Structural and Biological Characterization of Diverse Oligomers Derived from Abeta

项目摘要/摘要：了解β-淀粉样肽（Aβ）的聚集形成毒性 寡聚体的研究是理解阿尔茨海默病（AD）的分子基础的基础。尽管经过数十年的 研究表明，Aβ寡聚体的结构仍然是一个谜，构成了对AD理解的重大空白。 该提案旨在通过对生物样品进行结构、生物物理和生物特征分析， 一组不同的Aβ寡聚体模型以及这些模型与生物源性Aβ寡聚体的相关性。 我的实验室开发了一种方法，用于创建结构定义的Aβ寡聚体模型，该模型由以下组成： 来自Aβ的肽片段被限制在β-发夹中。这些Aβ β- 发夹肽揭示了肽可以形成的低聚物的结构和关键的分子间接触 这些肽在寡聚状态下产生的。这些接触揭示了可以交联的位点， 共价稳定的Aβ寡聚体模型，模拟晶体学寡聚体。研究交联的 寡聚体然后允许寡聚体结构与生物物理和生物学性质之间的详细关联。 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， 提供详细的见解，了解我们的Aβ寡聚体模型如何影响不同的脑细胞类型，从而帮助摆脱 阐明Aβ寡聚体结构与AD中发生的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。我们将评估下游 神经元、小胶质细胞和星形胶质细胞中Aβ寡聚体模型引起的生化和细胞效应， 评价治疗如何影响细胞凋亡、坏死、钙稳态、内质网应激， 氧化应激，神经突长度，tau蛋白磷酸化和聚集，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寡聚体模型的结构之间的关系， 生物源性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，然后检查 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寡聚体模型，通过创造新的Aβ β发夹肽， Aβ肽序列和交替β链比对。我们将通过以下方式创建新的交联Aβ寡聚体模型： 识别现有和新发现的Aβ寡聚体模型中的关键接触点，然后在二硫化物中进行工程化 键以稳定低聚物。为了表征新的Aβ的结构和寡聚性质， 寡聚体模型，我们将使用X射线晶体学和各种其他生物物理学， 实验，包括CD光谱法、SDS-PAGE、尺寸排阻色谱法（SEC）、分析 超离心（AUC）、NMR光谱和Förster共振能量转移（FRET）研究。的 将如上所述研究这些新的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.