SARS-CoV-2 N-protein induces the formation of composite α-synuclein/N-protein fibrils that transform into a strain of α-synuclein fibrils.

The presence of deposits of alpha-synuclein (αS) fibrils in the cells of the brain is a hallmark of several α-synucleinopathies, including Parkinson's disease. As most disease cases are not familial, it is likely that external factors play a role in the disease onset. One of the external factors that may influence the disease onset is viral infection. It has recently been shown in in vitro assays that in the presence of SARS-Cov-2 N-protein, αS fibril formation is faster and proceeds in an unusual two-step aggregation process. Here, we show that faster fibril formation is not due to the SARS-CoV-2 N-protein-catalysed formation of an aggregation-prone nucleus. Instead, aggregation starts with the formation of a population of mixed αS/N-protein fibrils with low affinity for αS. Mixed amyloid fibrils, composed of two different proteins, have not been observed before. After the depletion of N-protein, fibril formation comes to a halt, until a slow transformation into fibrils with characteristics of a pure αS fibril strain occurs. This transformation into a strain of αS fibrils subsequently results in a second phase of fibril growth until a new equilibrium is reached. We hypothesize that this fibril strain transformation may be of relevance in the cell-to-cell spread of the αS pathology and disease onset.

Quantitative Seed Amplification Assay: A Proof-of-Principle Study.

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.

Interactions between SARS-CoV-2 N-Protein and α-Synuclein Accelerate Amyloid Formation.

First cases that point at a correlation between SARS-CoV-2 infections and the development of Parkinson's disease (PD) have been reported. Currently, it is unclear if there is also a direct causal link between these diseases. To obtain first insights into a possible molecular relation between viral infections and the aggregation of α-synuclein protein into amyloid fibrils characteristic for PD, we investigated the effect of the presence of SARS-CoV-2 proteins on α-synuclein aggregation. We show, in test tube experiments, that SARS-CoV-2 spike protein (S-protein) has no effect on α-synuclein aggregation, while SARS-CoV-2 nucleocapsid protein (N-protein) considerably speeds up the aggregation process. We observe the formation of multiprotein complexes and eventually amyloid fibrils. Microinjection of N-protein in SH-SY5Y cells disturbed the α-synuclein proteostasis and increased cell death. Our results point toward direct interactions between the N-protein of SARS-CoV-2 and α-synuclein as molecular basis for the observed correlation between SARS-CoV-2 infections and Parkinsonism.

Cross-seeding of alpha-synuclein aggregation by amyloid fibrils of food proteins.

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 ß-lactoglobulin to cross-seed aSyn aggregation in the test tube. We observed that amyloid fibrils from lysozyme, but not ß-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 ß-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.

α-Synuclein Penetrates Mucin Hydrogels Despite Its Mucoadhesive Properties.

Recent research indicates that the progression of Parkinson's disease can start from neurons of the enteric nervous system, which are in close contact with the gastrointestinal epithelium: α-synuclein molecules can be transferred from these epithelial cells in a prion-like fashion to enteric neurons. Thin mucus layers constitute a defense line against the exposure of noninfected cells to potentially harmful α-synuclein species. We show that-despite its mucoadhesive properties-α-synuclein can translocate across mucin hydrogels, and this process is accompanied by structural rearrangements of the mucin molecules within the gel. Penetration experiments with different α-synuclein variants and synthetic peptides suggest that two binding sites on α-synuclein are required to accomplish this rearrangement of the mucin matrix. Our results support the notion that the translocation of α-synuclein across mucus barriers observed here might be a critical step in the infection of the gastrointestinal epithelium and the development of Parkinson's disease.

Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils.

Thioflavin-T (ThT) is the most commonly used fluorescent dye for following amyloid formation semi-quantitatively in vitro, specifically probing the fibrillar cross-ß-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 α-synuclein polymorphs. In ThT fluorescence assays the two polymorphs show type-specific fluorescence intensity behaviour although their ß-sheet content has been shown to be similar. Further, fluorescence lifetime measurements of fibril-bound 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.

