Determining How Amyloid-β Fibril Polymorphism Influences Cellular Toxicity

与阿尔茨海默病(AD)一样，脑淀粉样血管病(CAA)的病理特征是 形成由淀粉样蛋白-β(A-β)纤维组成的斑块。人们普遍假设， 纤维，称为多态，有助于CAA和AD的病理生理学。然而，目前还没有明确的 了解它们的分子结构如何诱导细胞毒活性。本应用程序的目标是 是利用新的化学成像和电化学传感方法来直接监测结构 不同Aβ纤维多态的动力学、异常相互作用和毒性活性。中环 假说是有毒的多晶型通过表现出更快的生长动力学，破坏 细胞膜，并通过活性氧化物种(ROS)诱导更高水平的氧化应激 一代。这一假设将通过追求三个具体目标来检验：1)表征分子 拉曼光谱研究β纤维的结构和生长机理2)采用新型激发剂 用拉曼化学成像方法直接观察β原纤维多态与蛋白质的相互作用 活细胞；以及3)使用电化学传感来直接评估活性氧物种(ROS)的水平 由A-β原丝多态诱导。在目标1和目标2下，普尼浩勒人开创了一种新的方法 被称为拉曼化学成像的研究小组将被用来直接监测不同的纤维多晶型是如何生长的， 在结构上进化，并改变细胞膜的流动性、完整性和化学成分。在AIM 3、对不同纤维多形性诱导的氧化应激进行监测。这将通过以下方式完成 快速电化学法测量ROS浓度的急性和慢性变化 由细胞产生。这些新方法的协同耦合是创新的，因为它们一起 提供连接分子水平相互作用所需的时间尺度上的结构和化学信息 在纤维多形性和具有细胞反应的细胞成分之间，包括产生 ROS和细胞毒标志物的快速释放。拟议的工作意义重大，因为它构建了 为更广泛的研究计划奠定基础，这将产生对分子如何 淀粉样蛋白纤维的结构是CAA和CAA患者临床症状和病理生理的基础 广告。最终，所获得的见解将指导治疗策略和药物的合理设计 限制有毒菌株Aβ纤维的形成，抑制其与细胞的异常相互作用，减轻氧化 损害，并有可能逆转与痴呆症相关的皮质组织的丧失和萎缩。

As with Alzheimer’s Disease (AD), the pathological hallmark of Cerebral Amyloid Angiopathy (CAA) is the formation of plaques composed of amyloid-β (Aβ) fibrils. It is widely hypothesized that structural variants of fibrils, termed polymorphs, contribute to the pathophysiology of CAA and AD. However, there is no clear understanding of how their molecular structure induces cytotoxic activity. The objective of this application is to employ novel chemical imaging and electrochemical sensing methods to directly monitor the structural dynamics, aberrant interactions, and toxic activities of different Aβ fibril polymorphs. The central hypothesis is that toxic polymorphs promote cytotoxicity by exhibiting faster growth kinetics, disrupting cellular membranes, and inducing higher levels of oxidative stress via reactive oxidative species (ROS) generation. This hypothesis will be tested by pursuing three specific aims: 1) Characterize the molecular structures and growth mechanisms of Aβ fibrils using Raman spectroscopy; 2) Employ novel stimulated Raman chemical imaging methods to directly visualize the interactions between Aβ fibril polymorphs and living cells; and 3) Use electrochemical sensing to directly assess levels of reactive oxygen species (ROS) induced by Aβ fibril polymorphs. Under Aims 1 and 2, a novel methodology pioneered by the Punihaole group called Raman Chemical Imaging, will be used to directly monitor how different fibril polymorphs grow, structurally evolve, and alter the fluidity, integrity, and chemical composition of cellular membranes. In Aim 3, oxidative stress induced by different fibril polymorphs will be monitored. This will be accomplished using fast electrochemical measurements to measure acute and chronic changes in the concentration of ROS generated by the cells. Synergistic coupling of these novel methods is innovative since they together provide structural and chemical information on time scales required to link molecular-level interactions between fibril polymorphs and cellular components with cellular responses, including the production of ROS and rapid release of cytotoxic markers. The proposed work is significant because it builds the foundation of a broader research program that will produce a holistic understanding of how the molecular structure of amyloid fibrils underlies the pathophysiology and clinical symptoms of patients with CAA and AD. Ultimately, the insights obtained will guide treatment strategies and the rational design of drugs to limit the formation of toxic strains of Aβ fibrils, inhibit their aberrant interactions with cells, mitigate oxidative damage, and potentially reverse the loss of cortical tissue and atrophy associated with dementia.