Disease-specific tau filaments assemble via polymorphic intermediates.

Intermediate species in the assembly of amyloid filaments are believed to play a central role in neurodegenerative diseases and may constitute important targets for therapeutic intervention1,2. However, structural information about intermediate species has been scarce and the molecular mechanisms by which amyloids assemble remain largely unknown. Here we use time-resolved cryogenic electron microscopy to study the in vitro assembly of recombinant truncated tau (amino acid residues 297-391) into paired helical filaments of Alzheimer's disease or into filaments of chronic traumatic encephalopathy3. We report the formation of a shared first intermediate amyloid filament, with an ordered core comprising residues 302-316. Nuclear magnetic resonance indicates that the same residues adopt rigid, ß-strand-like conformations in monomeric tau. At later time points, the first intermediate amyloid disappears and we observe many different intermediate amyloid filaments, with structures that depend on the reaction conditions. At the end of both assembly reactions, most intermediate amyloids disappear and filaments with the same ordered cores as those from human brains remain. Our results provide structural insights into the processes of primary and secondary nucleation of amyloid assembly, with implications for the design of new therapies.

Structure-based classification of tauopathies.

The ordered assembly of tau protein into filaments characterizes several neurodegenerative diseases, which are called tauopathies. It was previously reported that, by cryo-electron microscopy, the structures of tau filaments from Alzheimer's disease1,2, Pick's disease3, chronic traumatic encephalopathy4 and corticobasal degeneration5 are distinct. Here we show that the structures of tau filaments from progressive supranuclear palsy (PSP) define a new three-layered fold. Moreover, the structures of tau filaments from globular glial tauopathy are similar to those from PSP. The tau filament fold of argyrophilic grain disease (AGD) differs, instead resembling the four-layered fold of corticobasal degeneration. The AGD fold is also observed in ageing-related tau astrogliopathy. Tau protofilament structures from inherited cases of mutations at positions +3 or +16 in intron 10 of MAPT (the microtubule-associated protein tau gene) are also identical to those from AGD, suggesting that relative overproduction of four-repeat tau can give rise to the AGD fold. Finally, the structures of tau filaments from cases of familial British dementia and familial Danish dementia are the same as those from cases of Alzheimer's disease and primary age-related tauopathy. These findings suggest a hierarchical classification of tauopathies on the basis of their filament folds, which complements clinical diagnosis and neuropathology and also allows the identification of new entities-as we show for a case diagnosed as PSP, but with filament structures that are intermediate between those of globular glial tauopathy and PSP.

Novel tau filament fold in corticobasal degeneration.

Corticobasal degeneration (CBD) is a neurodegenerative tauopathy-a class of disorders in which the tau protein forms insoluble inclusions in the brain-that is characterized by motor and cognitive disturbances1-3. The H1 haplotype of MAPT (the tau gene) is present in cases of CBD at a higher frequency than in controls4,5, and genome-wide association studies have identified additional risk factors6. By histology, astrocytic plaques are diagnostic of CBD7,8; by SDS-PAGE, so too are detergent-insoluble, 37 kDa fragments of tau9. Like progressive supranuclear palsy, globular glial tauopathy and argyrophilic grain disease10, CBD is characterized by abundant filamentous tau inclusions that are made of isoforms with four microtubule-binding repeats11-15. This distinguishes such '4R' tauopathies from Pick's disease (the filaments of which are made of three-repeat (3R) tau isoforms) and from Alzheimer's disease and chronic traumatic encephalopathy (CTE) (in which both 3R and 4R isoforms are found in the filaments)16. Here we use cryo-electron microscopy to analyse the structures of tau filaments extracted from the brains of three individuals with CBD. These filaments were identical between cases, but distinct from those seen in Alzheimer's disease, Pick's disease and CTE17-19. The core of a CBD filament comprises residues lysine 274 to glutamate 380 of tau, spanning the last residue of the R1 repeat, the whole of the R2, R3 and R4 repeats, and 12 amino acids after R4. The core adopts a previously unseen four-layered fold, which encloses a large nonproteinaceous density. This density is surrounded by the side chains of lysine residues 290 and 294 from R2 and lysine 370 from the sequence after R4.

