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Molecules for inducibly modulating synaptic function

诱导调节突触功能的分子

基本信息

批准号：
7373673
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
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-03-01 至 2010-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7373673
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

项目摘要

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重大挑战。解决这一问题的典型方法包括损伤或局部药理学；这些方法是粗略的和非特异性的。最近，小鼠基因组计划和大规模表达研究，加上新兴的转基因技术，已经开始允许选择特定的神经元群体进行干预。但是一个靶点应该是什么来干预神经功能呢？理想情况下，干预机制应该是有条件的，即只有在实验者提示时才干预功能。这种诱导应该是迅速和迅速可逆的。干预也应该是微妙的，例如不需要重大的手术干预。最后，干预应该是具体的，从这个意义上说，它干扰了神经元功能的一个明确的方面，而不会产生无数的次要影响。我们将设计分子遗传学工具，允许突触传递的条件扰动。在没有内源性靶点的药物作用下，突触囊泡和/或对胞吐作用重要的蛋白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，这些结构域虽然本身不干扰蛋白质功能，但将赋予融合体与小化学交联剂(FK506和雷帕霉素的衍生物)相互作用的能力。这种细胞通透性二聚体的加入将诱导蛋白质-蛋白质相互作用，将必要的突触囊泡成分从其自然伙伴和/或功能部位隔离开来，导致突触传递的丧失。2.)将在培养的神经元和脑片上测试失活突触传递的系统，使用光学技术来监测囊泡循环，并使用电生理学来测量突触电流。3.)将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。系统将使用内部信号成像和电生理技术在体内进行测试。