Abstract
A major hallmark of Alzheimer's disease is the misfolding and aggregation of the amyloid- β peptide (Aβ). While early research pointed towards large fibrillar- and plaque-like aggregates as being the most toxic species, recent evidence now implicates small soluble Aβ oligomers as being orders of magnitude more harmful. Techniques capable of characterizing oligomer stoichiometry and assembly are thus critical for a deeper understanding of the earliest stages of neurodegeneration and for rationally testing next-generation oligomer inhibitors. While the fluorescence response of extrinsic fluorescent probes such as Thioflavin-T have become workhorse tools for characterizing large Aβ aggregates in solution, it is widely accepted that these methods suffer from many important drawbacks, including an insensitivity to oligomeric species. Here, we integrate several biophysics techniques to gain new insight into oligomer formation at the single-molecule level. We showcase single-molecule stepwise photobleaching of fluorescent dye molecules as a powerful method to bypass many of the traditional limitations, and provide a step-by-step guide to implementing the technique in vitro. By collecting fluorescence emission from single Aβ(1-42) peptides labelled at the N-terminal position with HiLyte Fluor 555 via wide-field total internal reflection fluorescence (TIRF) imaging, we demonstrate how to characterize the number of peptides per single immobile oligomer and reveal heterogeneity within sample populations. Importantly, fluorescence emerging from Aβ oligomers cannot be easily investigated using diffraction-limited optical microscopy tools. To assay oligomer activity, we also demonstrate the implementation of another biophysical method involving the ratiometric imaging of Fura-2-AM loaded cells which quantifies the rate of oligomer-induced dysregulation of intracellular Ca2+ homeostasis. We anticipate that the integrated single-molecule biophysics approaches highlighted here will develop further and in principle may be extended to the investigation of other protein aggregation systems under controlled experimental conditions.
Highlights
The amyloid-β peptide (Aβ) is derived via the proteolytic cleavage of the transmembrane amyloid precursor protein (APP) and ranges in length from 39 to 43 amino acids [1]
We describe the implementation of a wide-field, objective-based, total internal reflection fluorescence microscopy imaging method to access the stepwise-photobleaching trajectories obtained from single surface-immobilized oligomers composed of Aβ (1–42) peptides labelled at the N-terminal position with HiLyte Fluor 555 (Aβ555), a member of the cyanine family of fluorescent compounds
As evident from our results and others, single-molecule stepwise photobleaching measurements can provide unprecedented levels of detail and insight into the stoichiometry of single immobilized Aβ oligomers, providing important insight in particular when correlated with ratiometric information which details oligomer activity
Summary
193 (2021) 80–95 astrocytes with Aβ(25–35) fragments and serotonin has been shown to increase Ca2+ signaling [89], and recently Aβ(1–42) oligomers were found to aggravate the loss of store operated Ca2+ entry and increase the resting cytosolic Ca2+ concentration in aging neurons [90]. 6. To assay the effect of Aβ oligomers on cell viability, cells were treated for 2 h at 37 ◦C in KREBS buffer containing 4–10 μg/mL of Aβ(1–42) (unlabelled) or Aβ555 (labelled) in < 0.5% (v/v) HFIP and < 1% (v/v) DMSO, with 0.1 μM propidium iodide (PI) (Sigma Aldrich, UK, part number P4170). 7. To assay the dysregulation of intracellular Ca2+ by Aβ oligomers, cells are first incubated with 0.1 μM Fura-2-AM (Thermo Fisher Scientific, UK, part number F1201) and 0.001% pluronic acid in KREBS buffer for 20 min at 37 ◦C. After the third wash step, incubate the cells in KREBS buffer for 10 min at 37 ◦C prior to treatment with Aβ oligomers and live cell imaging. Intracellular calcium dysregulation is typically observed immediately after application of pre-formed HFIP-Aβ555 oligomers onto Fura-2-AM loaded cells, and followed via a singleexponential growth model with a kinetic rate constant of 0.028 ± 0.003 s−1 (χ2 = 0.97) (Fig. 13c). It is clear that instabilities in Ca2+ homeostasis appear early in AD pathogenesis, and the presented strategy may at least have some relevance to the disease process, and we expect the approach could aid basic science methods aimed at correcting or slowing Aβ-induced dysregulation of Ca2+ homeostasis
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