SURVIVAL AND DEATH OF NEURONS

神经元的生存和死亡

• 批准号: 2263031
• 负责人: Timothy J Timothy J Cunningham Cunningham
• 金额: $ 25.18万
• 依托单位: ALLEGHENY UNIVERSITY OF HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2263031
• 关键词: actins; age difference; cell death; cell growth regulation; developmental neurobiology; excitatory aminoacid; immunologic assay /test; laboratory rat; neurotoxins; neurotrophic factors; protein purification; protein sequence; thalamus

We have isolated a neuron survival factor from the developing neocortex of rats and from medium conditioned by a human cell line. This factor migrates as a 200kD band in both reduced and unreduced SDS gels. The 200kD factor is distributed in regions of high glutamate binding in the developing brain and protects axotomized neurons from degeneration in vitro. Initial amino acid sequences obtained from the 200kD band reveal sequences identical to gamma or beta actin. Biochemical characterization indicates that this protein is resistant to chemical reduction with beta- mercaptoethanol and continues to migrate at 200kD on SDS gels, indicating that the beta-actin segments are covalently associated in an apparent 200kD complex. A monoclonal antibody to the 200kD factor recognizes non- actin parts of the molecule but not any well known neurotrophic factors like NGF, CNTF, and acidic or basic FGF. This is a proposal to test the hypothesis that the 200 kD species is a cytoskeletally-associated regulatory molecule, comprised of actin covalently associated with non- actin sequences, that are also neurotrophic. Furthermore, we will examine the possibility that the factor prevents cell death and promotes cell growth by regulating neuronal responses to excitatory amino acids. The specific experiments proposed are designed to obtain additional amino acid sequences (specifically non-actin sequences) of the 200kD factor in order to more fully define this neurotrophic protein and provide the basis for future experiments involving gene cloning. To accomplish this characterization, the factor will be digested and non-actin peptides will be identified by high resolution peptide analysis. Along with these biochemical experiments, we will continue to test the neuron survival promoting properties of this factor using neonatal thalamic neurons in an in vitro bioassay. In particular, we will follow up preliminary observations that suggest the factor promotes neuron survival by acting at glutamate receptors including NMDA. In vitro studies of neonatal anterior thalamic neurons will test this hypothesis specifically. The neurotoxicity of NMDA and non NMDA glutamate receptor agonists will be investigated and the effects of the 200kD factor on this toxicity (and associated changes in intracellular calcium) will be determined.

我们已经从发展的新皮层中分离出神经元的生存因子 大鼠和由人类细胞系调节的培养基。这个因素 在减少和未还原的SDS凝胶中迁移为200kD频段。 200kd 因子分布在高谷氨酸结合的区域 发育大脑并保护轴位神经元免受变性 体外。从200kD带揭示的初始氨基酸序列 序列与伽马或β肌动蛋白相同。生化特征 表明该蛋白具有抗β-的化学还原性 胃乙醇并继续以200 kd的SDS凝胶迁移，表明 β-肌动蛋白片段在明显的 200KD复合体。对200KD因子的单克隆抗体识别非 - 分子的肌动蛋白部分，但没有任何知名的神经营养因素 例如NGF，CNTF，以及酸性或基本FGF。这是测试 假设200 kD物种是一种细胞骨骼相关的假设 调节分子，由与非 - 肌动蛋白序列，也是神经营养的。此外，我们将检查 该因子阻止细胞死亡并促进细胞的可能性 通过调节对兴奋性氨基酸的神经元反应的生长。 提出的特定实验旨在获得额外的氨基 200KD因子的酸序列（特别是非肌动蛋白序列） 为了更充分地定义这种神经营养蛋白并提供基础 用于将来涉及基因克隆的实验。实现这一目标 表征，该因子将被消化，非肌动蛋白肽将 可以通过高分辨率肽分析来识别。以及这些 生化实验，我们将继续测试神经元存活 使用新生儿丘脑神经元在 体外生物测定。特别是，我们将跟进初步 表明该因子通过作用在 包括NMDA在内的谷氨酸受体。新生儿前的体外研究 丘脑神经元将特别检验该假设。神经毒性 将研究NMDA和非NMDA谷氨酸受体激动剂，并 200KD因子对这种毒性的影响（以及相关的变化 在细胞内钙中）将被确定。