Aß40 has a subtle effect on Aß42 protofibril formation, but to a lesser degree than Aß42 concentration, in Aß42/Aß40 mixtures.

Recent findings suggest that the senile plaques in Alzheimer's disease may contain soluble amyloid-ß peptide (Aß) fibril precursors along with insoluble fibrils. These soluble Aß species, including oligomers and protofibrils, have been well-studied in vitro and are formed via non-covalent self-assembly of Aß monomers. While both 40- and 42-residue forms of Aß are observed in the human body, the majority of the Aß aggregation work has been conducted on Aß42 or Aß40 separately, with relatively few investigations of mixtures. In order to study the effect of different combinations of Aß40 and Aß42 on protofibril formation, mixtures of either dry solid peptide, or purified Aß40 and Aß42 monomer solutions were mixed together and protofibril/monomer distributions were quantified. Increases in the Aß42/Aß40 ratio increased protofibril formation but the presence of Aß40 in the mixed Aß solutions had a significant negative impact on protofibril formation compared to equivalent solutions of pure Aß42. Protofibril size was less affected, but ß-sheet structure increased with protofibrils formed from higher Aß42/Aß40 ratio solutions. Direct measurement of Aß42/Aß40 ratios by C-terminal-selective ELISA found very little Aß40 incorporated into protofibrils. The cumulative data emphasizes the critical importance of Aß42, yet establishes Aß40 as a regulator of Aß42 aggregation.

Biophysical comparison of soluble amyloid-ß(1-42) protofibrils, oligomers, and protofilaments.

Some of the pathological hallmarks of the Alzheimer's disease brain are senile plaques composed of insoluble amyloid-ß protein (Aß) fibrils. However, much of the recent emphasis in research has been on soluble Aß 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 Aß 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 Aß(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 Aß 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 Aß(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 Aß species.

Amyloid-ß(1-42) protofibrils stimulate a quantum of secreted IL-1ß despite significant intracellular IL-1ß accumulation in microglia.

Neuroinflammation is a characteristic feature of the Alzheimer's disease (AD) brain. Significant inflammatory markers such as activated microglia and cytokines can be found surrounding the extracellular senile plaques predominantly composed of amyloid-ß protein (Aß). Several innate immune pathways, including Toll-like receptors (TLRs) and the NLRP3 inflammasome, have been implicated in AD inflammation. Aß plays a primary role in activating these pathways which likely contributes to the progressive neurodegeneration in AD. In order to better understand the complexities of this interaction we investigated the inflammatory response of primary microglia to Aß(1-42) protofibrils. Aß(1-42) protofibrils triggered a time- and MyD88-dependent process that produced tumor necrosis factor alpha (TNFα) and interleukin-1ß (IL-1ß) mRNA, and intracellular pro and mature forms of IL-1ß protein. The accumulation of both IL-1ß forms indicated that Aß(1-42) protofibrils were able to prime and activate the NLRP3 inflammasome. Surprisingly, Aß-induced accumulation of intracellular mature IL-1ß did not translate into greater IL-1ß secretion. Instead, we found that Aß elicited a quantized burst of secreted IL-1ß and this process occurred even prior to Aß priming of the microglia suggesting a basal level of either pro or mature IL-1ß in the cultured primary microglia. The IL-1ß secretion burst was rapid but not sustained, yet could be re-evoked with additional Aß stimulation. The findings from this study demonstrated multiple sites of IL-1ß regulation by Aß(1-42) protofibrils including TLR/MyD88-mediated priming, NLRP3 inflammasome activation, and modulation of the IL-1ß secretory process. These results underscore the wide-ranging effects of Aß on the innate immune response.