Investigating structural heterogeneities in amyloid aggregates with multiscale infrared spectroscopic imaging

项目摘要 特定蛋白质的错误折叠和聚集成纤维状淀粉样沉积是 一大类疾病和神经退行性疾病，这是一个主要的公共卫生问题 全世界。然而，淀粉样蛋白聚集体在疾病发生和发展中的确切作用并不是很好。 已经成立了。引起神经毒性的确切聚集状态及其分布之间的关系 淀粉样蛋白聚集体中的二级结构与认知功能下降的关系尚不清楚。这个项目的目的是 体外和体外淀粉样蛋白聚集体中蛋白质二级结构的异质性研究 并确定它们与疾病进展的关系。我们的方法依赖于利用最先进的空间 分辨红外光谱，即纳米级红外光谱、超分辨红外显微镜 和共聚焦红外光谱成像，以绘制淀粉样蛋白沉积中的蛋白质二级结构。我们的目标是 集成这三种技术，开发一种空间和光谱自适应红外成像方法，将 实现对组织中光谱数据的多尺度测量。上述三种技术将进一步 用拉曼显微镜进行了增强。总而言之，这种方法将提供多模式、多尺度的结构和 对淀粉样蛋白聚集体的化学洞察，从它们在组织中的分布和化学亚型到结构 单原纤维内的变异。我们将重点研究淀粉样蛋白聚集体及其异质性。 开发和优化我们的方法，并随后旨在推广这些策略 研究其他疾病如帕金森氏病、乳腺癌和II型的淀粉样蛋白聚集物 糖尿病。这一努力背后的假设是，淀粉样蛋白沉积的结构异质性，而不仅仅是 特定的纤维结构，与疾病的进展相关。我们将利用光热AFM-IR，一种 用原子力显微镜增强红外光谱以获得纳米级聚集体专一性的技术 光谱。AD患者组织提取物的种子生长将使探测聚集的差异成为可能 与不同疾病阶段相关的途径和结构多态。组织中的淀粉样聚集体 将通过共聚焦红外和光热红外显微镜进行研究，这将允许识别淀粉样蛋白 在组织中沉积，然后以高次衍射空间分辨率研究单个斑块。这个 将分析组织光谱数据以基于其二级结构分布对淀粉样沉积进行分类， 此外，还将探讨不同类型的沉积物与疾病分期之间的相关性。独一无二的一面 这种方法的特点是它使用了空间分辨红外光谱和成像中的尖端技术 研究一种对一系列疾病的分子病理学至关重要的问题 治疗策略。

Project Summary The misfolding and aggregation of specific proteins into fibrillar amyloid deposits is the pathological hallmark of a wide class of diseases and neurodegenerative disorders, which represent a major public health concern worldwide. However, the precise role of amyloid aggregates in disease onset and progression is not well established. The exact aggregation states that cause neurotoxicity and relationship between distribution of secondary structures in amyloid aggregates and cognitive decline remains unclear. The aim of this project is to investigate the heterogeneities in protein secondary structure in amyloid aggregates both in-vitro and ex-vivo and identify their relationship with disease progression. Our approach relies on utilizing state-of-the-art spatially resolved infrared spectroscopy, namely nanoscale infrared (IR) spectroscopy, super-resolution IR microscopy and confocal IR spectroscopic imaging to map protein secondary structures in amyloid deposits. We aim to integrate these three techniques and develop a spatially and spectrally adaptive IR imaging approach that will enable multiscale measurement of spectral data in tissues. The above three techniques will be further augmented by Raman microscopy. Taken together, this approach will offer multimodal, multiscale structural and chemical insights on amyloid aggregates, from their distribution in tissues and chemical subtypes to structural variations within single fibrils. We will focus on studying amyloid aggregates and their heterogeneities in Alzheimer's disease (AD) to develop and optimize our methods, and subsequently aim to extend these strategies to investigate amyloid aggregates in other diseases such as Parkinson's disease, Breast Cancer and type-II diabetes. The hypothesis underlying this effort is that structural heterogeneities of amyloid deposits, and not just specific fibrillar structures, are correlated with disease progression. We will utilize photothermal AFM-IR, a technique that augments IR spectroscopy with Atomic Force Microscopy, to obtain nanoscale aggregate-specific spectra. Seeded growth from tissue extracts from AD patients will enable probing the differences in aggregation pathways and structural polymorphisms associated with different disease stages. Amyloid aggregates in tissues will be investigated through confocal IR and photothermal IR microscopies, which will allow for identifying amyloid deposits in tissues and then investigating individual plaques with high sub-diffraction spatial resolution. The tissue spectral data will be analyzed to classify amyloid deposits based on their secondary structure distributions, and the correlation between distinct classes of deposits and disease stages will be explored. The unique aspect of this approach is that it uses cutting edge technologies in spatially resolved IR spectroscopy and imaging to investigate a problem that is central to the molecular pathology of a wide range of diseases and development of therapeutic strategies.