Thioflavin T fluorescence anisotropy: an alternative technique for the study of amyloid aggregation

The process of amyloid polymerisation raises keen interest in particular because of the biomedical impact of this process. A variety of analytical methods have been developed to monitor amyloid formation. Thioflavin T (ThT) is the most commonly used dye for detection of amyloid aggregation. Nevertheless, ThT fluorescence enhancement is strongly dependent of fibril morphology. In this study using the HET-s prion fibril model, we show that amyloid formation can be monitored by measuring ThT fluorescence anisotropy. Kinetic parameters obtained by this method are identical to those determined by CD spectrometry. We propose that ThT anisotropy represent an interesting, simple and alternative technique to analyze the amyloid formation process.     

Inhibitory Role of β-Casein on the α-Synuclein Aggregation Associated with Parkinson’s Disease In Vitro

Large soluble oligomeric species are observed as probable intermediates during fibril formation in aggregations of Parkinson’s disease (PD). Fibrillar deposits occur in PD. Amyloid forms α-Synuclein is one of the main compounds aggregations. β-Casein, a member of the Casein family, has been demonstrated to inhibit α-Synuclein aggregation by chaperone-like activity. In this study, we investigated the effect of chaperone activity of β-Casein in preventing the aggregation of α-Synuclein protein. We have examined the effect of β-Casein in preventing α-Synuclein aggregation by using from Thioflavin T-binding assay, transmission electron microscopy, ANS-binding assay, circular dichroism spectroscopy and FTIR spectroscopy. Results from the ThT binding assay demonstrated an increase in the ThT fluorescence intensity of α-Synuclein incubated in absence of β-Casein but in its presence fluorescence intensity is decreased. Electron microscopy data also indicated that β-Casein decreased the aggregation content of α-Synuclein. ANS results also showed that β-Casein significantly decreased the the hydrophobic area in α-Synuclein incubated. Circular dichroism spectroscopy (CD) results also showed that β-sheet structures of α-Synuclein incubated change to structural α-helical and β-turn in presence of β-Casein. FTIR spectroscopy indicates the presence of β-sheet structures in α-Synuclein incubated in absence of β-Casein and β-sheet structures decreased in its presence. Thus, our results suggest that in vitro, β-Casein interacts with α-Synuclein fibrils, changes the α-Synuclein structure and prevents amyloid fibril formation. This means that β-Casein could be essential for therapies inhibiting aggregation and to be an important therapeutic drug against PD.     

Covalent α-Synuclein Dimers: Chemico-Physical and Aggregation Properties

The aggregation of α-synuclein into amyloid fibrils constitutes a key step in the onset of Parkinson''s disease. Amyloid fibrils of α-synuclein are the major component of Lewy bodies, histological hallmarks of the disease. Little is known about the mechanism of aggregation of α-synuclein. During this process, α-synuclein forms transient intermediates that are considered to be toxic species. The dimerization of α-synuclein could represent a rate-limiting step in the aggregation of the protein. Here, we analyzed four covalent dimers of α-synuclein, obtained by covalent link of the N-terms, C-terms, tandem cloning of two sequences and tandem juxtaposition in one protein of the 1–104 and 29–140 sequences. Their biophysical properties in solution were determined by CD, FT-IR and NMR spectroscopies. SDS-induced folding was also studied. The fibrils formation was analyzed by ThT and polarization fluorescence assays. Their morphology was investigated by TEM and AFM-based quantitative morphometric analysis. All dimers were found to be devoid of ordered secondary structure under physiological conditions and undergo α-helical transition upon interaction with SDS. All protein species are able to form amyloid-like fibrils. The reciprocal orientation of the α-synuclein monomers in the dimeric constructs affects the kinetics of the aggregation process and a scale of relative amyloidogenic propensity was determined. Structural investigations by FT IR spectroscopy, and proteolytic mapping of the fibril core did not evidence remarkable difference among the species, whereas morphological analyses showed that fibrils formed by dimers display a lower and diversified level of organization in comparison with α-synuclein fibrils. This study demonstrates that although α-synuclein dimerization does not imply the acquisition of a preferred conformation by the participating monomers, it can strongly affect the aggregation properties of the molecules. The results presented highlight a substantial role of the relative orientation of the individual monomer in the definition of the fibril higher structural levels.     

