Thioflavin-T (ThT) is the most commonly used fluorescent dye for following amyloid formation semiquantitatively in vitro, specifically probing the fibrillar cross-b-sheet content. In recent years, structural polymorphism of amyloid fibrils has been shown to be an important aspect of amyloid formation, both in vitro and in neurodegenerative diseases. Therefore, understanding ThT-amyloid interactions in the context of structural polymorphism of amyloids is necessary for correct interpretation of ThT fluorescence data. Here we study the influence of fibril morphology on ThT fluorescence and ThT binding sites, with two morphologically distinct but chemically identical a-synuclein polymorphs. In ThT fluorescence assays the two polymorphs show type-specific fluorescence intensity behaviour although their b-sheet content has been shown to be similar. Further, fluorescence lifetime measurements of fibrilbound ThT reveal the presence of at least two qualitatively different ThT binding sites on the polymorphs. The relative distributions of the binding sites on the fibril surfaces appear to be morphology dependent, thus determining the observed polymorph-specific ThT fluorescence intensities. These results, highlighting the role of fibril morphology in ThT-based amyloid studies, underline the relevance of polymorphs in ThT-amyloid interaction and can explain the variability often observed in ThT amyloid binding assays.

The aggregation of the protein α-synuclein (aSyn) into amyloid fibrils in the human brain is associated with the development of several neurodegenerative diseases, including Parkinson's disease. The previously observed prion-like spreading of aSyn aggregation throughout the brain and the finding that heterologous cross-seeding of amyloid aggregation occurs in vitro for some proteins suggest that exposure to amyloids in general may pose a risk for disease development. To elucidate which protein fibril characteristics determine if and how heterologous amyloid seeding can occur, we investigated the potential of amyloid fibrils formed from proteins found in food, hen egg white lysozyme, and bovine milk β-lactoglobulin to cross-seed aSyn aggregation in the test tube. We observed that amyloid fibrils from lysozyme, but not β-lactoglobulin, potently cross-seeded the aggregation of aSyn as indicated by a significantly shorter lag phase of aSyn aggregation in the presence of lysozyme fibrils. The cross-seeding effect of lysozyme was found to be primarily driven by a surface-mediated nucleation mechanism. The differential seeding effect of lysozyme and β-lactoglobulin on aSyn aggregation could be explained on the basis of binding affinity, binding site, and electrostatic interactions. Our results indicate that heterologous seeding of proteins may occur depending on the physicochemical characteristics of the seed protein fibril. Our findings suggest that heterologous seeding has the potential to determine the pathogenesis of neurodegenerative amyloid diseases.

Hydrogels of amyloid fibrils are a versatile biomaterial for tissue engineering and other biomedical applications. Their suitability for these applications has been partly ascribed to their excellent and potentially engineerable rheological properties. However, while in biomedical applications the gels have to function in compositionally complex physiological solutions, their rheological behavior is typically only characterized in simple buffers. Here we show that the viscoelastic response of networks of amyloid fibrils of the protein lysozyme in biologically relevant solutions substantially differs from the response in simple buffers. We observe enhanced energy dissipation in both cell culture medium and synovial fluid. We attribute this energy dissipation to interactions of the amyloid fibrils with other molecules in these solutions and especially to the adsorption of the abundantly present protein serum albumin. This finding provides the basis for a better understanding of the performance of amyloid hydrogels in biomedical applications.

Amyloid fibrils of the protein α-synuclein (αS) have recently been identified as a biomarker for Parkinson’s disease (PD). To detect the presence of these amyloid fibrils, seed amplification assays (SAAs) have been developed. SAAs allow for the detection of αS amyloid fibrils in biomatrices such as cerebral spinal fluid and are promising for PD diagnosis by providing a dichotomous (yes/no) response. The additional quantification of the number of αS amyloid fibrils may enable clinicians to evaluate and follow the disease progression and severity. Developing quantitative SAAs has been shown to be challenging. Here, we report on a proof-of-principle study on the quantification of αS fibrils in fibril-spiked model solutions of increasing compositional complexity including blood serum. We show that parameters derived from standard SAAs can be used for fibril quantification in these solutions. However, interactions between the monomeric αS reactant that is used for amplification and biomatrix components such as human serum albumin have to be taken into account. We demonstrate that quantification of fibrils is possible even down to the single fibril level in a model sample consisting of fibril-spiked diluted blood serum.

BackgroundA series of human diseases are caused by the misfolding and aggregation of specific proteins or peptides into amyloid fibrils; nine of these diseases, referred to as polyglutamine diseases, are associated with proteins carrying an expanded polyglutamine (polyQ) region. While the presence of this latter is thought to be the determinant factor for the development of polyQ diseases, the non-polyQ regions of the host proteins are thought to play a significant modulating role.MethodIn order to better understand the role of non-polyQ regions, the toxic effects of model proteins bearing different polyQ regions (containing up to 79 residues) embedded at two distinct locations within the β-lactamase (BlaP) host enzyme were evaluated in Caenorhabditis elegans. This small organism can be advantageous for the validation of in vitro findings, as it provides a multicellular context yet avoids the typical complexity of common studies relying on vertebrate models. Several phenotypic assays were performed in order to screen for potential toxic effects of the different BlaP-polyQ proteins.ResultsDespite the significant in vitro aggregation of BlaP-polyQ proteins with long polyQ regions, none of the BlaP-polyQ chimeras aggregated in the generated transgenic in vivo models.ConclusionThe absence of a toxic effect of the expression of BlaP-polyQ chimeras may find its cause in biochemical mechanisms present in vivo to cope with protein aggregation (e.g. presence of chaperones) or in C. elegans’ limitations such as its short lifespan. It is plausible that the aggregation propensities of the different BlaP chimeras containing embedded polyQ sequences are too low in this in vivo environment to permit their aggregation. These experiments emphasize the need for several comparative and in vivo verification studies of biologically relevant in vitro findings, which reveal both the strengths and limitations of widely used model systems.Electronic supplementary materialThe online version of this article (doi:10.1186/s12952-017-0080-5) contains supplementary material, which is available to authorized users.