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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 forms aggregates in the brains of Alzheimer patients. Here, the authors identify conditions, where the microtubule-binding repeats of Tau undergo a phosphorylation-dependent liquid–liquid phase separation, leading to molecular crowding in the formed Tau liquid droplets and characterize them by NMR and other biophysical methods.

Background Tau pathology in AD spreads in a hierarchical pattern, whereby it first appears in the entorhinal cortex, then spreads to the hippocampus and later to the surrounding areas. Based on this sequential appearance, AD can be classified into six stages (“Braak stages”). The mechanisms and agents underlying the progression of Tau pathology are a matter of debate. Emerging evidence indicates that the propagation of Tau pathology may be due to the transmission of Tau protein, but the underlying pathways and Tau species are not well understood. In this study we investigated the question of Tau spreading via small extracellular vesicles called exosomes. Methods Exosomes from different sources were analyzed by biochemical methods and electron microscopy (EM) and cryo-EM. Microfluidic devices that allow the culture of cell populations in different compartments were used to investigate the spreading of Tau. Results We show that Tau protein is released by cultured primary neurons or by N2a cells overexpressing different Tau constructs via exosomes. Neuron-derived exosomal Tau is hypo-phosphorylated, compared with cytosolic Tau. Depolarization of neurons promotes release of Tau-containing exosomes, highlighting the importance of neuronal activity. Using microfluidic devices we show that exosomes mediate trans-neuronal transfer of Tau depending on synaptic connectivity. Tau spreading is achieved by direct transmission of exosomes between neurons. In organotypic hippocampal slices, Tau-containing exosomes in conditioned medium are taken up by neurons and microglia, not astrocytes. In N2a cells, Tau assemblies are released via exosomes. They can induce inclusions of other Tau molecules in N2a cells expressing mutant human Tau. We also studied exosomes from cerebrospinal fluid in AD and control subjects containing monomeric and oligomeric Tau. Split-luciferase complementation reveals that exosomes from CSF can promote Tau aggregation in cultured cells. Conclusion Our study demonstrates that exosomes contribute to trans-synaptic Tau transmission, and thus offer new approches to control the spreading of pathology in AD and other tauopathies.

Significance Tau is an important microtubule-associated protein. Although the structure–function relationship of Tau has been intensively studied for many years primarily by molecular biology and biochemical approaches, little is still known about the molecular mechanisms by which Tau interacts with microtubules and promotes microtubule assembly. Here, we provide detailed insight into the Tau–microtubule association by using NMR spectroscopy and mass spectrometry. We show that Tau binds to microtubules by using small groups of residues, which are important for pathological aggregation of Tau. We further show that Tau stabilizes a straight protofilament conformation by binding to a hydrophobic pocket in between tubulin heterodimers. Our work is thus relevant to normal Tau action development and in Tau-related neurodegenerative diseases. The structure, dynamic behavior, and spatial organization of microtubules are regulated by microtubule-associated proteins. An important microtubule-associated protein is the protein Tau, because its microtubule interaction is impaired in the course of Alzheimer’s disease and several other neurodegenerative diseases. Here, we show that Tau binds to microtubules by using small groups of evolutionary conserved residues. The binding sites are formed by residues that are essential for the pathological aggregation of Tau, suggesting competition between physiological interaction and pathogenic misfolding. Tau residues in between the microtubule-binding sites remain flexible when Tau is bound to microtubules in agreement with a highly dynamic nature of the Tau–microtubule interaction. By binding at the interface between tubulin heterodimers, Tau uses a conserved mechanism of microtubule polymerization and, thus, regulation of axonal stability and cell morphology.

Cells form and use biomolecular condensates to execute biochemical reactions. The molecular properties of non-membrane-bound condensates are directly connected to the amino acid content of disordered protein regions. Lysine plays an important role in cellular function, but little is known about its role in biomolecular condensation. Here we show that protein disorder is abundant in protein/RNA granules and lysine is enriched in disordered regions of proteins in P-bodies compared to the entire human disordered proteome. Lysine-rich polypeptides phase separate into lysine/RNA-coacervates that are more dynamic and differ at the molecular level from arginine/RNA-coacervates. Consistent with the ability of lysine to drive phase separation, lysine-rich variants of the Alzheimer’s disease-linked protein tau undergo coacervation with RNA in vitro and bind to stress granules in cells. Acetylation of lysine reverses liquid–liquid phase separation and reduces colocalization of tau with stress granules. Our study establishes lysine as an important regulator of cellular condensation. Processing bodies (P-bodies) are non-membrane-bound protein/RNA granules in the cytosol. Here the authors combine bioinformatics, NMR and cell based assays and find that lysine is enriched in the disordered regions of P-body-associated proteins and show that lysine-rich polypeptides form highly dynamic lysine/RNA-coacervates and lysine acetylation reverses liquid-liquid phase separation.

