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.

Reconstitution of the Alzheimer's Disease Tau Core Structure from Recombinant Tau297-391 Yields Variable Quaternary Structures as Seen by Negative Stain and Cryo-EM.

The protein tau misfolds into disease-specific fibrillar structures in more than 20 neurodegenerative diseases collectively referred to as tauopathies. To understand and prevent disease-specific mechanisms of filament formation, in vitro models for aggregation that robustly yield these different end point structures will be necessary. Here, we used cryo-electron microscopy (cryo-EM) to reconstruct fibril polymorphs taken on by residues 297-391 of tau under conditions previously shown to give rise to the core structure found in Alzheimer's disease (AD). While we were able to reconstitute the AD tau core fold, the proportion of these paired helical filaments (PHFs) was highly variable, and a majority of filaments were composed of PHFs with an additional identical C-shaped protofilament attached near the PHF interface, termed triple helical filaments (THFs). Since the impact of filament layer quaternary structure on the biological properties of tau and other amyloid filaments is not known, the applications for samples of this morphology are presently uncertain. We further demonstrate the variation in the proportion of PHFs and PHF-like fibrils compared to other morphologies as a function of shaking time and AD polymorph-favoring cofactor concentration. This variation in polymorph abundance, even under identical experimental conditions, highlights the variation that can arise both within a lab and in different laboratory settings when reconstituting specific fibril polymorphs in vitro.

Fluorene-based tau fibrillation sensor and inhibitor with fluorogenic and photo-crosslinking properties.

Tau protein aggregation into neurofibrillary tangles often causes tauopathies. Herein, we report fluorene based sensors with fluorogenicity upon binding to tau proteins. Intriguingly, these sensors possess triplet state properties to inhibit tau fibrillation upon photo-induced crosslinking.

Dityrosine Cross-links are Present in Alzheimer's Disease-derived Tau Oligomers and Paired Helical Filaments (PHF) which Promotes the Stability of the PHF-core Tau (297-391) In Vitro.

A characteristic hallmark of Alzheimer's Disease (AD) is the pathological aggregation and deposition of tau into paired helical filaments (PHF) in neurofibrillary tangles (NFTs). Oxidative stress is an early event during AD pathogenesis and is associated with tau-mediated AD pathology. Oxidative environments can result in the formation of covalent dityrosine crosslinks that can increase protein stability and insolubility. Dityrosine cross-linking has been shown in Aß plaques in AD and α-synuclein aggregates in Lewy bodies in ex vivo tissue sections, and this modification may increase the insolubility of these aggregates and their resistance to degradation. Using the PHF-core tau fragment (residues 297 - 391) as a model, we have previously demonstrated that dityrosine formation traps tau assemblies to reduce further elongation. However, it is unknown whether dityrosine crosslinks are found in tau deposits in vivo in AD and its relevance to disease mechanism is unclear. Here, using transmission electron microscope (TEM) double immunogold-labelling, we reveal that neurofibrillary NFTs in AD are heavily decorated with dityrosine crosslinks alongside tau. Single immunogold-labelling TEM and fluorescence spectroscopy revealed the presence of dityrosine on AD brain-derived tau oligomers and fibrils. Using the tau (297-391) PHF-core fragment as a model, we further showed that prefibrillar tau species are more amenable to dityrosine crosslinking than tau fibrils. Dityrosine formation results in heat and SDS stability of oxidised prefibrillar and fibrillar tau assemblies. This finding has implications for understanding the mechanism governing the insolubility and toxicity of tau assemblies in vivo.

Structural study of the recognition mechanism of tau antibody Tau2r3 with the key sequence (VQIINK) in tau aggregation.

