Single Molecule Approach to Neurodegeneration in Alzheimer's Disease

由淀粉样β蛋白形成的寡聚体，A-β，已被证明是神经毒性的，有效地渗透生物膜，被认为是阿尔茨海默病(AD)的关键角色。应用传统的方法来研究这些低聚物及其毒性的来源是具有挑战性的，因为系综平均掩盖了少量的瞬时中间体，并阻碍了物种异质性的解决，特别是当与膜结合时。在这个由ARRA资助的为期两年的项目中，我们建议应用基于单分子显微镜(SMM)的新方法，专注于A-β与神经元来源的膜之间的相互作用，以解决以下特定目标：目标1：确定A-β低聚物在溶液中的时间演化，并确定与神经细胞膜结合并使其通透性的低聚物。我们将描述溶液生成的A-β寡聚物种与由大脑提取的脂类制成的膜制剂以及从神经元细胞中切除的膜片之间的相互作用。这些测量将检验这样一个假设，即在肽浓度升高时，有毒低聚物直接在溶液中形成，并与膜结合并使其通透性，而不需要额外的生长。目的2：确定神经细胞膜表面Aβ低聚体的组装机制，表征低聚体在通透性开始时的大小，并跟踪其随后的进化。对这一目标的工作将使我们能够：a)确定天然膜环境对低聚物组装过程和这些低聚物的渗透能力(相对于AIM 1中的溶液产生的低聚物)的影响；b)确定在低生理浓度的A-β时，膜如何促进有毒低聚物的形成。ARRA资金的主要任务之一是创造新的就业机会和保留现有的就业机会。在这项请求中，额外的支持将用于加快研究的节奏，并将使我们能够创造三个新的职位(两个GSRA和一个博士后)，并保留两名经验丰富的研究人员，否则他们将被解雇。

Oligomers formed by the amyloid beta peptide, A-beta, have been shown to be neurotoxic, potently permeabilize biological membranes and are believed to be critical players in Alzheimers disease (AD). Applying traditional approaches to study these oligomers and the origin of their toxicity is challenging because ensemble averaging masks low amounts of transient intermediates and hinders the resolution of species heterogeneity especially when bound to membranes. In this two year ARRA supported project we propose to apply novel approaches based on single molecule microscopy (SMM), that are uniquely suited for these studies, to focus on the interactions between A-beta and neuronally-derived membranes to address the following specific aims: Aim 1: To determine the time evolution of A-beta oligomers in solution and identify the oligomers that bind to neuronal membranes and permeabilize them. We will characterize the interactions between solution-generated A-beta oligomeric species and membrane preparations made from brain extracted lipids as well as membrane patches excised from neuronal cells. These measurements will test the hypothesis that at elevated peptide concentrations, toxic oligomers form directly in solution and bind to and permeabilize the membrane without the need for additional growth. Aim 2: To determine the mechanism of Abeta oligomer assembly on the surface of membranes from neuronal cells, characterize the size of the oligomers at the onset of permeabilization and follow their subsequent evolution. Work on this Aim will allow us to: a) determine the effect of the native membrane environment on the oligomer assembly process and on the permeabilization power of these oligomers (relative to the solution generated oligomers in aim 1); b) determine how the membrane facilitates the formation of toxic oligomers at the low physiological concentrations of A-beta. One of the major missions of the ARRA funding is the creation of new jobs and the retention of current ones. In this request the additional support will be used to speed up the tempo of research, and will allow us to create three new positions (two GSRA and one postdoctoral) and to retain two experienced researchers who would otherwise be laid off.