Monomeric Tau Research Articles

Rapid Cell Type-Dependent Uptake of 0N4R TAU Monomer is not Solely Heparin Sulfate Proteoglycan Dependen

The brains of Alzheimer's patients typically show a distribution of extracellular amyloid plaques, composed of Aβ and neurofibrillary tangles, composed of postranslationally-modified and abnormally aggregated tau protein. The mechanism underlying the initial conversion and spread of pathological tau aggregates remains unknown. Recent work suggests that tau species can be released into the extracellular space and be subsequently endocytosed via negatively-charged heparan sulfate proteoglycans (HSPGs) available on the cell surface. Here, we produced monomeric 0N4R tau in both nonphosphorylated and phosphorylated states via recombinant expression in E. coli and purified for testing in cell culture. Using live-cell confocal microscopy and flow cytometry, we show that soluble 0N4R monomer can be rapidly endocytosed by multiple cell types and that this uptake is independent of phosphorylation state. Furthermore, we demonstrate that HSPGs enhance, but are not essential, for cellular endocytosis of both phosphorylated and non-phosphorylated tau. Current studies are focused on the impact of isoform type and degree of aggregation on uptake rate.

First dual binder of microRNA-146a and monomeric tau: a novel approach for multitargeted therapeutics for neurodegenerative diseases.

We report a new approach for the development of multitargeted therapeutics for Alzheimer's disease (AD) based on dual targeting of monomeric tau and biogenesis of microRNA-146a. Compound MG-1102 displayed a superior neuroprotective activity, in comparison to mono-targeted therapeutics, which validates the likelihood of the success of this approach in AD drug development.

Tau-Cofactor Complexes as Building Blocks of Tau Fibrils.

The aggregation of the human tau protein into neurofibrillary tangles is directly diagnostic of many neurodegenerative conditions termed tauopathies. The species, factors and events that are responsible for the initiation and propagation of tau aggregation are not clearly established, even in a simplified and artificial in vitro system. This motivates the mechanistic study of in vitro aggregation of recombinant tau from soluble to fibrillar forms, for which polyanionic cofactors are the most commonly used external inducer. In this study, we performed biophysical characterizations to unravel the mechanisms by which cofactors induce fibrillization. We first reinforce the idea that cofactors are the limiting factor to generate ThT-active tau fibrils, and establish that they act as templating reactant that trigger tau conformational rearrangement. We show that heparin has superior potency for recruiting monomeric tau into aggregation-competent species compared to any constituent intermediate or aggregate “seeds.” We show that tau and cofactors form intermediate complexes whose evolution toward ThT-active fibrils is tightly regulated by tau-cofactor interactions. Remarkably, it is possible to find mild cofactors that complex with tau without forming ThT-active species, except when an external catalyst (e.g., a seed) is provided to overcome the energy barrier. In a cellular context, we propose the idea that tau could associate with cofactors to form a metastable complex that remains “inert” and reversible, until encountering a relevant seed that can trigger an irreversible transition to β-sheet containing species.

Seeding Activity-Based Detection Uncovers the Different Release Mechanisms of Seed-Competent Tau Versus Inert Tau via Lysosomal Exocytosis.

The pathological aggregation of tau characterizes a set of neurodegenerative diseases collectively referred to as tauopathies. Recent studies using cellular and animal models have suggested that tau pathology progresses by trans-cellular propagation. The process of propagation is mediated by certain species of extracellular tau, which are taken up by recipient cells and serve as a seed for tau aggregation. Tau propagation is currently one of the most active areas of research in dementia. Previous efforts to identify the specific tau molecules involved in propagation have suggested that multiple forms of tau with different molecular weights derived from recombinant tau or brain lysates exert seeding activity. Nonetheless, the molecular characteristics of the “extracellular” seed-competent tau as well as its release mechanisms remain to be elucidated. Given that tau is physiologically released into the extracellular space, it is critical to distinguish seed-competent tau from normal monomeric tau. Utilizing biosensor cells expressing P301S mutant tau fused to CFP/YFP, here we discriminated between seed-competent tau and inert monomer tau released from HEK293 cells. By analyzing the size-exclusion fractions of the media, we found that seed-competent tau was enriched in high molecular weight fractions of >2,000 kDa, while the majority of soluble tau in the media positively detected by ELISA was in low molecular weight fractions. We also found that lysosomal stress not only increased Ca2+-dependent release of seed-competent tau but also altered its molecular size. Inhibiting lysosomal exocytosis specifically decreased release of seed-competent tau without influencing total tau. These data underscore the differential response of seed-competent tau and inert tau to lysosomal stress and indicates the presence of distinct release mechanisms via lysosomes.

