Minimally invasive biomarkers are urgently needed to detect molecular pathology in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Here, we show that plasma extracellular vesicles (EVs) contain quantifiable amounts of TDP-43 and full-length tau, which allow the quantification of 3-repeat (3R) and 4-repeat (4R) tau isoforms. Plasma EV TDP-43 levels and EV 3R/4R tau ratios were determined in a cohort of 704 patients, including 37 genetically and 31 neuropathologically proven cases. Diagnostic groups comprised patients with TDP-43 proteinopathy ALS, 4R tauopathy progressive supranuclear palsy, behavior variant FTD (bvFTD) as a group with either tau or TDP-43 pathology, and healthy controls. EV tau ratios were low in progressive supranuclear palsy and high in bvFTD with tau pathology. EV TDP-43 levels were high in ALS and in bvFTD with TDP-43 pathology. Both markers discriminated between the diagnostic groups with area under the curve values >0.9, and between TDP-43 and tau pathology in bvFTD. Both markers strongly correlated with neurodegeneration, and clinical and neuropsychological markers of disease severity. Findings were replicated in an independent validation cohort of 292 patients including 34 genetically confirmed cases. Taken together, the combination of EV TDP-43 levels and EV 3R/4R tau ratios may aid the molecular diagnosis of FTD, FTD spectrum disorders and ALS, providing a potential biomarker to monitor disease progression and target engagement in clinical trials.

Low-invasive biomarkers are urgently needed for the detection of molecular pathology in Frontotemporal Dementia (FTD), FTD spectrum disorders and Amyotrophic Lateral Sclerosis (ALS). This is particularly true in behavior variant FTD (bvFTD), in which premortem biomarkers are missing to distinguish underlying Tau from TAR DNA binding protein (TDP-43) pathology. This lack of biomarkers prevents the stratification of patients for intervention trials and constitutes a major obstacle for the development of disease-modifying therapies. Extracellular vesicles (EVs) have been implicated in neurodegenerative disease pathology, contributing to the release and potentially to intercellular transmission of pathologically aggregated proteins. Here, we show that plasma EVs contain quantifiable amounts of TDP-43 and full-length Tau, which allows the quantification of 3 repeat (3R) and 4 repeat (4R) Tau isoforms. Plasma EV TDP-43 levels and EV 3R/4R Tau ratios were determined in a pilot and validation study of 704 patients, including 37 genetic and 31 neuropathologically proven cases. Diagnostic groups comprised patients with the TDP-43 proteinopathy ALS, the 4R tauopathy Progressive Supranuclear Palsy (PSP), bvFTD as a group with either Tau or TDP-43 pathology, and healthy controls (HC). Compared to HC, plasma EV 3R/4R Tau ratios were decreased in PSP, unchanged in ALS, and increased in a subset of bvFTD patients, consistent with Tau pathology in approximately 40% of cases with bvFTD. EV Tau ratio discriminated between PSP and bvFTD, ALS and healthy controls (AUC 0.96-0.99), and between bvFTD and ALS (AUC 0.90) as well as HC (AUC 0.91). Plasma EV TDP-43 levels were increased in ALS and in those bvFTD patients who did not display high EV Tau ratios. Plasma EV TDP-43 discriminated patients with ALS from HC (AUC 0.99), bvFTD (AUC 0.91) and PSP (AUC 0.99). The combination of EV Tau ratio and EV TDP-43 was reliably able to discriminate between TDP-43 and Tau pathology in bvFTD. This blood-based classification was confirmed in genetic and autopsy proven cases. Both markers strongly correlated with the neurodegeneration marker neurofilament light chain (NfL) as well as with clinical and neuropsychological markers of disease severity in ALS (TDP-43 with ECAS, ALS-FRS-R), bvFTD (TDP-43 and Tau ratio with CDR-SB, CDR plus NACC FTLD) and PSP (Tau ratio with PSP-RS). Taken together, the combination of both markers may aid the molecular diagnosis of FTD, FTD spectrum disorders and ALS, the stratification of patients for therapeutic trials and bears the potential of a biomarker to monitor disease progression and target engagement.

