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 是如何改变tau蛋白的氨基酸序列，沿着翻译后修饰（PTM）， 磷酸化和切割导致蛋白质错误折叠并破坏正常神经元功能。虽然许多 经过几十年的严格和集中的研究，目前还没有疾病修饰疗法 以治疗AD或相关的Tau蛋白病。最近，该领域开始了一个复杂但有希望的转变， 大的tau纤维（例如PHF和NFT）以破坏较小的非纤维状tau寡聚体。 虽然已经对晚期tau纤维进行了广泛的研究-包括最近的一系列高分辨率冷冻- EM结构-有一些工具来研究早期阶段的寡聚体，特别是在细胞中。结果，几乎没有 已知1）产生有毒的、非纤维状tau寡聚体的早期错误折叠事件; 2）这些 寡聚体共同选择蛋白质机制以引起细胞痛苦。为了开始填补这一空白，我们的2019年阿尔茨海默氏症& 痴呆论文建立了一套高分辨率，基于寿命FRET的生物传感器，可监测全长tau蛋白 细胞中的寡聚体。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， 其中原纤维结构中的折叠基序以及PTM最影响早期tau寡聚体。 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纳尔逊在2016年发表的《自然医学》论文显示， 半胱天冬酶-2（Casp 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。我们展示了有趣的证据，表明卵裂， 寡聚化和毒性。第二，tau和α-突触核蛋白（aSyn）在多种肿瘤中具有众所周知的共病性。 阿尔茨海默病相关痴呆，但它们在早期错误折叠中相互作用的生物物理学研究很差 明白我们提供了tau和aSyn之间优选结合方向的初步证据， 这表明了稳定的并因此是可靶向的结合基序。 该提案的两个主要目标是：1）确定在可用的tau中揭示了哪些结构基序 原纤维结构，以及哪些PTM对细胞中的早期寡聚化和病理学贡献最大;以及 2)以表征和抑制两种致病性tau相互作用：tau/Casp 2和tau/aSyn。在目标1中，我们分析了 最近可用的原纤维结构，并问：如何使用这些结构来解开否则难以捉摸 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， 在共同研究者Shauna Yuan的帮助下，我们将开发新的iPSC衍生的人皮质多巴胺能神经细胞系， 神经元表达我们的生物传感器。然后，在目标2和目标3中，我们研究tau蛋白之间的生物物理相互作用， 分别观察tau/Casp 2和tau/aSyn的寡聚化和毒性。在每种情况下，我们也将执行高- 通过小分子筛选来鉴定这两种病理性寡聚体组装的有效抑制剂。

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.