One of the histopathological features of Alzheimer's disease (AD) is higher order neurofibrillary tangles formed by abnormally aggregated tau protein. The sequence 275VQIINK280 in the microtubule-binding domain of tau plays a key role in tau aggregation. Therefore, an aggregation inhibitor targeting the VQIINK region in tau may be an effective therapeutic agent for AD. We have previously shown that the Fab domain (Fab2r3) of a tau antibody that recognizes the VQIINK sequence can inhibit tau aggregation, and we have determined the tertiary structure of the Fab2r3-VQIINK complex. In this report, we determined the tertiary structure of apo Fab2r3 and analyzed differences in the structures of apo Fab2r3 and Fab2r3-VQIINK to examine the ligand recognition mechanism of Fab2r3. In comparison with the Fab2r3-VQIINK structure, there were large differences in the arrangement of the constant and variable domains in apo Fab2r3. Remarkable structural changes were especially observed in the H3 and L3 loop regions of the complementarity determining regions (CDRs) in apo Fab2r3 and the Fab2r3-VQIINK complex. These structural differences in CDRs suggest that formation of hydrophobic pockets suitable for the antigen is important for antigen recognition by tau antibodies.

The Structure Biology of Tau and Clue for Aggregation Inhibitor Design.

Tau is a microtubule-associated protein that is mainly expressed in central and peripheral nerve systems. Tau binds to tubulin and regulates assembly and stabilization of microtubule, thus playing a critical role in neuron morphology, axon development and navigation. Tau is highly stable under normal conditions; however, there are several factors that can induce or promote aggregation of tau, forming neurofibrillary tangles. Neurofibrillary tangles are toxic to neurons, which may be related to a series of neurodegenerative diseases including Alzheimer's disease. Thus, tau is widely accepted as an important therapeutic target for neurodegenerative diseases. While the monomeric structure of tau is highly disordered, the aggregate structure of tau is formed by closed packing of ß-stands. Studies on the structure of tau and the structural transition mechanism provide valuable information on the occurrence, development, and therapy of tauopathies. In this review, we summarize recent progress on the structural investigation of tau and based on which we discuss aggregation inhibitor design.

Maturation of neuronal AD-tau pathology involves site-specific phosphorylation of cytoplasmic and synaptic tau preceding conformational change and fibril formation.

In Alzheimer's disease (AD), tau-protein undergoes a multi-step process involving the transition from a natively unfolded monomer to large, aggregated structures such as neurofibrillary tangles (NFTs). However, it is not yet clear which events initiate the early preclinical phase of AD tauopathy and whether they have impact on the propagation of tau pathology in later disease stages. To address this question, we analyzed the distribution of tau species phosphorylated at T231, S396/S404 and S202/T205, conformationally modified at the MC1 epitope and fibrillary tau detected by the Gallyas method (Gallyas-tau), in the brains of 15 symptomatic and 20 asymptomatic cases with AD pathology as well as of 19 nonAD cases. As initial tau lesions, we identified phosphorylated-T231-tau diffusely distributed within the somatodendritic compartment (IC-tau) and phosphorylated-S396/pS404-tau in axonal lesions of the white matter and in the neuropil (IN-tau). The subcellular localization of pT231-tau in the cell body and pS396/pS404-tau in the presynapse was confirmed in hP301L mutant Drosophila larvae. Phosphorylated-S202/T205-tau, MC1-tau and Gallyas-tau were negative for these lesions. IC- and IN-tau were observed in all analyzed regions of the human brain, including early affected regions in nonAD cases (entorhinal cortex) and late affected regions in symptomatic AD cases (cerebellum), indicating that tau pathology initiation follows similar processes when propagating into previously unaffected regions. Furthermore, a sequence of AD-related maturation of tau-aggregates was observed, initiated by the appearance of IC- and IN-tau, followed by the formation of pretangles exhibiting pT231-tau, pS396/pS404-tau and pS202/pT205-tau, then by MC1-conformational tau, and, finally, by the formation of Gallyas-positive NFTs. Since cases classified as nonAD [Braak NFT stages < I (including a-1b)] already showed IC- and IN-tau, our findings suggest that these lesions are a prerequisite for the development of AD.