Emerging experimental evidence suggests that the spread of tau pathology in the brain in Tauopathies reflects the propagation of abnormal tau species along neuroanatomically connected brain areas. This propagation could occur through a “prion-like” mechanism involving transfer of abnormal tau seeds from a “donor cell” to a “recipient cell” and recruitment of normal tau in the latter to generate new tau seeds. This review critically appraises the evidence that the spread of tau pathology occurs via such a “prion-like” mechanism and proposes a number of recommendations for directing future research. Recommendations for definitions of frequently used terms in the tau field are presented in an attempt to clarify and standardize interpretation of research findings. Molecular and cellular factors affecting tau aggregation are briefly reviewed, as are potential contributions of physiological and pathological post-translational modifications of tau. Additionally, the experimental evidence for tau seeding and “prion-like” propagation of tau aggregation that has emerged from cellular assays and in vivo models is discussed. Propagation of tau pathology using “prion-like” mechanisms is expected to incorporate several steps including cellular uptake, templated seeding, secretion and intercellular transfer through synaptic and non-synaptic pathways. The experimental findings supporting each of these steps are reviewed. The clinical validity of these experimental findings is then debated by considering the supportive or contradictory findings from patient samples. Further, the role of physiological tau release in this scenario is examined because emerging data shows that tau is secreted but the physiological function (if any) of this secretion in the context of propagation of pathological tau seeds is unclear. Bona fide prions exhibit specific properties, including transmission from cell to cell, tissue to tissue and organism to organism. The propagation of tau pathology has so far not been shown to exhibit all of these steps and how this influences the debate of whether or not abnormal tau species can propagate in a “prion-like” manner is discussed. The exact nature of tau seeds responsible for propagation of tau pathology in human tauopathies remains controversial; it might be tightly linked to the existence of tau strains stably propagating peculiar patterns of neuropathological lesions, corresponding to the different patterns seen in human tauopathies. That this is a property shared by all seed-competent tau conformers is not yet firmly established. Further investigation is also required to clarify the relationship between propagation of tau aggregates and tau-induced toxicity. Genetic variants identified as risks factors for tauopathies might play a role in propagation of tau pathology, but many more studies are needed to document this. The contribution of selective vulnerability of neuronal populations, as an alternative to prion-like mechanisms to explain spreading of tau pathology needs to be clarified. Learning from the prion field will be helpful to enhance our understanding of propagation of tau pathology. Finally, development of better models is expected to answer some of these key questions and allow for the testing of propagation-centred therapies.

Microtubule-associated proteins regulate microtubule dynamics, bundle actin filaments, and cross-link actin filaments with microtubules. In addition, aberrant interaction of the microtubule-associated protein Tau with filamentous actin is connected to synaptic impairment in Alzheimer’s disease. Here we provide insight into the nature of interaction between Tau and actin filaments. We show that Tau uses several short helical segments to bind in a dynamic, multivalent process to the hydrophobic pocket between subdomains 1 and 3 of actin. Although a single Tau helix is sufficient to bind to filamentous actin, at least two, flexibly linked helices are required for actin bundling. In agreement with a structural model of Tau repeat sequences in complex with actin filaments, phosphorylation at serine 262 attenuates binding of Tau to filamentous actin. Taken together the data demonstrate that bundling of filamentous actin and cross-linking of the cellular cytoskeleton depend on the metamorphic and multivalent nature of microtubule-associated proteins. The microtubule associated protein Tau also interacts with filamentous actin. Here the authors combine biophysical experiments and NMR studies to characterize the structural changes that occur in Tau upon binding to filamentous actin and show that phosphorylation of serine 262 attenuates actin binding of Tau.

The aggregation of the intrinsically disordered tau protein into highly ordered β-sheet-rich fibrils is implicated in the pathogenesis of a range of neurodegenerative disorders. The mechanism of tau fibrillogenesis remains unresolved, particularly early events that trigger the misfolding and assembly of the otherwise soluble and stable tau. We investigated the role the lipid membrane plays in modulating the aggregation of three tau variants, the largest isoform hTau40, the truncated construct K18, and a hyperphosphorylation-mimicking mutant hTau40/3Epi. Despite being charged and soluble, the tau proteins were also highly surface active and favorably interacted with anionic lipid monolayers at the air/water interface. Membrane binding of tau also led to the formation of a macroscopic, gelatinous layer at the air/water interface, possibly related to tau phase separation. At the molecular level, tau assembled into oligomers composed of ~ 40 proteins misfolded in a β-sheet conformation at the membrane surface, as detected by in situ synchrotron grazing-incidence X-ray diffraction. Concomitantly, membrane morphology and lipid packing became disrupted. Our findings support a general tau aggregation mechanism wherein tau’s inherent surface activity and favorable interactions with anionic lipids drive tau-membrane association, inducing misfolding and self-assembly of the disordered tau into β-sheet-rich oligomers that subsequently seed fibrillation and deposition into diseased tissues.