Contribution of syndecans to cellular uptake and fibrillation of \u03b1-synuclein and tau

Scientific evidence suggests that α-synuclein and tau have prion-like properties and that prion-like spreading and seeding of misfolded protein aggregates constitutes a central mechanism for neurodegeneration. Heparan sulfate proteoglycans (HSPGs) in the plasma membrane support this process by attaching misfolded protein fibrils. Despite of intense studies, contribution of specific HSPGs to seeding and spreading of α-synuclein and tau has not been explored yet. Here we report that members of the syndecan family of HSPGs mediate cellular uptake of α-synuclein and tau fibrils via a lipid-raft dependent and clathrin-independent endocytic route. Among syndecans, the neuron predominant syndecan-3 exhibits the highest affinity for both α-synuclein and tau. Syndecan-mediated internalization of α-synuclein and tau depends heavily on conformation as uptake via syndecans start to dominate once fibrils are formed. Overexpression of syndecans, on the other hand, reduces cellular uptake of monomeric α-synuclein and tau, yet exerts a fibril forming effect on both proteins. Data obtained from syndecan overexpressing cellular models presents syndecans, especially the neuron predominant syndecan-3, as important mediators of seeding and spreading of α-synuclein and tau and reveal how syndecans contribute to fundamental molecular events of α-synuclein and tau pathology.

Targeting the ensemble of heterogeneous tau oligomers in cells: A novel small molecule screening platform for tauopathies

ObjectiveUnderstanding the heterogeneous pathology in Alzheimer's disease and related tauopathies is one of the most urgent and fundamental challenges facing the discovery of novel disease-modifying therapies. Through monitoring ensembles of toxic and nontoxic tau oligomers spontaneously formed in cells, our biosensor technology can identify tool compounds that modulate tau oligomer structure and toxicity, providing much needed insight into the nature and properties of toxic tau oligomers. BackgroundTauopathies are a group of neurodegenerative disorders characterized by pathologic aggregation of the microtubule binding protein tau. Recent studies suggest that tau oligomers are the primary toxic species in tauopathies. New/Updated HypothesisWe hypothesize that tau biosensors capable of monitoring tau oligomer conformation are able to identify tool compounds that modulate the structure and conformation of these tau assemblies, providing key insight into the unique structural fingerprints of toxic tau oligomers. These fingerprints will provide gravely needed biomarker profiles to improve staging of early tauopathy pathology and generate lead compounds for potential new therapeutics. Our time-resolved fluorescence resonance energy transfer biosensors provide us an exquisitely sensitive technique to monitor minute structural changes in monomer and oligomer conformation. In this proof-of-concept study, we identified a novel tool compound, MK-886, which directly binds tau, perturbs the conformation of toxic tau oligomers, and rescues tau-induced cytotoxicity. Furthermore, we show that MK-886 alters the conformation of tau monomer at the proline-rich and microtubule binding regions, stabilizing an on-pathway oligomer. Major Challenges for the HypothesisOur approach monitors changes in the ensemble of assemblies that are spontaneously formed in cells but does not specifically isolate or enrich unique toxic tau species. However, time-resolved fluorescence resonance energy transfer does not provide high-resolution, atomic scale information, requiring additional experimental techniques to resolve the structural features stabilized by different tool compounds. Linkage to Other Major TheoriesOur biosensor technology is broadly applicable to other areas of tauopathy therapeutic development. These biosensors can be readily modified for different isoforms of tau, specific post-translational modifications, and familial Alzheimer's disease–associated mutations. We are eager to explore tau interactions with chaperone proteins, monitor cross-reactivity with other intrinsically disordered proteins, and target seeded oligomer pathology.