Novel tau filament fold in chronic traumatic encephalopathy encloses hydrophobic molecules.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative tauopathy that is associated with repetitive head impacts or exposure to blast waves. First described as punch-drunk syndrome and dementia pugilistica in retired boxers1-3, CTE has since been identified in former participants of other contact sports, ex-military personnel and after physical abuse4-7. No disease-modifying therapies currently exist, and diagnosis requires an autopsy. CTE is defined by an abundance of hyperphosphorylated tau protein in neurons, astrocytes and cell processes around blood vessels8,9. This, together with the accumulation of tau inclusions in cortical layers II and III, distinguishes CTE from Alzheimer's disease and other tauopathies10,11. However, the morphologies of tau filaments in CTE and the mechanisms by which brain trauma can lead to their formation are unknown. Here we determine the structures of tau filaments from the brains of three individuals with CTE at resolutions down to 2.3 Å, using cryo-electron microscopy. We show that filament structures are identical in the three cases but are distinct from those of Alzheimer's and Pick's diseases, and from those formed in vitro12-15. Similar to Alzheimer's disease12,14,16-18, all six brain tau isoforms assemble into filaments in CTE, and residues K274-R379 of three-repeat tau and S305-R379 of four-repeat tau form the ordered core of two identical C-shaped protofilaments. However, a different conformation of the ß-helix region creates a hydrophobic cavity that is absent in tau filaments from the brains of patients with Alzheimer's disease. This cavity encloses an additional density that is not connected to tau, which suggests that the incorporation of cofactors may have a role in tau aggregation in CTE. Moreover, filaments in CTE have distinct protofilament interfaces to those of Alzheimer's disease. Our structures provide a unifying neuropathological criterion for CTE, and support the hypothesis that the formation and propagation of distinct conformers of assembled tau underlie different neurodegenerative diseases.

Structures of filaments from Pick's disease reveal a novel tau protein fold.

The ordered assembly of tau protein into abnormal filamentous inclusions underlies many human neurodegenerative diseases1. Tau assemblies seem to spread through specific neural networks in each disease2, with short filaments having the greatest seeding activity3. The abundance of tau inclusions strongly correlates with disease symptoms4. Six tau isoforms are expressed in the normal adult human brain-three isoforms with four microtubule-binding repeats each (4R tau) and three isoforms that lack the second repeat (3R tau)1. In various diseases, tau filaments can be composed of either 3R or 4R tau, or of both. Tau filaments have distinct cellular and neuroanatomical distributions5, with morphological and biochemical differences suggesting that they may be able to adopt disease-specific molecular conformations6,7. Such conformers may give rise to different neuropathological phenotypes8,9, reminiscent of prion strains10. However, the underlying structures are not known. Using electron cryo-microscopy, we recently reported the structures of tau filaments from patients with Alzheimer's disease, which contain both 3R and 4R tau11. Here we determine the structures of tau filaments from patients with Pick's disease, a neurodegenerative disorder characterized by frontotemporal dementia. The filaments consist of residues Lys254-Phe378 of 3R tau, which are folded differently from the tau filaments in Alzheimer's disease, establishing the existence of conformers of assembled tau. The observed tau fold in the filaments of patients with Pick's disease explains the selective incorporation of 3R tau in Pick bodies, and the differences in phosphorylation relative to the tau filaments of Alzheimer's disease. Our findings show how tau can adopt distinct folds in the human brain in different diseases, an essential step for understanding the formation and propagation of molecular conformers.

The Enigma of Tau Protein Aggregation: Mechanistic Insights and Future Challenges.

