Single Molecule Approach to Neurodegeneration in Alzheimer's Disease

淀粉样β肽（a - β）形成的低聚物已被证明具有神经毒性，可渗透生物膜，并被认为是阿尔茨海默病（AD）的关键因素。应用传统的方法来研究这些低聚物及其毒性的起源是具有挑战性的，因为集合平均掩盖了少量的瞬态中间体，阻碍了物种异质性的分解，特别是当结合到膜上时。在这个为期两年的ARRA支持的项目中，我们建议应用基于单分子显微镜（SMM）的新方法，这是唯一适合于这些研究的方法，专注于a - β和神经元衍生膜之间的相互作用，以解决以下具体目标：目标1：确定溶液中a - β低聚物的时间演变，并确定与神经元膜结合并使其渗透的低聚物。我们将描述溶液生成的a - β寡聚物与由脑提取脂质制成的膜制剂以及从神经元细胞中切除的膜斑块之间的相互作用。这些测量将测试假设，即在肽浓度升高时，有毒低聚物直接在溶液中形成，并结合并渗透膜，而不需要额外的生长。目的2：确定Abeta低聚物在神经细胞膜表面组装的机制，表征低聚物在渗透开始时的大小，并跟踪其随后的进化。在这个目标上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的影响（相对于目标1中产生的低聚物的溶液）；b)确定在低生理浓度的a - β下，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并保留现有的就业机会。在这项申请中，额外的支持将用于加快研究的节奏，并允许我们创建三个新职位（两个GSRA和一个博士后），并保留两个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.