Polyphosphate Initiates Tau Aggregation through Intra- and Intermolecular Scaffolding

The aggregation and deposition of tau is a hallmark of a class of neurodegenerative diseases called tauopathies. Despite intensive study, cellular and molecular factors that trigger tau aggregation are not well understood. Here, we provide evidence for two mechanisms relevant to the initiation of tau aggregation in the presence of cytoplasmic polyphosphates (polyP): changes in the conformational ensemble of monomer tau and noncovalent cross-linking of multiple tau monomers. We identified conformational changes throughout full-length tau, most notably diminishment of long-range interactions between the termini coupled with compaction of the microtubule binding and proline- rich regions. We found that while the proline-rich and microtubule binding regions both contain polyP binding sites, the proline-rich region is a requisite for compaction of the microtubule binding region upon binding. Additionally, both the magnitude of the conformational change and the aggregation of tau are dependent on the chain length of the polyP polymer. Longer polyP chains are more effective at intermolecular, noncovalent cross-linking of tau. These observations provide an understanding of the initial steps of tau aggregation through interaction with a physiologically relevant aggregation inducer.

Tau Phosphorylation and Aggregation in the Developing Human Brain.

Tau hyperphosphorylation, mostly at serine (Ser) or threonine (Thr) residues, plays a key role in the pathogenesis of Alzheimer disease (AD) and other tauopathies. Rodent studies show similar hyperphosphorylation in the developing brain, which may be involved in regulating axonal growth and plasticity, but detailed human studies are lacking. Here, we examine tau phosphorylation by immunohistochemistry and immunoblotting in human fetal and adult autopsy brain tissue. Of the 20 cases with sufficient tissue preservation, 18 (90%) showed positive staining for S214 (pSer214), with the majority also positive for CP13 (pSer202), and PHF-1 (pSer396/pSer404). AT8 (pSer202/pThr205) and RZ3 (pThr231) were largely negative while PG5 (pSer409) was negative in all cases. Immunoblotting showed tau monomers with a similar staining pattern. We also observed phospho-tau aggregates in the fetal molecular layer, staining positively for S214, CP13, and PHF1 and negative for thioflavin S. These corresponded to high-molecular weight (∼150 kD) bands seen on Western blots probed with S214, PHF1, and PG5. We therefore conclude that fetal phosphorylation overlaps with AD in some residues, while others (e.g. T231, S409) appear to be unique to AD, and that tau is capable of forming nontoxic aggregates in the developing brain. These findings suggest that the fetal brain is resilient to formation of toxic aggregates, the mechanism for which may yield insights into the pathogenesis of tau aggregation and toxicity in the aging brain.

Nanobiocatalyst facilitated aglycosidic quercetin as a potent inhibitor of tau protein aggregation

Polyphenols have been suggested as potential therapeutic agents for the treatment of amyloidogenic diseases. In this work, we evaluate quercetin-rich onion extract for its ability to inhibit tau fibrillization. Considering the presence of polyphenols in multiple glycosidic and aglycosidic forms, a nanobiocatalyst-mediated approach has been used to extract quercetin from onion skins. The nanobiocatalysts facilitate greater release of quercetin compared to the use of free enzymes. Atomic force microscopy and fluorescence microscopy show that quercetin possesses a novel inhibitory character on tau-fibril aggregation. In contrast, quercetin-diglucoside does not have an inhibitory effect. Molecular Dynamics simulations reveal conformational changes in tau protein upon interaction with quercetin due to specific hydrogen bonding and hydrophobic interactions. The resulting conformational stability of tau monomer reduces propensity of the protein to aggregate. The ability of quercetin to inhibit tau fibrillization expands the paradigm for application of bioactive polyphenols.

