节Ca2+水平，这对于自我更新和分化至关重要。最后，在皮质的胚胎发育过程中，我们对神经干细胞以及神经元和神经胶质种群进行了大规模分类。将与Lynn Hudson博士的实验室合作对这些人群进行cDNA微阵列分析，以揭示其丰度与细胞谱系进展不同阶段相关的基因簇。