A Single Molecule Approach to neurodegeneration in AD

描述(由申请人提供)：了解导致阿尔茨海默病(AD)神经变性的神经毒性的来源对于开发干预措施至关重要。最近的研究指出，淀粉样β蛋白的早期寡聚体，A-β，在AD的病因学中起关键作用。应用传统方法来研究这些低聚物可能是具有挑战性的，因为整体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。我们建议应用最近发展的基于单分子光谱学(SMS)的方法来开发以下特定目标：目标1：应用单分子光谱学跟踪A-β齐聚物在溶液中形成的时间演变，并识别那些发展为有序结构(如前原纤维)和/或不溶纤维的瞬时低聚物。有明确的证据表明，在A-β结合的早期阶段，形成了高度不均匀的低聚物混合物，使病理物种的鉴定变得复杂。我们将使用荧光标记的A-β多肽的SM来跟踪低聚体的形成作为时间的函数，并检查A-β1-42聚集的明显倾向增加的基础。需要检验的具体假设是，可溶低聚物由多条反应途径形成，A-β1-42更容易形成原纤维/原纤维形成的初始核，从而降低了A-β1-42/1-40混合物中聚集的临界浓度。目的2：确定A-β齐聚物/原纤维在膜脂质体表面形成的反应顺序，检测膜通透性，初步表征膜通透性，鉴定影响膜通透性的低聚体。最近的观察表明，A-β的部分神经毒性可能与前纤维结构的膜结合有关。我们将启动SMS实验，以验证膜促进低聚物形成的假设，并研究低聚物形成的关键方面。我们还将确定结合的A-β寡聚体的一个亚类是否导致膜通透性，如果是，在什么时候会发生这种情况，是否存在一个独特的种类或大量的通透性低聚体。从长远来看，我们希望更好地了解A-β寡聚体进化的生物分子机制和相互作用，并研究它们在纤维形成和神经毒性中的作用。

DESCRIPTION (provided by applicant): Understanding the origin of neurotoxicity that leads to neurodegeneration in Alzheimers Disease (AD) is critical to the development of an intervention. Recent studies point to early oligomers of the amyloid beta peptide, A-beta, as critical players in the etiology of AD. Applying traditional approaches to study these oligomers can be challenging because ensemble averaging masks low amounts of transient intermediates and hinders the resolution of species heterogeneity. We propose to apply recently developed approaches, based on single molecule spectroscopy (SMS), that are uniquely suited for these studies to develop the following specific aims: Aim 1: To apply SMS to follow the time evolution of A-beta oligomer formation in solution and to identify those transient oligomers that develop into ordered structures (such as pre- protofibrils) and/or insoluble fibrils. There is clear evidence that during the early stages of A-beta association a highly heterogeneous mix of oligomers develops complicating the identification of the pathological species. We will use SMS of fluorescently labeled A-beta peptides to follow the formation of oligomers as a function of time and to examine the basis for the apparent increased propensity for aggregation of A-beta1-42. The specific hypothesis to be tested is that soluble oligomers form by multiple reaction pathways and that A- beta1-42 more readily forms the initial nucleus for protofibril/fibril formation thus reducing the critical concentration for aggregation in the A-beta 1-42/1-40 mix. Aim 2: To determine the reaction sequence for A- beta oligomer/protofibril formation on the surface of membrane liposomes, to detect membrane permeabilization, to make initial characterization of the pores created and to identify oligomers that affect permeabilization. Recent observations suggest that part of A-beta's neurotoxicity may be associated with membrane binding of pre-fibril structures. We will initiate SMS experiments to test the hypothesis that membranes facilitate the formation of oligomers and to study key aspects of their formation. We will also determine whether a sub-class of the bound A-beta oligomers leads to membrane permeabilization and, if so, at which point this happens and is there a unique species or a multitude of permeabilizing oligomers. Long-term we expect to develop a better understanding of the biomolecular mechanisms and interactions that underlie the evolution of A-beta oligomers, and examine their role in fibril formation and neurotoxicity.