Exploring the Aβ1-42 fibrillogenesis timeline by atomic force microscopy and surface enhanced Raman spectroscopy

Abstract

Introduction: Alzheimer's disease (AD) is a progressive debilitating neurological disorder representing the most common neurodegenerative disease worldwide. Although the exact pathogenic mechanisms of AD remain unresolved, the presence of extracellular amyloid-β peptide 1-42 (Aβ_1-42) plaques in the parenchymal and cortical brain is considered one of the hallmarks of the disease. Methods: In this work, we investigated the Aβ_1-42 fibrillogenesis timeline up to 48 h of incubation, providing morphological and chemo-structural characterization of the main assemblies formed during the aggregation process of Aβ_1-42, by atomic force microscopy (AFM) and surface enhanced Raman spectroscopy (SERS), respectively. Results: AFM topography evidenced the presence of characteristic protofibrils at early-stages of aggregation, which form peculiar macromolecular networks over time. SERS allowed to track the progressive variation in the secondary structure of the aggregation species involved in the fibrillogenesis and to determine when the β-sheet starts to prevail over the random coil conformation in the aggregation process. Discussion: Our research highlights the significance of investigating the early phases of fibrillogenesis to better understand the molecular pathophysiology of AD and identify potential therapeutic targets that may prevent or slow down the aggregation process.

Keywords: AFM; Alzheimer’s disease; SERS; amyloid-β peptide; fibrillogenesis; neurodegeneration.

FIGURE 1

Representative AFM height images acquired on Aβ_1-42 that was incubated for (A) 0 min (immediately after the initiation of Aβ_1-42 fibrillation), (B) 60 min and (C) 120 min (the colour-coded height bar is shown beside). The height profiles and the mean height values obtained by measuring along the cyan lines (indicated by the cyan arrows) are displayed beneath the corresponding image (where the ordinate axis indicates the height and the abscissa axis indicates the length). (A) also displays the representative width of a protofibril as measured along the white line.

FIGURE 2

Representative AFM height images acquired on Aβ_1-42 that was incubated for (A) 240 min and (B) 480 min (the colour-coded height bar is shown beside). The height profiles and the mean height values obtained by measuring along the cyan lines (indicated by the cyan arrows) are displayed beneath the corresponding image (where the ordinate axis indicates the height and the abscissa axis indicates the length).

FIGURE 3

Representative AFM height images acquired on Aβ_1-42 that was incubated for (A) 24 h and (B) 48 h (the colour-coded height bar is shown beside). The height profiles and the mean height values obtained by measuring along the cyan lines (indicated by the cyan arrows) are displayed beneath the corresponding image (where the ordinate axis indicates the height and the abscissa axis indicates the length). The panel on the bottom left of figure (A) has a narrower height range (0–2.5 nm) to highlight the presence of the protofibrils in the background. The green arrows in figure (B) indicate some mature fibrils displaying the periodic helical twist.

FIGURE 4

Aggregation kinetic of Aβ_1-42 (purple) versus control (fuchsia) monitored by ThT fluorescence assay (λex = 440 nm, λem = 485 nm).

FIGURE 5

SERS averaged spectra (λex = 633 nm) of Aβ_1-42 that was deposited on AgNWs substrate at different time points: 0 min, 60 min, 120 min, 240 min, 480 min, 24 h and 48 h. Each spectrum was calculated as average from 50 acquisitions. The spectra were staked for clarity.

FIGURE 6

Normalized curve fitting of the amide I vibration mode of Aβ_1-42 aggregates at different time points: 0 min, 60 min, 120 min, 240 min, 480 min, 24 h and 48 h. Three shoulder bands approximately centred at 1650, 1670 and 1680 cm^-1, associated with α-helix, β-sheet and random coil structures, respectively, are obtained from the fitting procedure (dashed lines) (A). Histogram displaying the percentage contribution of each secondary structure (α-helix, β-sheet, and random coil) to the amide I vibration band (B).