Tau protein misfolding and aggregation are pathological hallmarks of Alzheimer's disease and over twenty neurodegenerative disorders. However, the molecular mechanisms of tau aggregation in vivo remain incompletely understood. There are two types of tau aggregates in the brain: soluble aggregates (oligomers and protofibrils) and insoluble filaments (fibrils). Compared to filamentous aggregates, soluble aggregates are more toxic and exhibit prion-like transmission, providing seeds for templated misfolding. Curiously, in its native state, tau is a highly soluble, heat-stable protein that does not form fibrils by itself, not even when hyperphosphorylated. In vitro studies have found that negatively charged molecules such as heparin, RNA, or arachidonic acid are generally required to induce tau aggregation. Two recent breakthroughs have provided new insights into tau aggregation mechanisms. First, as an intrinsically disordered protein, tau is found to undergo liquid-liquid phase separation (LLPS) both in vitro and inside cells. Second, cryo-electron microscopy has revealed diverse fibrillar tau conformations associated with different neurodegenerative disorders. Nonetheless, only the fibrillar core is structurally resolved, and the remainder of the protein appears as a "fuzzy coat". From this review, it appears that further studies are required (1) to clarify the role of LLPS in tau aggregation; (2) to unveil the structural features of soluble tau aggregates; (3) to understand the involvement of fuzzy coat regions in oligomer and fibril formation.

Cryo-EM structures of tau filaments from Alzheimer's disease.

Alzheimer's disease is the most common neurodegenerative disease, and there are no mechanism-based therapies. The disease is defined by the presence of abundant neurofibrillary lesions and neuritic plaques in the cerebral cortex. Neurofibrillary lesions comprise paired helical and straight tau filaments, whereas tau filaments with different morphologies characterize other neurodegenerative diseases. No high-resolution structures of tau filaments are available. Here we present cryo-electron microscopy (cryo-EM) maps at 3.4-3.5 Å resolution and corresponding atomic models of paired helical and straight filaments from the brain of an individual with Alzheimer's disease. Filament cores are made of two identical protofilaments comprising residues 306-378 of tau protein, which adopt a combined cross-ß/ß-helix structure and define the seed for tau aggregation. Paired helical and straight filaments differ in their inter-protofilament packing, showing that they are ultrastructural polymorphs. These findings demonstrate that cryo-EM allows atomic characterization of amyloid filaments from patient-derived material, and pave the way for investigation of a range of neurodegenerative diseases.

In vitro 0N4R tau fibrils contain a monomorphic ß-sheet core enclosed by dynamically heterogeneous fuzzy coat segments.

Misfolding of the microtubule-binding protein tau into filamentous aggregates is characteristic of many neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Determining the structures and dynamics of these tau fibrils is important for designing inhibitors against tau aggregation. Tau fibrils obtained from patient brains have been found by cryo-electron microscopy to adopt disease-specific molecular conformations. However, in vitro heparin-fibrillized 2N4R tau, which contains all four microtubule-binding repeats (4R), was recently found to adopt polymorphic structures. Here we use solid-state NMR spectroscopy to investigate the global fold and dynamics of heparin-fibrillized 0N4R tau. A single set of 13C and 15N chemical shifts was observed for residues in the four repeats, indicating a single ß-sheet conformation for the fibril core. This rigid core spans the R2 and R3 repeats and adopts a hairpin-like fold that has similarities to but also clear differences from any of the polymorphic 2N4R folds. Obtaining a homogeneous fibril sample required careful purification of the protein and removal of any proteolytic fragments. A variety of experiments and polarization transfer from water and mobile side chains indicate that 0N4R tau fibrils exhibit heterogeneous dynamics: Outside the rigid R2-R3 core, the R1 and R4 repeats are semirigid even though they exhibit ß-strand character and the proline-rich domains undergo large-amplitude anisotropic motions, whereas the two termini are nearly isotropically flexible. These results have significant implications for the structure and dynamics of 4R tau fibrils in vivo.

Cryo-EM structures of tau filaments from Alzheimer's disease with PET ligand APN-1607.

