Unraveling the Early Events of Amyloid-β Protein (Aβ) Aggregation: Techniques for the Determination of Aβ Aggregate Size

Abstract

The aggregation of proteins into insoluble amyloid fibrils coincides with the onset of numerous diseases. An array of techniques is available to study the different stages of the amyloid aggregation process. Recently, emphasis has been placed upon the analysis of oligomeric amyloid species, which have been hypothesized to play a key role in disease progression. This paper reviews techniques utilized to study aggregation of the amyloid-β protein (Aβ) associated with Alzheimer’s disease. In particular, the review focuses on techniques that provide information about the size or quantity of oligomeric Aβ species formed during the early stages of aggregation, including native-PAGE, SDS-PAGE, Western blotting, capillary electrophoresis, mass spectrometry, fluorescence correlation spectroscopy, light scattering, size exclusion chromatography, centrifugation, enzyme-linked immunosorbent assay, and dot blotting.

Simultaneous measurement of a range of particle sizes during Aβ1–42 fibrillogenesis quantified using fluorescence correlation spectroscopy

“…Despite intense efforts there are currently no validated, reliable and sensitive means to detect Ab oligomers in CSF. The oligomerization and subsequent formation of protein fibrils of Ab is a complex interplay of many peptides and mechanisms and is not yet fully understood [16][17][18][19][20]. However, the majority of studies find that the mechanism for fibrillogenesis is consistent with a nucleation-dependent polymerization model [5,18,21] as illustrated in Fig.…”

“…Variability in the time before onset of oligomer formation can depend on preparation conditions [16], and/or additional stresses applied to the sample to speed up the process [25,[34][35][36]. Measurements were taken systematically over 48 h using consistent preparation methods to minimize these effects.…”

“…Few techniques can measure and track the components of such a heterogenous mixture of different sized species simultaneously. Several methods have been used to map the progression of peptide aggregation, from monomer to fibril, including, high performance liquid chromatography, gel electrophoresis, atomic force microscopy, transmission electron microscopy (TEM), Thioflavin T assays, dynamic light scattering and others [16]. Monomeric Ab has a hydrodynamic radius (R h ) of 0.9 ± 0.1 nm [23], while oligomeric and fibril forms range in size from several nanometers to several microns [16][17][18][19].…”

Opportunities for Conformation-Selective Antibodies in Amyloid-Related Diseases

“…Different forms of soluble Aβ have often been ambiguous, with overlapping definitions based on the method of detection (e.g., biochemical or immunohistochemical analysis) [46]. Soluble Aβ oligomers often exhibit profound variability in secondary structure comprising a wide range of conformations ranging from amorphous aggregates, micelles, protofibrils, prefibrillar aggregates, amyloid beta-derived diffusible ligands (ADDLs), Aβ*56, globulomers, amylospheroids, "tAβ" (toxic soluble Aβ), "paranuclei," to annular protofibrils [18,19,[45][46][47][48].…”

“…Different forms of soluble Aβ have often been ambiguous, with overlapping definitions based on the method of detection (e.g., biochemical or immunohistochemical analysis) [46]. Soluble Aβ oligomers often exhibit profound variability in secondary structure comprising a wide range of conformations ranging from amorphous aggregates, micelles, protofibrils, prefibrillar aggregates, amyloid beta-derived diffusible ligands (ADDLs), Aβ*56, globulomers, amylospheroids, "tAβ" (toxic soluble Aβ), "paranuclei," to annular protofibrils [18,19,[45][46][47][48]. It is therefore critical to identify forms closely associated with the pathology of AD, and conformational antibodies and antibody fragments can serve as tools for investigating these Aβ states, and their dynamics as well as offering potential for immunotherapy [49][50][51][52][53][54][55][56][57][58][59][60][61] (see Figure 2 and Table1).…”