Phosphorylation and Dephosphorylation of Tau Protein During Synthetic Torpor.

Tau protein is of primary importance for many physiological processes in neurons, where it affects the dynamics of the microtubule system. When hyperphosphorylated (PP-Tau), Tau monomers detach from microtubules and tend to aggregate firstly in oligomers, and then in neurofibrillary tangles, as it occurs in a group of neurodegenerative disorders named thauopathies. A hypothermia-related accumulation of PP-Tau, which is quickly reversed after the return to normothermia, has been shown to occur in the brain of hibernators during torpor. Since, recently, in our lab, a hypothermic torpor-like condition (synthetic torpor, ST) was pharmacologically induced in the rat, a non-hibernator, the aim of the present work was to assess whether ST can lead to a reversible PP-Tau accumulation in the rat brain. PP-Tau was immunohistochemically assessed by staining for AT8 (phosphorylated Tau) and Tau-1 (non-phosphorylated Tau) in 19 brain structures, which were chosen mostly due to their involvement in the regulation of autonomic and cognitive functions in relation to behavioral states. During ST, AT8 staining was strongly expressed throughout the brain, while Tau-1 staining was reduced compared to control conditions. During the following recovery period, AT8 staining progressively reduced close to zero after 6 h from ST. However, Tau-1 staining remained low even after 38 h from ST. Thus, overall, these results show that ST induced an accumulation of PP-Tau that was, apparently, only partially reversed to normal during the recovery period. While the accumulation of PP-Tau may only depend on the physicochemical characteristics of the enzymes regulating Tau phosphorylation, the reverse process of dephosphorylation should be actively regulated, also in non-hibernators. In conclusion, in this work a reversible and widespread PP-Tau accumulation has been induced through a procedure that leads a non-hibernator to a degree of reversible hypothermia, which is comparable to that observed in hibernators. Therefore, the physiological mechanism involved in this process can sustain an adaptive neuronal response to extreme conditions, which may however lead to neurodegeneration when particular intensities and durations are exceeded.

Tau Interacts with the C-Terminal Region of α-Synuclein, Promoting Formation of Toxic Aggregates with Distinct Molecular Conformations.

An increasing body of evidence suggests that aggregation-prone proteins associated with various neurodegenerative diseases synergistically promote their mutual aggregation, leading to the co-occurrence of multiple neurodegenerative diseases in the same patient. Here we investigated teh molecular basis of synergistic interactions between the two pathological proteins, tau and α-synuclein, using various biophysical techniques including transmission electron microscopy (TEM), circular dichroism (CD), and solution and solid-state NMR. Our biophysical analyses of α-synuclein aggregation in the absence and presence of tau reveal that tau monomers promote the formation of α-synuclein oligomers and subsequently fibril formation. Solution NMR results also indicate that monomeric forms of tau selectively interact with the C-terminal region of the α-synuclein monomer, accelerating α-synuclein aggregation. In addition, a combined use of TEM and solid-state NMR spectroscopy reveals that the synergistic interactions lead to the formation of toxic α-synuclein aggregates with a distinct morphology and molecular conformation. The filamentous α-synuclein aggregates as well as α-synuclein monomers were also able to induce tau aggregation.

Extracellular Monomeric Tau Is Internalized by Astrocytes

Tau is a microtubule-associated protein that is expressed in neurons. However, in a group of neurodegenerative diseases named tauopathies – characterized by an increase in aggregated and/or hyperphosphorylated Tau – the protein accumulates inside other cells, such as astrocytes and microglia. Given that these glial cells do not produce Tau, its presence can be explained by internalization from the extracellular medium and consequent formation of Tau aggregates. Among internalization mechanisms, heparan sulfate proteoglycans (HSPGs) have been proposed to be responsible for fibrillary Tau uptake in various cell types. Here we studied whether monomeric Tau, which has been observed to be internalized by glial cells such as microglia, was also taken up by astrocytes. Although this Tau form was internalized from the extracellular medium by these cells, the mechanism of uptake was found to be independent of HSPGs.