Key Points Tauopathies are classified into several groups based on the isoform composition of tau aggregates. The splicing of the gene encoding tau is regulated by multiple factors besides tau mutations. Tau is a natively unfolded protein that shows no tendency for aggregation by itself. Post-translational modifications may modify the processes of tau oligomerization, aggregation and tau-induced neurodegeneration. The polarized distribution of tau into the axonal compartment of neurons is determined by multiple mechanisms. In addition, part of tau is actively released into extracellular space. Besides stabilizing microtubules, regulating their dynamic instability and supporting axonal transport, tau can interact with various cell components and thus serves other functions in various other processes, including neuronal activity, neurogenesis, iron export and long-term depression. Pathological tau may induce neurotoxicity owing to its loss of function, toxic gain of function or its mislocalization, which mediates amyloid-β-induced toxicity. Several tau- or microtubule-based therapeutic approaches have been proposed, including tau aggregation inhibitors, inhibitors of kinases targeting tau, inhibition of tau acetylation, stabilization of microtubules, reduction of tau by antisense oligonucleotides, and immunotherapy using antibodies against tau or phosphorylated tau. Tau is a microtubule-stabilizing protein that aggregates in several neurodegenerative diseases. In this Review, Wang and Mandelkow describe the structure, expression and post-translational modifications of tau, and the functions of this protein in health and in disease. Tau is a microtubule-associated protein that has a role in stabilizing neuronal microtubules and thus in promoting axonal outgrowth. Structurally, tau is a natively unfolded protein, is highly soluble and shows little tendency for aggregation. However, tau aggregation is characteristic of several neurodegenerative diseases known as tauopathies. The mechanisms underlying tau pathology and tau-mediated neurodegeneration are debated, but considerable progress has been made in the field of tau research in recent years, including the identification of new physiological roles for tau in the brain. Here, we review the expression, post-translational modifications and functions of tau in physiology and in pathophysiology.

Protein aggregation plays a key role in neurodegenerative disease, giving rise to small oligomers that may become cytotoxic to cells. The fundamental microscopic reactions taking place during aggregation, and their rate constants, have been difficult to determine due to lack of suitable methods to identify and follow the low concentration of oligomers over time. Here we use single-molecule fluorescence to study the aggregation of the repeat domain of tau (K18), and two mutant forms linked with familial frontotemporal dementia, the deletion mutant ΔK280 and the point mutant P301L. Our kinetic analysis reveals that aggregation proceeds via monomeric assembly into small oligomers, and a subsequent slow structural conversion step before fibril formation. Using this approach, we have been able to quantitatively determine how these mutations alter the aggregation energy landscape. Alzheimer’s disease is characterized by the aggregation of the Abeta peptide and the tau protein. Here the authors track the formation of wild-type or mutant tau oligomers through to large aggregates and gain insights into the molecular basis of how tau mutations cause disease by altering the aggregation pathway.

The aggregation of hyperphosphorylated tau into amyloid fibrils is closely linked to the progression of Alzheimer’s disease. To gain insight into the link between amyloid structure and disease, the three-dimensional structure of tau fibrils has been studied using solid-state NMR (ssNMR) and cryogenic electron microscopy (cryo-EM). The proline-rich region of tau remains poorly defined in the context of tau amyloid structures, despite the clustering of several phosphorylation sites, which have been associated with Alzheimer’s disease. In order to gain insight into the contribution of the proline-rich region P2 of tau to amyloid fibrils, we studied in vitro aggregated amyloid fibrils of tau constructs, which contain both the proline-rich region P2 and the pseudo-repeats. Using ssNMR we show that the sequence 225 KVAVVRT 231 , the most hydrophobic patch within the P2 region, loses its flexibility upon formation of amyloid fibrils. The data suggest a contribution of the P2 region to tau amyloid fibril formation, which might account for some of the unassigned electron density in cryo-EM studies of tau fibrils and could be modulated by tau phosphorylation at the disease-associated AT180 epitope T231/S235.