Single Chain Variable Fragment Against Aβ Expressed in Baculovirus Inhibits Abeta Fibril Elongation and Promotes its Disaggregation

Alzheimer’s disease (AD) is the most common form of age-related dementia, and the most urgent problem is that it is currently incurable. Amyloid-β (Aβ) peptide is believed to play a major role in the pathogenesis of AD. We previously reported that an Aβ N-terminal amino acid targeting monoclonal antibody (MAb), A8, inhibits Aβ fibril formation and has potential as an immunotherapy for AD based on a mouse model. To further study the underlying mechanisms, we tested our hypothesis that the single chain fragment variable (scFv) without the Fc fragment is capable of regulating either Aβ aggregation or disaggregation in vitro. Here, a model of cell-free Aβ “on-pathway” aggregation was established and identified using PCR, Western blot, ELISA, transmission electron microscopy (TEM) and thioflavin T (ThT) binding analyses. His-tagged A8 scFvs was cloned and solubly expressed in baculovirus. Our data demonstrated that the Ni-NTA agarose affinity-purified A8 scFv inhibited the forward reaction of “on-pathway” aggregation and Aβ fibril maturation. The effect of A8 scFv on Aβ fibrillogenesis was markedly more significant when administered at the start of the Aβ folding reaction. Furthermore, the results also showed that pre-formed Aβ fibrils could be disaggregated via incubation with purified A8 scFv, which suggested that A8 scFv is involved in the reverse reaction of Aβ aggregation. Therefore, A8 scFv was capable of both inhibiting fibrillogenesis and disaggregating matured fibrils. Our present study provides valuable insight into the regulators of ultrastructural dynamics of cell-free “on-pathway” Aβ aggregation and will assist in the development of therapeutic strategies for AD.     

Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains

Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this work, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40–60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. This mutagenic approach helped to identify key regions implicated in λ3 AL.     

RNA Aptamers Generated against Oligomeric Aβ40 Recognize Common Amyloid Aptatopes with Low Specificity but High Sensitivity

Aptamers are useful molecular recognition tools in research, diagnostics, and therapy. Despite promising results in other fields, aptamer use has remained scarce in amyloid research, including Alzheimer''s disease (AD). AD is a progressive neurodegenerative disease believed to be caused by neurotoxic amyloid β-protein (Aβ) oligomers. Aβ oligomers therefore are an attractive target for development of diagnostic and therapeutic reagents. We used covalently-stabilized oligomers of the 40-residue form of Aβ (Aβ40) for aptamer selection. Despite gradually increasing the stringency of selection conditions, the selected aptamers did not recognize Aβ40 oligomers but reacted with fibrils of Aβ40, Aβ42, and several other amyloidogenic proteins. Aptamer reactivity with amyloid fibrils showed some degree of protein-sequence dependency. Significant fibril binding also was found for the naïve library and could not be eliminated by counter-selection using Aβ40 fibrils, suggesting that aptamer binding to amyloid fibrils was RNA-sequence-independent. Aptamer binding depended on fibrillogenesis and showed a lag phase. Interestingly, aptamers detected fibril formation with ≥15-fold higher sensitivity than thioflavin T (ThT), revealing substantial β-sheet and fibril formation undetected by ThT. The data suggest that under physiologic conditions, aptamers for oligomeric forms of amyloidogenic proteins cannot be selected due to high, non-specific affinity of oligonucleotides for amyloid fibrils. Nevertheless, the high sensitivity, whereby aptamers detect β-sheet formation, suggests that they can serve as superior amyloid recognition tools.     

FAD-deficient P187S mutation of NAD(P)H:quinone oxidoreductase 1 (NQO1*2) binds and accelerates β-amyloid aggregation

