Dynamic Structural Properties of Synapses

突触的动态结构特性

• 批准号: 10708615
• 负责人: Thomas S Reese
• 金额: $ 125.25万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10708615
• 关键词: 3-Dimensional; A kinase anchoring protein; AMPA Receptors; Acidic Region; Animals; Antibodies; Behavior; Behavior Control; Binding Sites; Brain; CRISPR/Cas technology; Cell Differentiation process; Cell physiology; Cells; Chemical Synapse; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Colorado; Complex; Cyclic AMP-Dependent Protein Kinases; Data; Electrophysiology (science); Endosomes; Evolution; Filament; Freeze Substitution; Genetic; Glean; Glutamate Receptor; Glutamates; Goals; Gravity Perception; Hippocampus (Brain); Image; Immunoelectron Microscopy; Individual; Knock-in; Knock-out; Label; Length; Light; Link; Location; Macromolecular Complexes; Maps; Measurement; Membrane; Memory; Methods; Microtomy; Modeling; Molecular; Molecular Conformation; Molecular Machines; Morphologic artifacts; Muscle; NMDA receptor A1; National Institute of Neurological Disorders and Stroke; Nerve; Nervous System; Neurons; Neuropeptides; Neurophysiology - biologic function; Neurotransmitters; Organelles; Organism; Peroxides; Phosphotransferases; Plastic Embedding; Population; Positioning Attribute; Problem Solving; Process; Property; Proteins; Publications; Publishing; Recycling; Regulation; Research; Resolution; Role; Scaffolding Protein; Scanning; Secretory Cell; Shapes; Signal Pathway; Signal Transduction; Site; Structural Models; Structure; Synapses; Synaptic Transmission; Synaptic plasticity; System; Thick; Tomogram; Universities; Vertebral column; Water; Work; calmodulin-dependent protein kinase II; cell type; density; information processing; insight; intercellular communication; interest; light microscopy; macrophage; molecular dynamics; nanobodies; nanocluster; neuroregulation; overexpression; palmitoylation; postsynaptic; postsynaptic density protein; presynaptic density protein 95; protein complex; receptor binding; reconstruction; success; synaptic function; tomography; trafficking

The postsynaptic density (PSD) at excitatory glutamatergic synapses is a large molecular machine that is known to be a key site of memory, information processing, and storage. To map the molecular organization of the PSD, we freeze-substitute hippocampal cultures and examine them in plastic embedded sections by EM tomography. This reveals individual protein complexes within the PSD. Our early tomography work revealed that the core of the PSD is an array of membrane-associated, vertically oriented filaments like PSD-95. This finding provided insight into the overall organization of the PSD. For instance, scaffolding proteins like PSD-95 have multiple common binding sites arrayed along their length, such that, regular arrays of vertically oriented PSD-95 filaments impose order on other PSD proteins, including the glutamate receptors, and provide an overall plan for the core structure of the PSD. The projects outlined below use and refine our fundamental insight into the organization of synaptic structures to explore the dynamics of molecular changes in the PSD and understand how these molecules contribute to synaptic function. We have several lines of ongoing research and collaboration to investigate specific synaptic proteins. Recently, we collaborated with Rumbaugh Lab to characterize the structural role of SynGAP, which negatively regulates the glutamate AMPA receptor binding to the PDZ domain of PSD-95 at the PSD. We have used immunoEM to map the location, orientation, and conformation of SynGAP at the PSD to arrive at a structural model of how SynGAP might regulate and control synaptic excitability. In collaboration with the Roger Nicoll Lab, we are studying the effects of overexpressing constitutively activated CaMKII on synaptic structure and function. To ameliorate potential artifacts due to overexpressing of CaMKII, we are also using a newly developed CaMKII CRISPR knock-in construct which allows expression and localization of