Structural and Biological Characterization of Diverse Oligomers Derived from Abeta

项目摘要/摘要：了解β-淀粉样多肽(Aβ)聚集形成毒性 寡聚体是了解阿尔茨海默病(AD)分子基础的基础。尽管几十年来 研究表明，Aβ寡聚体的结构仍然是一个谜，这构成了对AD的理解的一个重大差距。 这项建议寻求通过结构、生物物理和生物概况来解决这一知识鸿沟 一组不同的Aβ寡聚体模型及其与生物源Aβ寡聚体的相关性。 我的实验室已经开发了一种方法来创建结构定义的Aβ齐聚物模型，该模型包括 来自Aβ的多肽片段被限制在β发夹中。这些Aββ-的X-射线晶体结构 发夹型多肽揭示了多肽可以形成的低聚物的结构和关键的分子间联系 这些多肽在低聚状态下产生。这些联系人显示可以通过交叉链接创建的站点 共价稳定的Aβ低聚物模型，模拟晶体低聚物。研究交联剂 然后，低聚物允许低聚物结构与生物物理和生物学特性之间的详细关联。 我们将描述我们的Aβ寡聚体模型如何与神经元、小胶质细胞和星形胶质细胞相互作用并影响它们。 提供我们的Aβ寡聚体模型如何影响不同类型的脑细胞从而帮助脱脂的详细见解 Aβ寡聚体结构与AD细胞事件之间的关系。我们将使用 荧光显微镜和荧光辅助细胞分选(FACS)以可视化和量化相互作用 以及我们的Aβ寡聚体模型与神经元、小胶质细胞和星形胶质细胞的摄取。我们将对下游进行评估 β寡聚体模型对神经元、小胶质细胞和星形胶质细胞的生化和细胞效应 评估治疗如何影响细胞凋亡、坏死、钙稳态、内质网应激、 氧化应激、轴突长度、tau磷酸化和聚集以及促炎反应 小胶质细胞和星形胶质细胞。为了阐明我们的Aβ齐聚物模型的结构与 生物源Aβ寡聚体，我们将产生针对每个Aβ寡聚体模型的多克隆抗体，然后检测 这些抗体与5XFAD小鼠脑蛋白提取物和脑切片的免疫反应性。 我们将发现新的Aβ低聚物模型，方法是创建新的Aββ-发夹肽，它包含更多的 β多肽序列和交替的β链比对。我们将创建新的交联型Aβ齐聚物模型 在现有和新发现的A-β齐聚物模型中确定关键接触点，然后在二硫化物中进行工程 用债券来稳定低聚物。新型A-β的结构和齐聚性能表征 我们生成的齐聚物模型，我们将使用X射线结晶学和其他各种生物物理 实验包括CD光谱、SDS-PAGE、尺寸排除层析(SEC)、分析 超速离心(AUC)、核磁共振波谱和Förster共振能量转移(FRET)研究。这个 如上所述，我们将研究这些新的Aβ寡聚体模型的生物学和免疫学特性。

Project Summary/Abstract: Understanding the aggregation of the β-amyloid peptide (Aβ) to form toxic oligomers is fundamental to understanding the molecular basis of Alzheimer’s disease (AD). Despite decades of research, the structures of Aβ oligomers remain a mystery, constituting a significant gap in understanding AD. This proposal seeks to address this knowledge gap through the structural, biophysical, and biological profiling of a diverse group of Aβ oligomer models and correlation of these models with biogenic Aβ oligomers. My laboratory has developed an approach for create structurally defined Aβ oligomer models composed of peptide fragments from Aβ constrained into a β-hairpin. The X-ray crystallographic structures of these Aβ β- hairpin peptides reveal the structures of oligomers that the peptides can form and key intermolecular contacts that the peptides make in the oligomeric state. These contacts reveal sites that can be crosslinked to create covalently stabilized Aβ oligomer models that mimic the crystallographic oligomers. Studying the crosslinked oligomers then allows detailed correlation between oligomer structure and biophysical and biological properties. We will characterize how our Aβ oligomer models interact with and affect neurons, microglia, and astrocytes, to provide detailed insights into how our Aβ oligomer models impact different brain cell types and thus help shed light on the relationship between Aβ oligomer structure and cellular events that occur in AD. We will use fluorescence microscopy and fluorescence-assisted cell sorting (FACS) to visualize and quantify the interactions and uptake of our Aβ oligomer models with neurons, microglia, and astrocytes. We will evaluate downstream biochemical and cellular effects elicited by Aβ oligomer models in neurons, microglia, and astrocytes and evaluate how treatment affects apoptosis, necrosis, calcium homeostasis, endoplasmic reticulum stress, oxidative stress, neurite length, tau phosphorylation and aggregation, and proinflammatory responses in microglia and astrocytes. To elucidate the relationship between the structures of our Aβ oligomer models and biogenic Aβ oligomers, we will generate polyclonal antibodies against each Aβ oligomer model and then examine the immunoreactivity of these antibodies with brain protein extract and brain slices from 5XFAD mice. We will discover new Aβ oligomer models, by creating new Aβ β-hairpin peptides that contain more of the Aβ peptide sequence and alternate β-strand alignments. We will create new crosslinked Aβ oligomer models by identifying key contacts in existing and newly discovered Aβ oligomer models and then engineering in disulfide bonds to stabilize the oligomers. To characterize the structures and oligomerization properties of the new Aβ oligomer models that we generate, we will use X-ray crystallography and a variety of other biophysical experiments, including CD spectroscopy, SDS-PAGE, size exclusion chromatography (SEC), analytical ultracentrifugation (AUC), NMR spectroscopy, and Förster resonance energy transfer (FRET) studies. The biological and immunological properties of these new Aβ oligomer models will be studied as described above.