Determining the ultrastructural differences between dually and singly innervated dendritic spines and their changes following glutamate excitotoxicity using Cryo-Electron Tomography

使用冷冻电子断层扫描确定双重和单神经支配的树突棘之间的超微结构差异及其在谷氨酸兴奋性毒性后的变化

• 批准号: 10679214
• 负责人: Erik David Anderson
• 金额: $ 4.77万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2025-09-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679214
• 关键词: 3-Dimensional; Actins; Action Potentials; Affect; Age; Alzheimer' s Disease; Axon; Calcium; Cells; Cryo-electron tomography; Cryoelectron Microscopy; Cytoskeleton; Dendrites; Dendritic Spines; Diffusion; Dose; Excitatory Postsynaptic Potentials; Excitatory Synapse; Fluorescence; Fluorescence Microscopy; Glutamates; Head and neck structure; Image; Inhibitory Synapse; Injury; Ions; Learning; Macromolecular Complexes; Membrane; Memory; Microscopy; Modeling; Molecular; Morphology; Neck; Necrosis; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Neuronal Injury; Neurons; Process; Rattus; Research; Research Personnel; Resolution; Resources; Role; Secondary Protein Structure; Shock; Spatial Distribution; Stroke; Structure; Synapses; Techniques; Training; Transfection; Vertebral column; Visualization; age related; chemical fixation; excitotoxicity; interest; nanometer resolution; neural circuit; neuron apoptosis; prevent; protective effect; therapeutic target

PROJECT SUMMARY Glutamate excitotoxicity causes neuronal apoptosis and necrosis in a myriad of age-associated neurologic conditions such as stroke and Alzheimer’s disease. High dose glutamate stimulation of neurons causes a rapid loss of dendritic spines (DS), membranous protrusions that bud off dendrites. DS are critical for learning and memory, but the structural changes that result in this loss remain poorly understood due to their small size. Up to 10% of DS are dually innervated with inhibitory synapses (DiDS) and found to be more stable than singly innervated DS (SiDS) containing only an excitatory synapse. A process termed compartmentalization is also considered key to DS stability, whereby mature spines with large heads and narrow necks restrict molecules and ions from diffusion into and out of the dendrite. Recent evidence suggests an actin diffusion barrier within the DS neck and head-neck junction could be key to compartmentalization, but this remains poorly understood. Following excitation calcium influx causes actin network remodeling that drives DS morphologic change. Inhibitory synapses on DiDS have been found to dampen excitatory post synaptic potentials and calcium influx, and upwards of 86% contain a spine apparatus. Based on these findings, I hypothesize following glutamate excitotoxicity DiDS maintain a more stable DS structure than SiDS. To investigate I will use high resolution cryo-electron tomography paired with correlative fluorescence to compare DiDS and SiDS and elucidate structural changes that result in DS loss following glutamate excitotoxicity. Aim 1 will determine the ultrastructural differences between DiDS and SiDS actin networks under normal conditions. Aim 2 will determine the ultrastructural changes that occur between DiDS and SiDS following glutamate excitotoxicity. If successful, this project would determine what high resolution structural differences exist between DiDS and SiDS and whether DiDS are more stable following excitotoxic shock. This would also provide investigators of excitotoxicity high resolution structural evidence for why inhibitory synapses could be therapeutic targets to prevent neuronal injury.

项目概要 谷氨酸兴奋性毒性导致多种与年龄相关的神经系统中的神经元凋亡和坏死 高剂量的谷氨酸刺激神经元会导致中风和阿尔茨海默氏病等疾病。 树突棘（DS）的丧失，从树突上萌芽的膜状突起对于学习和发育至关重要。 记忆，但由于其体积小，导致这种损失的结构变化仍然知之甚少。 10% 的 DS 受抑制性突触 (DiDS) 双重支配，并且比单一神经支配更稳定 仅包含兴奋性突触的神经支配的 DS (SiDS) 也是一种称为区室化的过程。 被认为是 DS 稳定性的关键，因此具有大头和窄颈的成熟棘限制了分子 最近的证据表明，树突内部存在肌动蛋白扩散屏障。 DS 颈部和头颈连接处可能是分区的关键，但这仍然很差 激发后钙流入会导致肌动蛋白网络重塑，从而驱动 DS 形态。 已发现 DiDS 上的抑制性突触会抑制兴奋性突触后电位和 钙流入，并且超过 86% 含有脊柱装置 根据这些发现，我。 谷氨酸兴奋毒性后，DiDS 比 SiDS 保持更稳定的 DS 结构。 将使用高分辨率冷冻电子断层扫描结合相关荧光来比较 DiDS 和 SiDS 并阐明导致谷氨酸兴奋性毒性后 DS 损失的结构变化。 确定正常条件下 DiDS 和 SiDS 肌动蛋白网络之间的超微结构差异。 2 将确定谷氨酸后 DiDS 和 SiDS 之间发生的超微结构变化 如果成功，该项目将确定存在哪些高分辨率结构差异。 DiDS 和 SiDS 之间的关系以及 DiDS 在兴奋性毒性休克后是否更稳定。 为兴奋性毒性研究人员提供高分辨率结构证据，说明为什么抑制性突触可能是 预防神经损伤的治疗目标。