High-Speed Atomic Force Microscopy Reveals the Structural Dynamics of the Amyloid-β and Amylin Aggregation Pathways

Abstract

Individual Alzheimer’s disease (AD) patients have been shown to have structurally distinct amyloid-β (Aβ) aggregates, including fibrils, in their brain. These findings suggest the possibility of a relationship between AD progression and Aβ fibril structures. Thus, the characterization of the structural dynamics of Aβ could aid the development of novel therapeutic strategies and diagnosis. Protein structure and dynamics have typically been studied separately. Most of the commonly used biophysical approaches are limited in providing substantial details regarding the combination of both structure and dynamics. On the other hand, high-speed atomic force microscopy (HS-AFM), which simultaneously visualizes an individual protein structure and its dynamics in liquid in real time, can uniquely link the structure and the kinetic details, and it can also unveil novel insights. Although amyloidogenic proteins generate heterogeneously aggregated species, including transient unstable states during the aggregation process, HS-AFM elucidated the structural dynamics of individual aggregates in real time in liquid without purification and isolation. Here, we review and discuss the HS-AFM imaging of amyloid aggregation and strategies to optimize the experiments showing findings from Aβ and amylin, which is associated with type II diabetes, shares some common biological features with Aβ, and is reported to be involved in AD.

Inactivation of seeding activity of amyloid β‐protein aggregates in vitro

“…A sample–scanning type of HS‐AFM was operated in the amplitude modulation–tapping mode with a BL‐AC10DS‐A2 cantilever (Olympus Corp.) (Ando et al, 2001; Sahoo et al, 2019; Uchihashi et al, 2012; Watanabe‐Nakayama et al, 2016, 2020). The cantilever probes were prepared at the top of the cantilevers by electron‐beam deposition using the scanning electron microscope mode of ELS‐7500, which is an electron lithography system (Elionix Inc.).…”

“…The results of both the elongation speed of Aβ42 aggregates in HS‐AFM and the length of Aβ aggregates with fibril‐like structures in EM images were not normally distributed, and we used Brunner–Munzel tests to evaluate the statistical significance of the HS‐AFM datasets without assuming normal distribution and equal variance, as previously reported (Watanabe‐Nakayama et al, 2016, 2020). These tests were performed using a R software version 3.6.2.…”

“…Blinding was not performed and test for outliers was not conducted. In this study, no pre‐determined samples size calculation was applied, and the sample sizes were based on previous studies (Ono et al, 2012; Watanabe‐Nakayama et al, 2016, 2020).…”

“…The cantilever probes were prepared at the top of the cantilevers by electron-beam deposition using the scanning electron microscope mode of ELS-7500, which is an electron lithography system (Elionix Inc.). The operation methods of HS-AFM were based on a previous report (Watanabe -Nakayama et al, 2016-Nakayama et al, , 2020. Before the injection of Aβ42 with filtration into the sample chamber of HS-AFM, a 2 µl aliquot of each Aβ42 aggregate sample (untreated Aβ42 aggregates and autoclaved Aβ42 aggregates treated at 120°C for 30 min or 135°C for 120 min) was spotted onto mica to attach the Aβ42 aggregates.…”

“…The magnification of the EM images was 30 000× when we measured the width of Aβ aggregates with fibril-like structures and 5000-30 000× when we measured the length of Aβ aggregates with fibril-like structures. The results of both the elongation speed of Aβ42 aggregates in HS-AFM and the length of Aβ aggregates with fibril-like structures in EM images were not normally distributed, and we used Brunner-Munzel tests to evaluate the statistical significance of the HS-AFM datasets without assuming normal distribution and equal variance, as previously reported (Watanabe- Nakayama et al, 2016Nakayama et al, , 2020. These tests were performed using a R software version 3.6.2.…”