Alzheimer’s disease (AD) is one of the most prominent neurodegenerative diseases. Results from animal and cellular models suggest that FAD-deficient forms of NAD(P)H quinone oxidoreductase 1 (NQO1) may accelerate the aggregation of Alzheimer’s amyloid-β peptide (Aβ_1-42). Here, we examined in vitro whether NQO1 and its FAD-deficient P187S mutation (NQO1*2) directly interact with Aβ_1-42 and modify its rate of aggregation. When monitored using the fluorescence of either noncovalent thioflavin T (ThT) or HiLyte Fluor 647 (HF647) dye covalently attached to the Aβ_1-42 peptide, the aggregation kinetics of Aβ_1-42 were markedly more rapid in the presence of NQO1*2 than the wild-type (WT) NQO1. Experiments using apo-NQO1 indicate that this increase is linked to the inability of NQO1*2 to bind to FAD. Furthermore, dicoumarol, an NQO1 inhibitor that binds near the FAD-binding site and stabilizes NQO1*2, markedly decreased the aggregation kinetics of Aβ_1-42. Imaging flow cytometry confirmed in-vitro coaggregation of NQO1 isoforms and Aβ_1-42. Aβ_1-42 alone forms rod-shaped fibril structures while in the presence of NQO1 isoforms, Aβ_1-42 is incorporated in the middle of larger globular protein aggregates surrounded by NQO1 molecules. Isothermal titration calorimetry (ITC) analysis indicates that Aβ_1-42 interacts with NQO1 isoforms with a specific stoichiometry through a hydrophobic interaction with positive enthalpy and entropy changes. These data define the kinetics, mechanism, and shape of coaggregates of Aβ_1-42 and NQO1 isoforms and the potential relevance of FAD-deficient forms of NQO1 for amyloid aggregation diseases.     

PAMAM G4 dendrimers affect the aggregation of α-synuclein

α-Synuclein (ASN) aggregation plays a key role in neurodegenerative disorders including Parkinson's disease, and inhibition of fibril formation is a potential therapeutic strategy for these conditions. The aim of the present study was to investigate polyamidoamine (PAMAM) dendrimers (generations 4 and 3.5) as inhibitors of fibril formation in vitro by examining their interaction with ASN intrinsic tyrosine fluorescence. Furthermore, the effect of dendrimers on ASN aggregation was studied using circular dichroism (CD) spectroscopy and CD studies were complemented by a fluorescence assays using the dye thioflavin T (ThT). The PAMAM G4 dendrimer caused an increase in tyrosine residue fluorescence, and inhibited fibrillation of ASN; inhibited fibrillation was not observed with PAMAM G3.5 dendrimers.     

l-Arginine reduces thioflavin T fluorescence but not fibrillation of bovine serum albumin

This work examines the effects of l-arginine (l-Arg) on the aggregation and amyloid fibrillation of bovine serum albumin (BSA). We demonstrate that l-Arg dose-dependently reduces thioflavin T (ThT) fluorescence of BSA within the l-Arg concentration range used (0–1.4 M). However, as revealed by electron microscopy, size exclusion chromatography, and dynamic light scattering results, l-Arg does not prevent amyloid-like fibril formation by BSA. We conclude that l-Arg competes against ThT for binding sites on BSA amyloid-like fibrils, leading to biased results in ThT fluorescence measurements. Moreover, the use of ThT fluorescence assay to screen for potential inhibitors against amyloid fibrillation can give misleading results.     

Study on the binding of Thioflavin T to beta-sheet-rich and non-beta-sheet cavities

Amyloid fibril formation plays a role in more than 20 diseases including Alzheimer's disease. In vitro detection of these fibrils is often performed using Thioflavin T (ThT), though the ThT binding mode is largely unknown. In the present study, spectral properties of ThT in binding environments representing beta-sheet-rich and non-beta-sheet cavities were examined. Acetylcholinesterase and gamma-cyclodextrin induced a characteristic ThT fluorescence similar to that with amyloid fibrils, whereas beta-cyclodextrin and the beta-sheet-rich transthyretin did not. The cavities of acetylcholinesterase and gamma-cyclodextrin were of similar diameter and only these cavities could accommodate two ThT ions according to molecular modelling. Binding stoichiometry studies also showed a possible binding of two ThT ions. Thus, the characteristic ThT fluorescence is induced in cavities with a diameter of 8-9A and a length able to accommodate the entire length of the ThT ion. The importance of a cavity diameter capable of binding two ThT ions, among others, indicates that an excimer formation is a plausible mechanism for the characteristic fluorescence. We propose a similar ThT binding mode in amyloid fibrils, where cavities of an appropriate size running parallel to the fibril axis have previously been proposed in several amyloid fibril models.     

