Helical Filaments Research Articles

Tau, tau kinases, and tauopathies: An updated overview.

Tau is a macrotubule-associated protein primarily involved in the stabilization of the cytoskeleton. Under normal conditions, phosphorylation reduces the affinity of tau for tubulin, allowing the protein to detach from microtubules and ensuring the system dynamics in neuronal cells. However, hyperphosphorylated tau aggregates into paired helical filaments, the main constituents of neurofibrillary tangles found in the brains of patients with Alzheimer's disease and other tauopathies. In this review, we provide an overview of the structure of tau and the pathophysiological roles of tau phosphorylation. We also evaluate the major protein kinases involved and discuss the progress made in the development of drug therapies aimed at inhibiting tau kinases.

Selective In Vitro and Ex Vivo Staining of Brain Neurofibrillary Tangles and Amyloid Plaques by Novel Ethylene Ethynylene-Based Optical Sensors.

The identification of protein aggregates as biomarkers for neurodegeneration is an area of interest for disease diagnosis and treatment development. In this work, we present novel super luminescent conjugated polyelectrolyte molecules as ex vivo sensors for tau-paired helical filaments (PHFs) and amyloid-β (Aβ) plaques. We evaluated the use of two oligo-p-phenylene ethynylenes (OPEs), anionic OPE12- and cationic OPE24+, as stains for fibrillar protein pathology in brain sections of transgenic mouse (rTg4510) and rat (TgF344-AD) models of Alzheimer's disease (AD) tauopathy, and post-mortem brain sections from human frontotemporal dementia (FTD). OPE12- displayed selectivity for PHFs in fluorimetry assays and strong staining of neurofibrillary tangles (NFTs) in mouse and human brain tissue sections, while OPE24+ stained both NFTs and Aβ plaques. Both OPEs stained the brain sections with limited background or non-specific staining. This novel family of sensors outperformed the gold-standard dye Thioflavin T in sensing capacities and co-stained with conventional phosphorylated tau (AT180) and Aβ (4G8) antibodies. As the OPEs readily bind protein amyloids in vitro and ex vivo, they are selective and rapid tools for identifying proteopathic inclusions relevant to AD. Such OPEs can be useful in understanding pathogenesis and in creating in vivo diagnostically relevant detection tools for neurodegenerative diseases.

Rotational multimaterial printing of filaments with subvoxel control.

Helical structures are ubiquitous in nature and impart unique mechanical properties and multifunctionality1. So far, synthetic architectures that mimic these natural systems have been fabricated by winding, twisting and braiding of individual filaments1-7, microfluidics8,9, self-shaping1,10-13 and printing methods14-17. However, those fabrication methods are unable to simultaneously create and pattern multimaterial, helically architected filaments with subvoxel control in arbitrary two-dimensional (2D) and three-dimensional (3D) motifs from a broad range of materials. Towards this goal, both multimaterial18-23 and rotational24 3D printing of architected filaments have recently been reported; however, the integration of these two capabilities has yet to be realized. Here we report a rotational multimaterial 3D printing (RM-3DP) platform that enables subvoxel control over the local orientation of azimuthally heterogeneous architected filaments. By continuously rotating a multimaterial nozzle with a controlled ratio of angular-to-translational velocity, we have created helical filaments with programmable helix angle, layer thickness and interfacial area between several materials within a given cylindrical voxel. Using this integrated method, we have fabricated functional artificial muscles composed of helical dielectric elastomer actuators with high fidelity and individually addressable conductive helical channels embedded within a dielectric elastomer matrix. We have also fabricated hierarchical lattices comprising architected helical struts containing stiff springs within a compliant matrix. Our additive-manufacturing platform opens new avenues to generating multifunctional architected matter in bioinspired motifs.

Cryo-EM structures of amyloid-β filaments with the Arctic mutation (E22G) from human and mouse brains

