Postsynaptic Protein Trafficking and Synapse Remodeling

描述(申请人提供)：突触的可塑性对大脑的发育、学习和记忆是必不可少的。Hebbian型可塑性，如长时程增强和长时程抑制，是突触特异性的快速修饰。相比之下，动态平衡可塑性涉及突触的全局修饰，在更长的时间尺度上起作用，被认为对维持和协调神经元网络功能至关重要。Hebbian型可塑性主要通过AMPA受体的转运来调节，但对稳态可塑性的机制知之甚少。近年来，泛素-蛋白酶体系统在突触上的活性依赖性蛋白转运已成为包括稳态可塑性在内的多种突触可塑性的重要机制。然而，尚不清楚活动如何协调伴随的泛素化/降解和突触处特定蛋白质组的招募。在活性调节蛋白中，GKAP是突触后密度的主要支架蛋白之一，为PSD-95/NMDA受体复合体和Shank/Hmer提供了分子纽带。我们的初步研究表明，GKAP的活动通过钙依赖的蛋白激酶II(CaMKII)控制突触中GKAP的募集和移除。此外，我们发现GKAP的活性依赖的翻转是海马神经元突触伸缩所必需的。在这项研究中，我们将研究CaMKII控制GKAP泛素化/降解或募集到突触的分子机制，以及突触GKAP翻转在各种类型突触可塑性中的功能意义。目的1结合突变和生化分析，定位诱导GKAP泛素化的CaMKII磷酸化位点(S)和泛素化赖氨酸位点(S)。目的2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的GKAP交易。目的3通过使用缺乏活性依赖翻转的GKAP突变体，评估突触上GKAP移除/重新募集对于活性依赖的突触成分修饰和各种形式的突触可塑性的功能意义。由于突触的异常可塑性与多种神经和神经精神疾病有关，因此这些研究不仅可以让我们对突触组成发生长期变化的分子机制有新的、基本的认识，而且与这些脑部疾病有关。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。这项研究项目的主要主题GKAP与许多神经疾病有关，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的高级认知功能，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.