Molecules for inducibly modulating synaptic function

诱导调节突触功能的分子

• 批准号: 7191658
• 负责人: Karel Svoboda
• 金额: $ 32.55万
• 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/7191658
• 关键词: Actins; Animals; Behavioral Assay; Binding; Biological Neural Networks; Biological Process; Brain; Cell membrane; Cells; Chemicals; Chimeric Proteins; Chromosome Pairing; Computer information processing; Crosslinker; Cytoskeleton; Development; Dimerization; Electrophysiology (science); Engineering; Exocytosis; FK506; Feedback; Functional disorder; Goals; Heterodimerization; Homodimerization; Image; In Vitro; Intervention; Lead; Learning; Lesion; Measurement; Measures; Memory; Methods; Mitochondria; Molecular; Molecular Genetics; Monitor; Mus; Nature; Nervous System Physiology; Neurologic; Neurons; Neurosciences; Operative Surgical Procedures; Optics; Pharmacology; Population; Population Intervention; Presynaptic Terminals; Proteins; Role; Signal Transduction; Sirolimus; Site; Slice; Synapses; Synaptic Transmission; Synaptic Vesicles; System; Tacrolimus Binding Proteins; Techniques; Technology; Tertiary Protein Structure; Testing; Transgenic Organisms; Vesicle; Viral; Virus; base; crosslink; design; experience; gene therapy; in vitro Assay; in vivo; mouse genome; presynaptic; programs; protein function; protein protein interaction; recombinant virus; relating to nervous system; research study; synaptic function; tool

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）： 了解神经网络的连接性及其与神经系统功能和功能障碍的关系仍然是一个重大挑战。解决这个问题的典型方法包括损伤或局部药理学；这些方法都是粗暴且不具体的。最近，小鼠基因组计划和大规模表达研究，以及新兴的转基因技术，已经开始允许选择特定的神经元群体进行干预。但干预神经元功能的目标应该是什么呢？理想情况下，干预机制应该是有条件的，即仅在实验者提示时才干扰功能。这种诱导应该是快速的并且快速可逆的。干预也应该是微妙的，不需要例如大手术干预。最后，干预措施应该是具体的，即它干扰神经元功能的明确定义的方面，而不会产生无数的副作用。我们将设计允许突触传递有条件扰动的分子遗传工具。在没有内源性靶标的情况下，通过施用药物触发，突触小泡和/或对胞吐作用重要的蛋白质将被错误定位或固定，干扰突触传递。这将分三步完成。 1.) 我们将通过引入结构域（FK506 结合蛋白和雷帕霉素结合结构域的靶标）来设计参与胞吐作用的蛋白质的修饰版本，这些结构域本身不会干扰蛋白质功能，但会赋予融合物与小型化学交联剂（FK506 和雷帕霉素的衍生物）相互作用的能力。添加这种细胞可渗透的二聚体将诱导蛋白质-蛋白质相互作用，从而将重要的突触小泡成分与它们的天然伙伴和/或功能位点隔离开，导致突触传递的损失。 2.) 将在培养的神经元和脑切片中测试用于灭活突触传递的系统，使用光学技术监测囊泡循环，并使用电生理学测量突触电流。 3.) 使用病毒和转基因方法，将充分表征的系统引入小鼠神经元亚群。将使用内在信号成像和电生理学技术对系统进行体内测试。