Postsynaptic Protein Trafficking and Synapse Remodeling

描述（申请人提供）：突触可塑性对大脑发育、学习和记忆至关重要。Hebbian型可塑性如长时程增强和长时程抑制是快速的和突触特异性的修饰。相比之下，稳态可塑性涉及突触的全局修饰，在较长的时间尺度上运作，并且被认为对于维持和协调神经元网络功能至关重要。赫布型可塑性主要由AMPA受体的运输介导，但对稳态可塑性的机制知之甚少。近年来，泛素-蛋白酶体系统参与的突触活性依赖性蛋白质周转已成为突触可塑性包括稳态可塑性的重要机制。然而，目前尚不清楚活动如何协调伴随的泛素化/降解和特定蛋白质组在突触处的募集。在这些活性调节蛋白中，GKAP是突触后致密物中的主要支架蛋白之一，并为PSD-95/NMDA受体复合物和Shank/Homer提供分子连接。我们的初步研究表明，活动控制的招聘和清除GKAP从突触，都通过Ca 2+？依赖性蛋白激酶II（CaMK II）。此外，我们发现，GKAP的活性依赖性营业额所需的突触缩放在海马神经元。在这个提议中，我们将研究CaMK II控制泛素化/降解或GKAP向突触的募集的分子机制，以及GKAP在各种类型的突触可塑性中的突触周转的功能意义。目的1将使用诱变和生化测定的组合来定位诱导GKAP泛素化的CaMK II磷酸化位点和泛素化赖氨酸位点。目的2着重于通过分子遗传学方法了解DLC、MyoV和CaMKII在GKAP向突触募集中的作用。我们还将进行实时成像，以更高的时空分辨率了解动态GKAP贩运。目的3将通过使用缺乏活性依赖性周转的GKAP突变体，评估GKAP在突触处的去除/募集对于突触组成的活性依赖性修饰和各种形式的突触可塑性的功能意义。由于异常突触可塑性与多种神经系统和神经精神疾病有关，因此拟议的研究不仅使我们能够对突触组成的持久变化的分子机制获得新的和基本的见解，而且与这些脑疾病有关。公共卫生相关性：突触可塑性是神经元储存经验并形成学习和记忆基础的基本机制。该研究项目的主要课题GKAP涉及许多神经系统疾病，包括自闭症，精神分裂症和强迫症。因此，研究GKAP在突触可塑性中的作用不仅有助于理解人类高级认知功能，而且与疾病有直接关系。

DESCRIPTION (provided by applicant): Synaptic plasticity is essential for the development of brain, learning and memory. Hebbian-type plasticity such as long-term potentiation and long-term depression is rapid and synapse-specific modification. In contrast, homeostatic plasticity involves global modification of synapses, operates over longer timescales, and is believed to be crucial for the maintaining and orchestrating neuronal network function. Hebbian-type plasticity is mediated mainly by the trafficking of AMPA receptors but not much is known for the mechanisms of homeostatic plasticity. Recently, activity-dependent protein turnover at the synapses by ubiquitin-proteasome system has emerged as crucial mechanisms associated with various types of synaptic plasticity including homeostatic plasticity. However, it is unknown how activity orchestrates concomitant ubiquitination/degradation and recruitment of specific group of proteins at synapses. Among the activity-regulated proteins, GKAP is one of the major scaffolding proteins in the postsynaptic densities and provides a molecular link for PSD-95/NMDA receptor complex and Shank/Homer. Our preliminary studies suggest that activity controls the recruitment and removal of GKAP from synapses, both through Ca2????dependent protein kinase II (CaMKII). Further, we found that the activity-dependent turnover of GKAP is required for synaptic scaling in hippocampal neurons. In this proposal, we will investigate the molecular mechanisms by which CaMKII controls ubiquitination/degradation or recruitment of GKAP to synapses, and the functional significance of the GKAP turnover at the synapses in various types of synaptic plasticity. Aim 1 will map the CaMKII phosphorylation site(s) and ubiquitinated lys site(s) that induce ubiquitination of GKAP, using a combination of mutagenesis and biochemical assays. Aim 2 focuses on understanding the role of DLC, MyoV, and CaMKII for GKAP recruitment to synapses by molecular genetic approaches. We will also perform real-time imaging to understand dynamic GKAP trafficking with greater spatio-temporal resolution. Aim 3 will assess the functional significance of GKAP removal/recruitment at synapses for the activity-dependent modification of synapse compositions and various forms of synaptic plasticity, by using GKAP mutants lacking the activity- dependent turnover. Since aberrant synaptic plasticity is implicated for a variety of neurological and neuropsychiatric diseases, the proposed studies will not only allow us to gain novel and fundamental insight into the molecular mechanisms for long-lasting changes in synapse compositions but also are relevant to these brain diseases. PUBLIC HEALTH RELEVANCE: Synaptic plasticity is a fundamental mechanism by which neurons store experience and forms a foundation for learning and memory. The main subject of this research project, GKAP, is implicated for number of neurological diseases including autism, schizophrenia, and obsessive-compulsive disorder. Thus, studying the function of GKAP in synaptic plasticity not only help understanding the higher cognitive function of human but also is directly relevant to disease.