Tau and Aß assemblies of Alzheimer's disease (AD) can be visualized in living subjects using positron emission tomography (PET). Tau assemblies comprise paired helical and straight filaments (PHFs and SFs). APN-1607 (PM-PBB3) is a recently described PET ligand for AD and other tau proteinopathies. Since it is not known where in the tau folds PET ligands bind, we used electron cryo-microscopy (cryo-EM) to determine the binding sites of APN-1607 in the Alzheimer fold. We identified two major sites in the ß-helix of PHFs and SFs and a third major site in the C-shaped cavity of SFs. In addition, we report that tau filaments from posterior cortical atrophy (PCA) and primary age-related tauopathy (PART) are identical to those from AD. In support, fluorescence labelling showed binding of APN-1607 to intraneuronal inclusions in AD, PART and PCA. Knowledge of the binding modes of APN-1607 to tau filaments may lead to the development of new ligands with increased specificity and binding activity. We show that cryo-EM can be used to identify the binding sites of small molecules in amyloid filaments.

Protein Aggregation Landscape in Neurodegenerative Diseases: Clinical Relevance and Future Applications.

Intrinsic disorder is a natural feature of polypeptide chains, resulting in the lack of a defined three-dimensional structure. Conformational changes in intrinsically disordered regions of a protein lead to unstable ß-sheet enriched intermediates, which are stabilized by intermolecular interactions with other ß-sheet enriched molecules, producing stable proteinaceous aggregates. Upon misfolding, several pathways may be undertaken depending on the composition of the amino acidic string and the surrounding environment, leading to different structures. Accumulating evidence is suggesting that the conformational state of a protein may initiate signalling pathways involved both in pathology and physiology. In this review, we will summarize the heterogeneity of structures that are produced from intrinsically disordered protein domains and highlight the routes that lead to the formation of physiological liquid droplets as well as pathogenic aggregates. The most common proteins found in aggregates in neurodegenerative diseases and their structural variability will be addressed. We will further evaluate the clinical relevance and future applications of the study of the structural heterogeneity of protein aggregates, which may aid the understanding of the phenotypic diversity observed in neurodegenerative disorders.

From Posttranslational Modifications to Disease Phenotype: A Substrate Selection Hypothesis in Neurodegenerative Diseases.

A number of neurodegenerative diseases including prion diseases, tauopathies and synucleinopathies exhibit multiple clinical phenotypes. A diversity of clinical phenotypes has been attributed to the ability of amyloidogenic proteins associated with a particular disease to acquire multiple, conformationally distinct, self-replicating states referred to as strains. Structural diversity of strains formed by tau, α-synuclein or prion proteins has been well documented. However, the question how different strains formed by the same protein elicit different clinical phenotypes remains poorly understood. The current article reviews emerging evidence suggesting that posttranslational modifications are important players in defining strain-specific structures and disease phenotypes. This article put forward a new hypothesis referred to as substrate selection hypothesis, according to which individual strains selectively recruit protein isoforms with a subset of posttranslational modifications that fit into strain-specific structures. Moreover, it is proposed that as a result of selective recruitment, strain-specific patterns of posttranslational modifications are formed, giving rise to unique disease phenotypes. Future studies should define whether cell-, region- and age-specific differences in metabolism of posttranslational modifications play a causative role in dictating strain identity and structural diversity of strains of sporadic origin.

HDAC6 ZnF UBP as the Modifier of Tau Structure and Function.

Histone deacetylase 6 is a class II histone deacetylase primarily present in the cytoplasm and involved in the regulation of various cellular functions. It consists of two catalytic deacetylase domains and a unique zinc finger ubiquitin binding protein domain, which sets it apart from other HDACs. HDAC6 is known to regulate cellular activities by modifying the function of microtubules, HSP90, and cortactin through deacetylation. Apart from the catalytic activity of HDAC6, it interacts with other proteins through either the SE14 domain or the ZnF UBP domain to modulate their functions. Here, we have studied the role of the HDAC6 ZnF UBP domain as a modifier of Tau aggregation by its direct interaction with the polyproline region/repeat region of Tau. Interaction of HDAC6 ZnF UBP with Tau was found to reduce the propensity of Tau to self-aggregate and to disaggregate preformed aggregates in a concentration-dependent manner and also bring about the conformational changes in Tau protein. The interaction of HDAC6 ZnF UBP with Tau results in its degradation, suggesting either proteolytic activity of HDAC6 ZnF UBP or its role in enhancing autoproteolysis of Tau.

