Subsynaptic organization of the major NMDAR subtypes

主要 NMDAR 亚型的突触亚组织

• 批准号: 10620768
• 负责人: Michael C. Anderson
• 金额: $ 4.36万
• 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: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10620768
• 关键词: Address; Affect; Area; Biochemical; Biomedical Research; Brain; Brain region; Cell Death; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Cognition Disorders; Color; Complement; DLG4 gene; DNA; Darkness; Data; Degenerative Disorder; Development; Distant; Epilepsy; Event; Exhibits; Family; Fluorescence; Gene Expression; Genes; Glutamate Receptor; Glutamates; Hippocampus; Image; Kinetics; Knock-in; Knowledge; Label; Learning; Life; Link; Location; Maps; Measures; Mediating; Memory; Mental Depression; Methodology; Methods; Microscopy; Modeling; Molecular; Mood Disorders; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; NMDA receptor A1; Nerve Degeneration; Neuronal Plasticity; Neurons; Pathology; Peptides; Physiological; Play; Population; Positioning Attribute; Probability; Property; Proteins; Receptor Activation; Regulation; Reporting; Research; Research Personnel; Role; Scaffolding Protein; Schizophrenia; Series; Signal Transduction; Site; Stimulus; Synapses; Synaptic Receptors; Synaptic Transmission; Synaptic plasticity; Time; Training; Transfection; Universities; Validation; Vesicle; Visualization; Work; career; density; design; excitotoxicity; experimental study; glutamatergic signaling; insight; interest; nano; nanobodies; nanocluster; nanometer resolution; nanoscale; novel; novel strategies; postsynaptic; presynaptic; receptor; receptor function; scaffold; superresolution imaging; trafficking; vesicular release

Glutamatergic signaling via NMDA receptors (NMDARs) is critical in synaptic plasticity, excitotoxicity, and numerous degenerative and cognitive disorders. Each NMDAR comprises four subunits: two obligatory GluN1 subunits and two GluN2A-D subunits. Notably, NMDARs with different subunit compositions display different channel kinetics and protein interactions, supporting the widely held notion that subunit composition dictates NMDAR function. The subsynaptic organization of NMDARs influences the probability of their activation during synaptic transmission, and extrasynaptic receptors are thought to play roles in cell death, plasticity and gene expression. However, despite evidence for subunit-specific NMDAR nanoclustering, to date there is almost no information about the nanoscale distribution of specific NMDAR subunits within synapses or with respect to sites of vesicular release. Thus, to elucidate the organization of synaptic and extrasynaptic NMDARs with respect to key postsynaptic molecules and presynaptic release sites, in the first aim of my project, I will use a combination of CRISPR-mediated gene tagging and nanobody labeling with DNA-Exchange PAINT super-resolution imaging. These measures will provide a comprehensive map of major NMDAR subunits and provide insight into their roles in neurons. Next, though many NMDARs contain GluN1 and two identical GluN2 subunits, in many brain regions, NMDARs commonly are “triheteromeric” receptors containing both GluN2A and GluN2B with GluN1. However, despite their deduced importance, there are no methods to unambiguously distinguish triheteromeric NMDARs from other subtypes in neurons, and thus their distribution and subcellular trafficking remain mysterious. To overcome this, I have designed a method for direct visualization of triheteromeric NMDARs using bimolecular fluorescent complementation (BiFC). I 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). In the second aim of my proposal, following a series of validation experiments, I will use these split-tagged NMDARs to address two straightforward but longstanding questions surrounding NMDARs. First, though GluN2A and GluN2B traffic with different dynamics and target subsynaptic regions in part through differing interactions with scaffolding molecules, it is unknown which subunit dominates synaptic integration of triheteromeric receptors. Using my new probes, I will provide the first direct measure of triheteromeric synaptic enrichment. Second, the rate of NMDAR turnover in synapses is critical for determining synaptic strength and plasticity, yet the exchange rate and mobile fraction of triheteromeric receptors have not previously been possible to measure. Using FRAP, I will time determine if triheteromeric NMDARs display unique turnover compared to other NMDARs. Completion of this work will provide a training plan that combines deep and diverse training in methodologies and professional development that will leave me well- positioned to excel in my career as an academic researcher at a biomedical research-oriented university.

NMDA受体（NMDAR）的谷氨酸能信号传导在突触可塑性，兴奋性毒性和 许多退化和认知障碍。每个NMDAR都包含四个亚基：两个强制性的Glun1 亚基和两个glun2a-d亚基。值得注意的是，具有不同亚基组成的NMDAR显示不同 通道动力学和蛋白质相互作用，支持亚基组成的广泛持有的观念 NMDAR功能。 NMDAR的次突触组织会影响其激活期间激活的可能性 突触传播和突触外受体被认为在细胞死亡，可塑性和基因中起着作用 表达。但是，dospite证据表明亚基特异性NMDAR纳米群集，迄今为止几乎没有 有关突触中特定NMDAR亚基的纳米级分布的信息 囊泡释放。这是为了阐明有关突触和突触外NMDAR的组织 关键的突触后分子和突触前释放点，在我的项目的第一个目的中，我将使用一个组合 用DNA交换涂料超分辨率成像的CRISPR介导的基因标记和纳米型标记。 这些措施将为主要的NMDAR亚基提供全面的地图，并洞悉其角色 在神经元中。接下来，尽管许多NMDAR都包含Glun1和两个相同的Glun2亚基，但在许多大脑区域中，但 NMDAR通常是包含glun2a和glun2b的“三分球”受体。然而， 尽管它们的重要性被推论出来，但没有任何方法可以明确区分三个月室NMDAR 从神经元中的其他亚型中，它们的分布和亚细胞贩运仍然神秘。到 克服了这一点，我设计了一种使用双分子直接可视化三分态NMDAR的方法 荧光完成（BIFC）。我用修改的荧光灯标记了Glun2a和Glun2b 仅当NMDAR组装在一起时，蛋白质才能产生荧光 glun2a和glun2b亚基（分开标记的NMDAR）。在我的提议的第二个目标中，跟随一系列 验证实验，我将使用这些拆分标签的NMDAR来解决两个直接但长期存在的 围绕NMDAR的问题。首先，尽管具有不同动态和目标的Glun2a和Glun2b流量 突触区域部分通过区分与脚手架分子的相互作用，是未知的亚基 主导三位异构体受体的突触整合。使用我的新问题，我将提供第一个直接 三分机突触富集的度量。其次，突触中的NMDAR周转率对于 确定突触强度和可塑性，但汇率和移动率的分数 以前没有可能测量。使用FRAP，我会时间确定三分态NMDARS是否 与其他NMDAR相比，显示独特的营业额。这项工作的完成将提供一个培训计划 结合了方法和专业发展的深入而多样的培训，这将使我变得很好 在我在生物医学研究的大学中担任学术研究员的职业生涯中，他的职业生涯脱颖而出。