Xanthene food dye, as a modulator of Alzheimer's disease amyloid-beta peptide aggregation and the associated impaired neuronal cell function

Background

Alzheimer''s disease (AD) is the most common form of dementia. AD is a degenerative brain disorder that causes problems with memory, thinking and behavior. It has been suggested that aggregation of amyloid-beta peptide (Aβ) is closely linked to the development of AD pathology. In the search for safe, effective modulators, we evaluated the modulating capabilities of erythrosine B (ER), a Food and Drug Administration (FDA)-approved red food dye, on Aβ aggregation and Aβ-associated impaired neuronal cell function.

Methodology/Principal Findings

In order to evaluate the modulating ability of ER on Aβ aggregation, we employed transmission electron microscopy (TEM), thioflavin T (ThT) fluorescence assay, and immunoassays using Aβ-specific antibodies. TEM images and ThT fluorescence of Aβ samples indicate that protofibrils are predominantly generated and persist for at least 3 days. The average length of the ER-induced protofibrils is inversely proportional to the concentration of ER above the stoichiometric concentration of Aβ monomers. Immunoassay results using Aβ-specific antibodies suggest that ER binds to the N-terminus of Aβ and inhibits amyloid fibril formation. In order to evaluate Aβ-associated toxicity we determined the reducing activity of SH-SY5Y neuroblastoma cells treated with Aβ aggregates formed in the absence or in the presence of ER. As the concentration of ER increased above the stoichiometric concentration of Aβ, cellular reducing activity increased and Aβ-associated reducing activity loss was negligible at 500 µM ER.

Conclusions/Significance

Our findings show that ER is a novel modulator of Aβ aggregation and reduces Aβ-associated impaired cell function. Our findings also suggest that xanthene dye can be a new type of small molecule modulator of Aβ aggregation. With demonstrated safety profiles and blood-brain permeability, ER represents a particularly attractive aggregation modulator for amyloidogenic proteins associated with neurodegenerative diseases.     

Simultaneous Measurement of Amyloid Fibril Formation by Dynamic Light Scattering and Fluorescence Reveals Complex Aggregation Kinetics

An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington''s disease, in vitro. Huntington''s disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer''s and Parkinson''s disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, β-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports β-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.     

On the binding of Thioflavin-T to HET-s amyloid fibrils assembled at pH 2

Amyloid fibrils are ordered β-sheet protein or peptide polymers. The benzothiazole dye Thioflavin-T (ThT) shows a strong increase in fluorescence upon binding to amyloid fibrils and has hence become the most commonly used amyloid-specific dye. In spite of this widespread use, the mechanism underlying specific binding and fluorescence enhancement upon interaction with amyloid fibrils remains largely unknown. Recent contradictory reports have proposed radically different modes of binding. We have studied the interaction of ThT with fibrils of the prion forming domain of the fungal HET-s prion protein assembled at pH 2 in order to try to gain some insight into the general mechanism of ThT-binding and fluorescence. We found that ThT does not bind to HET-s(218–289) fibrils as a micelle as previously proposed in the case of insulin fibrils. We have measured binding kinetics, affinity and stoichiometry at pH values above and below the pI of the HET-s(218–289) fibrils and found that binding is dramatically affected by pH and ionic strength. Binding is poor at acidic pH, presumably as a result of repulsive electrostatic interaction between the positively charged ThT molecule and the fibril surface. Finally, we found that ThT acquires chiral properties when it is fibril-bound. These results are discussed in relation to the different ThT-binding modes that have been proposed.     

Binding mode of Thioflavin T in insulin amyloid fibrils

Amyloid fibrils share various common structural features and their presence can be detected by Thioflavin T (ThT). In this paper, the binding mode of ThT to insulin amyloid fibrils was examined. Scatchard analysis and isothermal titration calorimetry (ITC) showed at least two binding site populations. The binding site population with the strongest binding was responsible for the characteristic ThT fluorescence. This binding had a capacity of about 0.1 moles of ThT bound per mole of insulin in fibril form. The binding capacity was unaffected by pH, but the affinity was lowest at low pH. Notably, presence of a third binding process prior to the other processes was suggested by ITC. Binding of ThT resulted in only minor changes in the fibril structure according to the X-ray diffraction patterns, where a slightly more dominant equatorial reflection at 16A relative to the intersheet distance of 11A was observed. No change in the interstrand distance of 4.8A was observed. On the basis of our results, we propose that ThT binds in cavities running parallel to the fibril axis, e.g., between the protofilaments forming the fibrils. Such cavities have been proposed previously in insulin fibrils and several other amyloid fibril models.     