Aggregation Kinetics and Filament Structure of a Tau Fragment Are Influenced by the Sulfation Pattern of the Cofactor Heparin.

A pathological signature of Alzheimer's disease (AD) is the formation of neurofibrillary tangles comprising filamentous aggregates of the microtubule associated protein tau. Tau self-assembly is accelerated by polyanions including heparin, an analogue of heparan sulfate. Tau filaments colocalize with heparan sulfate proteoglycans (HSPGs) in vivo, and HSPGs may also assist the transcellular propagation of tau aggregates. Here, we investigate the role of the sulfate moieties of heparin in the aggregation of a recombinant tau fragment Δtau187, comprising residues 255-441 of the C-terminal microtubule-binding domain. The effects that the selective removal of the N-, 2-O-, and 6-O-sulfate groups from heparin have on the kinetics of tau aggregation, aggregate morphology, and protein structure and dynamics were examined. Aggregation kinetics monitored by thioflavin T (ThT) fluorescence revealed that aggregation is considerably slower in the presence of 2-O-desulfated heparin than with N- or 6-O-desulfated heparin. Transmission electron microscopy revealed that tau filaments induced by 2-O-desulfated heparin were more slender than filaments formed in the presence of intact heparin or 6-O-desulfated heparin. The 2-O-desulfated heparin-induced filaments had more extensive regions of flexibility than the other filaments, according to circular dichroism and solid-state NMR spectroscopy. These results indicate that the sulfation pattern of heparin regulates tau aggregation, not purely though electrostatic forces but also through conformational perturbations of heparin when the 2-O-sulfate is removed. These findings may have implications for the progression of AD, as the sulfation pattern of GAGs is known to change during the aging process, which is the main risk factor for the disease.

Structure-based inhibitors halt prion-like seeding by Alzheimer's disease-and tauopathy-derived brain tissue samples.

In Alzheimer's disease (AD) and tauopathies, tau aggregation accompanies progressive neurodegeneration. Aggregated tau appears to spread between adjacent neurons and adjacent brain regions by prion-like seeding. Hence, inhibitors of this seeding offer a possible route to managing tauopathies. Here, we report the 1.0 Å resolution micro-electron diffraction structure of an aggregation-prone segment of tau with the sequence SVQIVY, present in the cores of patient-derived fibrils from AD and tauopathies. This structure illuminates how distinct interfaces of the parent segment, containing the sequence VQIVYK, foster the formation of distinct structures. Peptide-based fibril-capping inhibitors designed to target the two VQIVYK interfaces blocked proteopathic seeding by patient-derived fibrils. These VQIVYK inhibitors add to a panel of tau-capping inhibitors that targets specific polymorphs of recombinant and patient-derived tau fibrils. Inhibition of seeding initiated by brain tissue extracts differed among donors with different tauopathies, suggesting that particular fibril polymorphs of tau are associated with certain tauopathies. Donors with progressive supranuclear palsy exhibited more variation in inhibitor sensitivity, suggesting that fibrils from these donors were more polymorphic and potentially vary within individual donor brains. Our results suggest that a subset of inhibitors from our panel could be specific for particular disease-associated polymorphs, whereas inhibitors that blocked seeding by extracts from all of the tauopathies tested could be used to broadly inhibit seeding by multiple disease-specific tau polymorphs. Moreover, we show that tau-capping inhibitors can be transiently expressed in HEK293 tau biosensor cells, indicating that nucleic acid-based vectors can be used for inhibitor delivery.

Elucidating Tau function and dysfunction in the era of cryo-EM.

