Nano Today
Volume 38, June 2021, 101125
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Spontaneous formation of β-sheet nano-barrels during the early aggregation of Alzheimer’s amyloid beta

https://doi.org/10.1016/j.nantod.2021.101125Get rights and content

Highlights

  • Soluble low-molecular-weight oligomers have been hypothesized as causative to neurodegeneration in Alzheimer’s disease.

  • The oligomers are challenging to characterize due to their polymorphic, heterogeneous and transient nature.

  • We performed the first synergic validation of the structure, dynamics and toxicity of full-length Aβ42 oligomers.

  • oligomers assumed nanosized β-barrels and impaired the cognitive function in a mouse model.

  • The occurrence of β-barrels supports their roles as the common toxic intermediates in Alzheimer’s and a therapeutic target.

Abstract

Soluble low-molecular-weight oligomers formed during the early aggregation of amyloid peptides have been hypothesized as a major toxic species of amyloidogenesis. Herein, we performed the first synergic in silico, in vitro and in vivo validations of the structure, dynamics and toxicity of Aβ42 oligomers. Aβ peptides readily assembled into β-rich oligomers comprised of extended β-hairpins and β-strands. Nanosized β-barrels were observed with certainty with simulations, transmission electron microscopy and Fourier transform infrared spectroscopy, corroborated by immunohistochemistry, cell viability, apoptosis, inflammation, autophagy and animal behavior assays. Secondary and tertiary structural properties of these oligomers, such as the sequence regions with high β-sheet propensities and inter-residue contact frequency patterns, were similar to the properties known for Aβ fibrils. The unambiguous spontaneous formation of β-barrels in the early aggregation of Aβ42 supports their roles as the common toxic intermediates in Alzheimer’s pathobiology and a target for Alzheimer’s therapeutics.

Graphical Abstract

Free-energy landscape of peptides in aggregation. Aβ monomers initially assemble into low β-sheet content oligomers, followed by conformational conversion into high β-sheet abundant oligomers (including β-barrels). Eventually, the β-sheet rich oligomers are converted into proto-fibrils by crossing a high free-energy barrier.

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Introduction

The misfolding and self-assembly of amyloid-β (Aβ) plays a key role in the pathogenesis of Alzheimer’s disease (AD) [1], [2], [3], [4]. Similar to type 2 diabetes (T2D) and Parkinson’s and Huntington’s diseases, a common hallmark across these pathologies is the formation of extracellular amyloid protein deposits [1], [5], [6], [7], [8], yet the pertinent toxicity mechanisms of these diseases are not fully understood. The amyloid assembly kinetics features a sigmoid curve with three phases, corresponding to the nucleation of monomers into oligomers and proto-fibrils, as well as the elongation and saturation of proto-fibrils into mature fibrils [7], [9], [10], [11]. Mounting data have demonstrated that soluble oligomers formed during the early aggregation (e.g., the nucleation phase) were more cytotoxic than mature fibrils [12], [13], [14]. For instance, the accelerated aggregation prompted by small molecules or nanoparticles can reduce amyloid cytotoxicity because of the reduced overall population of toxic oligomer species [12], [15], [16], [17]. Therefore, characterization of various oligomer structures during the early aggregation holds the key to understanding the cytotoxicity mechanism of Aβ assembly, which is central to the development of effective therapeutic strategies against AD, the primary form of neurological disorder with no cure currently available.

Aβ peptides ranging from 38 to 43 in residual number are cleaved off from the amyloid precursor protein by β and γ-secretases [2], [7], [8], [18]. Aβ40 and Aβ42 are the two major isoforms in senile plaques, with Aβ42 featuring a greater propensity for assembly and neurotoxicity [19]. Aβ monomers mainly adopt random coil and partial β-sheet structures in solution as determined by prior nuclear magnetic resonance (NMR) and circular dichroism (CD) experiments [20], [21], [22]. The amyloid fibrils of Aβ are polymorphic but share a common cross-β core, where the peptides form parallel in-register β-sheets according to numerous X-ray crystallography, solid-state NMR (ssNMR) or cryo-EM experiments [1], [4], [21], [23], [24], [25], [26], [27], [28], [29]. However, oligomers formed during the early assembly are heterogeneous and unstable, making their isolation, quantification, and structural determination experimentally challenging [13], [14]. Therefore, the assembly dynamics of Aβ and structures of various oligomer species, including the potentially cytotoxic intermediates, remain elusive.

Despite differences in their primary, secondary and tertiary structures, amyloid proteins are believed to share a similar mechanism in eliciting cytotoxicity. A well accepted hypothesis suggests that amyloid proteins disrupt membrane integrity and permeability by forming amyloid nanopores [30], [31], [32]. Based on the principle of structure-function relationship where a specific function of a protein or protein complex is determined by its unique conformation [33], [34], the toxic oligomers formed by different amyloid proteins should possess ubiquitous structures. Using an 11-residue slow-aggregating fragment from αB crystallin that entailed cytotoxicity, Laganowsky et al. observed a stable β-sheet oligomer in the shape of a cylinder (i.e., β-sheet nano-barrel, or β-barrel) using X-ray crystallography, and proposed it as the candidate for toxic oligomers [35]. A similar β-sheet nanobarrel conformation was suggested for the C-terminal fragments of Aβ by Thanh et al. based on the structural features determined by ion mobility-mass spectrometry (IM-MS) experiments along with computational modeling [36]. Recent experiments using Aβ fragments suggested that the formation of β-barrels induced membrane leakage [37], [38]. Moreover, the formation of β-sheet nano-barrel by full-length Aβ was also supported experimentally [39], [40], [41]. For example, Aβ42 was found to form specific β-barrel pores in a lipid mimicking environment [39], which were observed by native mass spectrometry in a recent study [40]. The formation of β-barrel oligomers by Aβ40 in a slightly basic solution was supported by top-down hydrogen exchange mass spectrometry [41]. Complementary to the experimental observations, multiple β-barrel models were proposed based on features of known barrel protein structures [42], [43] and molecular dynamics (MD) simulations have been used to evaluate their feasibility. For example, constructed using four Aβ peptides, the stability of a model β-sheet nanobarrel in both aqueous solution and lipid membranes was investigated by MD simulations, where the designed β-barrel formed by Aβ42 was more stable than Aβ40 [44], [45], and the D23N variant of Aβ42 rendered a lower β-barrel nanopore propensity than the wild type and its A2T variant [46]. However, due to the high degrees of freedom and macroscopic timescales of conformational conversion, prior all-atom MD simulations of Aβ were mainly focused on either the structures and dynamics of full-length monomer [47], [48], [49] and dimer [47], [50], [51], [52] or the oligomerization of multiple short fragments [36], [53], [54], [55], [56], [57]. Overall, the spontaneous formation of β-barrels by Aβ42 and their corresponding structural and dynamic properties remain elusive, especially in silico.

