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.

通过NMDA受体（NMDARS）通过NMDA受体（NMDAR）的谷氨酸信号在突触塑料，兴奋性毒性和 许多退化性和认知障碍。 亚基和两个值得注意的glun2a-d亚基。 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。 突触传播和突触外受体在细胞死亡，塑料和基因中起着作用 尽管有证据表明亚基特异性NMDAR纳米群 关于突触中特定NMDAR亚基的纳米级分布的信息或相对于站点 因此，囊泡释放。 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。 NMDAR通常是包含glun2a和glun2b的“三分球”受体。 尽管有必要的重要性，但没有任何方法可以明确区分三角体NMDAR 从神经元中的其他亚型中，因此分布和亚细胞贩运仍然神秘。 克服这一点，我设计了一种使用双分子直接可视化triperomeric nmdar的方法 荧光互补（BIFC）I标记Glun2a和Glun2b 仅当组装NMDAR才能产生荧光的蛋白质都会构成 Glun2a和Glun2b亚基（分裂标签的NMDAR）。 验证实验，我将使用标记的NMDAR来解决两个范围但长期存在 围绕NMDAR的问题。 与脚手架分子的一部分突触区域的突触区域，未知哪个亚基 使用我的新探针主导Triperomeric受体的突触整合，我将提供第一个直接 衡量三分态突触富集。 确定突触strengs的稳固性，但三个月室受体的汇率和移动分数 以前无法使用FRAP进行测量 与其他NMDAR相比，显示独特的营业额。 结合了方法论和专业发展的深入而多样的培训，这将使我变得很好 在我的职业生涯中担任生物医学研究的大学学术研究员的职业生涯中的位置。