Heterogeneity and Turnover of Intermediates during Amyloid‑β (Aβ) Peptide Aggregation Studied by Fluorescence Correlation Spectroscopy

Self-assembly of amyloid β (Aβ) peptide molecules into large aggregates is a naturally occurring process driven in aqueous solution by a dynamic interplay between hydrophobic interactions among Aβ molecules, which promote aggregation, and steric and overall electrostatic hindrance, which stifles it. Aβ self-association is entropically unfavorable, as it implies order increase in the system, but under favorable kinetic conditions, the process proceeds at appreciable rates, yielding Aβ aggregates of different sizes and structures. Despite the great relevance and extensive research efforts, detailed kinetic mechanisms underlying Aβ aggregation remain only partially understood. In this study, fluorescence correlation spectroscopy (FCS) and Thioflavin T (ThT) were used to monitor the time dependent growth of structured aggregates and characterize multiple components during the aggregation of Aβ peptides in a heterogeneous aqueous solution. To this aim, we collected data during a relatively large number of observation periods, 30 consecutive measurements lasting 10 s each, at what we consider to be a constant time point in the slow aggregation process. This approach enabled monitoring the formation of nanomolar concentrations of structured amyloid aggregates and demonstrated the changing distribution of amyloid aggregate sizes throughout the aggregation process. We identified aggregates of different sizes with molecular weight from 260 to more than 1 × 10⁶ kDa and revealed the hitherto unobserved kinetic turnover of intermediates during Aβ aggregation. The effect of different Aβ concentrations, Aβ:ThT ratios, differences between the 40 (Aβ40) and 42 (Aβ42) residue long variants of Aβ, and the effect of stirring were also examined

Ionic Strength Modulation of the Free Energy Landscape of Aβ₄₀ Peptide Fibril Formation

Protein misfolding and formation of cross-β structured amyloid fibrils are linked to many neurodegenerative disorders. Although recently developed quantitative approaches have started to reveal the molecular nature of self-assembly and fibril formation of proteins and peptides, it is yet unclear how these self-organization events are precisely modulated by microenvironmental factors, which are known to strongly affect the macroscopic aggregation properties. Here, we characterize the explicit effect of ionic strength on the microscopic aggregation rates of amyloid β peptide (Aβ40) self-association, implicated in Alzheimer’s disease. We found that physiological ionic strength accelerates Aβ40 aggregation kinetics by promoting surface-catalyzed secondary nucleation reactions. This promoted catalytic effect can be assigned to shielding of electrostatic repulsion between monomers on the fibril surface or between the fibril surface itself and monomeric peptides. Furthermore, we observe the formation of two different β-structured states with similar but distinct spectroscopic features, which can be assigned to an off-pathway immature state (F_β*) and a mature stable state (F_β), where salt favors formation of the F_β fibril morphology. Addition of salt to preformed F_β* accelerates transition to F_β, underlining the dynamic nature of Aβ40 fibrils in solution. On the basis of these results we suggest a model where salt decreases the free-energy barrier for Aβ40 folding to the F_β state, favoring the buildup of the mature fibril morphology while omitting competing, energetically less favorable structural states

Hydrophobicity and Conformational Change as Mechanistic Determinants for Nonspecific Modulators of Amyloid β Self-Assembly

The link between many neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases, and the aberrant folding and aggregation of proteins has prompted a comprehensive search for small organic molecules that have the potential to inhibit such processes. Although many compounds have been reported to affect the formation of amyloid fibrils and/or other types of protein aggregates, the mechanisms by which they act are not well understood. A large number of compounds appear to act in a nonspecific way affecting several different amyloidogenic proteins. We describe here a detailed study of the mechanism of action of one representative compound, lacmoid, in the context of the inhibition of the aggregation of the amyloid β-peptide (Aβ) associated with Alzheimer’s disease. We show that lacmoid binds Aβ(1–40) in a surfactant-like manner and counteracts the formation of all types of Aβ(1–40) and Aβ(1–42) aggregates. On the basis of these and previous findings, we are able to rationalize the molecular mechanisms of action of nonspecific modulators of protein self-assembly in terms of hydrophobic attraction and the conformational preferences of the polypeptide