Microtubule-associated protein tau is a naturally unfolded protein that can modulate a vast array of physiological processes through direct or indirect binding with molecular partners. Aberrant tau homeostasis has been implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. In this study, we performed an unbiased high-content protein profiling assay by incubating recombinant human tau on microarrays containing thousands of human polypeptides. Among the putative tau-binding partners, we identify SAH hydrolase-like protein 1/inositol 1,4,5-trisphosphate receptor (IP3R)-binding protein (AHCYL1/IRBIT), a member of the SAH hydrolase family and a previously described modulator of IP3R activity. Using coimmunoprecipitation assays, we show that endogenous as well as overexpressed tau can physically interact with AHCYL1/IRBIT in brain tissues and cultured cells. Proximity ligation assay experiments demonstrate that tau overexpression may modify the close localization of AHCYL1/IRBIT to IP3R at the endoplasmic reticulum. Together, our experimental evidence indicates that tau interacts with AHCYL1/IRBIT and potentially modulates AHCYL1/IRBIT function.

The functions of the neuronal microtubule-associated protein Tau in the central nervous system are regulated by manifold posttranslational modifications at more than 50 sites. Tau in healthy neurons carries multiple phosphate groups, mostly in its microtubule assembly domain. Elevated phosphorylation and aggregation of Tau are widely considered pathological hallmarks in Alzheimer's disease (AD) and other tauopathies, triggering the quest for Tau posttranslational modifications in the disease context. However, the phosphorylation patterns of physiological and pathological Tau are surprisingly similar and heterogenous, making it difficult to identify specific modifications as therapeutic targets and biomarkers for AD. We present a concise summary of - and view on - important previous and recent advances in Tau phosphorylation analysis in the context of AD.

Tau misfolding and assembly is linked to a number of neurodegenerative diseases collectively described as tauopathies, including Alzheimer’s disease (AD) and Parkinson’s disease. Anionic cellular membranes, such as the cytosolic leaflet of the plasma membrane, are sites that concentrate and neutralize tau, primarily due to electrostatic interactions with tau’s microtubule binding repeat domain (RD). In addition to electrostatic interactions with lipids, tau also has interactions with membrane proteins, which are important for tau’s cellular functions. Tau also interacts with lipid tails to facilitate direct translocation across the membrane and can form stable protein-lipid complexes involved in cell-to-cell transport. Concentrated tau monomers at the membrane surface can form reversible condensates, change secondary structures, and induce oligomers, which may eventually undergo irreversible crosslinking and fibril formation. These β-sheet rich tau structures are capable of disrupting membrane organization and are toxic in cell-based assays. Given the evidence for relevant membrane-based tau assembly, we review the emerging hypothesis that polyanionic membranes may serve as a site for phase-separated tau condensation. Membrane-mediated phase separation may have important implications for regulating tau folding/misfolding, and may be a powerful mechanism to spatially direct tau for native membrane-mediated functions.

AbstractDie zelluläre Kondensation von intrinsisch ungeordneten Proteinen (IDPs) durch Flüssig‐flüssig‐Phasentrennung (LLPS) ermöglicht die dynamische Kompartimentierung und Regulation biologischer Prozesse. Das IDP Tau, das den Aufbau von Mikrotubuli fördert und bei der Alzheimer‐Krankheit hyperphosphoryliert wird, kann in Lösung und auf der Oberfläche von Mikrotubuli LLPS‐Prozesse durchlaufen. Der Einfluss der Tau‐Phosphorylierung auf Tau‐LLPS‐vermittelte Tubulinpolymerisation ist jedoch weitestgehend unbekannt. Wir zeigen hier, dass unmodifiziertes Tau sowie an krankheitsassoziierten Epitopen phosphoryliertes Tau zu flüssigkeitsähnlichen Tröpfchen kondensieren. Obwohl sich Tubulin in allen Tau‐Tröpfchen in hohen Konzentrationen anreichert, ist es nicht in der Lage, aus dem Inneren von Tröpfchen, die durch am AT180‐Epitop phosphoryliertes Tau (T231/S235) gebildet wurden, zu Mikrotubuli zu wachsen. Im Gegensatz dazu hemmte weder die Phosphorylierung von Tyrosinresten in Tau noch die Phosphorylierung in der Repeatdomäne die Polymerisation von Tubulin aus Tau‐Tröpfchen. Da gezeigt wurde, dass LLPS von IDPs verschiedene Arten der Zytoskelettassemblierung fördert, legt unsere Studie nahe, dass die IDP‐Phosphorylierung ein weit verbreiteter Mechanismus für die Regulation der kondensatvermittelten Zytoskelettassemblierung sein könnte.