endogenous CaMKIIs in neurons. Electrophysiology measurements show that activated CaMKII expression enhances synaptic transmission, and we plan to analyze changes in spine sizes and PSD structure, using serial section EM or thick section STEM tomography. We are finishing up the work directly identifying NMDARs in the PSD in intact hippocampal synapses by using CRISPR-Cas9 construct developed in the Nicoll Lab. The knockout eliminates the required GluN1 subunit of NMDARs. We made 3Dreconstructions of the resulting PSDs with dark field scanning EM tomography. As result, we now have evidence that individual NMDARs and AMPARs can be identified by EM tomography, and their organization and connections with other molecules can be delineated. We are in the process of finishing up this work and preparing it for publication. Also, we just published a major study with M. DellAcqua's lab at the University of Colorado on the conformations and distribution of Anchoring Proteins (AKAPs) in the hippocampal synapse. In this work, we used immunoEM on thick sections and visualized them using STEM tomography. The results showed palmitoylation effects on AKAP150/79 membrane organization, trafficking, and mobility. Membrane-associated AKAPs are known to interact with PSD-95 MAGUKs and anchor several classes of kinases (PKA and PKC) important for synaptic plasticity (LTP and LTD). This work demonstrates that there is a conformational change in AKAPs in the PSD, different than that at the extrasynaptic membrane, and this distinction may have important functional implications in understanding the role of AKAPs in regulating AMPARs at the PSDs. We also found extensive AKAP association with recycling endosomes and that depalmitolylation appeared to diminish such association. This project opened an exciting new front in imaging AMPAR trafficking. EM tomography has allowed the creation of 3D reconstructions to delineate the organization of subsynaptic organelles, key synaptic proteins, and macromolecular complexes at synapses. Reconstructions provide the size, shape, and location of structures at 2-4 nm resolution but cannot guarantee unambiguous molecular identification of the individual structures. While we had success using immunogold to label endogenous and overexpressed GFP-tagged PSD-95, the large antibody complexes that also manifest as filamentous structures in tomograms confound the identification of the target PSD proteins. Now we have a major technical breakthrough with APEX2 and nanobody labeling to solve this problem. In one localization project, we are using the genetic tag APEX2 to localize CaMKII. The CaMKII-APEX2 construct in the presence of DAB and peroxide has revealed individual CaMKIIs. We are studying CaMKII in the spine and membrane in basal and high K stimulated conditions by EM tomography, and we are in the process of finishing up the work and preparing a publication. In the meantime, we are expanding the APEX2 work to Shank and Homer. Furthermore, another localization project uses an advance in nanobody labeling. In this parallel method, we developed a method to use nanobodies with EM tomography to directly identify PSD proteins in spines. This is a swift method, and we are expecting exciting new results in the coming years on a slew of synaptic molecules. Recently, a major issue has cropped up in the field regarding PSD-95. Several super-resolution light microscopy studies have suggested that PSD-95 forms 100 nm subsynaptic nanoclusters at the PSD. This is significantly smaller than the average size of a PSD, yet we considered PSD-95 to be uniformly distributed. Currently, we are re-examining the endogenous PSD-95 distribution at the PSD with thick section tomography to further study PSD-95 distribution and clustering at the PSD. We are also further analyzing the distribution of all vertical filaments in tomograms using the methods outlined above to see if any clustering of the vertical filaments ever exists within the PSD and if we can reconcile the super-resolution findings with