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

该提案的目的是表征相分离的结构和功能，在体外和神经元细胞生物学中的浓缩形式的tau蛋白。Tau是一种本质上无序的神经元特异性微管结合蛋白。虽然tau是非常可溶的，但它可以在不同性质的小细胞内tau“浓缩物”（例如应激颗粒、细胞可传递的错误折叠寡聚体、短暂轴突积聚）中发现，甚至在阿尔茨海默病（AD）和额颞叶痴呆（FTD）中的极其稳定的神经元淀粉样蛋白样聚集体中发现。最近，我们和其他人发现了较不有序的tau缩合形式，其可以被描述为通过液-液相分离（LLPS）形成的“蛋白液滴”或液体致密簇（LDCs）。它们可以被作为拥挤试剂的聚合物诱导，但有趣的是，也可以被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 LLPS，与RNA结合蛋白FUS、TDP-43和hnRNP 1类似，tau LDCs也可以转变为聚集体;这些甚至能够在细胞中接种tau聚集。因此，Tau LLPS可能是重要的病理聚集的tau在各种家族性和散发性neurodegenerative diseases.In这个合作和跨学科的研究项目的三个研究小组，我们要描述和了解的分子和细胞机制的液-液相分离的神经元tau蛋白。我们的目标是确定所有方面的tau LLPS，从光和X-射线结构表征的tau最不发达国家，生化和生物物理描述的tau凝聚过程，甚至神经元细胞的生物和病理作用的阶段分离的tau。利用互补的生物物理和生物化学方法（Mandelkow集团），创新的原位动态光散射和X射线散射技术（Betzel组）和各种神经生物学和细胞生物物理学技术（Wegmann集团），我们将：[i]使用重组蛋白在体外表征液体tau相的形成和成熟，（ii）描述在体外和细胞中tau缩合成LDC和聚集体期间的结构转化，（iii）表征调节tau LLPS的翻译后修饰，和（iv）研究额颞痴呆中tau突变对LLPS和随后聚集的影响。此外，我们将探索tau LLPS作为神经元应激反应和作为阿尔茨海默病和tau蛋白病中tau聚集的前状态的细胞作用。我们相信，拟议的研究对于理解tau液体细胞器的神经元功能及其作为前聚集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.