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 氨基酸序列以及翻译后修饰 (PTM)，例如 磷酸化和裂解会导致蛋白质错误折叠并破坏正常的神经元功能。虽然很多有 经过数十年严格而集中的研究，目前还没有疾病修饰疗法 治疗AD或相关tau蛋白病。最近，该领域开始了一个复杂但充满希望的转变，从瞄准 大的 tau 原纤维（例如 PHF 和 NFT）破坏较小的非原纤维 tau 寡聚体。 虽然晚期 tau 原纤维已被广泛研究，包括最近的一系列高分辨率冷冻技术 电镜结构——研究早期低聚物的工具很少，尤其是在细胞中。结果几乎什么都没有 已知 1) 早期错误折叠事件会产生有毒的非纤维状 tau 寡聚体；也不是2）这些如何 寡聚物利用蛋白质机制来引起细胞困扰。为了开始填补这一空白，我们的 2019 年阿尔茨海默病和 痴呆症论文建立了一套基于终生 FRET 的高分辨率生物传感器，可监测全长 tau 细胞中的寡聚物。在这里，我们提供了令人信服的初步数据，表明这些生物传感器可以描绘 原纤维结构中的折叠基序以及 PTM 对早期 tau 寡聚体影响最大。 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性 tau 相互作用。首先，共同 研究人员 Karen Ashe 和 Kathryn Nelson 在 2016 年《自然医学》杂志上发表的论文表明，tau 蛋白的裂解 caspase-2 (Casp2) 导致 tau 蛋白错误定位到树突棘，关闭 AMPA 受体并促进 小鼠的认知缺陷。我们展示了有趣的证据表明分裂之间存在复杂的反馈循环， 低聚和毒性。其次，tau 蛋白和 α-突触核蛋白 (aSyn) 在多种疾病中具有众所周知的共存疾病。 阿尔茨海默病与痴呆有关，但它们在早期错误折叠中相互作用的生物物理学知之甚少 明白了。我们提供了 tau 和 aSyn 之间首选结合方向的初步证据， 表明存在稳定且可靶向的结合基序。 该提案的两个主要目标是：1）确定可用 tau 蛋白中揭示的结构基序 原纤维结构以及哪些 PTM 对细胞早期寡聚化和病理学贡献最大；和 2) 表征并抑制两种致病性 tau 相互作用：tau/Casp2 和 tau/aSyn。在目标 1 中，我们分析 最近可用的原纤维结构并询问：如何使用这些结构来解开其他难以捉摸的结构 非纤维状 tau 寡聚体的结构细节？此外，为了加深我们调查的影响， 在联合研究员 Shauna Yuan 的帮助下，我们将开发 iPSC 衍生的人类皮质多巴胺能细胞的新系列 表达我们生物传感器的神经元。然后，在目标 2 和 3 中，我们研究了 tau 蛋白之间的生物物理相互作用。 分别观察 tau/Casp2 和 tau/aSyn 的寡聚化和毒性。在每种情况下，我们也将表现出色 通量小分子筛选，以确定这两种病理性寡聚组装体的有效抑制剂。

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.