immuno-labeling and tomographic EM. Outside our work in mammalian neurons, an ongoing collaboration with Carolyn Smith in the NINDS Light Microscopy Facility and Adriano Senatore (University of Toronto Mississauga) sheds light on the evolution of cell types and pre-neural regulations in a primitive animal, Trichoplax. Although lacking muscles, nerves, and synapses, Trichoplax demonstrates different types of behaviors indicative of neural function. We identified a cell that senses gravity and described the consequence of stages, which occurs during the differentiation of this cell. We also described a population of cells functioning as macrophages. Our collaborative effort provided evidence that Trichoplax can sense the pH of ambient water and demonstrates avoidance of low pH (acidic) regions. However, the regulatory mechanisms as well as the ways Trichoplax cells communicate with each other are not yet clear. Our previous results and elsewhere obtained data showed that this organism utilizes neuropeptide signaling pathways dependent on many of the same proteins found at synapses in higher animals. In our next steps, we will characterize different types of secretory cells in Trichoplax, which presumably regulate and integrate functional activities of the cells in the nerveless animal. Knowing exactly how these unconventional, nonsynaptic systems function to control behaviors is expected to provide previously overlooked information on non-synaptic signaling mechanisms in mammalian brains.

兴奋性谷氨酸突触的突触后密度（PSD）是一个大型分子机器，被认为是记忆、信息处理和存储的关键部位。为了绘制 PSD 的分子组织图，我们冷冻替代海马培养物，并通过 EM 断层扫描在塑料嵌入切片中检查它们。这揭示了 PSD 内的单个蛋白质复合物。我们早期的断层扫描工作表明，PSD 的核心是一系列与膜相关的垂直定向细丝，如 PSD-95。这一发现提供了对 PSD 整体组织的深入了解。例如，像 PSD-95 这样的支架蛋白具有沿其长度排列的多个常见结合位点，因此，垂直方向的 PSD-95 细丝的规则阵列对其他 PSD 蛋白（包括谷氨酸受体）施加了顺序，并为PSD 的核心结构。下面概述的项目利用并完善了我们对突触结构组织的基本见解，以探索 PSD 中分子变化的动态，并了解这些分子如何促进突触功能。 我们正在进行多项研究和合作，以研究特定的突触蛋白。最近，我们与 Rumbaugh 实验室合作表征了 SynGAP 的结构作用，它负向调节谷氨酸 AMPA 受体在 PSD 处与 PSD-95 的 PDZ 结构域的结合。我们使用immunoEM绘制了SynGAP在PSD处的位置、方向和构象图，以得出SynGAP如何调节和控制突触兴奋性的结构模型。我们与 Roger Nicoll 实验室合作，研究过表达组成型激活的 CaMKII 对突触结构和功能的影响。为了改善由于 CaMKII 过度表达而导致的潜在伪影，我们还使用新开发的 CaMKII CRISPR 敲入构建体，该构建体允许内源性 CaMKII 在神经元中表达和定位。电生理学测量表明，激活的 CaMKII 表达可增强突触传递，我们计划使用连续切片 EM 或厚切片 STEM 断层扫描来分析脊柱大小和 PSD 结构的变化。 我们正在完成使用 Nicoll 实验室开发的 CRISPR-Cas9 构建体直接识别完整海马突触 PSD 中 NMDAR 的工作。敲除消除了 NMDAR 所需的 GluN1 亚基。我们使用暗场扫描 EM 断层扫描对生成的 PSD 进行了 3D 重建。因此，我们现在有证据表明，可以通过电磁断层扫描识别单个 NMDAR 和 AMPAR，并且可以描绘它们的组织以及与其他分子的联系。我们正在完成这项工作并准备出版。此外，我们刚刚与科罗拉多大学 M. DellAcqua 实验室合作发表了一项关于海马突触中锚定蛋白 (AKAP) 的构象和分布的重大研究。在这项工作中，我们在厚切片上使用了免疫电镜，并使用 STEM 断层扫描对其进行可视化。结果显示棕榈酰化对 AKAP150/79 膜组织、运输和流动性有影响。已知膜相关 AKAP 与 PSD-95 MAGUK 相互作用，并锚定对突触可塑性（LTP 和 LTD）很重要的几类激酶（PKA 和 PKC）。这项工作表明，PSD 中的 AKAP 存在构象变化，与突触外膜上的构象变化不同，这种区别对于理解 AKAP 在调节 PSD 中 AMPAR 的作用可能具有重要的功能意义。我们还发现 AKAP 与循环内体有广泛的关联，而去棕榈酰化似乎会减弱这种关联。该项目在 AMPAR 贩运成像方面开辟了令人兴奋的新前沿。 EM 断层扫描可以创建 3D 重建来描绘突触下细胞器、关键突触蛋白和突触大分子复合物的组织。重建以 2-4 nm 分辨率提供结构的大小、形状和位置，但不能保证单个结构的明确分子识别。虽然我们成功地使用免疫金来标记内源性和过度表达的 GFP 标记的 PSD-95，但在断层扫描中也表现为丝状结构的大型抗体复合物混淆了目标 PSD 蛋白的识别。现在我们通过 APEX2 和纳米抗体标记取得了重大技术突破来解决这个问题。 在一个本地化项目中，我们使用遗传标签 APEX2 来本地化 CaMKII。在 DAB 和过氧化物存在下的 CaMKII-APEX2 构建体揭示了单个 CaMKII。我们正在通过 EM 断层扫描研究基础和高 K 刺激条件下脊柱和膜中的 CaMKII，我们正在完成这项工作并准备出版物。与此同时，我们正在将 APEX2 工作扩展到 Shank 和 Homer。 此外，另一个本地化项目使用了纳米抗体标记的先进技术。在这种并行方法中，我们开发了一种使用纳米抗体和 EM 断层扫描直接识别脊柱中的 PSD 蛋白的方法。这是一种快速的方法，我们期待在未来几年在大量突触分子上取得令人兴奋的新结果。 最近，该领域出现了一个有关 PSD-95 的重大问题。多项超分辨率光学显微镜研究表明，PSD-95 在 PSD 处形成 100 nm 的突触亚纳米簇。这明显小于 PSD 的平均大小，但我们认为 PSD-95 是均匀分布的。目前，我们正在利用厚切片断层扫描重新检查 PSD 处的内源性 PSD-95 分布，以进一步研究 PSD 处的 PSD-95 分布和聚类。我们还使用上述方法进一步分析断层图像中所有垂直丝的分布，看看 PSD 中是否存在任何垂直丝的聚集，以及我们是否可以将超分辨率发现与免疫标记和断层扫描 EM 相协调。 除了我们在哺乳动物神经元方面的工作之外，与 NINDS 光学显微镜设施的 Carolyn Smith 和 Adriano Senatore（多伦多大学密西沙加分校）正在进行的合作揭示了原始动物 Trichoplax 的细胞类型和前神经调节的进化。尽管缺乏肌肉、神经和突触，丝盘菌却表现出指示神经功能的不同类型的行为。我们鉴定了一种能够感知重力的细胞，并描述了该细胞分化过程中发生的各个阶段的结果。我们还描述了一群具有巨噬细胞功能的细胞。我们的合作提供了证据，证明 Trichoplax 可以感知环境水的 pH 值，并证明可以避免低 pH（酸性）区域。然而，调节机制以及毛盘细胞彼此通讯的方式尚不清楚。我们之前的结果和其他地方获得的数据表明，这种生物体利用神经肽信号传导途径，依赖于高等动物突触中发现的许多相同蛋白质。在下一步中，我们将描述毛盘菌中不同类型的分泌细胞的特征，这些细胞可能调节和整合无神经动物细胞的功能活动。准确地了解这些非常规的非突触系统如何发挥控制行为的作用，有望提供以前被忽视的有关哺乳动物大脑中非突触信号机制的信息。