In order to uncover the oligomerization dynamics of Aβ42 and assess the population of β-barrel oligomers during the early aggregation, we utilized atomistic discrete molecular dynamic (DMD) simulations with implicit solvent model - an efficient and predictive MD algorithm widely used to study protein dynamics, protein folding, misfolding and aggregation [54], [58], [59], [60], [61], [62], [63]. Using DMD simulations, we have previously demonstrated that peptide fragments derived from amyloid peptides whose monomer structures are either unstructured [54], [64], [65], [66] or helical [59] could form well-defined β-sheet nano-barrels as the aggregation intermediates before forming cross-β nano-fibrils. Since pentamers and hexamers have been experimentally determined as the stable oligomers in Aβ42 solutions [67], [68], we systematically studied the self-assembly dynamics of up to six Aβ42 peptides in DMD simulations. For each molecular system, a large number of independent DMD simulations were performed for increased sampling, and Aβ peptides mainly assembled into β-sheet rich oligomers with diverse extended β-hairpins and β-strands during the early aggregation. Regions with the highest β-sheet propensities were located at residues 11–21 and 31–41, corresponding to β-strands in most Aβ fibril structures featuring typical β-loop-β motifs4,24–31. The inter-residue interactions identified to stabilize oligomers were also widely observed in most fibril models4,24–31. Moreover, the formation of β-barrel oligomers was explicitly observed in our simulations as well as transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) spectroscopy. The toxicity of Aβ oligomers was further validated in vitro by immunohistochemistry (IHC) and cellular viability assays and in vivo with a mouse model. Our aggregation free-energy landscape analysis revealed that the β-barrel intermediates mainly reside in the free-energy basin of low β-sheet to high β-sheet content conversion. Together with the β-sheet nano-barrel observations in prior amyloid fragments [35], [36], [53], [54], [55], [56], [57], [59], [64], [65], [66] and full-length IAPP [60] aggregation studies, the current synergistic in silico, in vitro and in vivo study underscores the hypothesis of β-barrel oligomers as the common intermediates of amyloidogenesis. With well-defined three-dimensional structures and consistency to the amyloid pore hypothesis, the toxic β-sheet nano-barrels may serve as a definitive target for future AD therapeutics.

Section snippets

Aβ mainly assembled into extended β-strand and β-hairpin structures in early aggregation

We first examined the average secondary structure propensities of Aβ assemblies in each system. To avoid potential biases of initial states, the first 200 ns trajectories were not included and the last 300 ns simulation data were used for analysis. Aβ predominantly adopted random coils and β-sheets, although other structures including bends, turns and helices were also observed (Fig. 1a). These results were consistent with previous replicate exchange molecular dynamics (REMD) simulations of Aβ

Conclusion

In this study, we combined rapid all-atom DMD simulations with complementary in vitro (TEM, FTIR, IHC and cell viability) and in vivo characterizations (animal behavior and western blotting) to systematically investigate the aggregation of full-length Aβ42. First, the Aβ oligomers spontaneously formed during the early aggregation mainly adopted extended β-hairpin and β-strand conformations. Both secondary and tertiary structures of these oligomers – such as the β-sheet regions and inter-residue

Materials and methods

The sequence of Aβ42 is 1DAEFRHDSGY 11EVHHQKLVFF 21AEDVGSNKGA 30IIGLMVGGVV 40IA. The initial structure used in our simulation was taken from the protein data bank (PDB ID: 1z0q) obtained by an NMR study in aqueous media environment [88]. To investigate the nucleation kinetics and the oligomer conformation of Aβ, we systematically simulated six different molecular systems with the number of simulated peptides ranging from one to six. For each system, 50 independent DMD simulations, each lasted

Author contributions

Y. Sun, Y. Song, P.C. Ke and F. Ding conceived the project. Y. Sun, A. Kakinen, X. Wan, F. Ding and P.C. Ke wrote the article. Y. Sun and F. Ding performed DMD simulations and data analysis. A. Kakinen performed ThT, TEM and FTIR experiments and data analysis. Y. Li performed cellular viability assay. N. Andrikopoulos and A. Nandakumar carried out the immunohistochemistry assay. N. Moriarty and C.P.J. Hunt performed the mouse surgery and behavior assays. X. Wan and Y. Song performed western

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This work was supported in part by the National Natural Science Foundation of China under the Grant No. 11904189 (Sun), K.C.Wong Magna Fund in Ningbo University, China (Sun), US National Science Foundation CBET-1553945 (Ding), and US National Institutes of Health R35GM119691 (Ding). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NSFC, NIH and NSF. This work was performed in part at the Queensland node of the Australian

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