Imaging triheteromeric NMDAR distribution and trafficking

三异体 NMDAR 分布和贩运成像

基本信息

批准号：
10313352
负责人：
Thomas A Blanpied
金额：
$ 20.64万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10313352
关键词：
Address Adult Alzheimer&apos s Disease Behavior Biochemical Biological Assay Biophysics Brain C-terminal Cell Death Cell membrane Cell surface Cells Cognition Disorders Cognitive Complement Complex Data Development Disease Disease model Drug abuse Electrophysiology (science)Etiology Family Fluorescence Functional disorder Future Gene Expression Glutamate Receptor Glutamates Goals Human Image Individual Intellectual functioning disability Investigation Kinetics Knowledge Learning Life Link Location Maps Measures Mediating Mental disorders Methods Modeling Molecular Mutation N-Methyl-D-Aspartate Receptors N-Methylaspartate NMDA receptor A1 Nerve Degeneration Neuronal Plasticity Neurons Neurosciences Neurotransmitter Receptor Pathologic Peptides Performance Pharmacology Phosphotransferases Physiological Physiology Play Positioning Attribute Probability Property Proteins Research Resolution Role Scaffolding Protein Schizophrenia Series Signal Transduction Site Synapses Synaptic Receptors Synaptic Transmission Synaptic plasticity Tail Transfection Visualization Whole-Cell Recordings Work base biophysical properties brain cell experience experimental study follow-up hippocampal pyramidal neuron imaging approach insight nanoscale neuron loss novel receptor receptor function response single molecule stem tool trafficking transmission process

项目摘要

Activation of NMDA-type glutamate receptors (NMDARs) drives signaling and neuronal plasticity that mediates brain circuit wiring and learning. However, NMDAR overactivation can trigger neuronal cell death and is linked to neurodegeneration, and hypofunction of NMDARs is a leading model of the etiology of schizophrenia and other cognitive disorders. Further, the subsynaptic organization of NMDARs influences the probability of their activation during synaptic transmission, and extrasynaptic receptors are thought to play critical roles in cell death and gene expression. Thus, neurons utilize a variety of mechanisms to control the abundance of NMDARs on the cell surface and particularly in synapses. These mechanisms are complex in part because NMDARs are tetramers, formed from two obligatory GluN1 subunits and two subunits typically from the GluN2 family. Notably, NMDARs with different GluN2 compositions display different biophysical characteristics, and the GluN2 subunits also guide different protein interactions and signaling. Accordingly, different subtypes of NMDARs drive vastly different forms of physiological plasticity and are linked to different disorders. Thus, identifying how neurons control abundance of specific NMDAR subtypes in synapses has been a longstanding goal in neuroscience. Unfortunately, our understanding of these mechanisms has been crucially restricted by inability to visualize one of the key classes of NMDARs in neurons, the triheteromeric receptors, which contain GluN1 and two different GluN2 subunits (most commonly GluN2A+GluN2B). Triheteromeric receptors are thought to be the majority of NMDARs in adult brain, but there are as yet no tools to distinguish them from other NMDAR subtypes in neurons. Thus, their distribution with neurons remains mysterious, and the mechanisms controlling their subcellular trafficking remain almost totally unknown. To overcome this, we here introduce a new tool to visualize triheteromeric NMDARs in neurons. Our strategy is based on bimolecular complementation, and we tagged GluN2A and GluN2B with two parts of a modified fluorescent protein that complement to produce fluorescence only when an NMDAR is assembled containing both the GluN2A and GluN2B subunits (split-tagged NMDARs). Preliminary data demonstrate that split-tagged receptors traffic normally within neurons and accumulate strongly within synapses, and whole-cell recordings demonstrate that their activation by glutamate is unaltered by the presence of the tags. Proceeding from these results, we propose to develop and validate several versions of split-tagged triheteromeric NMDARs useful for different experiments. We will use confocal and super-resolution imaging to provide the first maps of triheteromeric NMDAR distribution in neurons. Finally, because the rate of NMDAR turnover in synapses is critical for determining synaptic strength and plasticity, we will use FRAP and single-molecule tracking to provide the first measures of synaptic exchange of triheteromeric NMDARs. This work will fill longstanding gaps in our knowledge of NMDARs and lay necessary groundwork for investigation of other aspects of triheteromeric NMDAR trafficking in healthy neurons and disease models.

NMDA 型谷氨酸受体 (NMDAR) 的激活驱动信号传导和介导的神经元可塑性脑回路连接和学习。然而，NMDAR 过度激活会引发神经元细胞死亡，并且与神经退行性变和 NMDAR 功能减退是精神分裂症病因学的主要模型其他认知障碍。此外，NMDAR 的突触亚组织会影响其出现的概率。突触传递过程中的激活和突触外受体被认为在细胞死亡中发挥关键作用和基因表达。因此，神经元利用多种机制来控制神经元上 NMDAR 的丰度。细胞表面，特别是突触中。这些机制很复杂，部分原因是 NMDAR 四聚体，由两个必需的 GluN1 亚基和两个通常来自 GluN2 家族的亚基形成。尤其，具有不同GluN2组成的NMDAR表现出不同的生物物理特征，并且GluN2亚基还指导不同的蛋白质相互作用和信号传导。因此，NMDAR 的不同亚型极大地驱动了不同形式的生理可塑性与不同的疾病有关。因此，识别神经元如何控制突触中特定 NMDAR 亚型的丰度一直是神经科学的长期目标。不幸的是，我们对这些机制的理解受到了严重限制，因为我们无法想象其中的一个机制。神经元中 NMDAR 的关键类别，即三异聚受体，其中包含 GluN1 和两种不同的 GluN2 亚基（最常见的是 GluN2A+GluN2B）。三异体受体被认为是大多数 NMDAR 存在于成人大脑中，但目前还没有工具可以将它们与神经元中的其他 NMDAR 亚型区分开来。因此，它们在神经元中的分布仍然是神秘的，控制它们的亚细胞的机制贩运活动仍然几乎完全不为人知。为了克服这个问题，我们在这里引入了一种新的工具来可视化神经元中的三异聚 NMDAR。我们的策略基于双分子互补，我们标记为 GluN2A 和 GluN2B 具有修饰荧光蛋白的两个部分，互补产生荧光仅当组装的 NMDAR 包含 GluN2A 和 GluN2B 亚基（分割标记的 NMDAR）时。初步数据表明，分裂标记受体在神经元内正常流通并强烈积累在突触内，全细胞记录表明，谷氨酸对突触的激活不会因标签的存在。根据这些结果，我们建议开发和验证几个版本分裂标记的三异聚 NMDAR 可用于不同的实验。我们将使用共焦和超分辨率成像提供了神经元中三异体 NMDAR 分布的第一张图。最后，因为速率突触中的 NMDAR 周转对于确定突触强度和可塑性至关重要，我们将使用 FRAP 和单分子追踪提供了三异体 NMDAR 突触交换的第一个测量。这这项工作将填补我们对 NMDAR 知识的长期空白，并为研究健康神经元和疾病模型中三异体 NMDAR 贩运的其他方面奠定必要的基础。