A Single Molecule Study of Amyloid Beta Neuronal Toxicity

β淀粉样蛋白神经元毒性的单分子研究

• 批准号: 7777812
• 负责人: ARI GAFNI
• 金额: $ 16.25万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2011-11-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7777812
• 关键词: Address; Affect; Age; Alzheimer' s Disease; Amyloid beta-Protein; Area; Binding; Biological; Biophysics; Brain; Calcium; Cell Aging; Cell membrane; Cell surface; Cells; Cellular Membrane; Complex; Development; Disease; Energy Transfer; Environment; Etiology; Event; Evolution; Extravasation; Foundations; Future; Goals; Heterogeneity; Image; Individual; Intervention; Knowledge; Lead; Life; Location; Masks; Membrane; Methods; Modeling; Molecular; Monitor; Neurons; Pathology; Peptides; Phospholipids; Photobleaching; Physiological; Pilot Projects; Play; Process; Public Health; Research; Role; Solutions; Specificity; Spectrum Analysis; Structure; Surface; Techniques; Testing; Time; Toxic effect; Work; abeta oligomer; base; brain tissue; calcium indicator; cell age; cytotoxic; cytotoxicity; design; dimer; fluidity; in vivo; insight; instrumentation; membrane assembly; membrane model; monomer; neurotoxicity; peptide A; public health relevance; research study; single molecule

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.

描述(申请人提供)：早期形成的淀粉样β多肽寡聚体被认为通过结合和渗透生物膜而在阿尔茨海默病(AD)中发挥关键作用，从而对神经元产生毒性。然而，对于观察到的许多Abeta物种中哪些在体内具有细胞毒性，毒性的潜在机制是什么，有毒聚集体是如何形成的，它们如何与细胞膜相互作用，为什么神经元特有的病理学以及为什么相互作用依赖于细胞的年龄(即，AD与年龄相关的分子/细胞基础是什么？)仍然缺乏了解。这些都是具有实验挑战性的问题，因为Abeta寡聚体是高度不均匀的(它们的大小从二聚体到数百个多肽单体)，而且也是高度亚稳的，因此只暂时存在。因此，通过使用大分子集合和相对较高的肽浓度的传统方法来研究Aβ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了少量瞬时中间体的事实的阻碍，并且不能有效地解决它们的动态异质性。我们的长期目标是通过应用单分子光谱学(SMS)来研究单个多肽低聚体的形成和与培养的神经细胞的相互作用，从而有助于了解Abeta的细胞毒性。实时表征单个细胞附着的低聚物之间的相互作用将使我们能够识别有毒物种并量化它们的细胞通透性。将SMS扩展到多肽诱导的细胞膜破坏的研究，如神经元一样复杂，也将在SMS和膜生物物理学方面取得重大进展。这一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.使SMS方法适用于研究Abeta与培养神经元的相互作用，并检验以下假设，即脑组织典型的A？浓度下形成多肽低聚物的主要机制是通过膜结合多肽物种的组装。我们将应用SMS来监测培养神经元表面单个Abeta寡聚物种的时间演变。目的2.验证Abeta对神经细胞膜的通透性高度依赖于寡聚体类型及其在细胞表面的定位的假说。为了对单个神经元加载荧光钙指示剂进行成像，我们将使用SMS来监测膜上的单个Abeta寡聚体，如上面的目标1所示，同时记录(在第二个波长，钙指示剂发射的地方)渗透事件，细胞表面每个孔的位置，并通过与之相关的钙内流强度来量化其渗透效率。然后，我们将跟踪钙渗漏通过每个单独的孔的时间演变，以得出例如，孔大小演变的动态轨迹。这些结果还将使我们能够确定神经元上的特定区域是否更容易受到寡聚体形成/渗透的影响，从而在未来的研究中确定特异性的来源。 与公共卫生相关：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，这是由于一种名为淀粉样β蛋白的多肽形成的有毒复合体所致。目前的研究将探索导致这些有毒结构在细胞表面形成的分子相互作用，以及它们发挥毒性的机制。这些基础知识将增进我们对AD的理解，并可为未来制定针对该疾病的干预策略服务。