Molecular and Cellular Determinants of Tau Protein Condensation into Dense Liquid Phases

这项建议的目的是在体外和神经细胞生物学中表征相分离的凝集型tau蛋白的结构和功能。Tau是一种具有神经元特异性的无序微管结合蛋白。尽管tau特别易溶，但它可以在不同性质的细胞内的小tau“凝聚体”中发现(例如，应激颗粒、细胞可传递的错误折叠低聚物、一过性轴突堆积)，甚至在阿尔茨海默病(AD)和额颞性痴呆(FTD)中非常稳定的神经元淀粉样聚集体中也能发现它。最近，我们和其他人发现了无序形式的tau凝聚，这种凝聚可以描述为由液-液相分离(LLP)形成的“蛋白质液滴”或液体致密团簇(LDC)。它们可以由充当拥挤试剂的聚合物诱导，但有趣的是，也可以由RNA诱导。磷酸化和FTD相关的常染色体显性tau突变可以催化tau LLP，类似于RNA结合蛋白FUS、TDP-43和hnRNP1，tau LDC也可以转变为聚集体；这些聚集体甚至能够在细胞中播种tau聚集。在这项由三个研究小组合作的跨学科研究项目中，我们想要描述和了解神经元tau蛋白液-液相分离的分子和细胞机制。我们的目标是确定tau LLP的各个方面，从tau最不发达国家的光和X射线结构特征，到tau缩合过程的生化和生物物理描述，甚至到相分离tau的神经细胞生物学和病理学作用。利用互补的生物物理和生化方法(Mandelkow组)、创新的原位动态光散射和X射线散射技术(Betzel组)以及各种神经生物学和细胞生物物理技术(Wegmann组)，我们将：[i)利用重组蛋白表征液体tau的形成和成熟；(Ii)描述tau在体外和细胞内缩合成LDC和聚集体过程中的结构转换；(Iii)表征tau LLPs的翻译后修饰；以及(Iv)研究额叶到颞叶痴呆中tau突变对LLPs和随后聚集的影响。此外，我们将探索tau LLPs在阿尔茨海默病和tau病中作为神经元应激反应和tau聚集的前状态所起的细胞作用。我们相信，所提出的研究对于了解tau液体细胞器的神经元功能及其作为聚集前状态的作用，以及作为治疗神经退行性疾病的潜在新药靶点是重要的。

The aim of this proposal is to characterize the structure and functions of phase separated, condensed forms of tau protein in vitro and in neuronal cell biology. Tau is an intrinsically disordered and neuron-specific microtubule binding protein. Although tau is exceptionally soluble, it can be found in small intracellular tau "condensates" of different nature (e.g. stress granules, cell-transmissible misfolded oligomers, transient axonal accumulations), and even in extremely stable neuronal amyloid-like aggregates in Alzheimer’s disease (AD) and Frontotemporal Dementia (FTD). Recently, we and others discovered less ordered forms of tau condensation that can be described as “protein droplets” or liquid dense clusters (LDCs) formed by liquid-liquid phase separation (LLPS). They can be induced by polymers acting as crowding reagents, but, interestingly, also by RNA. Phosphorylation and FTD-associated autosomal dominant tau mutations can catalyze tau LLPS, and similar to the RNA-binding proteins FUS, TDP-43, and hnRNP1, tau LDCs can also transition into aggregates; these are even capable of seeding tau aggregation in cells. Tau LLPS may therefore be of importance for the pathological aggregation of tau in various familial and sporadic neurodegenerative diseases.In this collaborative and interdisciplinary research project of three research groups, we want to describe and understand the molecular and cellular mechanisms underlying the liquid-liquid phase separation of the neuronal tau protein. We aim to determine all aspects of tau LLPS ranging from light and x-ray structural characterization of tau LDCs, to the biochemical and biophysical description of the tau condensation process, and even to the neuronal cell biological and pathological role of phase separated tau. Using complementary biophysical and biochemical methods (Mandelkow group), innovative in situ dynamic light scattering and X-ray scattering techniques (Betzel group), and various neurobiological and cell biophysical techniques (Wegmann group), we will: [i) characterize the formation and maturation of liquid tau phases in vitro using recombinant proteins, (ii) describe the structural conversions during condensation of tau into LDCs and aggregates both in vitro and in cells , (iii) characterize the post-translational modifications regulating tau LLPS , and (iv) study the impact of tau mutations in fronto-temporal dementias on LLPS and subsequent aggregation. Furthermore, we will explore the cellular role of tau LLPS as a neuronal stress response and as a pre-state for tau aggregation in Alzheimer’s disease and tauopathies.We believe that the proposed research is important to understand the neuronal function of tau liquid organelles and their role as pre-aggregation states, and as potential new drug targets in the treatment of neurodegenerative diseases.