Distinct Mechanisms Determine α-Synuclein Fibril Morphology during Growth and Maturation.

Amyloid polymorphs have become one of the focal points of molecular studies of neurodegenerative diseases like Parkinson's disease. Due to their distinct biochemical properties and prion-like characteristics, insights into the molecular origin and stability of amyloid polymorphs over time are crucial for understanding the potential role of amyloid polymorphism in these diseases. Here, we systematically study the fibrillization of recombinantly produced human α-synuclein (αSyn) over an extended period of time to unravel the origin and temporal evolution of polymorphism. We follow morphological changes in the same fibril sample with atomic force microscopy over a period of 1 year. We show that wild-type (wt) αSyn fibrils undergo a slow maturation over time after reaching the plateau phase of aggregation (as detected in a Thioflavin-T fluorescence assay). This maturation, visualized by changes in the fibril periodicity over time, is absent in the disease mutant fibrils. The ß-sheet content of the plateau phase and matured fibrils, obtained using Fourier transform infrared spectroscopy, is however similar for the αSyn protein sequences, suggesting that the morphological changes in wt αSyn fibrils are tertiary or quaternary in origin. Furthermore, results from a reversibility assay show that the plateau phase fibrils do not disassemble over time. Together, the observed changes in the periodicity distributions and stability of the fibrillar core over time point toward two distinct mechanisms that determine the morphology of wt αSyn fibrils: competitive growth between different polymorphs during the fibrillization phase followed by a process wherein fibrils undergo slow maturation or annealing.

Conformational Compatibility Is Essential for Heterologous Aggregation of α-Synuclein.

Under aggregation-prone conditions, soluble amyloidogenic protein monomers can self-assemble into fibrils or they can fibrillize on preformed fibrillar seeds (seeded aggregation). Seeded aggregations are known to propagate the morphology of the seeds in the event of cross-seeding. However, not all proteins are known to cross-seed aggregation. Cross-seeding has been proposed to be restricted either because of differences in the protein sequences or because of conformations between the seeds and the soluble monomers. Here, we examine cross-seeding efficiency between three α-synuclein sequences, wild-type, A30P, and A53T, each varying in only one or two amino acids but forming morphologically distinct fibrils. Results from bulk Thioflavin-T measurements, monomer incorporation quantification, single fibril fluorescence microscopy, and atomic force microscopy show that under the given solution conditions conformity between the conformation of seeds and monomers is essential for seed elongation. Moreover, elongation characteristics of the seeds are defined by the type of seed.

Parkinson's Protein α-Synuclein Binds Efficiently and with a Novel Conformation to Two Natural Membrane Mimics.

Binding of human α-Synuclein, a protein associated with Parkinson's disease, to natural membranes is thought to be crucial in relation to its pathological and physiological function. Here the binding of αS to small unilamellar vesicles mimicking the inner mitochondrial and the neuronal plasma membrane is studied in situ by continuous wave and pulsed electron paramagnetic resonance. Local binding information of αS spin labeled by MTSL at positions 56 and 69 respectively shows that also helix 2 (residues 50-100) binds firmly to both membranes. By double electron-electron resonance (DEER) on the mutant spin labeled at positions 27 and 56 (αS 27/56) a new conformation on the membrane is found with a distance of 3.6 nm/ 3.7 nm between residues 27 and 56. In view of the low negative charge density of these membranes, the strong interaction is surprising, emphasizing that function and pathology of αS could involve synaptic vesicles and mitochondria.

Solution conditions define morphological homogeneity of α-synuclein fibrils.

