Atomic Force Microscopy Analysis of EPPS-Driven Degradation and Reformation of Amyloid-β Aggregates

Abstract

Amyloid-β (Aβ) peptides can be aggregated into β-sheet rich fibrils or plaques and deposited on the extracellular matrix of brain tissues, which is a hallmark of Alzheimer's disease. Several drug candidates have been designed to retard the progression of the neurodegenerative disorder or to eliminate toxic Aβ aggregates. Recently, 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS) has emerged as a promising drug candidates for elimination of toxic Aβ aggregates. However, the effect of EPPS on the degradation of Aβ aggregates such as fibrils has not yet been fully elucidated. In this article, we investigate the EPPS-driven degradative behavior of Aβ aggregates at the molecular level by using high-resolution atomic force microscopy. We synthesized Aβ fibrils and observed degradation of fibrils following treatment with various concentrations (1-50 mM) of EPPS for various time periods. We found that degradation of Aβ fibrils by EPPS increased as a function of concentration and treatment duration. Intriguingly, we also found regeneration of Aβ aggregates with larger sizes than original aggregates at high concentrations (10 and 50 mM) of EPPS. This might be attributed to a shorter lag phase that facilitates reformation of Aβ aggregates in the absence of clearance system.

Keywords: 4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS); Alzheimer’s disease; Aβ aggregates; amyloid-β; atomic force microscopy; fibril degradation.

Fig.1

AFM images of Aβ fibrils. (a) 50 μM of Aβ fibrils in 10 mM of hydrochloric acid (HCl) solution (approximately adjusted to pH 2). (b) 50 μM of Aβ fibrils after dialysis in distilled water (H₂O) for 60 h. (c) 5 μM of Aβ fibrils diluted from (b). (d, e) AFM images of 5 mM (d) and 10 mM (e) of EPPS treated Aβ fibrils with different incubation times (6–24 h).

Fig.2

AFM images of EPPS treated Aβ fibrils (5 μM) with different concentrations of EPPS (1–50 mM). (a) 1 mM of EPPS. (b) 5 mM of EPPS. (c) 10 mM of EPPS. (d) 50 mM of EPPS. Incubation time: 1 day. (e) Cross section profiles of Aβ fibrils on images (a-d). We sectioned across the white dot line on images. (f) Differences of surface roughness by various EPPS treatment to Aβ fibrils.

Fig.3

AFM images of EPPS (1 mM) treated Aβ fibrils (50 μM) for different incubation times (1–3 days). (a) 1 day. (b) 2 days. (c) 3 days. White arrows indicate the Aβ aggregates. (d-f) Cross section profiles of Aβ fibrils on images (a-c). We sectioned across the white dot line on images.

Fig.4

AFM images of Aβ fibrils (50 μM) which are treated to different concentrations of EPPS (1–50 mM) for 1 day. (a) 1 mM of EPPS. (b) 5 mM of EPPS. (c) 10 mM of EPPS. (d) 50 mM of EPPS. White arrows indicate the Aβ aggregates. We sectioned across the white dot line on images. (e) Cross section profiles of Aβ aggregate on images (a–d). (f) Differences of surface roughness by various EPPS treatment to Aβ fibrils.

Fig.5

AFM images of Aβ fibrils (50 μM) which are treated to EPPS for different incubation times (6–24 h) and concentrations of EPPS (5 and 10 mM). (a-c) 5 mM. (d) Differences of surface roughness by 5 mM EPPS treatment at different incubation time. (e-g) 10 mM. White arrow shows the Aβ aggregates. (h) Differences of surface roughness by 10 mM EPPS treatment at different incubation time.

Fig.6

Characterization of structural information of re-formed Aβ aggregates depending on EPPS treatment by using AFM images. Topological images of Aβ aggregates by (a) 10 and (b) 50 mM EPPS treatment for 1 day. (c) Average height depth of Aβ aggregates. Amplitude error images of Aβ aggregates by (d) 10 and (e) 50 mM EPPS treatment for 1 day. (f) Average amplitude error depth of Aβ aggregates.

Fig.7

Schematic diagram of our proposed model describing the mechanism of degradation and reformation of Aβ aggregates.