Imaging triheteromeric NMDAR distribution and trafficking

三异体 NMDAR 分布和贩运成像

• 批准号: 10434923
• 负责人: Thomas A Blanpied
• 金额: $ 19.27万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 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/10434923
• 关键词: Address; Adult; Alzheimer' 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

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神经元可塑性，介导 脑电路接线和学习。但是，NMDAR过度活化会触发神经元细胞死亡，并连接 对于神经变性，NMDAR的功能障碍是精神分裂症的病因的主要模型 其他认知障碍。此外，NMDAR的突触次触及组织会影响其的可能性 突触传播期间的激活和外突触受体被认为在细胞死亡中起关键作用 和基因表达。因此，神经元利用多种机制来控制NMDAR的丰度 细胞表面，尤其是突触。这些机制很复杂，部分原因是NMDAR是 四聚体由两个强制性的Glun1亚基和两个亚基形成，通常来自Glun2家族。尤其， 具有不同GLUN2组成的NMDAR显示出不同的生物物理特性，而Glun2亚基 还指导不同的蛋白质相互作用和信号传导。因此，NMDAR的不同亚型驱动 不同形式的生理可塑性，与不同的疾病有关。因此，确定神经元如何 突触中特定的NMDAR亚型的控制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至关重要地限制 在神经元中NMDAR的关键类别中，含有glun1和两个不同的三个地分体受体 glun2亚基（最常见的是glun2a+glun2b）。三分态受体被认为是大多数 成人大脑中的NMDAR，但尚无将其与神经元中其他NMDAR亚型区分开的工具。 因此，它们在神经元中的分布仍然神秘，并且控制其亚细胞的机制 贩运几乎仍然是完全未知的。为了克服这一点，我们在这里引入了一个新工具来可视化 神经元中的三个月室NMDAR。我们的策略是基于双分子互补的，我们标记了 glun2a和glun2b，带有改性荧光蛋白的两个部分，它们补充可产生荧光 只有在组装NMDAR时，同时包含glun2a和glun2b亚基（分裂标记的NMDAR）。 初步数据表明，分裂标记的受体在神经元内通常会流量，并强烈积累 在突触中，整个细胞记录表明，谷氨酸的激活并没有改变 标签的存在。从这些结果开始，我们建议开发和验证多个版本的 分裂标签的三分态NMDAR对不同的实验有用。我们将使用共聚焦和超分辨率 成像以提供神经元中三分态NMDAR分布的第一张地图。最后，因为 突触中的NMDAR更新对于确定突触强度和可塑性至关重要，我们将使用FRAP和 单分子跟踪以提供三分体NMDAR的突触交换的首次度量。这 工作将填补我们对NMDAR的知识的长期空白，并为健康的神经元和疾病模型中的三分态NMDAR贩运的其他方面提供必要的基础。