Amyloid Fibrils Research Articles

Fiber complex‐stabilized high‐internal‐phase emulsion for allicin encapsulation: microstructure, stability, and thermal‐responsive properties

AbstractBACKGROUNDStimuli‐responsive emulsions have garnered significant attention for their ability to enhance sensory qualities and control the release of encapsulated nutrient in emulsion‐based products. However, the characteristics of synthetic materials of fabricating stimuli‐responsive emulsions have been a crucial limitation in the food industry. Regulating the behavior of molecules at the interface could potentially achieve the desired stimuli‐responsive behavior, but currently there is limited information available.RESULTSHigh‐internal‐phase emulsions (HIPEs) were fabricated for the encapsulation of allicin, stabilized by a complex of 20 g kg−1 whey protein amyloid fibrils (WPF) and 20 g kg−1 glycyrrhizin fibers (GA). The intermolecular interactions between WPF and GA in the fiber complexes were predominantly governed by hydrophobic and electrostatic forces. These complexes adsorbed and stacked around the oil droplets, forming a protective interfacial film that enhanced droplet stability. An increased proportion of WPF (WPF = 3:1 or 4:1) surrounding the oil droplets enhanced the accelerated storage stability of HIPEs, with instability indexes approaching 0.2. Additionally, HIPEs displayed a temperature‐dependent modulus, with the emulsion stabilized by a WPF ratio of 3:1 showing the highest modulus at 85 °C. The encapsulation efficiency of allicin in HIPEs ranged from 88.69 ± 6.62% to 101 ± 1.37% at 25 °C, and from 31.95 ± 1.92% to 78.69 ± 4.63% after incubation at 85 °C for 8 h. The release profile of allicin from the HIPEs exhibited thermal responsiveness, depending on the interfacial content of GA.CONCLUSIONThese findings indicated that the thermal‐responsive properties of HIPEs can be strategically engineered by manipulating their interfacial characteristics. © 2024 Society of Chemical Industry.

Mutation/metal deficiency in the "electrostatic loop" enhanced aggregation process in apo/holo SOD1 variants: implications for ALS diseases

Despite the many mechanisms it has created to prevent unfolding and aggregation of proteins, many diseases are caused by abnormal folding of proteins, which are called misfolding diseases. During this process, proteins undergo structural changes and become stable, insoluble beta-sheet aggregates called amyloid fibrils. Mutations/disruptions in metal ion homeostasis in the ALS-associated metalloenzyme superoxide dismutase (SOD1) reduce conformational stability, consistent with the protein aggregation hypothesis for neurodegenerative diseases. However, the exact mechanism of involvement is not well understood. Hence, to understand the role of mutation/ metal deficiency in SOD1 misfolding and aggregation, we investigated the effects of apo/holo SOD1 variants on structural properties using biophysical/experimental techniques. The MD results support the idea that the mutation/metal deficiency can lead to a change in conformation. The increased content of β-sheet structures in apo/holo SOD1 variants can be attributed to the aggregation tendency, which was confirmed by FTIR spectroscopy and dictionary of secondary structure in proteins (DSSP) results. Thermodynamic studies of GdnHCl showed that metal deficiency/mutation/intramolecular S–S reduction together are required to initiate misfolding/aggregation of SOD1. The results showed that apo/holo SOD1 variants under destabilizing conditions induced amyloid aggregates at physiological pH, which were detected by ThT/ANS fluorescence, as well as further confirmation of amyloid/amorphous species by TEM. This study confirms that mutations in the electrostatic loop of SOD1 lead to structural abnormalities, including changes in hydrophobicity, reduced disulfide bonds, and an increased propensity for protein denaturation. This process facilitates the formation of amyloid/amorphous aggregates ALS-associated.

Modulation of AIE and Intramolecular Charge Transfer of a Pyrene-Based Probe for Discriminatory Detection and Imaging of Oligomers and Amyloid Fibrils.

