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。以往的研究指出，肌动蛋白动力学和黏附在突触形成和可塑性中起着关键作用。这些过程可以被GIT1结合在一起，GIT1是一种最近确定的分子，位于海马神经元的突触前和突触后终末。GIT1是一个连接RAC效应器和调节器以及其他突触分子的适配器。我们的初步工作已经导致了工作假说，这项提议的目标是使用分离的海马神经元培养和切片制备来严格检验特定目标中概述的这些假说。具体目的I将验证GIT1通过组装由RAC、PIX和PAK组成的信号模块来调节突触可塑性的假设，该信号模块定位于树突棘中。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突变体文库来确定。在特定的目标II中，我们将检验整合素信号激活突触中RAC的假设。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。在特定的目标III中，我们将使用来自海马体的切片培养来将我们的观察扩展到类似活体的环境。这些研究将加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.