CRCNS: Regulation of assembly and disassembly of the postsynaptic density during synaptic plasticity and its effect on AMPAR trapping

快速突触传递是基于一个精确而复杂的分子组织， 需要控制突触后位点AMPA型谷氨酸受体（AMPAR）的数量 树突棘上的突触。AMPAR的数量随着预处理和 突触的突触后激活历史。现在已经很好地描述了突触可以改变它们的 AMPAR的数量，因此，它们通过突触的生化机制的响应特性， 可塑性。这样，信息就存储在大脑中。本项目的总体目标是使用定量 模型和实验来回答两个基本问题的作用，丰富的突触后 synGAP蛋白在AMPAR数量调节中的作用。在完整的神经元中进行的大量实验 揭示了synGAP在突触处的表达水平与突触内的突触数量呈负相关。 synGAP有助于调节AMPAR数量的变化， 在突触可塑性中。synGAP的酶促GAP结构域作为棘轮来调节 从枝晶表面添加和去除AMPAR。SynGAP还包含一个视线， 通过其三个蛋白结合PDZ结构域与主要支架蛋白PSD-95紧密结合。重要的 作为NIMH的心理健康使命，SynGAP在大脑的学习和记忆中起着关键作用， SynGAP的突变与认知障碍有关。该项目分为两大目标。在Aim中 1，我们将回答这个问题：synGAP控制AMPA量的机制是什么 受体的突触后密度（PSD）-通过控制表面量和/或通过控制的可用性 突触中的PDZ结构域结合位点？我们将改进我们现有的计算模型， synGAP和AMPAR之间的竞争结合PSD-95，通过将其纳入我们的模型， AMPAR走私。我们将利用遗传学和复杂的分子工程学 解开这两个机制。对AMPAR的纳米组织的影响将通过超级 分辨率荧光显微镜和电生理学。这些实验的结果将用于 限制了我们的AMPAR交易模式。目标2，通过实验和计算的协同作用， 方法，我们将解决的问题：如何形成的凝结之间的synGAP 和PSD-95，以及另外的PDZ结构域结合蛋白（GluN 2受体亚基， 神经连接素、nNOS、CRIPT等）影响AMPAR-TARP在PSD基底中的纳米组织 状态和突触可塑性期间？ 相关性（参见说明）： 我们提出了一个计算和实验工作的结合，这将有助于澄清synGAP的作用 在中枢神经系统突触中AMPAR的调节，包括其在精神疾病中的作用。这项工作将影响A 称为“SynGAP单倍不足”或“MRD 5”的特定医学病症，其中SynGAP在 约1%的儿童患有伴有自闭症和/或癫痫的散发性非综合征性认知障碍。 这项工作的医学影响可能是相当重要的，因为它可以帮助指出具体的 分子干预疗法可以改善这些患者的生活。

Fast glutamatergic synaptic transmission is based on a precise and complex molecular organization which requires the control of the number of AMPA-type glutamate receptors (AMPARs) at the postsynaptic sites of glutamatergic synapses on dendritic spines. The number of AMPARs varies as a function of pre- and postsynaptic activation history of the synapse. It is now well described that synapses can change their number of AMPARs and therefore, their response properties through biochemical mechanisms of synaptic plasticity. In this way, information is stored in the brain. The overall goal of this project is to use quantitative models and experiments to answer two fundamental questions about the role of an abundant postsynaptic protein, synGAP, in regulation of the numbers of AMPARs. Numerous experiments in intact neurons have revealed that the level of synGAP expressed at synapses is inversely correlated with the amount of AMPARs available at the synapses, and that synGAP helps to regulate changes in AMPAR numbers during synaptic plasticity. The enzymatic GAP domain of synGAP acts as a ratchet to adjust the rates of addition and removal of AMPARs from the surface of the dendrite. SynGAP also contains a sight that binds tightly to the major scaffold protein PSD-95 via its three protein-binding PDZ domains. Important to the mental health mission of the NIMH, SynGAP plays a critical role in learning and memory in the Brain and mutation of SynGAP is implicated in cognitive disabilities. The project is divided into two broad Aims. In Aim 1, we will answer the question: What are the mechanisms by which synGAP controls the amount of AMPA receptor in the postsynaptic density (PSD) - by control of surface amount and/or by control of availability of PDZ domain binding sites in the synapse? We will improve our existing computational model of the competition between synGAP and AMPARs for binding to PSD-95 by incorporating it into our model of AMPAR trafficking. We will use genetics and sophisticated molecular engineering to experimentally disentangle the two mechanisms. Effects on the nano-organization of AMPARs will be measured by super- resolution fluorescence microscopy and electrophysiology. Results of these experiments will be used to constrain our model of AMPAR trafficking. Aim 2, Through the synergy of experimental and computational approaches, we will address the questions: How does the formation of the condensate between synGAP and PSD-95, and the presence of additional PDZ domain-binding proteins (GluN2 receptor subunits, neuroligin, nNOS, CRIPT, etc.) influence the nano-organization of AMPAR-TARPs in the PSD in the basal state and during synaptic plasticity? RELEVANCE (See instructions): We propose a combination of computational and experimental work that will help clarify the role of synGAP in regulation of AMPARs in CNS synapses, including its role in mental illness. The work will impact a specific medical condition termed “SynGAP haploinsufficiency” or “MRD5”, in which SynGAP is mutated in ~1% of children with sporadic non-syndromic cognitive disability accompanied by autism and/or epilepsy. The medical impacts of this work are potentially quite significant as it could help to point toward specific molecular interventions with therapeutics that could improve the lives of patients with these afflictions.