Abnormal changes of neuronal Tau protein, such as phosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer's disease. Abnormal phosphorylation is thought to precede aggregation and therefore to promote aggregation, but the nature and extent of phosphorylation remain ill-defined. Tau contains ∼85 potential phosphorylation sites, which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, methodological limitations (e.g. in MS of phosphopeptides, or antibodies against phosphoepitopes) led to conflicting results regarding the extent of Tau phosphorylation in cells. Here we present results from a new approach based on native MS of intact Tau expressed in eukaryotic cells (Sf9). The extent of phosphorylation is heterogeneous, up to ∼20 phosphates per molecule distributed over 51 sites. The medium phosphorylated fraction Pm showed overall occupancies of ∼8 Pi (± 5) with a bell-shaped distribution; the highly phosphorylated fraction Ph had 14 Pi (± 6). The distribution of sites was highly asymmetric (with 71% of all P-sites in the C-terminal half of Tau). All sites were on Ser or Thr residues, but none were on Tyr. Other known posttranslational modifications were near or below our detection limit (e.g. acetylation, ubiquitination). These findings suggest that normal cellular Tau shows a remarkably high extent of phosphorylation, whereas other modifications are nearly absent. This implies that abnormal phosphorylations at certain sites may not affect the extent of phosphorylation significantly and do not represent hyperphosphorylation. By implication, the pathological aggregation of Tau is not likely a consequence of high phosphorylation.

Tau aggregation into amyloid fibers based on the cross-beta structure is a hallmark of several Tauopathies, including Alzheimer Disease (AD). Trans-cellular propagation of Tau with pathological conformation has been suggested as a key disease mechanism. This is thought to cause the spreading of Tau pathology in AD by templated conversion of naive Tau in recipient cells into a pathological state, followed by assembly of pathological Tau fibers, similar to the mechanism of nucleated polymerization proposed for prion pathogenesis. In cell cultures, the process is often monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (TauRD) with a pro-aggregant mutation, fused to GFP-based FRET pairs. Since the size of the reporter GFP (barrel of ~ 3 nm × 4 nm) is ~ 7 times larger than the β-strand distance (0.47 nm), this points to a potential steric clash. Hence, we investigated the influence of the GFP tag on TauFL or TauRD aggregation. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of TauRD-GFP was severely inhibited and incompatible with that of Alzheimer filaments. These observations argue against the hypothesis that the propagation of Tau pathology in AD is caused by the prion-like templated aggregation of Tau protein, transmitted via cell-to-cell spreading of Tau. Thus, even though the observed local increase of FRET in recipient cells may be a valid hallmark of a pathological reaction, our data argue that it is caused by a process distinct from assembly of TauRD filaments.

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.

Cellular condensation of intrinsically disordered proteins (IDPs) through liquid-liquid phase separation (LLPS) allows dynamic compartmentalization and regulation of biological processes. The IDP tau, which promotes the assembly of microtubules and is hyperphosphorylated in Alzheimer's disease, undergoes LLPS in solution and on the surface of microtubules. Little is known, however, about the influence of tau phosphorylation on its ability to nucleate microtubule bundles in conditions of tau LLPS. Herein, we show that unmodified tau as well as tau phosphorylated at disease-associated epitopes condense into liquid-like droplets. Although tubulin partitioned into and reached high concentrations inside all tau droplets, it was unable to grow into microtubules form the inside of droplets formed by tau phosphorylated at the AT180 epitope (T231/S235). In contrast, neither phosphorylation of tau in the repeat domain nor at its tyrosine residues inhibited the assembly of tubulin from tau droplets. Because LLPS of IDPs has been shown to promote different types of cytoskeletal assembly, our study suggests that IDP phosphorylation might be a broadly used mechanism for the modulation of condensate-mediated cytoskeletal assembly.

Deutsches Zentrum für Neurodegenerative E 37075 Göttingen, Germany. E-mail: mazw@ Max-Planck-Institut für Biophysikalische Ch Germany NMR & Structural Chemistry Division, CSIR Hyderabad, 500007, India Deutsches Zentrum für Neurodegenerative Allee 2, 53175 Bonn, Germany CAESAR Research Center, Bonn, MPI for Me 22607 Hamburg, Germany † Electronic supplementary informa 10.1039/c9sc00531e Cite this: Chem. Sci., 2019, 10, 6503

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.

