A Single Molecule Approach to neurodegeneration in AD

描述（由申请方提供）：了解导致阿尔茨海默病（AD）神经变性的神经毒性的起源对于开发干预措施至关重要。最近的研究指出淀粉样β肽A-β的早期寡聚体是AD病因学中的关键参与者。应用传统的方法来研究这些低聚物可能具有挑战性，因为系综平均掩盖了少量的瞬态中间体，并阻碍了物种异质性的解决方案。我们建议应用最近开发的方法，基于单分子光谱（SMS），这是唯一适合于这些研究开发以下具体目标：目的1：应用SMS遵循的时间演变的A-β寡聚体形成在溶液中，并确定那些瞬态寡聚体发展成有序的结构（如前原纤维）和/或不溶性原纤维。有明确的证据表明，在A-β结合的早期阶段，低聚物的高度异质混合物的发展使病理物种的鉴定复杂化。我们将使用荧光标记的A-β肽的SMS来跟踪作为时间函数的寡聚体的形成，并检查A-β 1 -42聚集倾向明显增加的基础。待检验的具体假设是可溶性低聚物通过多种反应途径形成，并且A-β 1 -42更容易形成用于原纤维/原纤维形成的初始核，从而降低A-β 1-42/1-40混合物中聚集的临界浓度。目标二：确定膜脂质体表面A-β寡聚体/原纤维形成的反应顺序，检测膜透化，对所产生的孔进行初步表征，并鉴别影响透化的寡聚体。最近的观察表明，A-β的神经毒性的一部分可能与前原纤维结构的膜结合。我们将启动SMS实验来验证膜促进低聚物形成的假设，并研究其形成的关键方面。我们还将确定结合的A-β寡聚体的亚类是否导致膜透化，如果是，则在哪一点发生，以及是否存在独特的种类或大量的透化寡聚体。从长远来看，我们希望能更好地了解A-β寡聚体进化的生物分子机制和相互作用，并研究它们在原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.