REGULATION OF SYNAPTIC DEVELOPMENT AND FUNCTION BY NEUROTROPHIC FACTORS

神经营养因子对突触发育和功能的调节

• 批准号: 6495432
• 负责人: Louis French Reichardt
• 金额: $ 22.55万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6495432
• 关键词: PC12 cells; Xenopus; Xenopus oocyte; biological signal transduction; brain mapping; cell differentiation; chimeric proteins; developmental neurobiology; exocytosis; green fluorescent proteins; growth factor receptors; integrins; laboratory mouse; neural plasticity; neuromuscular junction; neuropeptide receptor; neurotrophic factors; phosphatidylinositol 3 kinase; protein sequence; receptor expression; retinal ganglion; superior colliculus; synaptogenesis

The goal of this research is to understand the roles in regulating synapse formation, function and plasticity of neurotrophins, their receptors trkA, B, and C and their downstream signaling molecules. To examine the role of neurotrophins in synapse formation, we will generate DNA constructs encoding fusion proteins in which constituents of different elements of the synapse (release zone, synaptic vesicle), such as VAMP, synapsin I, and the Ca channel beta subunit are fused to Green Fluorescent Protein (GFP). These will be injected into Xenopus embryo cells whose descendents include retinal ganglion cells. After grafting of "tagged" retinal anlage from these to fresh embryos, retinal axon and synapse development will be studied in the optic tectum. Effects on synapse formation of neurotrophins and dominant negative constructs of the BDNF receptor trkB will be studied. Signaling pathways important in differentiation will be determined by injecting trkA constructs defective in interactions with individual downstream signaling molecules and determining effects of exogenous NGF. After insertion of the GFP chimeras into a defective Adenovirus, infections of murine retina will permit examination of synapse development in normal and neurotrophin-deficient mutant mice. The second project will seek to understand the molecular bases of neurotrophin regulation of exocytosis, observed by others at the Xenopus neuromuscular junction and Schaeffer collateral CA 1 synapses in the hippocampus. We will determine effects of NGF application on synaptic transmission at the Xenopus NMJ after injection of precursors with trkA. To dissect downstream signaling pathways, trkA receptors defective in activation of specific signalling pathways will be used in the same model. To further delineate a pathway, effects of dominant negative constructs of downstream signalling molecules, such PI-3 kinase and ras, will be examined. Efforts will be made to extend this analysis will be extended to a biochemically amenable system using PC12 cells expressing receptors with signalling pathway deficiencies where effects of neurotrophin application on phosphorylation of proteins associated with the exocytotic apparatus will be examined and functional consequences can be assessed.

这项研究的目的是了解调节突触中的作用 神经营养蛋白的形成，功能和可塑性，其受体TRKA， B，C及其下游信号分子。检查 神经营养蛋白是突触形成的，我们将生成DNA构建体 编码融合蛋白，其中不同元素的成分 突触（释放区，突触囊泡），例如vamp，Sytapsin I， 和CA通道β亚基融合到绿色荧光蛋白 （GFP）。这些将被注入后代的爪蟾胚胎细胞 包括视网膜神经节细胞。嫁接“标记”的视网膜闭合 从这些到新鲜的胚胎，视网膜轴突和突触的发育将是 在视觉底面进行了研究。对神经营养蛋白突触形成的影响 BDNF受体TRKB的主要负面结构将是 研究。在分化中重要的信号通路将是 通过注射TRKA构建体在与 单个下游信号分子和确定的影响 外源性NGF。将GFP嵌合体插入后 腺病毒，鼠视网膜的感染将允许检查突触 正常和神经营养不良的突变小鼠的发育。第二个 项目将寻求了解神经营养素的分子碱基 胞吐作用的调节，由其他人观察到爪蟾神经肌肉 海马中的交界处和Schaeffer侧支Ca 1突触。我们 将确定NGF应用对在 用TRKA注射前体后NMJ Xenopus NMJ。剖析下游 信号通路，TRKA受体在特定的激活中有缺陷 信号通路将在同一模型中使用。进一步描述 途径，下游主要负面构造的影响 信号分子（此类PI-3激酶和RAS）将进行检查。努力 将扩展此分析将扩展到生化 使用PC12细胞表达具有信号的受体的系统 途径缺陷，神经营养蛋白应用对 与胞吐剂相关的蛋白质的磷酸化将 可以检查并可以评估功能后果。