Single Molecule Approach to Neurodegeneration in Alzheimer's Disease

由淀粉样β肽A-β形成的寡聚体已被证明是神经毒性的，有效地渗透生物膜，并且被认为是阿尔茨海默病（AD）的关键参与者。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，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：目的1：确定A-β寡聚体在溶液中的时间演变，并确定结合到神经元膜和透化它们的寡聚体。我们将表征溶液生成的A-β寡聚物物质与由脑提取的脂质制成的膜制剂以及从神经元细胞切除的膜贴片之间的相互作用。这些测量将检验这样的假设，即在升高的肽浓度下，毒性寡聚体直接在溶液中形成，并结合到膜上并使膜透化，而不需要额外的生长。目标二：确定Abeta寡聚体在神经元细胞膜表面组装的机制，表征透化开始时寡聚体的大小，并跟踪其后续演变。关于该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对这些低聚物的透化能力的影响（相对于目标1中的溶液产生的低聚物）; B）确定膜如何在低生理浓度的A-β下促进毒性低聚物的形成。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。在这一请求中，额外的支持将用于加快研究克里思，并将使我们能够创建三个新职位（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.