The Arctic mutation, encoding E693G in the amyloid precursor protein (APP) gene [E22G in amyloid-β (Aβ)], causes dominantly inherited Alzheimer’s disease. Here, we report the high-resolution cryo-EM structures of Aβ filaments from the frontal cortex of a previously described case (AβPParc1) with the Arctic mutation. Most filaments consist of two pairs of non-identical protofilaments that comprise residues V12–V40 (human Arctic fold A) and E11–G37 (human Arctic fold B). They have a substructure (residues F20–G37) in common with the folds of type I and type II Aβ42. When compared to the structures of wild-type Aβ42 filaments, there are subtle conformational changes in the human Arctic folds, because of the lack of a side chain at G22, which may strengthen hydrogen bonding between mutant Aβ molecules and promote filament formation. A minority of Aβ42 filaments of type II was also present, as were tau paired helical filaments. In addition, we report the cryo-EM structures of Aβ filaments with the Arctic mutation from mouse knock-in line AppNL−G−F. Most filaments are made of two identical mutant protofilaments that extend from D1 to G37 (AppNL−G−F murine Arctic fold). In a minority of filaments, two dimeric folds pack against each other in an anti-parallel fashion. The AppNL−G−F murine Arctic fold differs from the human Arctic folds, but shares some substructure.

Probing the Interactions of LRP1 Ectodomain-Derived Peptides with Fibrillar Tau Protein and Its Impact on Cellular Internalization

Cellular internalization and the spreading of misfolded tau have become increasingly important for elucidating the mechanism of Tau pathology involved in Alzheimer’s disease (AD). The low-density lipoprotein-related receptor 1 (LRP1) has been implicated in the internalization of fibrillar tau. In this work, we utilized homology modeling to model the Cluster 2 domain of LRP1 and determined that a 23-amino-acid sequence is involved in binding to paired helical filaments (PHF) of Tau. Fourteen short peptide segments derived from this ectodomain region were then designed and docked with PHF Tau. Molecular dynamics studies of the optimal peptides bound to PHF Tau demonstrated that the peptides formed critical contacts through Lys and Gln residues with Tau. Based on the computational results, flow cytometry, AFM, SPR analysis and CD studies were conducted to examine binding and cellular internalization. The results showed that the peptide sequence TauRP (1–14) (DNSDEENCES) was not only associated with fibrillar Tau but was also able to mitigate its cellular internalization in LRP1-expressed HEK-293 cells. Preliminary docking studies with Aβ (1–42) revealed that the peptides also bound to Aβ (1–42). While this study focused on the CCR2 domain of LRP1 to design peptide sequences to mitigate Tau internalization, the work can be extended to other domains of the LRP1 receptor or other receptors to examine if the cellular internalization of fibrillar Tau can be deterred. These findings show that short peptides derived from the LRP1 receptor can alter the internalization of its ligands.

High-Resolution Rotation Assay of the Bacterial Flagellar Motor Near Zero Loads Using a Mutant Having a Rod-Like Straight Hook.

The bacterial flagellar motor is embedded within the cell envelop and rotates the long helical filament, which acts as a molecular screw to propel the bacterium. The flagellar motor comprises a rotor and a dozen stator units, converting ion flux through the stator unit into torque. However, the energy coupling mechanism has not been fully understood. Various methods for rotation measurement have been developed to understand the rotation mechanism of the flagellar motor, but the most preferred method in recent studies is a bead assay, which tracks the rotation of a micron to submicron bead attached to the partially sheared flagellar filament at high temporal and spatial resolutions. The bead assay allows us to assess the motor rotation over a wide range of external load, but the elasticity of the axial parts of the flagellum, such as the hook and filament, limits the spatiotemporal resolution. In this chapter, we describe a bead assay optimized for the analysis of the flagellar motor dynamics at near zero load.

Bacteria-inspired robotic propulsion from bundling of soft helical filaments at low Reynolds number.

The bundling of flagella is known to create a "run" phase, where the bacteria moves in a nearly straight line rather than making changes in direction. Historically, mechanical explanations for the bundling phenomenon intrigued many researchers, and significant advances were made in physical models and experimental methods. Contributing to the field of research, we present a bacteria-inspired centimeter-scale soft robotic hardware platform and a computational framework for a physically plausible simulation model of the multi-flagellated robot under low Reynolds number (∼10-1). The fluid-structure interaction simulation couples the discrete elastic rods algorithm with the method of regularized Stokeslet segments. Contact between two flagella is handled by a penalty-based method. We present a comparison between our experimental and simulation results and verify that the simulation tool can capture the essential physics of this problem. Preliminary findings on robustness to buckling provided by the bundling phenomenon and the efficiency of a multi-flagellated soft robot are compared with the single-flagellated counterparts. Observations were made on the coupling between geometry and elasticity, which manifests itself in the propulsion of the robot by nonlinear dependency on the rotational speed of the flagella.

Live-Cell Imaging of the Assembly and Ejection Processes of the Bacterial Flagella by Fluorescence Microscopy.

