Molecules for inducibly modulating synaptic function

诱导调节突触功能的分子

• 批准号: 7026465
• 负责人: Karel Svoboda
• 金额: $ 33.52万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7026465
• 关键词: biotechnology; cell population study; crosslink; electrophysiology; exocytosis; genetically modified animals; laboratory mouse; laboratory rat; molecular genetics; molecular probes; neural transmission; peptidylprolyl isomerase; protein engineering; protein protein interaction; sirolimus; synaptic vesicles; technology /technique development; tissue /cell culture

DESCRIPTION (provided by applicant): Understanding the connectivity of neural networks and its relationships to nervous system function and dysfunction remains a major challenge. Typical approaches to this problem include lesions or focal pharmacology; these methods are crude and non-specific. Recently, the mouse genome project and largescale expression studies, together with emerging transgenic technologies, have begun to allow selection of specific neuronal populations for intervention. But what should one target to intervene in neuronal function? Ideally a mechanism for intervention should be conditional, i.e. only interfere with function when prompted by the experimenter. Such induction should be rapid and rapidly reversible. Intervention should also be delicate, not requiring, for example, major surgical intervention. Finally, the intervention should be specific, in the sense that it interferes with a well-defined aspect of neuronal function without a myriad of secondary effects. We will design molecular genetic tools that allow conditional perturbation of synaptic transmission. Triggered by administration of pharmacological agents without endogenous targets, synaptic vesicles and/or proteins important for exocytosis will be mislocalized or immobilized, interfering with synaptic transmission. This will be accomplished in three steps. 1.) We will engineer modified versions of proteins involved in exocytosis by introducing domains (FK506 binding protein and target of rapamycin binding domain) that, while by themselves do not interfere with protein function, will confer on the fusions the ability to interact with small chemical crosslinkers (derivatives of FK506 and rapamycin). Addition of such cell-permeable dimerizers will induce protein-protein interactions that will sequester essential synaptic vesicle components away from their natural partners and/or the site of function, resulting in loss of synaptic transmission. 2.) Systems for inactivating synaptic transmission will be tested in cultured neurons and brain slices using optical techniques to monitor vesicle cycling and electrophysiology to measure synaptic currents. 3.) Well-characterized systems will be introduced into subpopulations of neurons in mice using viruses and transgenic approaches. Systems will be tested in vivo using intrinsic signal imaging and electrophysiological techniques.

描述（由申请人提供）： 了解神经网络的连通性及其与神经系统功能和功能障碍的关系仍然是一个主要挑战。解决此问题的典型方法包括病变或局灶性药理学；这些方法是粗略的，非特异性的。最近，小鼠基因组项目和LARGESCALE表达研究以及新兴的转基因技术已开始允许选择特定的神经元种群进行干预。但是，要干预神经元功能的目标应该是什么？理想情况下，干预机制应是有条件的，即仅在实验者提示时才干扰功能。这种诱导应该是快速且快速可逆的。干预也应该是微妙的，不需要例如主要的手术干预。最后，干预应该是特定的，从某种意义上说，它会干扰神经元功能的明确方面而没有多种次要效应。我们将设计分子遗传工具，允许有条件的突触传播扰动。通过无内源性靶标的药理学剂触发，突触囊泡和/或蛋白质对胞吐作用很重要的蛋白质会被错误定位或固定，从而干扰突触传播。这将通过三个步骤完成。 1.）我们将通过引入域（FK506结合蛋白和雷帕霉素结合域的靶标）来设计参与外吞作用的蛋白质的修饰版本，尽管它本身不会干扰蛋白质功能，但它将赋予融合融合的能力与小型化学交叉链接（FK506和RAPAPAPAMCIN和RAPAPAMCIN）相互作用。这种可渗透细胞的二聚体的添加将诱导蛋白质 - 蛋白质相互作用，这些蛋白质蛋白质相互作用将使必需的突触囊泡成分从其自然伴侣和/或功能部位隔离，从而导致突触传播的损失。 2.）使用光学技术在培养的神经元和脑切片中测试灭活突触传播的系统，以监测囊泡循环和电生理学以测量突触电流。 3.）使用病毒和转基因方法将特征良好的系统引入小鼠的神经元亚群中。系统将使用内在信号成像和电生理技术在体内进行测试。