Alzheimer Peptides Aggregate into Transient Nanoglobules That Nucleate Fibrils

Protein/peptide oligomerization, cross-β strand fibrillation, and amyloid deposition play a critical role in many diseases, but despite extensive biophysical characterization, the structural and dynamic details of oligomerization and fibrillation of amyloidic peptides/proteins remain to be fully clarified. Here, we simultaneously monitored the atomic, molecular, and mesoscopic states of aggregating Alzheimer’s amyloid β (Aβ) peptides over time, using a slow aggregation protocol and a fast aggregation protocol, and determined the cytotoxicity of the intermediate states. We show that in the early stage of fast fibrillation (the lag phase) the Aβ peptides coalesced into apparently unstructured globules (15–200 nm in diameter), which slowly grew larger. Then a sharp transition occurred, characterized by the first appearance of single fibrillar structures of approximately ≥100 nm. These fibrils emerged from the globules. Simultaneously, an increase was observed for the cross-β strand conformation that is characteristic of the fibrils that constitute mature amyloid. The number and size of single fibrils rapidly increased. Eventually, the fibrils coalesced into mature amyloid. Samples from the early lag phase of slow fibrillation conditions were especially toxic to cells, and this toxicity sharply decreased when fibrils formed and matured into amyloid. Our results suggest that the formation of fibrils may protect cells by reducing the toxic structures that appear in the early lag phase of fibrillation

Endogenous Polyamines Reduce the Toxicity of Soluble Aβ Peptide Aggregates Associated with Alzheimer’s Disease

Polyamines promote the formation of the Aβ peptide amyloid fibers that are a hallmark of Alzheimer’s disease. Here we show that polyamines interact with nonaggregated Aβ peptides, thereby reducing the peptide’s hydrophobic surface. We characterized the associated conformational change through NMR titrations and molecular dynamics simulations. We found that even low concentrations of spermine, sperimidine, and putrescine fully protected SH-SY5Y (a neuronal cell model) against the most toxic conformational species of Aβ, even at an Aβ oligomer concentration that would otherwise kill half of the cells or even more. These observations lead us to conclude that polyamines interfere with the more toxic prefibrillar conformations and might protect cells by promoting the structural transition of Aβ toward its less toxic fibrillar state that we reported previously. Since polyamines are present in brain fluid at the concentrations where we observed all these effects, their activity needs to be taken into account in understanding the molecular processes related to the development of Alzheimer’s disease

The Manganese Ion of the Heterodinuclear Mn/Fe Cofactor in <i>Chlamydia trachomatis</i> Ribonucleotide Reductase R2c Is Located at Metal Position 1

The essential catalytic radical of Class-I ribonucleotide reductase is generated and delivered by protein R2, carrying a dinuclear metal cofactor. A new R2 subclass, R2c, prototyped by the <i>Chlamydia trachomatis</i> protein was recently discovered. This protein carries an oxygen-activating heterodinuclear Mn­(II)/Fe­(II) metal cofactor and generates a radical-equivalent Mn­(IV)/Fe­(III) oxidation state of the metal site, as opposed to the tyrosyl radical generated by other R2 subclasses. The metal arrangement of the heterodinuclear cofactor remains unknown. Is the metal positioning specific, and if so, where is which ion located? Here we use X-ray crystallography with anomalous scattering to show that the metal arrangement of this cofactor is specific with the manganese ion occupying metal position 1. This is the position proximal to the tyrosyl radical site in other R2 proteins and consistent with the assumption that the high-valent Mn­(IV) species functions as a direct substitute for the tyrosyl radical

Specific Binding of a β-Cyclodextrin Dimer to the Amyloid β Peptide Modulates the Peptide Aggregation Process

Alzheimer’s disease involves progressive neuronal loss. Linked to the disease is the amyloid β (Aβ) peptide, a 38–43-amino acid peptide found in extracellular amyloid plaques in the brain. Cyclodextrins are nontoxic, cone-shaped oligosaccharides with a hydrophilic exterior and a hydrophobic cavity making them suitable hosts for aromatic guest molecules in water. β-Cyclodextrin consists of seven α-d-glucopyranoside units and has been shown to reduce the level of fibrillation and neurotoxicity of Aβ. We have studied the interaction between Aβ and a β-cyclodextrin dimer, consisting of two β-cyclodextrin monomers connected by a flexible linker. The β-cyclodextrin monomer has been found to interact with Aβ(1–40) at sites Y10, F19, and/or F20 with a dissociation constant (<i>K</i>_D) of 3.9 ± 2.0 mM. Here ¹H–¹⁵N and ¹H–¹³C heteronuclear single-quantum correlation nuclear magnetic resonance (NMR) spectra show that in addition, the β-cyclodextrin monomer and dimer bind to the histidines. NMR translational diffusion experiments reveal the increased affinity of the β-cyclodextrin dimer (apparent <i>K</i>_D of 1.1 ± 0.5 mM) for Aβ(1–40) compared to that of the β-cyclodextrin monomer. Kinetic aggregation experiments based on thioflavin T fluorescence indicate that the dimer at 0.05–5 mM decreases the lag time of Aβ aggregation, while a concentration of 10 mM increases the lag time. The β-cyclodextrin monomer at a high concentration decreases the lag time of the aggregation. We conclude that cyclodextrin monomers and dimers have specific, modulating effects on the Aβ(1–40) aggregation process. Transmission electron microscopy shows that the regular fibrillar aggregates formed by Aβ(1–40) alone are replaced by a major fraction of amorphous aggregates in the presence of the β-cyclodextrin dimer