Oligomers and amyloid fibrils formed at different stages of protein aggregation are important biomarkers for a variety of neurodegenerative diseases including Alzheimer's and Parkinson's diseases. The development of probes for the sensitive detection of oligomeric species is important for early stage diagnosis of amyloidogenic diseases. Many small molecular dyes have been developed to probe the dynamic growth of amyloid fibrils. However, there is a lack of discriminatory detection strategies to monitor the dynamics of both oligomers and amyloid fibrils based on the differential modulation of the photophysical properties of a single dye. Here we report a pyrene-based intramolecular charge transfer (ICT) dye with large Stokes shifted red-emitting aggregation induced emission (AIE) for monitoring the dynamic populations of both oligomers and fibrils during the aggregation of hen egg white lysozyme (HEWL) protein. At the early stage of protein aggregation, the accumulation of HEWL oligomers results in a rapid and substantial increase in the red AIE intensity at 660 nm. Later, as the oligomers transform into mature fibrils, the dye exhibits a distinct photophysical change. Binding of the dye to HEWL fibrils strongly suppresses the red AIE and enhances ICT emission. This is evidenced by a gradual decrease in the AIE intensity (∼660 nm) and an increase in LE (∼490 nm) and ICT (∼540 nm) emission intensities during the later stages of protein aggregation. Thus, the dye provides simultaneous measurements of the population dynamics of both HEWL oligomers and fibrils during protein aggregation based on the discriminatory modulation of AIE and ICT of the dye. The dye also enables imaging of both HEWL oligomers and fibrils simultaneously using different emission channels in super-resolution confocal fluorescence microscopy.

Spatiotemporal formation of a single liquid-like condensate and amyloid fibrils of α-synuclein by optical trapping at solution surface

Liquid-like protein condensates have recently attracted much attention due to their critical roles in biological phenomena. They typically show high fluidity and reversibility for exhibiting biological functions, while occasionally serving as sites for the formation of amyloid fibrils. To comprehend the properties of protein condensates that underlie biological function and pathogenesis, it is crucial to study them at the single-condensate level; however, this is currently challenging due to a lack of applicable methods. Here, we demonstrate that optical trapping is capable of inducing the formation of a single liquid-like condensate of α-synuclein in a spatiotemporally controlled manner. The irradiation of tightly focused near-infrared laser at an air/solution interface formed a condensate under conditions coexisting with polyethylene glycol. The fluorescent dye-labeled imaging showed that the optically induced condensate has a gradient of protein concentration from the center to the edge, suggesting that it is fabricated through optical pumping-up of the α-synuclein clusters and the expansion along the interface. Furthermore, Raman spectroscopy and thioflavin T fluorescence analysis revealed that continuous laser irradiation induces structural transition of protein molecules inside the condensate to β-sheet rich structure, ultimately leading to the condensate deformation and furthermore, the formation of amyloid fibrils. These observations indicate that optical trapping is a powerful technique for examining the microscopic mechanisms of condensate appearance and growth, and furthermore, subsequent aging leading to amyloid fibril formation.

Structural Basis for Monoclonal Antibody Therapy for Transthyretin Amyloidosis.

The disease of transthyretin (TTR) amyloidosis (ATTR) has been known since the 1960s, and during the past 60 or so years, there has been a sustained period of steady discoveries that have led to the current model of ATTR pathogenesis. More recent research has achieved major advances in both diagnostics and therapeutics for ATTR, which are having a significant impact on ATTR patients today. Aiding these recent achievements has been the remarkable ability of cryo-electron microscopy (EM) to determine high-resolution structures of amyloid fibrils obtained from individual patients. Here, we will examine the cryo-EM structures of transthyretin amyloid fibrils to explore the structural basis of the two monoclonal antibody therapies for ATTR that are in clinical trials, ALXN-2220 and Coramitug, as well as to point out potential applications of this approach to other systemic amyloid diseases.

Salt-Induced Hydrophobic C-Terminal Region of α-Synuclein Triggers Its Fibrillation under the Mimic Physiologic Condition.

α-Synuclein (αS) causes Parkinson's disease due to the structural alteration into amyloid fibrils that form after the interaction with synaptic membranes in neurons. To understand the alternation mechanism, the effect of salt (NaCl) on the interaction of αS with synaptic mimic membrane was characterized at the molecular level because salt triggered the amyloid fibril formation. The membrane-bound conformation (or the initial conformation before fibrillation) showed that NaCl decreased the number of helical structures and Tyr residues interacting with the membrane surface compared to when NaCl was absent, implying an increase in solvent-exposed regions. The N-terminal region of αS interacted with the membrane, forming the helical structures regardless of NaCl, while the C-terminal region formed a random structure with weak membrane interaction, but NaCl inhibited the interaction of its hydrophobic area, suggesting that salt promoted amyloid fibril formations by exposing the hydrophobic C-terminal region, which can intermolecularly interact with free αS.

An evolutionarily conserved function of C-reactive protein is to prevent the formation of amyloid fibrils.

