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

摘要 淀粉样蛋白(Aβ，Aβ)被认为是阿尔茨海默病(AD)的主要毒物。为了开发AD疗法，一个 迫切需要更好地了解Aβ的毒性机制。在大脑中，β被发现最多 在可能被神经元吸收的细胞外沉积中。这项提议的目的是测试中央 假设神经元Aβ摄取和毒性是相关联的。 β形成具有不同神经毒性特征的不同聚集体。关于结构和聚集体是如何- 运动状态影响神经元对A-β的摄取和毒性。这使得制定拦截策略变得非常困难 这些致病过程。努力确定β的构象和聚集状态如何影响其 到目前为止，由于缺乏(A)生产稳定样品的方法，神经元摄取和毒性受到阻碍。 结构分析和(B)准确的工具，以量化不同的Aβ聚集体的神经元摄取。近期 拉斯卡托夫实验室基于手性的方法已经产生了一组稳定的寡聚和纤维状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。建议的研究 是跨学科和协作性的：它包括与艾森伯格博士和季奇科博士的结构性合作； 格拉布博士将为在拉斯卡托夫实验室进行的神经生物学实验提供咨询。 目标1的目的是通过单核磁共振完成外消旋Aβ纤维的结构鉴定，然后使用 这些结构性的见解，以设计更小的，更像药物的，低聚物到纤维的转化器，并测试工作 假设低聚物到纤维的转化减少了神经元对Aβ的摄取，从而抑制了其毒性。这 将使用基于C14的放射性定量工具与各种细胞培养分析相结合来完成 测量Aβ对神经元的快速和缓慢毒性作用。目标2将检验工作假设 Aβ42-E22e和Aβ42-S26的毒性差异是由于它们的神经元摄取不同所致。 还将测试另一种假设，即多肽交通到不同的亚细胞位置，并且 多肽毒性的差异是由于这一点。稳定的β42-E22e和β42-S26s的低温电子显微镜结构 将寻找低聚物，以确定导致它们毒性差异的结构基序。AIM 3将测试 高度聚集倾向的N末端截短Aβ相关肽p3的工作假说 促进Aβ中寡聚体到纤维的转化，从而降低Aβ的摄取效率，减少其神经毒性。 成功的完成将在神经元摄取和不同Aβ形式的毒性之间产生定量联系。 它可能会产生世界上第一个Aβ齐聚物结构，以及一种无毒的Aβ纤维结构。它可能会屈服 更小的D-肽A-β寡聚物到纤维的转化器将在未来转化为新的AD疗法，以及 它还将揭示P3加成是如何抑制Aβ毒性的。最后，拟议的研究可能会揭示 蛋白质聚集如何影响神经元摄取和毒性的一般结构洞察。

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.