Bacterial flagella are molecular machines used for motility and chemotaxis. The flagellum consists of a thin extracellular helical filament as a propeller, a short hook as a universal joint, and a basal body as a rotary motor. The filament is made up of more than 20,000 flagellin molecules and can grow to several micrometers long but only 20 nanometers thick. The regulation of flagellar assembly and ejection is important for bacterial environmental adaptation. However, due to the technical difficulty to observe these nanostructures in live cells, our understanding of the flagellar growth and loss is limited. In the last three decades, the development of fluorescence microscopy and fluorescence labeling of specific cellular structure has made it possible to perform the real-time observation of bacterial flagellar assembly and ejection processes. Furthermore, flagella are not only critical for bacterial motility but also important antigens stimulating host immune responses. The complete understanding of bacterial flagellar production and ejection is valuable for understanding macromolecular self-assembly, cell adaptation, and pathogen-host interactions.

A numerical method for the locomotion of bi-flagellated bacteria in viscous fluid

Flagellated bacteria propel themselves by rotating flexible flagella driven by independent motors. Depending on the rotation direction of the motors and the handedness of the helical filaments, the flagella either pull or push the cell body. Motivated by experimental observations of Magnetococcus marinus, we develop an elastohydrodynamic model to study the locomotion of a bi-flagellated bacterium with one puller flagellum and one pusher flagellum. In this model, the boundary integral technique and Kirchhoff rod model are employed respectively to calculate the hydrodynamic forces on the swimmer and model the elastic deformations of the flagella. Our numerical results demonstrate that the model bacterium travels along a double helical trajectory, which is consistent with the experimental observations. Varying the stiffness, orientations or positions of the flagella significantly changes the swimming characteristics. Notably, when either the applied torque is higher than a critical value or the flagellum stiffness is lower than a critical stiffness, the pusher flagellum exhibits overwhirling motion, resulting in a more complicated swimming style and a lower swimming speed. For a moderate flagellum stiffness, the swimming speed is insensitive to the rest configuration orientation over a wide range of orientation angles as the flagella deform to maintain alignment with the swimming direction.

Direct Observation of Archaellar Motor Rotation by Single-Molecular Imaging Techniques.

Single-molecular techniques have characterized dynamics of molecular motors such as flagellum in bacteria and myosin, kinesin, and dynein in eukaryotes. We can apply these techniques to a motility machine of archaea, namely, the archaellum, composed of a thin helical filament and a rotary motor. Although the size of the motor hinders the characterization of its motor function under a conventional optical microscope, fluorescence-labeling techniques allow us to visualize the architecture and function of the archaellar filaments in real time. Furthermore, a tiny polystyrene bead attached to the filament enables the visualization of motor rotation through the bead rotation and quantification of biophysical properties such as speed and torque produced by the rotary motor imbedded in the cell membrane. In this chapter, I describe the details of the above biophysical method based on an optical microscope.

Structural basis of CHMP2A-CHMP3 ESCRT-III polymer assembly and membrane cleavage.

The endosomal sorting complex required for transport (ESCRT) is a highly conserved protein machinery that drives a divers set of physiological and pathological membrane remodeling processes. However, the structural basis of ESCRT-III polymers stabilizing, constricting and cleaving negatively curved membranes is yet unknown. Here we present cryo-EM structures of membrane-coated CHMP2A-CHMP3 filaments from Homo sapiens of two different diameters at 3.3 and 3.6 Å resolution. The structures reveal helical filaments assembled by CHMP2A-CHMP3 heterodimers in the open ESCRT-III conformation, which generates a partially positive charged membrane interaction surface, positions short N-terminal motifs for membrane interaction and the C-terminal VPS4 target sequence toward the tube interior. Inter-filament interactions are electrostatic, which may facilitate filament sliding upon VPS4-mediated polymer remodeling. Fluorescence microscopy as well as high-speed atomic force microscopy imaging corroborate that VPS4 can constrict and cleave CHMP2A-CHMP3 membrane tubes. We therefore conclude that CHMP2A-CHMP3-VPS4 act as a minimal membrane fission machinery.

Helical vortices with small cross-section for 3D incompressible Euler equation

In this article, we construct traveling-rotating helical vortices with small cross-section to the 3D incompressible Euler equations in an infinite pipe, which tend asymptotically to singular helical vortex filament evolved by the binormal curvature flow. The construction is based on studying a general semilinear elliptic problem in divergence form{−ε2div(K(x)∇u)=(u−q|ln⁡ε|)+p,x∈Ω,u=0,x∈∂Ω, for small values of ε. Helical vortex solutions concentrating near several helical filaments with polygonal symmetry are also constructed.