C-reactive protein (CRP) binds to phosphocholine (PCh)-containing substances and subsequently activates the complement system to eliminate the ligand. The PCh-binding function of CRP has been conserved throughout evolution from arthropods to humans. Human CRP, in its structurally altered conformation at acidic pH, also binds to amyloid-β (Aβ) and prevents the formation of Aβ fibrils. It is unknown whether the Aβ-binding function of CRP has also been evolutionarily conserved. The aim of this study was to determine whether CRP isolated from American horseshoe crab Limulus polyphemus was also anti-amyloidogenic and whether this function required structural alteration of Limulus CRP (Li-CRP). Two CRP species Li-CRP-I and Li-CRP-II were purified from hemolymph by employing PCh-affinity chromatography and phosphoethanolamine-affinity chromatography, respectively. Both Li-CRP-I and Li-CRP-II bound to immobilized Aβ at physiological pH. Unlike human CRP, Li-CRP did not require any changes in its overall structure to bind to Aβ. Both Li-CRP-I and Li-CRP-II bound to Aβ in the fluid phase also and prevented the fibrillation of Aβ. Additionally, ion-exchange chromatography of purified Li-CRP indicated that a variety of Li-CRP molecules of different subunit compositions were present in Limulus hemolymph, raising the possibility that the presence of various Li-CRP species in hemolymph facilitates the recognition of a range of proteins with differing amyloidogenicity. We conclude that the binding of CRP to Aβ is an ancient function of CRP. In invertebrates, the Aβ-binding function of CRP can protect the host from toxicity caused by amyloidogenic and pathogenic proteins. In humans, the Aβ-binding function of CRP can protect against inflammatory diseases in which the host proteins are ectopically deposited on either host cells or foreign cells in an inflammatory milieu since immobilized proteins may expose Aβ-like structures after deposition at places where they are not supposed to be.

Integrative determination of the atomic structure of mutant huntingtin exon 1 fibrils implicated in Huntington's disease.

Neurodegeneration in Huntington's disease (HD) is accompanied by the aggregation of fragments of the mutant huntingtin protein, a biomarker of disease progression. A particular pathogenic role has been attributed to the aggregation-prone huntingtin exon 1 (HTTex1), generated by aberrant splicing or proteolysis, and containing the expanded polyglutamine (polyQ) segment. Unlike amyloid fibrils from Parkinson's and Alzheimer's diseases, the atomic-level structure of HTTex1 fibrils has remained unknown, limiting diagnostic and treatment efforts. We present and analyze the structure of fibrils formed by polyQ peptides and polyQ-expanded HTTex1 in vitro. Atomic-resolution perspectives are enabled by an integrative analysis and unrestrained all-atom molecular dynamics (MD) simulations incorporating experimental data from electron microscopy (EM), solid-state NMR, and other techniques. Alongside the use of prior data, we report new magic angle spinning NMR studies of glutamine residues of the polyQ fibril core and surface, distinguished via hydrogen-deuterium exchange (HDX). Our study provides a new understanding of the structure of the core as well as surface of aggregated HTTex1, including the fuzzy coat and polyQ-water interface. The obtained data are discussed in context of their implications for understanding the detection of such aggregates (diagnostics) as well as known biological properties of the fibrils.

Clinical and biochemical characterization of asymptomatic carriers and symptomatic patients with hereditary transthyretin amyloidosis caused by TTR V30L mutation.

Hereditary transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by amyloid fibril deposition. The TTR c.148G > T mutation (V30L) in ATTR is rarely reported, and its biochemical properties are unknown. Seven patients and two asymptomatic carriers from two unrelated families diagnosed with V30L variant of ATTR were included. Data on clinical manifestations, laboratory examination, electrophysiology, ophthalmological corneal confocal microscopy (CCM), pathology and molecular biological experiments was collected and analyzed. Most patients initially experienced paresthesia, with varying degrees of peripheral neuropathy, autonomic dysfunction, and cardiac involvement. Nerve conduction studies showed extensive motor and sensory nerve involvement in upper and lower limbs. CCM revealed reduced corneal nerve density and fiber length. Sural nerve biopsies indicated loss of myelinated nerve fibers, with neurogenic patterns in gastrocnemius muscle biopsies. Asymptomatic carriers had nearly normal electrophysiology but mild reductions in corneal nerve fiber density and length. Sural nerve biopsies in carriers showed mild reductions in small myelinated nerve fibers. V30L mutation impaired thermodynamic and kinetic stability of the mutant protein. Plasma TTR tetramer concentration was lower in ATTR V30L patients compared to healthy donors. Small molecule stabilizers failed to exhibit satisfactory inhibition on fibril formation of V30L mutation in vitro. This study highlights the multisystem involvement in ATTR V30L patients, including neuropathy and cardiac issues. Both patients and carriers showed abnormalities in nerve conduction, corneal microscopy, and pathology. The V30L mutation impaired protein stability and reduced plasma TTR tetramer levels. Small molecule stabilizers were ineffective, indicating a need for alternative treatments.

