MOLECULAR CHARACTERIZATION OF GLUTAMATE RECEPTOR EXPRESSION IN BRAIN

脑中谷氨酸受体表达的分子特征

• 批准号: 3756674
• 负责人: A BUONANNO
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/3756674
• 关键词: Adenoviridae; NMDA receptors; RNase protection assay; developmental genetics; developmental neurobiology; gene expression; genetic regulatory element; genetic transcription; genetic transduction; genetically modified animals; in situ hybridization; laboratory mouse; molecular cloning; neurogenesis; nucleic acid sequence; polymerase chain reaction; receptor expression; reporter genes; transfection /expression vector

Activity-dependent neural plasticity during development and in the adult is largely mediated by N-methyl-D-aspartate receptor (NMDAR) activation; the natural ligand for this excitatory amino acid receptor is glutamate. During synaptogenesis, NMDARs have been found to play a major role in stabilizing neural connections in the visual system of mammals and lower vertebrates, and in registering somatosensory maps. In the adult, NMDA receptors have been implicated in the induction of long-term potentiation, a process characterized by the increase of synaptic efficacy in response to electrical stimulation. For these reasons, NMDARs provide an interesting target for understanding the maleable properties of the CNS. As a first step to elucidating the molecular mechanisms that target the expression of these family of receptors to specific neural populations during development, we have begun to analyze the expression of different receptor subunits during development and to dissect the transcriptional regulatory elements that modulate NMDAR gene expression. Using in situ hybridization histochemistry, we and others found that the epsilonsubunits are differentially expressed during neurodevelopment. To begin elucidating the molecular mechanisms regulating NMDAR expression, the upstream region of the epsilon2 gene was cloned, sequenced and characterized. Multiple transcription initiation sites were mapped by RNase protection and a PCR technique based on primer extension. Transgenic mice harboring a chloramphenicol acetyltransferase (CAT) construct, driven by epsilon2 upstream sequences, expressed the reporter specifically in the brain. Experiments are in progress to delineate the cis-acting elements conferring developmental and neuron-specific transcription. The use of replication-deficient adenovirus vectors to introduce expression constructs into neurons has been investigated. This type of work provides the foundation for the future use of epsilon2 regulatory sequences in gene therapy experiments.

发育中和成人活动依赖的神经可塑性 主要由N-甲基-D-天冬氨酸受体(NMDAR)激活介导； 这种兴奋性氨基酸受体的天然配体是谷氨酸。 在突触发生过程中，NMDAR被发现在 稳定哺乳动物和低等动物视觉系统中的神经连接 脊椎动物，以及在登记体感地图方面。在成人中，NMDA 受体已被牵连在诱导长期的 增强，以突触增加为特征的过程 对电刺激的反应的有效性。出于这些原因，NMDAR 为理解易受攻击的属性提供了一个有趣的目标 中枢神经系统。作为阐明分子机制的第一步， 将这些受体家族的表达靶向特定的神经 在人口发展过程中，我们已经开始分析表达 不同受体亚基在发育过程中的差异，并解剖 调控NMDAR基因表达的转录调控元件。 利用原位杂交组织化学，我们和其他人发现 在神经发育过程中，表观亚基的表达存在差异。至 开始阐明调控NMDAR表达的分子机制， 克隆了epsilon2基因的上游区域，并对其进行了测序。 特色化的。多个转录起始点被定位 核糖核酸酶保护和基于引物延伸的聚合酶链式反应。 携带氯霉素乙酰转移酶(CAT)的转基因小鼠 构建，由epsilon 2上游序列驱动，记者表达 尤其是在大脑中。实验正在进行中，以描绘出 影响发育和神经元特异性的顺式作用元件 抄写。复制缺陷型腺病毒载体的应用 将表达结构引入神经元已有研究。这 工作类型为将来使用epsilon2奠定了基础 基因治疗实验中的调控序列。