The intrinsically disordered human α-synuclein (αSyn) protein exhibits considerable heterogeneity in in vitro fibrillization reactions. Using atomic force microscopy (AFM) we show that depending on the solvent conditions, A140C mutant and wild-type αSyn can be directed to reproducibly form homogeneous populations of fibrils exhibiting regular periodicity. Results from Thioflavin-T fluorescence assays, determination of residual monomer concentrations and native polyacrylamide gel electrophoresis reveal that solvent conditions including EDTA facilitate incorporation of a larger fraction of monomers into fibrils. The fibrils formed in 10mM Tris-HCl, 10mM NaCl and 0.1mM EDTA at pH7.4 display a narrow distribution of periodicities with an average value of 102±6nm for the A140C mutant and 107±9nm for wt αSyn. The ability to produce a homogeneous fibril population can be instrumental in understanding the detailed structural features of fibrils and the fibril assembly process. Moreover, the availability of morphologically well-defined fibrils will enhance the potential for use of amyloids as biological nanomaterials.

Inhibition of α-synuclein aggregation by small heat shock proteins.

The fibrillization of α-synuclein (α-syn) is a key event in the pathogenesis of α-synucleinopathies. Mutant α-syn (A53T, A30P, or E46K), each linked to familial Parkinson's disease, has altered aggregation properties, fibril morphologies, and fibrillization kinetics. Besides α-syn, Lewy bodies also contain several associated proteins including small heat shock proteins (sHsps). Since α-syn accumulates intracellularly, molecular chaperones like sHsps may regulate α-syn folding and aggregation. Therefore, we investigated if the sHsps αB-crystallin, Hsp27, Hsp20, HspB8, and HspB2B3 bind to α-syn and affect α-syn aggregation. We demonstrate that all sHsps bind to the various α-syns, although the binding kinetics suggests a weak and transient interaction only. Despite this transient interaction, the various sHsps inhibited mature α-syn fibril formation as shown by a Thioflavin T assay and atomic force microscopy. Interestingly, HspB8 was the most potent sHsp in inhibiting mature fibril formation of both wild-type and mutant α-syn. In conclusion, sHsps may regulate α-syn aggregation and, therefore, optimization of the interaction between sHsps and α-syn may be an interesting target for therapeutic intervention in the pathogenesis of α-synucleinopathies.

Neurotoxicity of Alzheimer's disease Aß peptides is induced by small changes in the Aß42 to Aß40 ratio.

The amyloid peptides Aß(40) and Aß(42) of Alzheimer's disease are thought to contribute differentially to the disease process. Although Aß(42) seems more pathogenic than Aß(40), the reason for this is not well understood. We show here that small alterations in the Aß(42):Aß(40) ratio dramatically affect the biophysical and biological properties of the Aß mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aß(42):Aß(40) ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aß species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aß peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti-amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non-toxic intermediates.

Single-molecule FRET reveals structural heterogeneity of SDS-bound alpha-synuclein.

SDS-concentration-dependent alpha-synuclein structure: Upon interaction with SDS, alpha Syn folds into a structure with two antiparallel alpha-helices. We show from single-molecule FRET that alpha Synn adopts this conformation in an all-or-none fashion below the SDS critical micelle concentration. Population of the folded species is directly coupled to an increase in alpha-helix content; this suggests that the entire N terminus is involved in the transaction.

Concentration dependence of alpha-synuclein fibril length assessed by quantitative atomic force microscopy and statistical-mechanical theory.

The initial concentration of monomeric amyloidogenic proteins is a crucial factor in the in vitro formation of amyloid fibrils. We use quantitative atomic force microscopy to study the effect of the initial concentration of human alpha-synuclein on the mean length of mature alpha-synuclein fibrils, which are associated with Parkinson's disease. We determine that the critical initial concentration, below which low-molecular-weight species dominate and above which fibrils are the dominant species, lies at approximately 15 muM, in good agreement with earlier measurements using biochemical methods. In the concentration regime where fibrils dominate, we find that their mean length increases with initial concentration. These results correspond well to the qualitative predictions of a recent statistical-mechanical model of amyloid fibril formation. In addition, good quantitative agreement of the statistical-mechanical model with the measured mean fibril length as a function of initial protein concentration, as well as with the fibril length distributions for several protein concentrations, is found for reasonable values of the relevant model parameters. The comparison between theory and experiment yields, for the first time to our knowledge, an estimate of the magnitude of the free energies associated with the intermolecular interactions that govern alpha-synuclein fibril formation.