Modification of methods to use Congo-red stain to simultaneously visualize amyloid plaques and tangles in human and rodent brain tissue sections.

Although there are multiple histochemical tracers available to label plaques and tangles in the brain to evaluate neuropathology in Alzheimer disease (AD), few of them are versatile in nature and compatible with immunohistochemical procedures. Congo Red (CR) is an anisotropic organic stain discovered to label amyloid beta (Aß) plaques in the brain. Unfortunately, its use is underappreciated due to its low resolution and brightness as stated in previous studies using bright field microscopy. Here, we modified a previous method to localize both plaques and tangles in brains from humans and a transgenic rodent model of AD for fluorescence microscopic visualization. The plaque staining affinities displayed by CR were compared with fibrillar pattern labeling seen with Thioflavin S. This study summarizes the optimization of protocols in which various parameters have been finetuned. To determine the target CR potentially binds, we have performed double labeling with different antibodies against Aß as well as phosphorylated Tau. The plaque staining affinities exhibited by CR are compared with those associated with the diffuse pattern of labeling seen with antibodies directed against different epitopes of Aß. Neither CP13, TNT2 or TOC1 binds all the neurofibrillary tangles as revealed by CR labeling in the human brain. Additionally, we also evaluated double labeling with AT8, AT180, and PHF1. Interestingly, PHF-1 shows 40% colocalization and AT8 shows 15% colocalization with NFT. Thus, CR is a much better marker to detect AD pathologies in human and rodent brains with higher fluorescence intensity relative to other conventional fluorescence markers.

Early but not late conformational changes of tau in association with ubiquitination of neurofibrillary pathology in Alzheimer's disease brains.

In Alzheimer's disease, tau protein undergoes post-translational modifications including hyperphosphorylation and truncation, which promotes two major conformational changes associated with progressive N-terminal folding. Along with the development of the disease, tau ubiquitination was previously shown to emerge in the early and intermediate stages of the disease, which is closely associated with early tau truncation at aspartic acid 421, but not with a subsequently truncated tau molecule at glutamic acid 391. In the same group of cases, using multiple immunolabeling and confocal microscopy, a possible relationship between the ubiquitin-targeting of tau and the progression of conformational changes adopted by the N-terminus of this molecule was further studied. A comparable number of neurofibrillary tangles was found displaying ubiquitin, an early conformation recognized by the Alz-50 antibody, and a phosphorylation. However, a more reduced number of neurofibrillary tangles were immunoreactive to Tau-66 antibody, a late tau conformational change marker. When double-labeling profiles of neurofibrillary tangles were assessed, ubiquitination was clearly demonstrated in tau molecules undergoing early N-terminal folding, but was barely observed in late conformational changes of the N-terminus adopted by tau. The same pattern of colocalization was visualized in neuritic pathology. Overall, these results indicate that a more intact conformation of the N-terminus of tau may facilitate tau ubiquitination, but this modification may not occur in a late truncated and more compressed folding of the N-terminus of the tau molecule.

DJ-1 can form ß-sheet structured aggregates that co-localize with pathological amyloid deposits.

The loss of native function of the DJ-1 protein has been linked to the development of Parkinson's (PD) and other neurodegenerative diseases. Here we show that DJ-1 aggregates into ß-sheet structured soluble and fibrillar aggregates in vitro under physiological conditions and that this process is promoted by the oxidation of its catalytic Cys106 residue. This aggregation resulted in the loss of its native biochemical glyoxalase function and in addition oxidized DJ-1 aggregates were observed to localize within Lewy bodies, neurofibrillary tangles and amyloid plaques in human PD and Alzheimer's (AD) patients' post-mortem brain tissue. These findings suggest that the aggregation of DJ-1 may be a critical player in the development of the pathology of PD and AD and demonstrate that loss of DJ-1 function can happen through DJ-1 aggregation. This could then contribute to AD and PD disease onset and progression.