Tyrosine Autofluorescence as a Measure of Bovine Insulin Fibrillation

The traditional approach to investigating the partial unfolding and fibrillation of insulin, and proteins at large, has involved use of the dyes 1-anilinonaphthalene-8-sulphonic acid (ANS) and Thioflavin T (ThT), respectively. We compare the kinetic profiles of ThT, ANS, light scattering, and intrinsic Tyr fluorescence during insulin fibrillation. The data reveal that the sequence of structural changes (dimers → monomers → partially unfolded monomers → oligomeric aggregates → fibrils) accompanying insulin fibrillation can be detected directly using intrinsic Tyr fluorescence. The results indicate that at least two distinguishable structural intermediates precede fibril development. There is no evidence of tyrosinate or dityrosine during insulin aggregation. Obtaining such critical information from the protein itself is complementary to existing aggregation probes and affords the advantage of directly examining structural changes that occur at the molecular level, providing concrete details of the early events preceding fibrillation.     

The anti-Parkinsonian drug selegiline delays the nucleation phase of α-synuclein aggregation leading to the formation of nontoxic species

Parkinson's disease (PD) is a movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of intraneuronal inclusions called Lewy bodies, which are composed mainly of α-synuclein (α-syn). Selegiline (Sel) is a noncompetitive monoamino oxidase B inhibitor that has neuroprotective effects and has been administered to PD patients as monotherapy or in combination with l-dopa. Besides its known effect of increasing the level of dopamine (DA) by monoamino oxidase B inhibition, Sel induces other effects that contribute to its action against PD. We evaluated the effects of Sel on the in vitro aggregation of A30P and wild-type α-syn. Sel delays fibril formation by extending the lag phase of aggregation. In the presence of Sel, electron microscopy reveals amorphous heterogeneous aggregates, including large annular species, which are innocuous to a primary culture enriched in dopaminergic neurons, while their age-matched counterparts are toxic. The inhibitory effect displayed by Sel is abolished when seeds (small fibril pieces) are added to the aggregation reaction, reinforcing the hypothesis that Sel interferes with early nuclei formation and, to a lesser extent, with fibril elongation. NMR experiments indicate that Sel does not interact with monomeric α-syn. Interestingly, when added in combination with DA (which favors the formation of toxic protofibrils), Sel overrides the inhibitory effect of DA and favors fibrillation. Additionally, Sel blocks the formation of smaller toxic aggregates by perturbing DA-dependent fibril disaggregation. These effects might be beneficial for PD patients, since the sequestration of protofibrils into fibrils or the inhibition of fibril dissociation could alleviate the toxic effects of protofibrils on dopaminergic neurons. In nondopaminergic neurons, Sel might slow the fibrillation, giving rise to the formation of large nontoxic aggregates.     

Manganese promotes α-synuclein amyloid aggregation through the induction of protein phase transition

α-Synuclein (α-Syn) is the major protein component of Lewy bodies, a key pathological feature of Parkinson’s disease (PD). The manganese ion Mn²⁺ has been identified as an environmental risk factor of PD. However, it remains unclear how Mn²⁺ regulates α-Syn aggregation. Here, we discovered that Mn²⁺accelerates α-Syn amyloid aggregation through the regulation of protein phase separation. We found that Mn²⁺ not only promotes α-Syn liquid-to-solid phase transition but also directly induces soluble α-Syn monomers to form solid-like condensates. Interestingly, the lipid membrane is integrated into condensates during Mn²⁺-induced α-Syn phase transition; however, the preformed Mn²⁺/α-syn condensates can only recruit lipids to the surface of condensates. In addition, this phase transition can largely facilitate α-Syn amyloid aggregation. Although the Mn²⁺-induced condensates do not fuse, our results demonstrated that they could recruit soluble α-Syn monomers into the existing condensates. Furthermore, we observed that a manganese chelator reverses Mn²⁺-induced α-Syn aggregation during the phase transition stage. However, after maturation, α-Syn aggregation becomes irreversible. These findings demonstrate that Mn²⁺ facilitates α-Syn phase transition to accelerate the formation of α-Syn aggregates and provide new insights for targeting α-Syn phase separation in PD treatment.     