Cavity Lasing of Thioflavin T in the Condensed Phase for Discrimination between Surface Interaction and β-Sheet Groove Binding in Alzheimer-Linked Peptides.

This study investigates the lasing effects in a Fabry-Perot cavity to discern the binding interactions of thioflavin T (ThT) with various peptides associated with Alzheimer's disease, including Aβ(1-42), KLVFFA, and diphenylalanine (FF) in the condensed phase. Utilizing kinetic lasing measurements, the research explores ThT emission enhancements due to specific groove binding in β-sheet structures and highlights additional contributions from weak surface interactions and solvent-solute interactions. Lasing spectroscopy reveals a lack of transition of the FF system from its native state to an amyloid-like structure, challenging traditional ThT assay interpretations. These findings show the potential of lasing spectroscopy in elucidating the molecular basis of amyloid fibril formation and the development of diagnostic tools for amyloidogenic diseases.

Energy gap of conformational transition related with temperature for the NACore of α-synuclein.

Pathological aggregation of α-synuclein (α-syn) into amyloid fibrils is a major feature of Parkinson's disease (PD). The self-assembly of α-syn is mainly governed by a non-amyloid-β component core (NACore). However, the effects of concentrations and temperatures on their conformational transition remain unclear. To answer this question, we investigated the aggregation kinetics of NACore oligomers in silico by performing several independent all-atom molecular dynamics simulations. The simulation results show that tetramers are more prone to form β-sheets at 300 K than dimers and octamers. We also found that the NACore oligomers had higher β-sheet and β-barrel contents at 310 K. The inter-chain hydrophobic interactions, the backbone hydrogen bonding, the residue-residue interactions between V70-V77 as well as V77-V77 play important roles in the aggregation tendency of NACore octamers at 310 K. Interestingly, the energy gap analysis revealed that the conformational transition of NACore oligomers from intermediate states (β-barrel conformation) to stable structures (β-sheet layers) was dependent on the temperatures. In short, our study provides insight into the kinetic and thermodynamic mechanisms of the conformational transition of NACore at different concentrations and temperatures, contributing to a better understanding of the aggregation process of α-syn in Parkinson's disease.

In cell NMR reveals cells selectively amplify and structurally remodel amyloid fibrils.

Amyloid forms of α-synuclein adopt different conformations depending on environmental conditions. Advances in structural biology have accelerated fibril characterization. However, it remains unclear which conformations predominate in biological settings because current methods typically not only require isolating fibrils from their native environments, but they also do not provide insight about flexible regions. To address this, we characterized α-syn amyloid seeds and used sensitivity enhanced nuclear magnetic resonance to investigate the amyloid fibrils resulting from seeded amyloid propagation in different settings. We found that the amyloid fold and conformational preferences of flexible regions are faithfully propagated in vitro and in cellular lysates. However, seeded propagation of amyloids inside cells led to the minority conformation in the seeding population becoming predominant and more ordered, and altered the conformational preferences of flexible regions. The examination of the entire ensemble of protein conformations in biological settings that is made possible with this approach may advance our understanding of protein misfolding disorders and facilitate structure-based drug design efforts.

Double cross-linked chitosan sponge encapsulated with ZrO2/soy protein isolate amyloid fibrils nanoparticles for the fluoride ion removal from water

