GIT1 Regulates Spine Morphogenesis and Synapse Formation

描述（由申请人提供）：突触的形成和可塑性是正常认知功能的基础，如学习和记忆。先前的研究指出，肌动蛋白动力学和粘附在突触形成和可塑性中的调节起着关键作用。这些过程可以通过GIT1聚集在一起，GIT1是一种最近被发现的分子，它存在于海马体神经元突触前和突触后末端。GIT1作为连接Rac效应器和调节因子以及其他突触分子的适配器。我们的初步工作已经产生了一些可行的假设，本提案的目的是严格检验这些假设，如在具体目标中概述的那样，使用游离海马神经元的培养和切片制备。我将验证GIT1通过组装树突棘中由Rac、PIX和PAK组成的信号模块，将Rac靶向到突触，从而调节突触可塑性的假设。活性Rac将使用FRET生物传感器以及我们正在开发的一种新的基于定位的检测方法进行检测。GIT1的功能将使用我们的GIT、PIX和PAK突变体库来确定。在Specific Aim II中，我们将验证整合素信号激活突触中的Rac的假设。该方法是确定整合素信号的关键调节因子，如FAK、Src和MAP激酶，是否调节突触可塑性。在Specific Aim III中，我们将使用海马体切片培养将我们的观察扩展到体内样环境。这些研究将增强我们对突触形成和可塑性背后的分子因素的理解。

DESCRIPTION (provided by applicant): Synapse formation and plasticity underlie normal cognitive functions, such as learning and memory. Previous studies point to a critical role for the regulation of actin dynamics and adhesion in synapse formation and plasticity. These processes can be brought together by GIT1, a recently characterized molecule that resides in pre and postsynaptic terminals of hippocampal neurons. GIT1 serves as an adapter that binds Rac effectors and regulators and other synaptic molecules. Our preliminary work has led to working hypotheses and the objective of this proposal is to rigorously test these hypotheses as outlined in the specific aims using cultures of dissociated hippocampal neurons and slice preparations. Specific Aim I will test the hypothesis that GIT1 can regulate synaptic plasticity by targeting Rac to synapses through the assembly of a signaling module comprised of Rac, PIX, and PAK that localizes in dendritic spines. Active Rac will be assayed using FRET biosensors as well as a new localization based assay that we are developing. The function of GIT1 will be determined using our library of GIT, PIX, and PAK mutants. In Specific Aim II, we will test the hypothesis that integrin signaling activates Rac in synapses. The approach is to determine whether key regulators of integrin signaling, such as FAK, Src, and MAP kinase, modulate synaptic plasticity. In Specific Aim III, we will use slice cultures from the hippocampus to extend our observation to an in vivo like environment. These studies will enhance our understanding of the molecular factors that underlie synapse formation and plasticity.