A Single Molecule Study of Amyloid Beta Neuronal Toxicity

描述（由申请人提供）：据信淀粉样蛋白β肽的早期形成寡聚体通过结合生物膜并使生物膜透化从而对神经元有毒而在阿尔茨海默病（AD）中起关键作用。然而，仍然缺乏对观察到的许多Abeta种类中的哪些在体内具有细胞毒性、毒性的潜在机制是什么、毒性聚集体如何形成、它们如何与细胞膜相互作用、为什么神经元特异性的病理以及为什么相互作用取决于细胞的年龄（即，AD的年龄相关性的分子/细胞基础是什么？）。这些是实验上具有挑战性的问题，因为Abeta寡聚体是高度异质的（它们的大小范围从二聚体到数百个肽单体），并且也是高度亚稳定的，因此仅瞬时存在。因此，通过使用大分子集合和相对高的肽浓度的传统方法研究Abeta寡聚体不仅具有有限的生理意义，而且固有地受到集合平均掩盖少量瞬时中间体并且不能有效地解决其动态异质性的事实的阻碍。我们的长期目标是通过应用单分子光谱（SMS）来研究单个肽寡聚体与培养的神经元细胞的形成和相互作用，从而有助于理解Abeta的细胞毒性。实时表征单细胞附着的寡聚体的相互作用将使我们能够识别有毒物质并量化其细胞透化功效。将SMS扩展到像神经元一样复杂的细胞中肽诱导的膜破坏的研究，也将在SMS和膜生物物理学方面取得重大进展。这种新的能力将使我们能够解决以下两个假设驱动的目标：目标1。调整SMS方法以研究Abeta与培养的神经元的相互作用，并检验以下假设：在脑组织典型的A？浓度下，肽寡聚体形成的主要机制是膜结合肽物质的组装。我们将应用SMS来监测培养的神经元表面上的单个Abeta寡聚物种类的时间演变。目标二。检验Abeta对神经元膜的透化作用高度依赖于寡聚体类型及其在细胞表面上的定位的假设。用荧光钙指示剂对单个神经元进行成像，我们将使用SMS监测膜上的单个Abeta寡聚体，如上述目标1所述，并同时记录（在Ca指示剂发射的第二波长下）透化事件，细胞表面上每个孔的位置，并通过与之相关的钙流入强度来量化其透化功效。通过每个单独的孔的钙渗漏的演变，以导出例如孔径演变的动态轨迹。这些结果还将使我们能够确定神经元上的特定结构域是否更容易受到寡聚体形成/透化的影响，从而在未来的研究中确定特异性的起源。 公共卫生相关性：阿尔茨海默病（AD）的主要病因是大脑某些区域的神经细胞丢失。有强有力的证据表明，这是由于一种称为淀粉样蛋白β的肽形成的有毒复合物。目前的研究将探索导致细胞表面形成这些毒性结构的分子相互作用以及它们发挥毒性的机制。这些基础知识将提高我们对AD的认识，并可在未来为疾病的干预策略的设计服务。

DESCRIPTION (provided by applicant): Early forming oligomers of the amyloid beta peptide are believed to play a critical role Alzheimer's disease (AD) by binding to, and permeabilizing, biological membranes thus being toxic to neurons. However, understanding is still lacking of which of the many Abeta species observed are cytotoxic in vivo, what is the underlying mechanism for toxicity, how do the toxic aggregates form, how do they interact with cellular membranes, why the pathology specific to neurons and why do the interactions depend on the cell's age (i.e., what is the molecular/cellular basis for the age-relatedness of AD?). These are experimentally challenging problems since the Abeta oligomers are highly heterogeneous (their sizes range from dimers to hundreds of peptide monomers) and are also highly metastable and therefore exist only transiently. As a result, studies of the Abeta oligomers by traditional approaches that use large ensembles of molecules and relatively high peptide concentrations, are not only of limited physiological significance, but also inherently hampered by the fact that ensemble-averaging masks low amounts of transient intermediates and cannot effectively resolve their dynamic heterogeneity. Our long-term goal is to contribute to the understanding of Abeta`s cellular toxicity by applying single molecule spectroscopy (SMS) to investigate the formation and interaction of individual peptide oligomers with cultured neuronal cells. Characterizing the interactions of single cell-attached oligomers in real-time will allow us to identify the toxic species and to quantify their cell permeabilization efficacy. Extending SMS to the study of peptide-induced membrane disruption in cells as complex as neurons will also create a major advance in SMS and membrane biophysics. This new capability will allow us to address the following two hypothesis-driven aims: Aim 1. To adapt the SMS approach to the study of Abeta interaction with cultured neurons and to test the hypothesis that the predominant mechanism for the formation of peptide oligomers at A¿ concentrations typical for brain tissue, is by assembly of membrane-bound peptide species. We will apply SMS to monitor the time evolution of individual Abeta oligomeric species on the surface of cultured neurons. Aim 2. To test the hypothesis that permeabilization of the neuronal membrane by Abeta is highly dependent on oligomer type and on its localization on the cell surface. Imaging single neurons loaded with a fluorescent calcium indicator, we will use SMS to monitor individual Abeta oligomers on the membrane, as in Aim 1 above, and simultaneously record (at a second wavelength, where the Ca indicator emits) permeabilization events, the location of each pore on the cell surface and quantify its permeabilizing efficacy via the intensity of calcium influx associated with it. We will then follow the time evolution of calcium leakage through each individual pore to derive, for example, a dynamic trace of the evolution of pore size. The results will also allow us to determine whether specific domains on the neuron are more susceptible to oligomer formation/permeabilization allowing, in future studies, to identify the origin of the specificity. PUBLIC HEALTH RELEVANCE: The main etiology of Alzheimer's disease (AD) is the loss of nerve cells in certain areas of the brain. There is strong evidence that this is due to toxic complexes formed by a peptide termed amyloid beta. The current study will explore the molecular interactions that lead to the formation of these toxic structures on the cell surface and the mechanism by which they exert their toxicity. This basic knowledge will enhance our understanding of AD and can serve in the future for the design of intervention strategies for the disease.