Understanding molecular features of aggregation-resistant tau conformer using oxidized monomer

BackgroundAggregation of tau into paired helical filament (PHF) is a hallmark of Alzheimer's disease (AD), and Cys-mediated disulfide bond formation plays a vital role in tau fibrillation. While intermolecular disulfide bond between Cys residues in microtubule-binding repeat (MTBR) region facilitates tau aggregation, intramolecular disulfide bond attenuates the same, though the molecular basis for such phenomenon remains obscure. Thus intramolecular disulfide-bonded tau monomer might be an excellent model to understand the unique features of aggregation-resistant tau conformer. MethodsWe synthesized the Cys cross-linked tau40 monomer by oxidation and characterized the altered conformational dynamics in the molecule by Hydrogen-deuterium exchange, limited proteolysis and fluorescence quenching. ResultsDeuterium exchange study showed that rigidity was imparted in the core PHF region of oxidized tau40 in MTBR segment, consisting of the fundamental PHF6 motif. Conformational rigidity was prominent in C-terminal tail region also. Limited proteolysis supported reduced accessibility of MTBR region in the molecule. ConclusionsPHF formation of oxidized tau40 might be attenuated either by induction of intramolecular H-bonding between the regions of high β-structure propensity in second and third MTBR (R2, R3), thus preventing intermolecular interaction between them, or by imparted rigidity in R2–R3, preventing the formation of extended β-structure preceding fibrillation. Data indicated plausible effect of conformational adaptation on the nucleation process of oxidized tau40 assembly. General significanceOur findings unravel the essential molecular features of aggregation-resistant tau conformer. Therapeutics stabilizing such conformers in vivo might be of high benefit in arresting tau assembly during AD and other tauopathies.

The role of annealing and fragmentation in human tau aggregation dynamics

Alzheimer's disease pathogenesis is associated with the conversion of monomeric tau protein into filamentous aggregates. Because both toxicity and prion-like spread of pathogenic tau depend in part on aggregate size, the processes that underlie filament formation and size distribution are of special importance. Here, using a combination of biophysical and computational approaches, we investigated the fibrillation dynamics of the human tau isoform 2N4R. We found that tau filaments engage in a previously uncharacterized secondary process involving end-to-end annealing and that rationalization of empirical aggregation data composed of total protomer concentrations and fibril length distributions requires inclusion of this process along with filament fragmentation. We noted that annealing of 2N4R tau filaments is robust, with an intrinsic association rate constant of a magnitude similar to that mediating monomer addition and consistent with diffusion-mediated protein-protein interactions in the absence of long-range attractive forces. In contrast, secondary nucleation on the surface of tau filaments did not detectably contribute to tau aggregation dynamics. These results indicate that tau filament ends engage in a range of homotypic interactions involving monomers, oligomers, and filaments. They further indicate that, in the case of tau protein, fibril annealing and fragmentation along with primary nucleation and elongation are the major processes controlling filament size distribution.

