Liquid–liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau

Abstract

The protein Tau aggregates into tangles in the brain of patients with Alzheimer’s disease. In solution, however, Tau is intrinsically disordered, highly soluble, and binds to microtubules. It is still unclear what initiates the conversion from an innocuous phase of high solubility and functionality to solid-like neurotoxic deposits. Here, we show that the microtubule-binding repeats of Tau, which are lysine-rich, undergo liquid–liquid phase separation in solution. Liquid–liquid demixing causes molecular crowding of amyloid-promoting elements of Tau and drives electrostatic coacervation. Furthermore, we demonstrate that three-repeat and four-repeat isoforms of Tau differ in their ability for demixing. Alternative splicing of Tau can thus regulate the formation of Tau-containing membrane-less compartments. In addition, phosphorylation of Tau repeats promotes liquid–liquid phase separation at cellular protein conditions. The combined data propose a mechanism in which liquid droplets formed by the positively charged microtubule-binding domain of Tau undergo coacervation with negatively charged molecules to promote amyloid formation.

Tau protein liquid–liquid phase separation can initiate tau aggregation

“…Furthermore, we found that the C‐terminal half of p‐tau441 alone, p‐tauCt (aa 242–441), was able to undergo phase separation in a NaCl‐insensitive (≤ 1 M NaCl) manner as well (Fig EV4). Similar observations were very recently reported for the phase separation of the tau repeat domain only (Ambadipudi et al , 2017). To evaluate whether interactions of the repeat domain were essential for LLPS of tau, we also expressed the N‐terminal (aa 1–256) half of tau441 in Sf9 cells, p‐tau256, and tested its LLPS characteristics (Fig EV5, Movie EV10).…”

“…The spontaneous formation of ThioS‐positive and aggregation seeding tau aggregates in and on the droplets supports this hypothesis. Furthermore, these findings are consistent with the recently reported in vitro LLPS of the tau repeat domain (Ambadipudi et al , 2017). Importantly, though, the contribution of the N‐terminal protein half to intracellular tau LLPS has to be considered as another regulatory entity that could play an important role for either the inhibition or stabilization of LLPS in the brain.…”

“…Therefore, in the cell, the association of tau with other factors like polyanionic RNA, for example, in stress granules (Vanderweyde et al , 2016), or the array of tubulin C‐termini on the microtubule surface (Ackmann et al , 2000) may play an important role for tau LLPS; this idea is consistent with our data showing tau LLPS induced by heparin and RNA, as well as recent independent reports from three other laboratories (Ambadipudi et al , 2017; Hernandez‐Vega et al , 2017; Zhang et al , 2017). …”

Protein Phase Separation: A New Phase in Cell Biology

“…For example, Tau phosphorylation is a hallmark of pathology in Alzheimer’s disease [115], and interestingly, tau phosphorylation promotes aggregation and phase separation in vitro [116]. …”

Mechanisms of Cell-to-Cell Transmission of Pathological Tau

“…14 Recent publications implicate a liquid-liquid phase separation that forms droplets of tau induced by electrostatic interactions that provide a molecular crowding environment to promote fibril formation. 15 The formation of higher-order aggregates of tau, whether in oligomers or fibrils, leads to neuron death through gain-of-function toxicity and/or the loss of function of microtubule stabilization. 14 Tau is also implicated as a mediator of β-amyloid–induced neurodegeneration, in that tau reduction rescued β-amyloid–induced axonal transport defects in primary neurons, and crossing mice that express both human amyloid protein precursor and β-amyloid with tau knockout mice resulted in the rescue of the dendritic hyperexcitability phenotype and restored network synchronicity.…”