Imaging signal transduction in single dendritic spines

单树突棘的成像信号转导

• 批准号: 8097332
• 负责人: Ryohei Yasuda
• 金额: $ 38.22万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8097332
• 关键词: Actins; Autistic Disorder; Biochemical; Brain; Calcium; Cell Line; Data; Dendrites; Dendritic Spines; Development; Diffusion; Disease; Energy Transfer; Excitatory Synapse; Fluorescence; Fluorescence Resonance Energy Transfer; Glutamates; Growth; Health; Image; Imaging Techniques; Individual; Invaded; Kinetics; Length; Link; Measures; Mediating; Mental Retardation; Microscopy; Modeling; Molecular; Morphogenesis; Morphology; Mutation; N-Methyl-D-Aspartate Receptors; Neck; Neuraxis; Neurons; Optics; Parents; Pattern; Photons; Play; Proteins; Psyche structure; Public Health; Reaction; Regulation; Resolution; Role; Sensitivity and Specificity; Shapes; Signal Pathway; Signal Transduction; Slice; Surface; Synapses; Synaptic plasticity; Techniques; Time; Vertebral column; base; calmodulin-dependent protein kinase II; disease-causing mutation; genetic inhibitor; insight; novel; novel therapeutics; postsynaptic; protein profiling; rho; rho GTP-Binding Proteins; sensor; spatiotemporal; synaptic function; tool

DESCRIPTION (provided by applicant): Dendritic spines are small (0.1 - 0.01 femtolitter) excitatory postsynaptic compartments emanating from the dendritic surface. Ca2+ influx into spines activates signaling networks consisting of hundreds of species of proteins that induce diverse forms of synaptic plasticity. Rho GTPase proteins, particularly Rac1, RhoA and Cdc42, are critical components of these signaling networks, and their activation plays an important role in regulating the morphology and function of dendritic spines. Consistent with the important role of Rho signaling in spine morphology and function, mutations in Rho signaling pathways are associated with many forms of mental retardation and autism. In this study, we will develop a technique to measure Rho signaling in single dendritic spines while they undergo morphological and functional plasticity in brain slices. To do so, we will combine 2-photon fluorescence lifetime imaging microscopy (2pFLIM) with fluorescent resonance energy transfer-based Rho activity sensors extensively optimized for 2pFLIM. Our preliminary data demonstrates that the activity of Cdc42 is restricted to spines undergoing synaptic plasticity, while Rac1 and RhoA activation spreads along dendrites over ~10 5m and invades neighboring spines. These results suggest that each Rho signaling pathway functions on a different length scale. We will study the mechanisms and roles of the spatial spreading of Rho GTPase proteins by measuring and perturbing the spatiotemporal dynamics of Rho signaling. The specific aims of this project are to 1) establish techniques to image Rho signaling in individual spines, 2) elucidate the mechanisms and roles of spatiotemporal dynamics of Rho during synaptic plasticity, and 3) identify signaling pathways connecting calcium with Rho GTPase activation and synaptic plasticity. This study will illuminate the molecular mechanisms of morphological and functional plasticity of dendritic spines, and will provide insights into mental diseases caused by mutations in Rho signaling pathways. PUBLIC HEALTH RELEVANCE: The shape and function of synapses are regulated by signaling mediated by Rho proteins. Many forms of mental retardation and autism are caused by abnormal Rho signaling. This project will develop a novel technique to measure the activity of Rho proteins in single synapses to elucidate the mechanisms linking the activity of Rho proteins and the morphology and function of synapses. This will facilitate understanding of mental retardation and autism.

描述（由申请人提供）：树枝状棘是从树枝状表面发出的小（0.1 - 0.01毫微微升）兴奋性突触后隔室。Ca 2+流入棘激活由数百种蛋白质组成的信号网络，这些蛋白质诱导不同形式的突触可塑性。Rho GT3蛋白，特别是Rac 1、RhoA和Cdc 42，是这些信号网络的关键组成部分，它们的激活在调节树突棘的形态和功能中起着重要作用。与Rho信号在脊柱形态和功能中的重要作用一致，Rho信号通路中的突变与许多形式的精神发育迟滞和自闭症相关。在这项研究中，我们将开发一种技术来测量单个树突棘中的Rho信号，同时它们在脑切片中经历形态和功能可塑性。为此，我们将结合联合收割机2光子荧光寿命成像显微镜（2 pFLIM）与荧光共振能量转移为基础的Rho活性传感器广泛优化2 pFLIM。我们的初步数据表明，Cdc 42的活动仅限于突触可塑性的棘，而Rac 1和RhoA激活传播沿着树突超过10 5米，并侵入相邻的棘。这些结果表明，每个Rho信号通路的功能在不同的长度尺度。我们将通过测量和扰动Rho信号的时空动力学来研究Rho GTdR蛋白的空间扩散的机制和作用。本项目的具体目标是：1）建立单个脊髓中Rho信号的成像技术，2）阐明Rho在突触可塑性过程中的时空动力学机制和作用，3）识别钙与Rho GT3激活和突触可塑性之间的信号通路。这项研究将阐明树突棘形态和功能可塑性的分子机制，并将为Rho信号通路突变引起的精神疾病提供见解。公共卫生相关性：突触的形状和功能由Rho蛋白介导的信号调节。许多形式的精神发育迟滞和自闭症是由异常的Rho信号传导引起的。本项目将开发一种新的技术来测量单个突触中Rho蛋白的活性，以阐明Rho蛋白的活性与突触的形态和功能之间的联系机制。这将有助于理解智力迟钝和自闭症。