Tau is a microtubule-associated protein involved in the regulation of axonal microtubules in neurons. In pathological conditions, it forms fibrils that are molecular hallmarks of neurological disorders known as tauopathies. In the last 2 years, cryo-EM has given unprecedented high-resolution views of Tau in both physiological and pathological conditions. We review here these new findings and put them into the context of the knowledge about Tau before this structural breakthrough. The first structures of Tau fibrils, a molecular hallmark of Alzheimer's disease (AD), were based on fibrils from the brain of an individual with AD and, along with similar patient-derived structures, have set the gold standard for the field. Cryo-EM structures of Tau fibers in three distinct diseases, AD, Pick's disease, and chronic traumatic encephalopathy, represent the end points of Tau's molecular trajectory. We propose that the recent Tau structures may call for a re-examination of databases that link different Tau variants to various forms of dementia. We also address the question of how this structural information may link Tau's functional and pathological aspects. Because this structural information on Tau was obtained in a very short period, the new structures should be viewed in light of earlier structural observations and past and present functional data to shed additional light on Tau function and dysfunction.

Distinct microscopic mechanisms for the accelerated aggregation of pathogenic Tau mutants revealed by kinetic analysis.

The self-assembly of Tau protein into amyloid structures is associated with Alzheimer's disease and other tauopathies. Dominant familial mutations in the Tau gene, such as P301L and P301S, increase the propensity of the Tau protein to aggregate abnormally into filaments. A quantitative description of the fibrillization process of Tau will facilitate the understanding of the cytotoxicity of Tau aggregates and their intercellular spreading. Here, we investigated the aggregation kinetics of Tau and disease-associated P301L and P301S mutants by combined thioflavin T assay and kinetic modeling, which revealed the rate constants of individual microscopic steps in the process of amyloid formation. Compared to WT Tau, P301L shows a larger primary nucleation rate while P301S has higher elongation and fragmentation rates and a more apparent fibril annealing process. Cross-seeding assays and FRET experiments indicate that the structures of the fibrillar nuclei of the three variants are distinct. These results provide detailed insights into how the amyloid aggregation mechanism of Tau protein is affected by the familial mutations P301L and P301S, and relates the physical properties of Tau mutants to their pathogenic mechanism.

HspB1 and Hsc70 chaperones engage distinct tau species and have different inhibitory effects on amyloid formation.

The microtubule-associated protein tau forms insoluble, amyloid-type aggregates in various dementias, most notably Alzheimer's disease. Cellular chaperone proteins play important roles in maintaining protein solubility and preventing aggregation in the crowded cellular environment. Although tau is known to interact with numerous chaperones, it remains unclear how these chaperones function mechanistically to prevent tau aggregation and how chaperones from different classes compare in terms of mechanism. Here, we focused on the small heat shock protein HspB1 (also known as Hsp27) and the constitutive chaperone Hsc70 (also known as HspA8) and report how each chaperone interacts with tau to prevent its fibril formation. Using fluorescence and NMR spectroscopy, we show that the two chaperones inhibit tau fibril formation by distinct mechanisms. HspB1 delayed tau fibril formation by weakly interacting with early species in the aggregation process, whereas Hsc70 was highly efficient at preventing tau fibril elongation, possibly by capping the ends of tau fibrils. Both chaperones recognized aggregation-prone motifs within the microtubule-binding repeat region of tau. However, HspB1 binding remained transient in both aggregation-promoting and non-aggregating conditions, whereas Hsc70 binding was significantly tighter under aggregation-promoting conditions. These differences highlight the fact that chaperones from different families play distinct but complementary roles in the prevention of pathological protein aggregation.

Protein structural and surface water rearrangement constitute major events in the earliest aggregation stages of tau.