In Vitro and Mechanistic Studies of an Antiamyloidogenic Self-Assembled Cyclic d,l‑α-Peptide Architecture

Misfolding of the Aβ protein and its subsequent aggregation into toxic oligomers are related to Alzheimer’s disease. Although peptides of various sequences can self-assemble into amyloid structures, these structures share common three-dimensional features that may promote their cross-reaction. Given the significant similarities between amyloids and the architecture of self-assembled cyclic d,l-α-peptide, we hypothesized that the latter may bind and stabilize a nontoxic form of Aβ, thereby preventing its aggregation into toxic forms. By screening a focused library of six-residue cyclic d,l-α-peptides and optimizing the activity of a lead peptide, we found one cyclic d,l-α-peptide (<b>CP-2</b>) that interacts strongly with Aβ and inhibits its aggregation. In transmission electron microscopy, optimized thioflavin T and cell survival assays, <b>CP-2</b> inhibits the formation of Aβ aggregates, entirely disassembles preformed aggregated and fibrillar Aβ, and protects rat pheochromocytoma PC12 cells from Aβ toxicity, without inducing any toxicity by itself. Using various immunoassays, circular dichroism spectroscopy, photoinduced cross-linking of unmodified proteins (PICUP) combined with SDS/PAGE, and NMR, we probed the mechanisms underlying <b>CP-2</b>’s antiamyloidogenic activity. NMR spectroscopy indicates that <b>CP-2</b> interacts with Aβ through its self-assembled conformation and induces weak secondary structure in Aβ. Upon coincubation, <b>CP-2</b> changes the aggregation pathway of Aβ and alters its oligomer distribution by stabilizing small oligomers (1–3 mers). Our results support studies suggesting that toxic early oligomeric states of Aβ may be composed of antiparallel β-peptide structures and that the interaction of Aβ with <b>CP-2</b> promotes formation of more benign parallel β-structures. Further studies will show whether these kinds of abiotic cyclic d,l-α-peptides are also beneficial as an intervention in related in vivo models

The Neuronal Tau Protein Blocks <i>in Vitro</i> Fibrillation of the Amyloid‑β (Aβ) Peptide at the Oligomeric Stage

In Alzheimer’s disease, amyloid-β (Aβ) plaques and tau neurofibrillary tangles are the two pathological hallmarks. The co-occurrence and combined reciprocal pathological effects of Aβ and tau protein aggregation have been observed in animal models of the disease. However, the molecular mechanism of their interaction remain unknown. Using a variety of biophysical measurements, we here show that the native full-length tau protein solubilizes the Aβ₄₀ peptide and prevents its fibrillation. The tau protein delays the amyloid fibrillation of the Aβ₄₀ peptide at substoichiometric ratios, showing different binding affinities toward the different stages of the aggregated Aβ₄₀ peptides. The Aβ monomer structure remains random coil in the presence of tau, as observed by nuclear magnetic resonance (NMR), circular dichroism (CD) spectroscopy and photoinduced cross-linking methods. We propose a potential interaction mechanism for the influence of tau on Aβ fibrillation

Cellular Polyamines Promote Amyloid-Beta (Aβ) Peptide Fibrillation and Modulate the Aggregation Pathways

The cellular polyamines spermine, spermidine, and their metabolic precursor putrescine, have long been associated with cell-growth, tumor-related gene regulations, and Alzheimer’s disease. Here, we show by in vitro spectroscopy and AFM imaging, that these molecules promote aggregation of amyloid-beta (Aβ) peptides into fibrils and modulate the aggregation pathways. NMR measurements showed that the three polyamines share a similar binding mode to monomeric Aβ(1–40) peptide. Kinetic ThT studies showed that already very low polyamine concentrations promote amyloid formation: addition of 10 μM spermine (normal intracellular concentration is ∼1 mM) significantly decreased the lag and transition times of the aggregation process. Spermidine and putrescine additions yielded similar but weaker effects. CD measurements demonstrated that the three polyamines induce different aggregation pathways, involving different forms of induced secondary structure. This is supported by AFM images showing that the three polyamines induce Aβ(1–40) aggregates with different morphologies. The results reinforce the notion that designing suitable ligands which modulate the aggregation of Aβ peptides toward minimally toxic pathways may be a possible therapeutic strategy for Alzheimer’s disease