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

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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.