New diagnostic method for Alzheimer's disease based on the toxic conformation theory of amyloid ß.

Recent investigations suggest that soluble oligomeric amyloid ß (Aß) species may be involved in early onset of Alzheimer's disease (AD). Using systematic proline replacement, solid-state NMR, and ESR, we identified a toxic turn at position 22 and 23 of Aß42, the most potent neurotoxic Aß species. Through radicalization, the toxic turn can induce formation of the C-terminal hydrophobic core to obtain putative Aß42 dimers and trimers. Synthesized dimer and trimer models showed that the C-terminal hydrophobic core plays a critical role in the formation of high molecular weight oligomers with neurotoxicity. Accordingly, an anti-toxic turn antibody (24B3) that selectively recognizes a toxic dimer model of E22P-Aß42 was developed. Sandwich enzyme-linked immunosorbent assay with 24B3 and 82E1 detected a significantly higher ratio of Aß42 with a toxic turn to total Aß42 in cerebrospinal fluid of AD patients compared with controls, suggesting that 24B3 could be useful for early onset of AD diagnosis.

Multi-site dynamic recording for Aß oligomers-induced Alzheimer's disease in vitro based on neuronal network chip.

Alzheimer's disease (AD) is a chronic central neurodegenerative disease. The pathological features of AD are the extracellular deposition of senile plaques formed by amyloid-ß oligomers (AßOs) and the intracellular accumulation of neurofibrillary tangles. However, due to the lack of effective method and experimental models to study the cognitive decline, communication at cell resolution and the implementation of interventions, the diagnosis and treatment on AD still progress slowly. In this paper, we established a pathological model of AD in vitro based on AßOs-induced hippocampal neuronal network chip for multi-site dynamic analysis of the neuronal electrical activity and network connection. The multiple characteristic parameters, including positive and negative spike intervals, firing rate and peak-to-peak values, were extracted through the analysis of spike signals, and two firing patterns from the interneurons and pyramidal neurons were recorded. The spatial firing patterns mapping and cross-correlation between channels were performed to validate the degeneration of neuronal network connectivity. Moreover, an electrical stimulation with frequency at 40 Hz was exerted to preliminarily explore the therapeutic effect on the pathological model of AD. This neuronal network chip enables the implementation of AD models in vitro for studying basic mechanisms of neurodegeneration within networks and for the parallel testing of various potential therapies. It can be a novel technique in the research of AD pathological model in vitro.

Tau and TDP-43 accumulation of the basal nucleus of Meynert in individuals with cerebral lobar infarcts or hemorrhage.

A previous study reported that a massive cerebral infarct in the territory of the middle cerebral artery (MCA) may be associated with development of neurofibrillary tangles (NFTs) in the ipsilateral basal nucleus of Meynert (BNM). We analyzed 19 cases of an MCA territory infarct and 12 with a putaminal hemorrhage (mean age 82.5 years; female/male ratio 8/23; mean time from stroke onset to autopsy 4182 days). In both groups, 74-100% had a significantly higher rate of phosphorylated tau immunoreactive or Gallyas Braak silver stain-positive neurons on the BNM-affected side than on the BNM-unaffected side. These NFTs were immunoreactive for anti-RD3 and anti-RD4 antibodies, and a triple-band pattern was observed by immunoblot analysis with anti-tau antibody. Most NFTs might be formed within the 5-10 years after stroke onset. There were significantly more TAR DNA-binding protein 43 (TDP43) immunoreactive structures on the BNM-affected side than on the BNM-unaffected side. We showed that many NFTs with TDP43-immunoreactive structures were observed in the ipsilateral BNM associated with a massive cerebral infarct in the MCA territory or a putaminal hemorrhage.

