Elucidating the biophysics of pre-fibrillar, toxic tau oligomers: from amino acid motifs to neuronal dysfunction

摘要 阿尔茨海默病(AD)和相关痴呆(ADRD)研究中最紧迫的问题之一 是如何改变tau的氨基酸序列，以及翻译后修饰(PTM)，如 磷酸化和切割，导致蛋白质错误折叠，扰乱正常的神经元功能。虽然很多人都有 经过几十年的严格和专注的研究，目前还没有改变疾病的疗法 用于治疗AD或相关的肌萎缩侧索硬化症。最近，该领域开始了一场复杂但有希望的转变，从目标转向 从大的tau纤维(如PHF和NFT)到破坏较小的非纤维tau低聚物。 虽然已经对晚期的tau原纤维进行了广泛的研究--包括最近一系列高分辨率的冷冻-- EM结构-几乎没有工具来研究早期低聚物，特别是细胞中的低聚物。结果，几乎什么都没有 已知的是1)产生有毒的非纤维状tau寡聚体的早期错误折叠事件；或者2)这些 寡聚体利用蛋白质机制来造成细胞痛苦。为了开始填补这一空白，我们的2019年阿尔茨海默氏症 痴呆症的一篇论文建立了一套基于高分辨率、终身FRET的生物传感器，用于监测全长tau 细胞中的低聚物。在这里，我们提出了令人信服的初步数据，表明这些生物传感器可以描绘出 纤维结构中的哪些折叠基序以及PTM对早期tau寡聚体的影响最大。 这些生物传感器还使我们能够研究细胞中两种不同的病理tau相互作用。首先，共同- 研究人员凯伦·阿什和凯瑟琳·纳尔逊在2016年的《自然医学》论文中显示，tau被 Caspase-2(Caspase-2)导致tau错误定位于树突棘，关闭AMPA受体并促进 小鼠的认知缺陷。我们展示了耐人寻味的证据，表明卵裂和卵裂之间存在复杂的反馈循环， 齐聚和毒性。其次，tau和α-突触核蛋白(ASyn)在多发性疾病中有众所周知的共同发病。 阿尔茨海默病相关痴呆，但它们在早期折叠错误中相互作用的生物物理学很差 明白了。我们提供了tau和aSyn之间优先结合取向的初步证据， 暗示了一个稳定的、因此具有靶向性的结合基序。 这项建议的两个主要目标是：1)确定在可用tau中揭示的结构基序 纤维结构，以及其中的PTM，对细胞的早期齐聚作用和病理学贡献最大； 2)研究和抑制两种致病的tau相互作用：tau/Casp2和tau/aSyn。在目标1中，我们分析了 最近可用的纤维结构，并问：这些结构如何才能用来解开否则难以捉摸的 非纤维状tau齐聚物的结构细节？此外，为了加深我们调查的影响，并与 在合作研究员袁少娜的帮助下，我们将开发新的IPSC来源的人类皮质多巴胺能 表达我们生物传感器的神经元。然后，在目标2和目标3中，我们研究了tau之间的生物物理相互作用。 Tau/Casp2和tau/aSyn的齐聚和毒性。在每一种情况下，我们也将执行高- 通过小分子筛选来确定这两种病理性低聚体组装的有效抑制物。

Abstract One of the most pressing questions in the study of Alzheimer’s disease (AD) and related dementias (ADRD) is how alterations in the amino-acid sequence of tau, along with post-translational modifications (PTMs) such as phosphorylation and cleavage, lead the protein to misfold and disrupt normal neuronal function. While much has been learned over decades of rigorous and focused research, there are currently no disease modifying therapies to treat AD or related tauopathies. Recently, the field has begun a complicated but promising shift from targeting large tau fibrils (e.g. PHFs and NFTs) to disrupting smaller, non-fibrillar tau oligomers. While late-stage tau fibrils have been studied extensively—including a flurry of recent high-resolution cryo- EM structures—there are few tools to study early-stage oligomers, especially in cells. As a result, almost nothing is known about 1) early misfolding events that produce toxic, non-fibrillar tau oligomers; nor 2) how these oligomers co-opt protein machinery to cause cellular distress. To begin to fill this void, our 2019 Alzheimer’s & Dementia paper established a set of high-resolution, lifetime-FRET based biosensors that monitor full-length tau oligomers in cells. Here, we present compelling preliminary data showing that these biosensors can delineate which folding motifs in the fibril structures, as well as PTMs, most affect early-stage tau oligomers. These biosensors have also enabled us to study two distinct pathological tau interactions in cells. First, co- Investigators Karen Ashe and Kathryn Nelson’s 2016 Nature Medicine paper showed that cleavage of tau by caspase-2 (Casp2) causes tau to mislocalize to dendritic spines, shut down AMPA receptors and promote cognitive defects in mice. We show intriguing evidence to suggest a complex feedback loop between cleavage, oligomerization and toxicity. Second, tau and alpha-Synuclein (aSyn) have well-known co-morbidity in multiple Alzheimer’s Disease related dementias, but the biophysics of their interaction in early-stage misfolding is poorly understood. We provide preliminary evidence of a preferred binding orientation between tau and aSyn, suggesting a stable and hence targetable binding motif. The two major goals of this proposal are to: 1) determine which structural motifs revealed in the available tau fibril structures, and which PTMs, contribute most to early-stage oligomerization in cells, and to pathology; and 2) to characterize and inhibit two pathogenic tau interactions: tau/Casp2 and tau/aSyn. In Aim 1, we analyze the recently available fibril structures and ask: how can these structures be used to unravel otherwise elusive structural details of non-fibrillar tau oligomers? Additionally, to deepen the impact of our investigations, and with the help of co-Investigator Shauna Yuan, we will develop new lines of iPSC-derived human cortical dopaminergic neurons expressing our biosensors. Then, in Aims 2 and 3, we study the biophysical interplay between tau oligomerization and toxicity of tau/Casp2 and tau/aSyn respectively. In each case, we will also perform high- throughput small-molecule screens to identify potent inhibitors of these two pathological, oligomeric assemblies.