Structure of a RecT/Redβ family recombinase in complex with a duplex intermediate of DNA annealing

Some bacteriophage encode a recombinase that catalyzes single-stranded DNA annealing (SSA). These proteins are apparently related to RAD52, the primary human SSA protein. The best studied protein, Redβ from bacteriophage λ, binds weakly to ssDNA, not at all to dsDNA, but tightly to a duplex intermediate of annealing formed when two complementary DNA strands are added to the protein sequentially. We used single particle cryo-electron microscopy (cryo-EM) to determine a 3.4 Å structure of a Redβ homolog from a prophage of Listeria innocua in complex with two complementary 83mer oligonucleotides. The structure reveals a helical protein filament bound to a DNA duplex that is highly extended and unwound. Native mass spectrometry confirms that the complex seen by cryo-EM is the predominant species in solution. The protein shares a common core fold with RAD52 and a similar mode of ssDNA-binding. These data provide insights into the mechanism of protein-catalyzed SSA.

Shapeshifting tau: from intrinsically disordered to paired-helical filaments.

Tau is an intrinsically disordered protein that has the ability to self-assemble to form paired helical and straight filaments in Alzheimer's disease, as well as the ability to form additional distinct tau filaments in other tauopathies. In the presence of microtubules, tau forms an elongated form associated with tubulin dimers via a series of imperfect repeats known as the microtubule binding repeats. Tau has recently been identified to have the ability to phase separate in vitro and in cells. The ability of tau to adopt a wide variety of conformations appears fundamental both to its biological function and also its association with neurodegenerative diseases. The recently highlighted involvement of low-complexity domains in liquid-liquid phase separation provides a critical link between the soluble function and the insoluble dysfunctional properties of tau.

Solid-state NMR of paired helical filaments formed by the core tau fragment tau(297-391).

Aggregation of the tau protein into fibrillar cross-β aggregates is a hallmark of Alzheimer's diseases (AD) and many other neurodegenerative tauopathies. Recently, several core structures of patient-derived tau paired helical filaments (PHFs) have been solved revealing a structural variability that often correlates with a specific tauopathy. To further characterize the dynamics of these fibril cores, to screen for strain-specific small molecules as potential biomarkers and therapeutics, and to develop strain-specific antibodies, recombinant in-vitro models of tau filaments are needed. We recently showed that a 95-residue fragment of tau (from residue 297 to 391), termed dGAE, forms filaments in vitro in the absence of polyanionic co-factors often used for in vitro aggregation of full-length tau. Tau(297-391) was identified as the proteolytic resistant core of tau PHFs and overlaps with the structures characterized by cryo-electron microscopy in ex vivo PHFs, making it a promising model for the study of AD tau filaments in vitro. In the present study, we used solid-state NMR to characterize tau(297-391) filaments and show that such filaments assembled under non-reducing conditions are more dynamic and less ordered than those made in the presence of the reducing agent DTT. We further report the resonance assignment of tau(297-391)+DTT filaments and compare it to existing core structures of tau.

Exploring mechanisms of synaptic vulnerability and resilience in Primary Age Related Tauopathy

AbstractBackgroundPrimary Age Related Tauopathy (PART) is a sporadic dementia‐related illness, characterized by pathological tau accumulation limited to the hippocampal‐entorhinal region in the absence of Aβ plaques. Studying PART thus presents a unique opportunity to 1) characterize toxic tau species, namely oligomeric (tauO) tau, and to 2) explore molecular mechanisms underlying regional synaptic vulnerability (i.e. hippocampus) and resilience (i.e. neocortex) within a singular tauopathy—such as low‐density lipoprotein receptor 1 (LRP1), recently uncovered as a key mediator of tau uptake and trans‐synaptic spreading.MethodsWe compared levels of tauO, total tau, and LRP1 between the hippocampus and superior medial temporal cortex (SMT) in 7 post‐mortem PART patients. Total homogenate and synaptosomal fractions were prepared for Western Blot; both fractions were probed for total tau (Tau13), and LRP1. TauO were ascribed to high molecular weight tau bands using the total tau probe. p‐tau (AT180) and paired helical filaments (PHF13.6) were also probed in the total homogenate fraction to assess PART neuropathology. The Wilcoxon matched‐pairs signed rank test was utilized to compare regional differences.ResultsWe found elevated levels of p‐tau and PHF in the hippocampus vs. SMT (total homogenate), thus validating previously reported PART neuropathology. We observed a trend towards increased tauO in the hippocampus vs. SMT in PART total homogenate (p=0.11) and synaptosome fractions (p=0.06). Of note, no regional differences were found in total tau levels in either the fractions. In the total homogenate, we found no statistically significant regional differences in LRP1 (p=0.30). Interestingly however, we observed decreased LRP1 in the hippocampus vs. SMT in 6 out of 7 PART patients. On the orher hand, we found no significant differences or notable trends in LRP1 between the hippocampus vs. SMT in the synaptosomal fraction.ConclusionsThis investigation of PART neuropathology is the first to demonstrate accumulation of tauO, restricted to the hippocampus and its synapses. We further report lower levels of LRP1 in the hippocampus of PART patients, a seemingly extra‐synaptic phenomenon, that could potentially mediate regional vulnerability to pathological tau species. Further biochemical and functional assays to characterize tau toxicity and spread are currently under investigation.