Fluoride ion pollution in water has become a serious threat to the water environment and human health. Adsorption is a promising means of fluoride removal, but it also faces challenges such as the difficult separation and recovery of powdered particles, the leaching of modified coatings from adsorbents, and the structural disintegration of macroscopic adsorbents. For addressing the above challenges, glutaraldehyde/polyvinyl alcohol co-crosslinked ZrSAF/chitosan spongy composites (ZrS/GPCS) were prepared by utilizing encapsulation strategies and cross-linking. ZrS/GPCS-1, ZrS/GPCS-3 and ZrS/GPCS-4 were prepared due to the different amounts of cross-linking agents. The results showed that their fluoride ion adsorption capacities were 42.02, 44.44 and 39.84 mg/g, respectively. The removal of fluoride ions by ZrS/GPCS was maintained at >80 % in the pH range of 4–10. The addition of glutaraldehyde and polyvinyl alcohol affected the contact efficiency of fluoride ions with chitosan and ZrSAF, influencing the adsorption rate and adsorption effect. Glutaraldehyde, polyvinyl alcohol and ZrSAF improved the thermal stability, mechanical properties and structural integrity of chitosan matrix. Both the chitosan matrix and the internal ZrSAF played an important role in fluoride removal, and the removal mechanisms included electrostatic interaction, hydrogen bonding, and complexation.

Valorization of Protein Materials Through Mechanochemistry and Self-Assembly.

The concept of combining mixing of solids by milling (a type of mechanochemistry) with aqueous self-assembly provides interesting possibilities for energy efficient production of advanced nanomaterials. Many proteins are outstanding building blocks for self-assembly, a prominent example being the conversion of proteins into protein nanofibrils (PNFs) - a structure related to amyloid fibrils. PNFs have attractive mechanical properties and have a tendency to form ordered materials. They are accordingly of interest as materials for bioplastics and potentially also for more high-tech applications. In this concept article we highlight our effort on valorization of such proteins with hydrophobic organic compounds such an organic dyes and drug molecules, by developing scalable methodology combining mechanochemistry and self-assembly. Compared to more established methodology, mechanochemical methodology is a valuable complement as it allows potential scalable production of hybrids between e. g. proteins and highly hydrophobic compounds - a class of hybrid material that is difficult to access by other means. This may allow for development of sustainable processes for fabrication of advanced protein-based materials derivable from renewable source materials.

Lysosomal TMEM106B interacts with galactosylceramidase to regulate myelin lipid metabolism

TMEM106B is an endolysosomal transmembrane protein not only associated with multiple neurological disorders including frontotemporal dementia, Alzheimer’s disease, and hypomyelinating leukodystrophy but also potentially involved in COVID-19. Additionally, recent studies have identified amyloid fibrils of C-terminal TMEM106B in both aged healthy and neurodegenerative brains. However, so far little is known about physiological functions of TMEM106B in the endolysosome and how TMEM106B is involved in a wide range of human conditions at molecular levels. Here, we performed lipidomic analysis of the brain of TMEM106B-deficient mice. We found that TMEM106B deficiency significantly decreases levels of two major classes of myelin lipids, galactosylceramide and its sulfated derivative sulfatide. Subsequent co-immunoprecipitation assay showed that TMEM106B physically interacts with galactosylceramidase. We also found that galactosylceramidase activity was significantly increased in TMEM106B-deficient brains. Thus, our results suggest that TMEM106B interacts with galactosylceramidase to regulate myelin lipid metabolism and have implications for TMEM106B-associated diseases.

Controlled preparation of tannic acid-derived carbonized dotsand their use to inhibit amyloid aggregation and promote aggregate disaggregation.

Tannic acid (TA)-derived carbon dots (TACDs) were synthesized for the first time via a solvothermal method using TA as one of the raw materials, which may effectively inhibit amyloid fibril aggregation and disaggregate mature fibril. The fluorescent property of TACDs were modulated by adjusting the ratio of TA to o-phenylenediamine (oPD), and TACDs fabricated with the precursor ratio as 1:1 showed the best fluorescent property. Circular dichroism spectra (CD) showed that the structure of β-sheet decreased as the concentration of TACDs increased. The inhibition efficiency, as confirmed by thioflavin T (ThT) and transmission electron microscopy (TEM), is extraordinary at 98.16%, whereas disaggregation efficiency is noteworthy at 97.97%, and the disaggregated lysozyme fibrils did not reaggregate after 7 days. More critically, TACDs can also alleviate the cellular toxicity caused by Aβ fibrils and improve cell viability. This work offers a new perspective on the design of scavengers for amyloid plaques.

Converting the Amyloidogenic Islet Amyloid Polypeptide into a Potent Nonaggregating Peptide Ligand by Side Chain-to-Side Chain Macrocyclization.

