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Amyloid β (Aβ) peptide accumulation in the brain is considered to be one of the hallmarks of Alzheimer's disease. Here, we compare two analytical techniques for detecting neurotoxic Aβ1-42 oligomers - Quartz Crystal Microbalance with Dissipation (QCM-D) and Single Molecule Array (Simoa). Both detection methods exploit a feature of the monoclonal antibody bapineuzumab, which targets N-terminal residues 1–5 of Aβ with high affinity and use it as both a capture and detection reagent. Assays developed with the two methods allow us to specifically recognize neurotoxic Aβ1-42 oligomers and higher aggregates such as fibrils but discriminate against Aβ1-42 monomer species. We find that for detection of Aβ1-42 oligomers, Simoa was roughly 500 times more sensitive than the QCM-D technique with limits of detection of 0.22 nM and 125 nM, respectively.

The histopathological hallmark of Alzheimer's disease (AD) is the aggregation and accumulation of the amyloid beta peptide (Aβ) into misfolded oligomers and fibrils. Here we examine the biophysical properties of a protective Aβ variant against AD, A2T, and a causative mutation, A2T, along with the wild type (WT) peptide. The main finding here is that the A2V native monomer is more stable than both A2T and WT, and this manifests itself in different biophysical behaviors: the kinetics of aggregation, the initial monomer conversion to an aggregation prone state (primary nucleation), the abundances of oligomers, and extended conformations. Aggregation reaction modeling of the conversion kinetics from native monomers to fibrils predicts the enhanced stability of the A2V monomer, while ion mobility spectrometry-mass spectrometry measures this directly confirming earlier predictions. Additionally, unique morphologies of the A2T aggregates are observed using atomic force microscopy, providing a basis for the reduction in long term potentiation inhibition of hippocampal cells for A2T compared with A2V and the wild type (WT) peptide. The stability difference of the A2V monomer and the difference in aggregate morphology for A2T (both compared with WT) are offered as alternate explanations for their pathological effects.

The discoveries that non-native proteins have a role in amyloidosis and that multiple protein misfolding diseases can occur concurrently suggest that cross-seeding of amyloidogenic proteins may be central to misfolding. To study this process, a synthetic chimeric amyloidogenic protein (YEHK21-YE8) composed of two components, one that readily folds to form fibrils (YEHK21) and one that does not (YE8), was designed. Secondary structural conformational changes during YEHK21-YE8 aggregation demonstrate that, under the appropriate conditions, YEHK21 is able to induce fibril formation of YE8. The unambiguous demonstration of the induction of folding and fibrillation within a single molecule illuminates the factors controlling this process and hence suggests the importance of those factors in amyloidogenic diseases.

In vitro fibril formation typically exhibits a lag phase followed by a rapid elongation phase. Soluble prefibrilar oligomers form as multiple assembly states occur during the lag phase and, after forming a nucleus, rapidly propagate into amyloid aggregates and fibrils. The structure and morphology of amyloid fibrils have been extensively characterized over the last decades, while little is known about the structural organization of the prefibrilar oligomers or their multiple assembly states. The main difficulty in structural characterization of prefibrilar aggregates is their low concentration (pmolar) and their continual reactive conversion. Herein we overcome these difficulties by utilizing Surface-Enhanced Raman Spectroscopy (SERS) with a model amyloid peptide, insulin. SERS is a powerful analytic tool that is able to provide detection of small molecules down to a single-molecule level. Using SERS we found that during the 3 lag phase before the onset of insulin fibril formation, the amount of insulin oligomers increased more than twice after the first hour of incubation under fibrillation conditions (pH 1.6, 65°C) and then slowly decreased with time. The latter finding is kinetically linked to the conversion of the prefibrilar oligomers into fibril species. This study provides valuable new information about the time-dependent structural organization of insulin oligomers and demonstrates the power and potential of SERS for detection and structural characterization of biological specimens present at low concentrations.

The antiallergy and potential anticancer drug tranilast has been patented for treating Alzheimer's disease (AD), in which amyloid β-protein (Aβ) plays a key pathogenic role. We used solution NMR to determine that tranilast binds to Aβ40 monomers with ∼300 μM affinity. Remarkably, tranilast increases Aβ40 fibrillation more than 20-fold in the thioflavin T assay at a 1:1 molar ratio, as well as significantly reducing the lag time. Tranilast likely promotes fibrillation by shifting Aβ monomer conformations to those capable of seed formation and fibril elongation. Molecular docking results qualitatively agree with NMR chemical shift perturbation, which together indicate that hydrophobic interactions are the major driving force of the Aβ-tranilast interaction. These data suggest that AD may be a potential complication for tranilast usage in elderly patients.