Seeding human tau knock‐in neurons with human derived pathological tau results in the formation of pathogenic conformations of tau

AbstractBackgroundOne of the pathological hallmarks of Alzheimer’s disease (AD) is the accumulation of pathological tau inclusions. Conformational exposure of an N‐terminal motif in tau known as the phosphatase‐activating domain (PAD) occurs early in AD and other tauopathies and aberrant exposure of this motif disrupts axonal transport. Tau is capable of seeding aggregation in cells; however, the extent to which disease‐associated pathogenic conformations can be effectively seeded remains an area of active investigation. To help address this gap, we seeded aggregation in primary neurons and assessed known pathogenic tau conformations.MethodPrimary hippocampal neurons from human tau knock‐in mice were treated with pathological tau aggregates from human AD‐brain (AD‐tau) according to methods described in Narasimhan and Lee, 2017. The same purification protocol was used with non‐demented human brain tissue (CO) as a control. The AD‐tau and CO samples were characterized by transmission electron microscopy and biochemical assays that measure total tau and pathological modifications, such as phosphorylation (PHF1) and a conformation specific antibody (TNT1) that labels display of PAD. Neurons treated at 5 days in vitro were maintained for up to 28 days post‐treatment. Markers of pathological species of tau (PAD exposed and phospho‐tau) were used to assess the formation of disease‐associated forms of tau and changes in the numbers of neurons (MAP2) and astrocytes (GFAP) were measured.ResultThe AD‐derived material showed morphologies consistent with small pieces of the characteristic paired helical filaments of AD, phosphorylation at the PHF1 site, and the presence of oligomeric tau and PAD‐exposed tau species. We observed a robust dose and time‐dependent increase in TNT1 in AD‐tau‐treated neurons and ongoing studies are evaluating the presence of other pathological tau species. There were no significant effects on cell number in seeded cultures out to 28 days post‐treatment.ConclusionOur results suggest that primary neurons seeded with AD‐tau form inclusions consisting of PAD‐exposed tau, but overt degeneration does not occur within 28 days of seeding. The presence of PAD‐exposed tau but lack of cell loss could indicate that more time is needed and/or that the consequences of these tau species are more subtle.

Negative Correlation of Corpora Amylacea with pTau in the Hippocampus of People with AD

AbstractBackgroundRecent studies suggest corpora amylacea (CA) activity may be involved in modulating AD pathology. Normally, CA function in the clearance of brain waste products by amassing cellular debris and extruding it into the cerebrospinal fluid. Disruption of this process may play a role in AD pathogenesis, and its effect might be influenced by degree of tau pathology and genetic risk. In this study, we determined if the abundance of CA was correlated with paired helical filament tau pathology in the CA1 region of the hippocampus in AD postmortem tissue samples. Tau pathology was assessed using both total pTau and total NFTs. Additionally, we investigated whether that correlation was influenced by APOE genotype.MethodPostmortem brain hippocampal tissue sections from 11 brain donors with pre‐mortem cognitive impairment and neuropathologically confirmed AD were double‐stained with Periodic‐acid Schiff (PAS) stain to identify CA and AT8 antibody to identify hyperphosphorylated Tau protein (pTau). Using the Motic slide scanner, stained sections were digitized at 40x. QuPath 0.3.2 was used to extract the CA1 hippocampal region and export to ImageJ for analysis. ImageJ 1.8 color thresholding was used to quantify the number and area of CA and pTau. Neurofibrillary tangle (NFT) pathology was classified by pTau aggregations larger than 150μm2. The percent area occupied (%AO) was calculated by dividing the sum of all positively stained objects by the area of the image, then multiplying by 100. Pearson correlations between CA %AO, pTau %AO, and NFT %AO were calculated in Python 3.7.ResultWhile not statistically significant, we found negative correlations of CA %AO with pTau %AO (‐0.14), and NFT %AO (‐0.28) in the CA1 of the hippocampus. These correlations (‐0.68 and ‐0.54 respectively) were stronger in the 4 individuals who did not carry the AD risk‐increasing APOE‐4 allele.ConclusionThese results suggest that as CA decline, pTau accumulates, which is consistent with prior studies that described a decline in CA from intermediate to advanced Braak Stages (III‐VI) in the dentate gyrus. These changes are consistent with our