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 ß2 and ß3 and ends at ß8. However, in SF, it is simultaneously dissociated from ß1 and ß8 and ends at ß5. 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, ß8 first interacts with the template by hydrophobic interaction. Then ß7, ß6, ß5, ß4, and ß1 sequentially bind to the template by electrostatic and hydrophobic interactions. After ß1 binds to the template, ß2 and ß3 very quickly bind to the template through hydrophobic interaction. For SF, ß5 of the monomer first interacts with the template by electrostatic attraction. Then ß4 and ß6, ß3 and ß7, and ß2 and ß8 bind to the template in turn. Finally, ß1 and ß8 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.

Structure of NFT: Biochemical Approach.

Neurofibrillary tangle (NFT), bundle of paired helical filaments in neurons is one of the defining features of Alzheimer's disease (AD) and their spreads well correlate with disease symptoms and progression of AD. Using the unusual insolubility, NFTs were partially purified and the antibodies were produced. Characterization of these antibodies and biochemical studies of tau in AD revealed that a hyperphosphorylated tau protein is the major component of NFTs. In 1998, mutations in the tau gene were discovered in FTDP-17, demonstrating that abnormalities of tau cause accumulation of tau and neurodegeneration. Abnormal tau pathology occurs not only in AD, but also in other neurodegenerative dementing disorders, such as Pick's disease (PiD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). The tau isoforms accumulated in these inclusions are different among the diseases. Biochemical and proteinchemical analyses of these pathological tau proteins in these tauopathies demonstrated that the protease-resistant cores of the tau aggregates are composed of different microtubule binding regions and distinct between the diseases. Recent Cryo-EM analyses revealed the core structures of tau filaments in AD and PiD, confirming our biochemical observations. Further studies of tau and other abnormal proteins will provide important insights into molecular mechanisms of protein aggregation and prion-like propagation in neurodegenerative diseases.

Cysteine-Independent Inhibition of Alzheimer's Disease-like Paired Helical Filament Assembly by Leuco-Methylthioninium (LMT).

Alzheimer's disease is a tauopathy characterized by pathological fibrillization of tau protein to form the paired helical filaments (PHFs), which constitute neurofibrillary tangles. The methylthioninium (MT) moiety reverses the proteolytic stability of the PHF core and is in clinical development for treatment of Alzheimer's disease in a stable reduced form as leuco-MT. It has been hypothesized that MT acts via oxidation of cysteine residues, which is incompatible with activity in the predominantly reducing environment of living cells. We have shown recently that the PHF-core tau unit assembles spontaneously in vitro to form PHF-like filaments. Here we describe studies using circular dichroism, SDS-PAGE, transmission electron microscopy and site-directed mutagenesis to elucidate the mechanism of action of the MT moiety. We show that MT inhibitory activity is optimal in reducing conditions, that the active moiety is the reduced leuco-MT form of the molecule and that its mechanism of action is cysteine independent.

Purification and Characterization of Low-n Tau Oligomers.

Deposition of Tau aggregates in patient's brains is a hallmark of several neurodegenerative diseases collectively called Tauopathies. One of the most studied Tauopathies is Alzheimer disease (AD) in which Tau protein aggregates into filaments and coalesces into neurofibrillary tangles. The distribution of Tau filaments is a reliable indicator of the clinical stages of AD (Braak stages), but intermediate oligomeric assemblies of Tau are considered to be more directly toxic to neurons than late stage filaments. Studying the elusive role of Tau oligomers has been difficult because of their dynamic nature and paucity of methods to purify them in vitro. In this chapter, we describe methods to purify Tau oligomers to near homogeneity and to characterize them by hydrophobic interaction chromatography and biophysical methods such as fluorescence spectrophotometry, dynamic light scattering, atomic force microscopy, and others. Functional characterization includes the assessment of synapses and toxicity assays which show that oligomers can damage synapses locally but show little toxicity to neurons globally.

Distinct differences in prion-like seeding and aggregation between Tau protein variants provide mechanistic insights into tauopathies.

