Molecular Mechanisms Underlying PSD-MAGUK/NMDA Receptor Interactions

描述(申请人提供)：我的建议的总体目标是了解谷氨酸受体，特别是NMDAR(NMDAR)与突触支架蛋白相互作用的分子机制，以及这些相互作用如何塑造突触传递。具体地说，我将研究突触后密度-95样膜相关鸟氨酸激酶(PSD-MAGUK)蛋白家族，特别是SAP97蛋白是如何运输NMDA受体亚基并改变其生理功能的。PSD-MAGUKs和NMDA受体在基础突触传递和学习记忆中起关键作用，并与多种神经疾病有关，从自闭症、精神分裂症等发育障碍到阿尔茨海默氏症等退行性疾病。我的研究目标概括为两个特定目标：特定目标1：SAP97控制AMPA和NMDA受体的运输和突触形态。我假设SAP97在早期发育过程中将AMPA和NMDAR运输到突触，并特别促进含有GluN2A的NMDAR。其次，我假设SAP97介导的信号也控制发育中神经元的树突和突触形态。我将在体内操纵SAP97蛋白水平，并使用电生理学和共聚焦成像来测量该蛋白在突触传递和神经元解剖中的作用。具体目标2：PSD-MAGUK的分子差异是NMDAR动力学和亚基转换的基础。首先，我假设PSD-93、-95和SAP97共有的特定蛋白结合域促进含有GluN2A的NMDARs的突触运输，而SAP102中的不同基序促进含有GluN2B的受体。其次，我假设PSD-MAGUK也直接影响NMDAR的生理学，每个PSD-MAGUK与NMDAR有不同的相互作用，并塑造突触电流。我将在体内设计并在NMDAR亚单位条件性基因敲除小鼠中过表达PSD-Maguk嵌合蛋白，并利用电生理学测量对NMDAR的影响。我还将使用异源表达系统来测量这些蛋白质之间的直接相互作用。因此，我将定义负责PSD-Maguk/NMDAR相互作用的蛋白质结构域，以及这些相互作用如何改变NMDAR生理。这些实验对一个重要的科学问题采取了多维的方法，结合了尖端的分子遗传学、生理学和解剖学技术，并将加强我们对突触传递和学习记忆的基本分子机制的理解。 与公共健康相关：我的实验研究了谷氨酸受体和在突触组织它们的蛋白质家族之间的相互作用。这些研究将揭示谷氨酸能突触传递的基本机制，这是神经信号的主要形式，以及突触可塑性，突触可塑性是学习和记忆的细胞基础。突触传递障碍是大多数神经系统疾病的症状。因此，我的结果将对基础科学家和临床医生都有意义，并将为未来学习和记忆以及缓解各种神经疾病的遗传原因和药物疗法的研究指明方向。

DESCRIPTION (provided by applicant): The overall objectives of my proposal are to understand the molecular mechanisms by which glutamate receptors, particularly NMDA receptors (NMDARs), interact with synaptic scaffolding proteins and how these interactions shape synaptic transmission. Specifically, I will study how the postsynaptic density-95-like membrane associated guanylate kinase (PSD-MAGUK) protein family, in particular the protein SAP97, traffic NMDA receptors subunits and change their physiology. PSD-MAGUKs and NMDA receptors play a critical role in basal synaptic transmission and learning and memory, and have been implicated in a wide variety of neurological diseases, ranging from developmental disorders such as autism, schizophrenia, to degenerative diseases such as Alzheimer's. My research goals are outlined in two Specific Aims: Specific Aim 1: SAP97 controls AMPA and NMDA receptor trafficking and synaptic morphology. I hypothesize that SAP97 traffics AMPA and NMDARs to synapses during early development and specifically promotes GluN2A-containing NMDARs. Second, I hypothesize that SAP97-mediated signaling also controls dendrite and synapse morphology in developing neurons. I will manipulate SAP97 protein levels in vivo and use electrophysiology and confocal imaging to measure the role of this protein in synaptic transmission and neuronal anatomy. Specific Aim 2: Molecular differences in PSD-MAGUKs underlie NMDAR kinetics and subunit switching. First, I hypothesize that specific protein binding domains shared by PSD-93, -95, and SAP97 promote synaptic trafficking of GluN2A-containing NMDARs while different motifs in SAP102 promote GluN2B- containing receptors. Second, I hypothesize that PSD-MAGUKs also directly influence NMDAR physiology, with each PSD-MAGUK differentially interacting with NMDARs and shaping synaptic currents. I will design and overexpress chimeric PSD-MAGUK proteins in vivo, in NMDAR subunit conditional knockout mice, and measure the effect on NMDARs using electrophysiology. I will also use a heterologous expression system to measure direct interactions between these proteins. Thus, I will define the protein domains responsible for PSD-MAGUK/NMDAR interactions and how these interactions alter NMDAR physiology. These experiments take a multi-dimensional approach to a vital scientific question, combining cutting edge molecular genetic, physiologocial, and anatomical techniques and will enhance our understanding of fundamental molecular mechanisms of synaptic transmission and learning and memory. PUBLIC HEALTH RELEVANCE: My experiments study the interactions between glutamate receptors and the family of proteins that organize them at synapses. These studies will uncover fundamental mechanisms of glutamatergic synaptic transmission, the major form of neural signaling, and synaptic plasticity, the cellular basis of learning and memory. Deficits in synaptic transmission are symptomatic of most neurological diseases. Thus, my results will be relevant both to basic scientists and to clinicians and will guide the way for future studies of learning and memory and the genetic causes of and pharmacological therapies for the alleviation of a variety of neurological diseases.