Nanoscale imaging of pT217‐tau in aged rhesus macaque: Trans‐synaptic propagation and seeding of tau pathology in entorhinal cortex

AbstractBackgroundTau pathology in Alzheimer’s Disease (AD) targets higher cortical circuits, with evidence that phosphorylated tau propagates from the entorhinal cortex (ERC) to “seed” pathology throughout the neuronal network. Recent discoveries indicate that tau phosphorylated at threonine 217 (pT217‐tau) can be captured in cerebrospinal fluid (CSF) and plasma as an early biomarker of ensuing disease. However, the role of pT217‐tau in brain tau pathology is unknown, especially as soluble tau species are dephosphorylated postmortem in humans. Rhesus macaques naturally develop the same qualitative pattern and sequence of tau and amyloid pathology, with neurofibrillary tangles comprised of paired helical filaments, identical to human AD. Perfusion fixation of monkey tissue preserves phosphorylation state and allows imaging of molecular location and interactions with nanometer resolution not possible in humans due to postmortem degradation. The current study examined the ultrastructural localization of pT217‐tau in layer II ERC of the aged rhesus macaques, focusing on potential evidence of propagation between neurons, and exposure to the extracellular space.MethodWe used immunohistochemistry paired with high spatial‐resolution immunoelectron microscopy (immunoEM) in aged rhesus macaques (18‐31 years) to localize pT217‐tau in the stellate cell islands in ERC layer II, which show the earliest signatures of tau pathology in AD.ResultpT217‐tau immunolabeling was predominantly observed in postsynaptic compartments in macaque ERC layer II. pT217‐tau accumulated on the calcium‐storing smooth endoplasmic reticulum spine apparatus near axospinous asymmetric glutamatergic synapses in dendritic spines. We observed extensive, trans‐synaptic pT217‐tau trafficking between interconnected neurons within omega‐shaped bodies in ERC layer II, specifically near excitatory, but not inhibitory synapses. Within dendritic shafts, pT217‐tau aggregated on microtubules often in concordance with autophagic vacuoles indicative of neurite dystrophy.ConclusionpT217‐tau accumulates in ERC layer II subcompartments known to be the earliest to show pathology in humans. The data provide the first evidence of pT217‐tau trafficking between neurons to “seed” tau pathology in higher brain circuits, potentially interfacing with the extracellular space to become readily accessible and captured in CSF and blood as a robust AD biomarker. Illuminating patterns of neurodegeneration with pT217‐tau could potentially guide earlier intervention of therapeutics that might mitigate tau hyperphosphorylation in AD.

Subtle change of fibrillation condition leads to substantial alteration of recombinant Tau fibril structure.

Invitro assembly of amyloid fibrils that recapitulate those in human brains is very useful for fundamental and applied research on the amyloid formation, pathology, and clinical detection. Recent success in the assembly of Tau fibrils invitro enables the recapitulation of the paired helical filament (PHF) of Tau extracted from brains of patients with Alzheimer's disease (AD). However, following the protocol, we observed that Tau constructs including 297-391 and a mixture of 266-391 (3R)/297-391, which are expected to predominantly form PHF-like fibrils, form highly heterogeneous fibrils instead. Moreover, the seemingly PHF-like fibril formed by Tau 297-391 exhibits a distinctive atomic structure with a spindle-like fold, that is neither PHF-like or similar to any known Tau fibril structures revealed by cryo-electron microscopy (cryo-EM). Our work highlights the high sensitivity of amyloid fibril formation to subtle conditional changes and suggests high-resolution structural characterization to invitro assembled fibrils prior to further laboratory use.