Using Chirality to Understand and Control Amyloid Beta Neuronal Uptake and Toxicity

摘要 β淀粉样蛋白（Aβ）是阿尔茨海默病（AD）的关键毒性因子。为了开发AD疗法， 目前迫切需要进一步了解Aβ毒性的机制。在大脑中，Aβ主要存在于 细胞外沉积物中的蛋白质。这项提案的目的是测试中央 假设神经元Aβ摄取和毒性相关。 Aβ形成具有不同神经毒性特征的不同聚集体。关于结构和聚集体的性质知之甚少- 神经元对Aβ的摄取和毒性受不同状态的影响。这使得很难制定策略来阻止 这些致病过程。努力确定Aβ的构象和聚集状态如何影响它的结构， 迄今为止，神经元摄取和毒性受到以下因素的阻碍：（a）缺乏产生用于 结构分析和（B）定量不同Aβ聚集体神经元摄取的精确工具。最近 Raskatov实验室基于手性的方法已经产生了一系列稳定的寡聚和非甾体Aβ 这些表格将在这里用作工具，目的是缩小这一重要的知识差距。拟议研究 是跨学科和协作：它包括与博士艾森伯格和博士Tycko结构合作; 博士Glabe将为Raskatov实验室的神经生物学实验提供咨询。 目的1是通过ssNMR完成外消旋Aβ纤维的结构解析，然后使用 这些结构的见解，设计更小，更像药物，寡聚体到原纤维转换器，并测试工作 假设寡聚体到原纤维的转化减少了Aβ进入神经元的摄取，从而抑制了其毒性。这 将使用基于C14的放射性定量工具结合各种细胞培养测定来完成， 测量Aβ对神经元的快速和缓慢毒性作用。目标2将检验以下工作假设： Aβ42-E22 e和Aβ42-S26 s之间的毒性差异是由于其神经元摄取的差异， 并且还将检验另一种假设，即肽运输到不同的亚细胞位点， 肽毒性的差异是由于这一点。稳定的Aβ42-E22 e和Aβ42-S26 s的CryoEM结构 将寻找低聚物，以鉴定导致其毒性差异的结构基序。目标3将测试 工作假设，高度聚集倾向，N-末端截短的Aβ相关肽p3 促进Aβ中寡聚体向原纤维的转化，从而降低Aβ摄取效率，降低其神经毒性。 成功完成将产生不同Aβ形式的神经元摄取和毒性之间的定量联系。 它可能产生世界上第一个Aβ寡聚体结构，以及无毒Aβ原纤维结构。它可能会产生 更小的D肽Aβ寡聚体-原纤维转化体，将来可转化为新型AD治疗剂， 这也将揭示β 3的加入如何抑制Aβ毒性。最后，拟议的研究可能会揭示 关于蛋白质聚集如何影响神经元摄取和毒性的一般结构见解。

ABSTRACT Amyloid β (Aβ) is a believed key toxic agent of Alzheimer’s Disease (AD). To develop AD therapeutics, an improved understanding of the mechanisms of Aβ toxicity is urgently needed. In the brain, Aβ is found mostly in extracellular deposits that may be taken up by neurons. The purpose of this proposal is to test the central hypothesis that neuronal Aβ uptake and toxicity are linked. Aβ forms diverse aggregates with varied neurotoxic profiles. Little is known about how structure and aggrega- tion state affect neuronal uptake and toxicity of Aβ. This makes it very difficult to devise strategies to block these pathogenic processes. Efforts to determine how conformation and aggregation state of Aβ affects its’ neuronal uptake and toxicity were hampered thus far by the lack of (a) methods to produce stable samples for structural analysis and (b) accurate tools to quantify neuronal uptake of different Aβ aggregates. Recent chirality-based approaches of the Raskatov lab have produced a set of stabilized oligomeric and fibrillary Aβ forms that will be used here as tools, with the goal to close this important knowledge gap. Proposed research is cross-disciplinary and collaborative: it includes structural collaborations with Dr. Eisenberg and Dr. Tycko; Dr. Glabe will consult on neurobiology experiments done in the Raskatov lab. The purpose of Aim 1 is to complete the structural elucidation of racemic Aβ fibrils by ssNMR, to then use those structural insights to devise smaller, more drug-like, oligomer-to-fibril converters, and to test the working hypothesis that oligomer-to-fibril conversion reduces Aβ uptake into neurons, thus suppressing its toxicity. This will be accomplished using C14-based radioquantitation tools in combination with various cell culture assays to measure both rapid and slow toxic actions of Aβ against neurons. Aim 2 will test the working hypothesis that the differences in toxicity between Aβ42-E22e and Aβ42-S26s are due to differences in their neuronal uptake, and will also test the alternative hypothesis that the peptides traffic to different sub-cellular sites, and that the differences in peptide toxicity are due to that. CryoEM structures of Aβ42-E22e and Aβ42-S26s stabilized oligomers will be sought, to identify the structural motifs responsible for their toxicity differences. Aim 3 will test the working hypothesis that the highly aggregation-prone, N-terminally truncated Aβ-related peptide p3 promotes oligomer-to-fibril conversion in Aβ, thus reducing Aβ uptake efficiency and making it less neurotoxic. Successful completion will yield a quantitative link between neuronal uptake and toxicity of different Aβ forms. It may yield the world’s first Aβ oligomer structures, as well as a structure of non-toxic Aβ fibrils. It may yield smaller, D-peptidic Aβ oligomer-to-fibril converters to be translated to novel AD therapeutics in the future, and it will also reveal how Aβ toxicity is suppressed by p3 addition. Finally, the proposed studies may uncover general structural insights on how protein aggregation affects neuronal uptake and toxicity.