Dural lymphatics regulate clearance of extracellular tau from the CNS

BackgroundAlzheimer’s disease is characterized by two main neuropathological hallmarks: extracellular plaques of amyloid-β (Aβ) protein and intracellular aggregates of tau protein. Although tau is normally a soluble monomer that bind microtubules, in disease it forms insoluble, hyperphosphorylated aggregates in the cell body. Aside from its role in AD, tau is also involved in several other neurodegenerative disorders collectively called tauopathies, such as progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), some forms of frontotemporal dementia, and argyrophilic grain disease (AGD). The prion hypothesis suggests that after an initial trigger event, misfolded forms of tau are released into the extracellular space, where they spread through different brain regions, enter cells, and seeding previously normal forms. Thus understanding mechanisms regulating the clearance of extracellular tau from the CNS is important. The discovery of a true lymphatic system in the dura and its potential role in mediating Aβ pathology prompted us to investigate its role in regulating extracellular tau clearance.MethodsTo study clearance of extracellular tau from the brain, we conjugated monomeric human tau with a near-infrared dye cypate, and injected this labeled tau in the parenchyma of both wild-type and K14-VEGFR3-Ig transgenic mice, which lack a functional CNS lymphatic system. Following injection we performed longitudinal imaging using fluorescence molecular tomography (FMT) and quantified fluorescence to calculate clearance of tau from the brain. To complement this, we also measured tau clearance to the periphery by measuring plasma tau in both groups of mice.ResultsOur results show that a significantly higher amount of tau is retained in the brains of K14-VEGFR3-Ig vs. wild type mice at 48 and 72 h post-injection and its subsequent clearance to the periphery is delayed. We found that clearance of reference tracer human serum albumin (HSA) was also significantly delayed in the K14-VEGFR3-Ig mice.ConclusionsThe dural lymphatic system appears to play an important role in clearance of extracellular tau, since tau clearance is impaired in the absence of functional lymphatics. Based on our baseline characterization of extracellular tau clearance, future studies are warranted to look at the interaction between tau pathology and efficiency of lymphatic function.

Filamentous Aggregates Are Fragmented by the Proteasome Holoenzyme

SummaryFilamentous aggregates (fibrils) are regarded as the final stage in the assembly of amyloidogenic proteins and are formed in many neurodegenerative diseases. Accumulation of aggregates occurs as a result of an imbalance between their formation and removal. Here we use single-aggregate imaging to show that large fibrils assembled from full-length tau are substrates of the 26S proteasome holoenzyme, which fragments them into small aggregates. Interestingly, although degradation of monomeric tau is not inhibited by adenosine 5’-(3-thiotriphosphate) (ATPγS), fibril fragmentation is predominantly dependent on the ATPase activity of the proteasome. The proteasome holoenzyme also targets fibrils assembled from α-synuclein, suggesting that its fibril-fragmenting function may be a general mechanism. The fragmented species produced by the proteasome shows significant toxicity to human cell lines compared with intact fibrils. Together, our results indicate that the proteasome holoenzyme possesses a fragmentation function that disassembles large fibrils into smaller and more cytotoxic species.

Understanding the Kinetic Roles of 14-3-3ζ during Tau Filament Formation

The microtubule associated protein tau and 14-3-3ζ protein are two proteins highly expressed in the central nervous system. Tau protein promotes microtubule assembly and aberrant accumulation of tau and formation of amyloid fibrils are pathological hallmarks of neurodegenerative tauopathies including Alzheimer's disease. Extensive studies have suggested that polyanionic inducers, such as heparin, interact with tau directly and induce specific conformational changes within tau, accelerating the amyloidogenesis process. 14-3-3ζ proteins form homodimers and bind their targets through specific pSer/pThr motifs. It has been found that tau protein contains two 14-3-3ζ-binding motifs, i.e., pSer214 and pSer324, and tau protein interacts with 14-3-3ζ protein directly. Furthermore, 14-3-3ζ is able to induce tau aggregation, although it's still controversial whether this process depends on tau phosphorylation. Here, we investigated the kinetic roles of 14-3-3ζ during tau filament formation. We used the K18 constructs of tau that consists of the four microtubule repeats and the pSer324 14-3-3ζ-binding motifs. Our results showed that 14-3-3ζ induced tau K18 aggregation at a rate much larger than that from full-length tau. Kinetics analyses showed that the lag time and rate of aggregation were dependent on the stoichiometry of tau to 14-3-3ζ. Although phosphorylation at Ser324 increased the binding affinity of tau K18 to 14-3-3ζ, phosphorylation only showed marginal effect on 14-3-3ζ mediated tau aggregation. Our results suggest that the formation of the 2:1 complex of tau monomer and 14-3-3ζ dimer plays an important role in the 14-3-3ζ mediated tau aggregation process.