Protein aggregation plays a critical role in the pathogenesis of neurodegenerative diseases, and the mechanism of its progression is poorly understood. Here, we examine the structural and dynamic characteristics of transiently evolving protein aggregates under ambient conditions by directly probing protein surface water diffusivity, local protein segment dynamics, and interprotein packing as a function of aggregation time, along the third repeat domain and C terminus of Δtau187 spanning residues 255-441 of the longest isoform of human tau. These measurements were achieved with a set of highly sensitive magnetic resonance tools that rely on site-specific electron spin labeling of Δtau187. Within minutes of initiated aggregation, the majority of Δtau187 that is initially homogeneously hydrated undergoes structural transformations to form partially structured aggregation intermediates. This is reflected in the dispersion of surface water dynamics that is distinct around the third repeat domain, found to be embedded in an intertau interface, from that of the solvent-exposed C terminus. Over the course of hours and in a rate-limiting process, a majority of these aggregation intermediates proceed to convert into stable ß-sheet structured species and maintain their stacking order without exchanging their subunits. The population of ß-sheet structured species is >5% within 5 min of aggregation and gradually grows to 50-70% within the early stages of fibril formation, while they mostly anneal block-wisely to form elongated fibrils. Our findings suggest that the formation of dynamic aggregation intermediates constitutes a major event occurring in the earliest stages of tau aggregation that precedes, and likely facilitates, fibril formation and growth.

Tau filaments from multiple cases of sporadic and inherited Alzheimer's disease adopt a common fold.

The ordered assembly of tau protein into abnormal filaments is a defining characteristic of Alzheimer's disease (AD) and other neurodegenerative disorders. It is not known if the structures of tau filaments vary within, or between, the brains of individuals with AD. We used a combination of electron cryo-microscopy (cryo-EM) and immuno-gold negative-stain electron microscopy (immuno-EM) to determine the structures of paired helical filaments (PHFs) and straight filaments (SFs) from the frontal cortex of 17 cases of AD (15 sporadic and 2 inherited) and 2 cases of atypical AD (posterior cortical atrophy). The high-resolution structures of PHFs and SFs from the frontal cortex of 3 cases of AD, 2 sporadic and 1 inherited, were determined by cryo-EM. We also used immuno-EM to study the PHFs and SFs from a number of cortical and subcortical brain regions. PHFs outnumbered SFs in all AD cases. By cryo-EM, PHFs and SFs were made of two C-shaped protofilaments with a combined cross-ß/ß-helix structure, as described previously for one case of AD. The higher resolution structures obtained here showed two additional amino acids at each end of the protofilament. The immuno-EM findings, which indicated the presence of repeats 3 and 4, but not of the N-terminal regions of repeats 1 and 2, of tau in the filament cores of all AD cases, were consistent with the cryo-EM results. These findings show that there is no significant variation in tau filament structures between individuals with AD. This knowledge will be crucial for understanding the mechanisms that underlie tau filament formation and for developing novel diagnostics and therapies.

Fracture and Growth Are Competing Forces Determining the Fate of Conformers in Tau Fibril Populations.

Tau fibrils are pathological aggregates that can transfer between neurons and then recruit soluble Tau monomers by template-assisted conversion. The propagation of different fibril polymorphs is thought to be a contributing factor to phenotypic diversity in Alzheimer disease and other Tauopathies. We found that a homogeneous population of Tau fibrils composed of the truncated version K18 (residues 244-372) gradually converted to a new set of fibril conformers when subjected to multiple cycles of seeding and growth. Using double electron-electron resonance (DEER) spectroscopy, we observed that the distances between spin labels at positions 311 and 328 in the fibril core progressively decreased. The findings were corroborated by changes in turbidity, morphology, and protease sensitivity. Fibrils that were initially formed under stirring conditions exhibited an increased fragility compared with fibrils formed quiescently after multiple cycles of seeding. The quiescently formed fibrils were marked by accelerated growth. The difference in fragility and growth between the different conformers explains how the change in incubation condition could lead to the amplification of a minor subpopulation of fibrils. Under quiescent conditions where fibril breakage is minimal, faster growing fibrils have a selective advantage. The findings are of general importance as they suggest that changes in selective pressures during fibril propagation in the human brain could result in the emergence of new fibril conformers with varied clinicopathological consequences.