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

快速谷氨酸能突触传递建立在精确而复杂的分子组织基础上， 需要控制突触后部位的AMPA型谷氨酸受体(AMPAR)的数量 树突棘上的谷氨酸能突触。AMPAR的数量随Pre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的数目及其通过突触生化机制的反应特性 可塑性。通过这种方式，信息存储在大脑中。这个项目的总体目标是使用量化 关于丰富的突触后突触作用的两个基本问题的模型和实验 蛋白SynGAP，参与AMPAR数目的调节。在完整神经元中进行的大量实验已经 研究发现，突触表达的SynGAP水平与突触数量呈负相关 突触上有AMPAR，而SynGAP有助于调节AMPAR数量的变化 在突触可塑性过程中。SynGAP的酶促GAP结构域起棘轮的作用，调节 在树枝晶表面添加和去除AMPAR。SynGAP还包含一个绑定 通过其三个蛋白结合的PDZ结构域与主要支架蛋白PSD-95紧密结合。重要的是 NIMH的精神健康使命，SynGAP在大脑和大脑的学习和记忆中发挥着关键作用 SynGAP突变与认知障碍有关。该项目分为两大目标。在AIM 1，我们将回答这个问题：SynGAP通过什么机制控制AMPA的量 突触后密度(PSD)中的受体-通过控制表面数量和/或通过控制可获得性 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之间通过将其整合到我们的模型中来竞争绑定到PSD-95 安帕尔贩卖。我们将利用遗传学和复杂的分子工程来进行实验 将这两种机制分开。AMPAR对纳米组织的影响将通过超 分辨荧光显微镜和电生理学。这些实验的结果将被用于 限制我们的AMPAR贩运模式。目标2，通过实验和计算的协同作用 方法，我们将解决这些问题：SynGAP之间的凝析油是如何形成的 和PSD-95，以及存在额外的PDZ结构域结合蛋白(GluN2受体亚单位， 神经连接素、nNOS、CRIPT等)基质对PSD中AMPAR-TARPS纳米结构的影响 在突触可塑性的状态下？ 相关性(请参阅说明)： 我们提出了一个计算和实验相结合的工作，这将有助于阐明SynGAP的作用 在调节中枢神经系统突触中的AMPAR，包括它在精神疾病中的作用。这项工作将对 称为“SynGAP单倍体功能不全”或“MRD5”的特定疾病，其中SynGAP突变在 约1%的散发性非综合征性认知障碍儿童伴有自闭症和/或癫痫。 这项工作的医学影响可能是相当重大的，因为它可能有助于指出具体的 分子干预疗法可以改善这些疾病患者的生活。

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.