Disclosing the Template-Induced Misfolding Mechanism of Tau Protein by Studying the Dissociation of the Boundary Chain from the Formed Tau Fibril Based on a Steered Molecular Dynamics Simulation.

The level of tau aggregation into neurofibrillary tangles, including paired helical filament (PHF) and straight filament (SF), is closely associated with Alzheimer's disease. Despite the pathological importance of misfolding and aggregation of tau, the corresponding mechanism remains unclear. Therefore, to uncover the misfolding mechanism of the tau monomer upon induction of formed PHF and SF, in this study, a conventional molecular dynamics simulation combined with a steered molecular dynamics simulation was performed to study the dissociation of the boundary chain. Interestingly, our results show that the dissociation mechanisms of the boundary chain in PHF and SF are different. In PHF, the boundary chain begins to dissociate from regions β2 and β3 and ends at β8. However, in SF, it is simultaneously dissociated from β1 and β8 and ends at β5. The dissociation of the boundary chain is the reverse of template-induced misfolding of the monomer. Therefore, we can deduce the misfolding mechanism of the monomer upon induction of the template. For PHF, β8 first interacts with the template by hydrophobic interaction. Then β7, β6, β5, β4, and β1 sequentially bind to the template by electrostatic and hydrophobic interactions. After β1 binds to the template, β2 and β3 very quickly bind to the template through hydrophobic interaction. For SF, β5 of the monomer first interacts with the template by electrostatic attraction. Then β4 and β6, β3 and β7, and β2 and β8 bind to the template in turn. Finally, β1 and β8 are fully bound to the template by hydrophobic interaction. The obtained results will be vital for understanding the earlier events during misfolding and aggregation of tau.

Liquid-Liquid Phase Separation of Tau Protein in Neurobiology and Pathology.

Tau is an intrinsically unfolded protein that, aside from its important role in the regulation of microtubule stability, harbors an emerging number of other functions. In order to find explanations for some longtime unsolved aspects of neuronal tau biology in the brain, we may have to step aside from observing tau molecules in dilute solutions, and from assuming a mono-molecular physicochemical behavior of molecules in the cell. Liquid condensed phases of tau proteins, which form through the biophysical process of liquid-liquid phase separation (LLPS), behave like liquids and thereby offer a new regime of interactions in the cell. So far, there is evidence that tau condensates (i) play a role for neurodegenerative diseases by transitioning into aggregated forms of tau, (ii) are involved in microtubule binding, nucleation, and bundling, and (iii) are interacting with RNA molecules, which could impact RNA homeostasis and transcription. Likewise the functions of monomeric tau, also tau condensation is regulated by post-translational modifications and can be influenced by the local environment, for example in neuronal sub-compartments. However, we are just beginning to understand the physicochemistry of tau LLPS, and the biological role of tau condensation has to be explored in the next years.

Tau monomer encodes strains.

Tauopathies have diverse presentation, progression, and neuropathology. They are linked to tau prion strains, self-replicating assemblies of unique quaternary conformation, whose origin is unknown. Strains can be propagated indefinitely in cultured cells, and induce unique patterns of transmissible neuropathology upon inoculation into mice. DS9 and DS10 cell lines propagate different synthetic strains that derive from recombinant tau. We previously observed that tau monomer adopts two conformational states: one that is inert (Mi) and one that is seed-competent (Ms) (Mirbaha et al., 2018). We have now found that Ms itself is comprised of multiple stable ensembles that encode unique strains. DS9 monomer inoculated into naive cells encoded only DS9, whereas DS10 monomer encoded multiple sub-strains. Sub-strains each induced distinct pathology upon inoculation into a tauopathy mouse model (PS19). Ms purified from an Alzeimer's disease brain encoded a single strain. Conversely, Ms from a corticobasal degeneration brain encoded three sub-strains, in which monomer from any one re-established all three upon inoculation into cells. Seed competent tau monomer thus adopts multiple, stable seed-competent conformations, each of which encodes a limited number of strains. This provides insight into the emergence of distinct tauopathies, and may improve diagnosis and therapy.