May 5, 2019April 9, 2019Free AccessStructure and Pro-toxic Mechanism of the Human Hsp90/PPIase/Tau Complex (P1.1-006)Javier Oroz, Bliss Chang, Piotr Wysoczanski, Chung-tien Lee, Angel Perez Lara, Pijush Chakraborty, Romina Hofele, … Show All … , Jeremy Baker, Laura Blair, Jacek Biernat, Henning Urlaub, Eckhard Mandelkow, Chad Dickey, and Markus Zweckstetter Show FewerAuthors Info & AffiliationsApril 9, 2019 issue92 (15_supplement)https://doi.org/10.1212/WNL.92.15_supplement.P1.1-006 Letters to the Editor

Liquid-liquid phase separation (LLPS) of proteins enables the formation of non-membrane-bound organelles in cells and is associated with cancer and neurodegeneration. Little is known however about the structure and dynamics of proteins in LLPS conditions, because of the polymorphic nature of liquid-like protein droplets. Using carbon-detected NMR experiments we here show that the conversion of the aggregation-prone repeat region of the Alzheimer's-related protein tau from the dispersed monomeric state to phase-separated liquid-like droplets involves tau's aggregation-prone hexapeptides and regulatory KXGS motifs. Droplet dissolution in presence of 1,6-hexanediol revealed that chemical shift perturbations in the hexapeptide motifs are temperature driven, while those in KXGS motifs report on phase separation. Residue-specific secondary structure analysis further indicated that tau's repeat region exists in extended conformation in the dispersed state and attains transient β-hairpin propensity upon LLPS. Taken together our work shows that NMR spectroscopy can provide high-resolution insights into LLPS-induced changes in intrinsically disordered proteins.

The microtubule-associated protein Tau promotes the polymerization of tubulin and modulates the function of microtubules. As a consequence of the dynamic nature of the Tau-tubulin interaction, the structural basis of this complex has remained largely elusive. By using NMR methods optimized for ligand-receptor interactions in combination with site-directed mutagenesis we demonstrate that the flanking domain downstream of the four microtubule-binding repeats of Tau binds competitively to a site on the α-tubulin surface. The binding process is complex, involves partial coupling of different interacting regions, and is modulated by phosphorylation at Y394 and S396. This study strengthens the hypothesis of an intimate relationship between Tau phosphorylation and tubulin binding and highlights the power of the INPHARMA NMR method to characterize the interaction of peptides derived from intrinsically disordered proteins with their molecular partners.

The molecular chaperone Hsp90 is critical for the maintenance of cellular homeostasis and represents a promising drug target. Despite increasing knowledge on the structure of Hsp90, the molecular basis of substrate recognition and pro-folding by Hsp90/co-chaperone complexes remains unknown. Here, we report the solution structures of human full-length Hsp90 in complex with the PPIase FKBP51, as well as the 280 kDa Hsp90/FKBP51 complex bound to the Alzheimer’s disease-related protein Tau. We reveal that the FKBP51/Hsp90 complex, which synergizes to promote toxic Tau oligomers in vivo, is highly dynamic and stabilizes the extended conformation of the Hsp90 dimer resulting in decreased Hsp90 ATPase activity. Within the ternary Hsp90/FKBP51/Tau complex, Hsp90 serves as a scaffold that traps the PPIase and nucleates multiple conformations of Tau’s proline-rich region next to the PPIase catalytic pocket in a phosphorylation-dependent manner. Our study defines a conceptual model for dynamic Hsp90/co-chaperone/client recognition.

Das Mikrotubuli‐assoziierte Protein Tau fördert die Polymerisation von Tubulin und beeinflusst die Mikrotubuli‐Funktion. Aufgrund einer inhärenten Flexibilität in der Tau‐Tubulin‐Wechselwirkung sind die strukturellen Grundlagen dieser Interaktion weitestgehend unbekannt. Mithilfe von Liganden‐Rezeptor‐spezifischen NMR‐Methoden und positionsspezifischer Mutagenese wird nachgewiesen, dass die Region N‐terminal zu den vier Mikrotubuli‐bindenden Wiederholungssequenzen an die Oberfläche von α‐Tubulin bindet. Der Bindungsprozess ist komplex, umfasst eine partielle Kupplung verschiedener Tau‐Aminosäuren und wird durch Tau‐Phosphorylierung an Y394 und S396 beeinflusst. Die Untersuchungen stärken die Hypothese einer engen Verbindung zwischen Tau‐Phosphorylierung und ‐Bindung an Tubulin. Ferner unterstreichen die Ergebnisse die Stärke der INPHARMA‐NMR‐Methode für die Analyse von Wechselwirkungen zwischen intrinsisch ungeordneten Peptiden/Proteinen und ihren molekularen Partnern.

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.

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.