The difficulty in identifying the toxic agents in all amyloid-related diseases is likely due to the complicated kinetics and thermodynamics of the nucleation process and subsequent fibril formation. The slow progression of these diseases suggests that the formation, incorporation, and/or action of toxic agents are possibly rate limiting. Candidate toxic agents include precursors (some at very low concentrations), also called oligomers and protofibrils, and the fibrils. Here, we investigate the kinetic and thermodynamic behavior of human insulin oligomers (imaged by cryo-EM) under fibril-forming conditions (pH 1.6 and 65 degrees C) by probing the reaction pathway to insulin fibril formation using two different types of experiments-cooling and seeding-and confirm the validity of the nucleation model and its effect on fibril growth. The results from both the cooling and seeding studies confirm the existence of a time-changing oligomer reaction process prior to fibril formation that likely involves a rate-limiting nucleation process followed by structural rearrangements of intermediates (into beta-sheet rich entities) to form oligomers that then form fibrils. The latter structural rearrangement step occurs even in the absence of nuclei (i.e., with added heterologous seeds). Nuclei are formed at the fibrillation conditions (pH 1.6 and 65 degrees C) but are also continuously formed during cooling at pH 1.6 and 25 degrees C. Within the time-scale of the experiments, only after increasing the temperature to 65 degrees C are the trapped insulin nuclei and resultant structures able to induce the structural rearrangement step and overcome the energy barrier to form fibrils. This delay in fibrillation and accumulation of nuclei at low temperature (25 degrees C) result in a decrease in the mean length of the fibers when placed at 65 degrees C. Fits of an empirical model to the data provide quantitative measures of the delay in the lag-time during the nucleation process and subsequent reduction in fibril growth rate resulting from the cooling. Also, the seeding experiments, within the time-scale of the measurements, demonstrate that fibers can initiate fast fibrillation with dissolved insulin (fresh or taken during the lag-period) but not with other fibers. Qualitatively this is explained with a conjectual free-energy space plot.

The role and mechanism of amyloid proteins in neurodegenerative diseases remains an unsolved problem. Recent findings have shown that low-molecular-weight oligomers, generated during the early stage of the nucleation process, are the most neurotoxic species – for example, Aβ peptide oligomers for Alzheimer’s disease. To address this challenge, researchers have attempted to isolate, identify, quantify and characterize these small oligomeric species using a series of characterization and high-resolution separation methods. Here we present an overview of the progress reported during the past decade regarding oligomers of a model amyloid protein, insulin. Although the behavior and properties of these oligomers during conversion from the folded insulin monomer through oligomers to fibers is not widely accepted, several conclusions are possible. Insulin proceeds to aggregate in vitro under harsh conditions (e.g. pH 1.6, 65°C) along a universal path independent of kinetics, from monomers to dimers to larger entities, such as nuclei comprising hexamers, and then to fibrils. Amyloidogenic oligomers occur in very low concentrations and, in some cases, at undetectable levels. Their aggregation is strongly influenced by different initial conditions. None of the techniques reviewed here was able to provide a detailed and complete description of the oligomeric species present because each technique suffered from intrinsic limitations. In summary, isolation and purification of these ephemeral oligomer species has proved to be very difficult due to their low number and strong inherent tendency to aggregate under increased shear, ionic strength, temperature and hydrogen ion concentration.

Amyloid diseases, including Alzheimer's and Parkinson's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, beta-sheet rich fibrils. There are many theories on the species that causes toxicity in amyloid diseases, mainly focused on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, which makes their full characterization difficult. We have isolated a toxic protein species that acts like an oligomer and may provide evidence of a stable oligomer. This oligomer was isolated by dissolving amyloid fibrils at high pH, then purified and concentrated by diafiltration. It has a mass greater than 100 kDa and a diameter ranging of 48 ± 15 nm. The oligomer seeds the formation of fibrils in a dose dependent manner and exhibits Thioflavin-T fluorescence, but circular dichroism and deep UV resonance Raman spectroscopy did not find any evidence of an increase in beta-sheet structure. It appears that this oligomer is largely unstructured protein. We hypothesize that the oligomer does not decompose at high pH and maintains its structure in solution. All of the insulin, however, that joined the oligomer to elongate the beta-sheet rich fibrils folded in a different conformation and could be removed from the fibril and returned to native, dissolved insulin. This is the first time that a stable oligomer of an amyloid reaction has been separated and characterized without genetically engineering the protein or having additives in the fibrillation media. It appears that the oligomer does not have the same structure as the fibrils and is possibly intrinsically unfolded. This may make the search for a stable amyloid oligomer or nucleus even more difficult since methods to detect beta-sheets are often employed in the search for these structures.