The formation of amyloid-like fibrils of α-chymotrypsin in different aqueous organic solvents

The formation of amyloid-like fibrils of α-chymotrypsin was studied in aqueous ethanol, methanol, tertbutanol, dimethylformamide and acetonitrile. Thioflavin T (ThT), Congo red (CR) and 1-anilino-8-naphthalenesulfonic acid (ANS) binding, turbidity, intrinsic fluorescence and far-UV circular dichroism measurements were employed to characterize the amyloid fibril formation. The greatest extent of fibril formation after incubation for 24 h at pH 7.0 and at 24 °C was in ethanol at 55%, in methanol and dimethylformamide (DMF) at 60-70% and in tert-butanol at 60-80%. The ANS binding and intrinsic fluorescence results showed that the hydrophobic residues are more solvent-exposed in the aggregated form of α-chymotrypsin. The ThT, CR binding and far-UV CD measurements indicated that the formation of the cross-β structure of α-chymotrypsin depends on the polarity of the organic solvent. To determine the role of surface charges in the aggregation, chemically modified forms of α-chymotrypsin were prepared. The citraconylated and succinylated enzymes exhibited a higher and the enzyme forms modified with aliphatic aldehydes a lower propensity for aggregation. These results suggest the important role of surface charges in the aggregation of α-chymotrypsin.     

Investigation of the kinetics of insulin amyloid fibrils formation

Today, the investigation of the structure of ordered protein aggregates-amyloid fibrils, the influence of the native structure of the protein and the external conditions on the process of fibrillation-is the subject of intense investigations. The aim of the present work is to study the kinetics of formation of insulin amyloid fibrils at low pH values (conditions that are used at many stages of the isolation and purification of the protein) using the fluorescent probe thioflavin T. It is shown that the increase of the fluorescence intensity of ThT during the formation of amyloid fibrils is described by a sigmoidal curve, in which three areas can be distinguished: the lag phase, growth, and a plateau, which characterize the various stages of fibril formation. Despite the variation in the length of the lag phase at the same experimental conditions (pH and temperature), it is seen to drop during solution stirring and seeding. Data obtained by electron microscopy showed that the formed fibrils are long, linear filaments ～20 nm in diameter. With increasing incubation time, the fibril diameter does not change, while the length increases to 2–3 μm, which is accompanied by a significant increase in the number of fibril aggregates. All the experimental data show that, irrespective of the kinetics of formation of amyloid fibrils, their properties after the completion of the fibrillation process are identical. The results of this work, together with the previous studies of insulin amyloid fibrils, may be important for clarification the mechanism of their formation, as well as for the treatment of amyloidosis associated with the aggregation of insulin.     

Structural and mechanistic insights into modulation of α-Synuclein fibril formation by aloin and emodin

α-Synuclein (α-Syn) aggregation/fibrillation is a leading cause of neuronal death and is one of the major pathogenic factors involved in the progression of Parkinson's' disease (PD). Against this backdrop, discovering new molecules as inhibitors or modulators of α-Syn aggregation/fibrillation is a subject of enormous research. In this study, we have shown modulation, disaggregation, and neuroprotective potential of aloin and emodin against α-Syn aggregation/fibrillation. Thioflavin T (ThT) fluorescence assay showed an increase in lag phase from (51.14 ± 2) h to (68.58 ± 2) h and (74.14 ± 3) h in the presence of aloin and emodin respectively. ANS binding assay represents a modulatory effect of these molecules on hydrophobicity which is crucial for aggregates/fibril formation. NMR spectroscopy and tyrosine quenching studies reveal the binding of aloin/emodin with monomeric α-Syn. TEM and DLS micrographs illustrate the attenuating effect of aloin/emodin against the development of large aggregates/fibrils. Our seeding experiments suggest aloin/emodin generate seeding incompetent oligomers that direct the off-pathway aggregation/fibrillation. Also, aloin/emodin capably reduces the fibrils-induced cytotoxicity and disassembles the preexisting amyloid fibrils. These findings provide deep insight into the modulatory mechanism of α-Syn aggregation/fibrillation in the presence of aloin and emodin, thereby suggesting their potential roles as promising therapeutic molecules against aggregation/fibrillation related disorders.