Biophysical Comparison of Soluble Amyloid-β(1–42) Protofibrils, Oligomers, and Protofilaments

Abstract

Some of the pathological hallmarks of the Alzheimer's disease brain are senile plaques composed of insoluble amyloid-β protein (Aβ) fibrils. However, much of the recent emphasis in research has been on soluble Aβ aggregates in response to a growing body of evidence that shows that these species may be more neurotoxic than fibrils. Within this subset of soluble aggregated Aβ are protofibrils and oligomers. Although each species has been widely investigated separately, few studies have directly compared and contrasted their physical properties. In this work, we examined well-recognized preparations of Aβ(1-42) oligomers and protofibrils with multiangle (MALS) and dynamic (DLS) light scattering in line with, or following, size-exclusion chromatography (SEC). Multiple SEC-MALS analyses of protofibrils revealed molecular weight (Mw) gradients ranging from 200 to 2600 kDa. Oligomeric Aβ species are generally considered to be a smaller and more nascent than protofibrils. However, oligomer Mw values ranged from 225 to 3000 kDa, larger than that for protofibrils. Root-mean-square radius (Rg) values correlated with the Mw trends with protofibril Rg values ranging from 16 to 35 nm, while oligomers produced one population at 40-43 nm with a more disperse population from 22 to 39 nm. Hydrodynamic radius (RH) measurements by DLS and thioflavin T fluorescence measurements indicated that protofibrils and oligomers had commonalities, yet electron microscopy revealed morphological differences between the two. SEC-purified Aβ(1-42) monomer at lower concentrations was slower to nucleate but formed protofibrils (1500 kDa) or soluble protofilaments (3000 kDa) depending on the buffer type. The findings from these studies shed new light on the similarities and differences between distinct soluble aggregated Aβ species.

Reduced Lipid Bilayer Thickness Regulates the Aggregation and Cytotoxicity of Amyloid-β

“…It may also increase the likelihood of A␤ fibrils to fragment to compensate for the increased side chain flexibility, explaining the truncated fibril structures observed by TEM (61,62). These two modes of disruption would be relatively independent of the main chain interactions and result in the maintenance of the conventional ␤-strand structure, resulting in an aggregate species that resembles a protofibrillar structure (63,64).…”

Aβ40 has a subtle effect on Aβ42 protofibril formation, but to a lesser degree than Aβ42 concentration, in Aβ42/Aβ40 mixtures

“…We have previously detailed the preparation and characterization of the biophysical and proinflammatory properties of protofibrils rapidly formed by Aβ42 and to a slower extent by Aβ40 [44, 45, 49]. A variety of solution and microscopic techniques indicated that Aβ42 protofibrils were short, curvilinear species less than 100 nm in length, rich in β-sheet structure, yet relatively small and quite soluble (hydrodynamic radius, R H 20–25 nm and molecular weight, M w , 200–2600 kDa).…”

“…A solution of Aβ42 alone yielded a typical 2-peak elution profile on Superdex 75 (fractionation range 3–70 kD) with roughly equivalent amounts of protofibrils and monomers (Figure 1A). Our previous work showed that protofibrils were larger than 70 kD [49], so protofibrils eluted in the void volume. The calculated distribution was 56% protofibrils and 44% monomers (Figure 1B).…”

“…A total Aβ (Aβ42 + Aβ40) concentration of 40 μM was selected so that 0.5 mL (90 μg Aβ) could be loaded for in-line SEC-MALS analysis and post-elution evaluation of fractions. This amount was the realistic minimum for sample detection and analysis [49]. Aβ42 and Aβ40 monomers were isolated in aCSF pH 7.8 containing 30 mM NaCl concentration, rather than 130 mM to encourage soluble aggregate formation and discourage insoluble fibril formation [49].…”

Thiosemicarbazone modification of 3-acetyl coumarin inhibits Aβ peptide aggregation and protect against Aβ-induced cytotoxicity

“…35 We used HFIP/NaOH treatment for the preparation monomeric Aβ peptide. Briefly, lyophilized Aβ(1-42) peptide was dissolved in HFIP and stored at -80 °C.…”