Imaging signal transduction in single dendritic spines

描述（由申请人提供）：树突棘是小的（0.1 - 0.01飞飞）兴奋性突触后室，从树突表面发出。钙离子流入脊柱激活由数百种蛋白质组成的信号网络，诱导不同形式的突触可塑性。Rho GTPase蛋白，特别是Rac1、RhoA和Cdc42是这些信号网络的重要组成部分，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信号通路在脊柱形态和功能中的重要作用一致，Rho信号通路的突变与多种形式的智力迟钝和自闭症有关。在这项研究中，我们将开发一种技术来测量大脑切片中单个树突棘在形态和功能可塑性过程中的Rho信号。为此，我们将结合2光子荧光寿命成像显微镜（2pFLIM）和基于荧光共振能量转移的Rho活度传感器，广泛优化2pFLIM。我们的初步数据表明，Cdc42的活性仅限于发生突触可塑性的棘，而Rac1和RhoA的激活沿树突扩散约10 5m，并侵入邻近的棘。这些结果表明，每种Rho信号通路在不同的长度尺度上起作用。我们将通过测量和干扰Rho信号的时空动态来研究Rho GTPase蛋白的空间扩散机制和作用。本项目的具体目标是：1)建立个体脊柱中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态机制和作用；3)确定钙与Rho GTPase激活和突触可塑性之间的信号通路。本研究将阐明树突棘形态和功能可塑性的分子机制，并为Rho信号通路突变引起的精神疾病提供新的见解。公共卫生相关性：突触的形状和功能是由Rho蛋白介导的信号调节的。许多形式的智力迟钝和自闭症都是由Rho信号异常引起的。本项目将开发一种新的技术来测量Rho蛋白在单个突触中的活性，以阐明Rho蛋白活性与突触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。

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.