The accumulation of aberrantly aggregated MAPT (microtubule-associated protein Tau) defines a spectrum of tauopathies, including Alzheimer's disease. Mutations in the MAPT gene cause frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), characterized by neuronal pathological Tau inclusions in the form of neurofibrillary tangles and Pick bodies and in some cases glial Tau pathology. Increasing evidence points to the importance of prion-like seeding as a mechanism for the pathological spread in tauopathy and other neurodegenerative diseases. Herein, using a cell culture model, we examined a multitude of genetic FTDP-17 Tau variants for their ability to be seeded by exogenous Tau fibrils. Our findings revealed stark differences between FTDP-17 Tau variants in their ability to be seeded, with variants at Pro301 and Ser320 showing robust aggregation with seeding. Similarly, we elucidated the importance of certain Tau protein regions and unique residues, including the role of Pro301 in inhibiting Tau aggregation. We also revealed potential barriers in cross-seeding between three-repeat and four-repeat Tau isoforms. Overall, these differences alluded to potential mechanistic differences between wildtype and FTDP-17 Tau variants, as well as different Tau isoforms, in influencing Tau aggregation. Furthermore, by combining two FTDP-17 Tau variants (either P301L or P301S with S320F), we generated aggressive models of tauopathy that do not require exogenous seeding. These models will allow for rapid screening of potential therapeutics to alleviate Tau aggregation without the need for exogenous Tau fibrils. Together, these studies provide novel insights in the molecular determinants that modulate Tau aggregation.

Common fibrillar spines of amyloid-ß and human islet amyloid polypeptide revealed by microelectron diffraction and structure-based inhibitors.

Amyloid-ß (Aß) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues and are associated with Alzheimer's disease (AD) and type II diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aß and hIAPP. Using the cryoEM method microelectron diffraction, we determined the atomic structures of 11-residue segments from both Aß and hIAPP, termed Aß(24-34) WT and hIAPP(19-29) S20G, with 64% sequence similarity. We observed a high degree of structural similarity between their backbone atoms (0.96-Å root mean square deviation). Moreover, fibrils of these segments induced amyloid formation through self- and cross-seeding. Furthermore, inhibitors designed for one segment showed cross-efficacy for full-length Aß and hIAPP and reduced cytotoxicity of both proteins, although by apparently blocking different cytotoxic mechanisms. The similarity of the atomic structures of Aß(24-34) WT and hIAPP(19-29) S20G offers a molecular model for cross-seeding between Aß and hIAPP.

Hyperspectral Raman imaging of neuritic plaques and neurofibrillary tangles in brain tissue from Alzheimer's disease patients.

Neuritic plaques and neurofibrillary tangles are crucial morphological criteria for the definite diagnosis of Alzheimer's disease. We evaluated 12 unstained frontal cortex and hippocampus samples from 3 brain donors with Alzheimer's disease and 1 control with hyperspectral Raman microscopy on samples of 30 × 30 µm. Data matrices of 64 × 64 pixels were used to quantify different tissue components including proteins, lipids, water and beta-sheets for imaging at 0.47 µm spatial resolution. Hierarchical cluster analysis was performed to visualize regions with high Raman spectral similarities. The Raman images of proteins, lipids, water and beta-sheets matched with classical brain morphology. Protein content was 2.0 times, the beta-sheet content 5.6 times and Raman broad-band autofluorescence was 2.4 times higher inside the plaques and tangles than in the surrounding tissue. The lipid content was practically equal inside and outside. Broad-band autofluorescence showed some correlation with protein content and a better correlation with beta-sheet content. Hyperspectral Raman imaging combined with hierarchical cluster analysis allows for the identification of neuritic plaques and neurofibrillary tangles in unstained, label-free slices of human Alzheimer's disease brain tissue. It permits simultaneous quantification and distinction of several tissue components such as proteins, lipids, water and beta-sheets.