Tissue transglutaminase modulates alpha-synuclein oligomerization.

We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's disease-associated alpha-synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant alpha-synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective alpha-synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/alpha-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all alpha-synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal alpha-synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal beta-sheet-containing oligomers, the tTG/alpha-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant alpha-synuclein variants. We propose that tTG cross-linking imposes structural constraints on alpha-synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species.

Quantitative characterization of protein nanostructures using atomic force microscopy.

The aggregation of proteins into fibrillar structures called amyloid is a characteristic of many diseases, including several neurodegenerative disorders. Although amyloid formation is inherent to several serious diseases, the mechanism of fibril formation and the modes of toxicity are not yet known. High concentrations of fibrillar aggregates of ¿-synuclein protein are found in the brains of patients suffering from Parkinson's disease. We exploit different contrast modes of high resolution atomic force microscopy (AFM) on fibrils formed by the wild-type alpha-synuclein protein, and by the familial disease-related A30P, E46K and A53T variants, to get more insight into the in vitro process of fibril assembly. From quantitative analysis of height images measured in tapping mode AFM, we obtained data that are compatible with a twisted hierarchical assembly model [1] for all protein variants. The E46K mutant displays the most distinct and smallest periodicity. The modulation depth for all mutants is very similar, and is smaller for wild-type protein commensurate with the lower fibril height. The detailed morphology observed in phase images indicates however that fibrils may also be formed through the association of fibril segments. To study the mechanical properties of fibrils we applied force while scanning in contact mode, resulting in characteristic deformation of protein fibrils with a periodicity corresponding to the modulation observed in tapping mode. Our observations suggest that the hierarchical assembly model may not be the exclusive mechanism of alpha-synuclein fibril assembly, but that multiple modes of fibril assembly play a role in alpha-synuclein fibril formation.

Quantitative morphological analysis reveals ultrastructural diversity of amyloid fibrils from alpha-synuclein mutants.

High resolution atomic force microscopy is a powerful tool to characterize nanoscale morphological features of protein amyloid fibrils. Comparison of fibril morphological properties between studies has been hampered by differences in analysis procedures and measurement error determination used by various authors. We describe a fibril morphology analysis method that allows for quantitative comparison of features of amyloid fibrils of any amyloidogenic protein measured by atomic force microscopy. We have used tapping mode atomic force microscopy in liquid to measure the morphology of fibrillar aggregates of human wild-type alpha-synuclein and the disease-related mutants A30P, E46K, and A53T. Analysis of the images shows that fibrillar aggregates formed by E46K alpha-synuclein have a smaller diameter (9.0 +/- 0.8 nm) and periodicity (mode at 55 nm) than fibrils of wild-type alpha-synuclein (height 10.0 +/- 1.1 nm; periodicity has a mode at 65 nm). Fibrils of A30P have smaller diameter still (8.1 +/- 1.2 nm) and show a variety of periodicities. This quantitative analysis procedure enables comparison of the results with existing models for assembly of amyloid fibrils.

Prism-based excitation wavelength selection for multicolor fluorescence coincidence measurements.

We have designed and constructed a prism setup for multiple-color confocal fluorescence experiments. The prism setup permits easy selection of any color or any combination of colors from a multicolor light source, such as a mixed-gas argon-krypton-ion laser. The selected colors emerging from the prism setup are, by design, optimally overlapped in the focus of a high-numerical-aperture objective, such as that commonly used in single-molecule fluorescence experiments. The various excitation powers can be easily adjusted in this setup. We will exemplify the potential of this setup in single-molecule fluorescence emission spectroscopy.