The islet amyloid polypeptide (IAPP), also known as amylin, is a hormone playing key physiological roles. However, its aggregation and deposition in the pancreatic islets are associated with type 2 diabetes. While this peptide adopts mainly a random coil structure in solution, its secondary conformational conversion into α-helix represents a critical step for receptor activation and contributes to amyloid formation and associated cytotoxicity. Considering the large conformational landscape and high amyloidogenicity of the peptide, as well as the complexity of the self-assembly process, it is challenging to delineate the delicate interplay between helical folding, peptide aggregation, and receptor activation. In the present study, we probed the roles of helical folding on the function-toxicity duality of IAPP by restricting its conformational ensemble through side chain-to-side chain stapling via azide-alkyne cycloaddition. Intramolecular macrocyclization (i; i + 4) constrained IAPP into α-helix and inhibited its aggregation into amyloid fibrils. These helical derivatives slowed down the self-assembly of unmodified IAPP. Site-specific macrocyclization modulated the capacity of IAPP to perturb lipid bilayers and cell plasma membrane and reduced, or even fully inhibited, the cytotoxicity associated with aggregation. Furthermore, the α-helical IAPP analogs showed moderate to high potency toward cognate G protein-coupled receptors. Overall, these results indicate that macrocyclization represents a promising strategy to protect an amyloidogenic peptide hormone from aggregation and associated toxicity, while maintaining high receptor activity.

Comparison of formation and structural characteristics of amyloid fibrils of peanut protein isolate after hydration treatment at different pH

In this paper, the process of fibrillation and the structure of fibrils formed from peanut protein after hydration treatment at pH 5.0 (group Q) and pH 2.0 (group S) were studied. The results showed that compared with the peanut protein obtained after hydration at pH 5.0, the particle size of the peanut protein obtained after hydration at pH 2.0 increased, and the surface hydrophobicity and intrinsic fluorescence intensity decreased, indicating that the protein aggregated. During fibrillation, the maximum Thioflavin T fluorescence intensity of protein in group Q was higher than that in group S, and the time for group S protein to reach the maximum fluorescence intensity was slower than that in group Q protein. Besides, the group Q protein first formed short worm-like fibrils and then long flexible fibrils, and the group S protein first formed short rigid fibrils and then worm-like fibrils. In conclusion, hydration treatment at pH 2.0 led to the aggregation of peanut protein, reduced the fibrils forming ability of peanut protein and affected subsequent self-assembly processes. This study could provide new insights of the fibril formation mechanism and structural properties of peanut protein after hydration treatment at different pH.

Protective Effect of Quercetin on Amyloid-Induced Alterations in Lipid Bilayer Integrity

The present study employs molecular dynamics simulations to investigate the interactions between quercetin, amyloid fibrils, and POPC lipid bilayers. The results demonstrate that quercetin does not significantly affect the molecular organization of the bilayer, while IAPP fibrils induce substantial structural changes, particularly in the outer monolayer. Quercetin mitigates these effects by reducing the impact on headgroup and glycerol regions and causing a more superficial positioning of IAPP. Additionally, quercetin slightly decreases the order of sn-2 acyl chains, indicating a disordering effect. In a ternary system with POPC, quercetin, and IAPP, the reduction in the deuterium order parameter of sn-2 acyl chains is less pronounced, underscoring quercetin's protective role. Unlike IAPP, ApoAI and insulin fibrils undergo significant structural reorganization in the membrane-bound state. Quercetin attenuative effects are observed only with ApoAI, highlighting its potential as a protective agent against amyloid-induced membrane disruption. These findings provide valuable insights into the interactions between polyphenols, amyloid fibrils, and lipid membranes, contributing to the understanding of membrane-associated amyloid pathologies.

Methods for high throughput discovery of fluoroprobes that recognize tau fibril polymorphs.

Aggregation of microtubule-associated protein tau (MAPT/tau) into conformationally distinct fibrils underpins neurodegenerative tauopathies. Fluorescent probes (fluoroprobes), such as thioflavin T (ThT), have been essential tools for studying tau aggregation; however, most of them do not discriminate between amyloid fibril conformations (polymorphs). This gap is due, in part, to a lack of high-throughput methods for screening large, diverse chemical collections. Here, we leverage advances in protein adaptive differential scanning fluorimetry (paDSF) to screen the Aurora collection of 300+ fluorescent dyes against multiple synthetic tau fibril polymorphs. This screen, coupled with orthogonal secondary assays, revealed pan-fibril binding chemotypes, as well as fluoroprobes selective for subsets of fibrils. One fluoroprobe recognized tau pathology in ex vivo brain slices from Alzheimer's disease patients. We propose that these scaffolds represent entry points for development of selective fibril ligands and, more broadly, that high throughput, fluorescence-based dye screening is a platform for their discovery.