Maghemite (γ-Fe2O3) magnetic nanoparticles of 15.0 ± 2.1 nm were formed by nucleation followed by controlled growth of maghemite thin films on gelatin-iron oxide nuclei. Human insulin amyloid fibrils were formed by incubating the monomeric insulin dissolved in aqueous continuous phase at pH 1.6 and 65°C. Magnetic human insulin amyloid fibrils/γ-Fe2O3 nanoparticle assemblies were prepared by interacting the γ-Fe2O3 nanoparticles with the insulin amyloid fibrils during or after their formation. The nanoparticles attached selectively to the insulin fibrils in both cases. The kinetics of the insulin fibrillation process in the absence and the presence of the γ-Fe2O3 nanoparticles was elucidated. The insulin amyloid fibrils/γ-Fe2O3 nanoparticle assemblies were easily extracted from the aqueous phase via a magnetic field. We hypothesize that this selective extraction approach may also be applicable for the removal of other amyloidogenic proteins that lead to neurodegenerative diseases (e.g., Alzheimer's, Parkinson's, Huntington's, mad cow, and prion diseases) from their continuous phase, e.g. milk, blood, neurological fluid, etc. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009

To help identify the etiological agents for amyloid-related diseases, attention is focused here on the fibrillar precursors, also called oligomers and protofibrils, and on modeling the reaction kinetics of the formation of the amyloid nucleus. Insulin is a favored model for amyloid formation, not only because amyloidosis can be a problem in diabetes, but also because aggregation and fibrillation causes problems during production, storage, and delivery. Small angle neutron scattering (SANS) is used to measure the temporal formation of insulin oligomers in H2O- and D2O-based solvents and obtain consistent evidence of the composition of the insulin nucleus that comprised three dimers or six monomers similar to that recently proposed in the literature. A simple molecular structural model that describes the growth of oligomers under a wide range of environmental conditions is proposed. The model first involves lengthening or end-on-end association of dimers to form three-dimer nuclei, and then exhibits broadening or side-on-side association of nuclei. Using different additives to demonstrate their influence on the kinetics of oligomer formation, we showed that, although the time required to form the nucleus was dependent on a specific system, they all followed a universal pathway for nucleus and precursor formation. The methods and analyses presented here provide the first experimental molecular size description of the details of amyloid nucleus formation and subsequent propagation to fibril precursors independent of kinetics. Proteins 2009. © 2008 Wiley-Liss, Inc.

BACKGROUND: In spite of our extensive knowledge of the more than 20 proteins associated with different amyloid diseases, we do not know how amyloid toxicity occurs or how to block its action. Recent contradictory reports suggest that the fibrils and/or the oligomer precursors cause toxicity. An estimate of their temporal concentration may broaden understanding of the amyloid aggregation process. METHODOLOGY/PRINCIPAL FINDINGS: Assuming that conversion of folded protein to fibril is initiated by a nucleation event, we back-calculate the distribution of nuclei concentration. The temporal in vitro concentration of nuclei for the model hormone, recombinant human insulin, is estimated to be in the picomolar range. This is a conservative estimate since the back-calculation method is likely to overestimate the nuclei concentration because it does not take into consideration fibril fragmentation, which would lower the amount of nuclei CONCLUSIONS: Because of their propensity to form aggregates (non-ordered) and fibrils (ordered), this very low concentration could explain the difficulty in isolating and blocking oligomers or nuclei toxicity and the long onset time for amyloid diseases.

Amyloid diseases, including Alzheimer's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, β-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a β-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils.

The molecular rearrangement of soluble proteins into fibers is a common attribute of amyloid diseases: Alzheimer's disease, Parkinson's disease, spongiform encephalopathies (including mad cow disease), and other prion diseases. In the past century considerable progress has been made in characterizing amyloid diseases, but the connection between amyloidosis and the disease is still unclear: Contradictory reports suggest that the fibrils and/or the oligomer precursors cause toxicity. The debate is still open.Here, we offer the first attempt to “reverse-estimate” the concentrations of nuclei, starting from a distribution of fibril lengths. Assuming the nucleation model is valid, with a few reasonable assumptions, a fibril length distribution and a set of seeding experiments, we estimated the in vitro concentration of nuclei for the model hormone, recombinant human insulin, to be in the picomolar range. Fibril lengths are measured with an atomic force microscope and seeding shows that fibrils of different lengths exhibit similar growth rates. Because of their propensity to form aggregates (non-ordered) and fibrils (ordered), this very low concentration could explain the difficulty in fractionating, isolating and blocking nuclei toxicity. Moreover, this theoretical approach, based on our measurements and a structural fibril model recently published by David Eisenberg's group at UCLA, is general and could be used for other amyloid proteins.