Specific Domains of Aβ Facilitate Aggregation on and Association with Lipid Bilayers

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Abstract

A hallmark of Alzheimer's disease, a late-onset neurodegenerative disease, is the deposition of neuritic amyloid plaques composed of aggregated forms of the β-amyloid peptide (Aβ). Aβ forms a variety of nanoscale, toxic aggregate species ranging from small oligomers to fibrils. Aβ and many of its aggregate forms strongly interact with lipid membranes, which may represent an important step in several toxic mechanisms. Understanding the role that specific regions of Aβ play in regulating its aggregation and interaction with lipid membranes may provide insights into the fundamental interaction between Aβ and cellular surfaces. We investigated the interaction and aggregation of several Aβ fragments (Aβ1–11, Aβ1–28, Aβ10–26, Aβ12–24, Aβ16–22, Aβ22–35, and Aβ1–40) in the presence of supported model total brain lipid extract (TBLE) bilayers. These fragments represent a variety of chemically unique domains within Aβ, that is, the extracellular domain, the central hydrophobic core, and the transmembrane domain. Using scanning probe techniques, we elucidated aggregate morphologies for these different Aβ fragments in free solution and in the presence of TBLE bilayers. These fragments formed a variety of oligomeric and fibrillar aggregates under free solution conditions. Exposure to TBLE bilayers resulted in distinct aggregate morphologies compared to free solution and changes in bilayer stability dependent on the Aβ sequence. Aβ10–26, Aβ16–22, Aβ22–35, and Aβ1–40 aggregated into a variety of distinct fibrillar aggregates and disrupted the bilayer structure, resulting in altered mechanical properties of the bilayer. Aβ1–11, Aβ1–28, and Aβ12–24 had minimal interaction with lipid membranes, forming only sparse oligomers.

Graphical Abstract

Highlights

► The toxicity of Aβ may be mediated via lipid membranes. ► Aβ fragments formed distinct aggregates in free solution and on lipid surfaces. ► Mechanical properties of supported lipid bilayers were modulated by Aβ fragments. ► The interaction of Aβ with lipids is facilitated by domains within the peptide.

Introduction

Conformational or ‘protein misfolding’ diseases are defined by the rearrangement of specific proteins to non-native conformations, promoting the formation and deposition of toxic, nanoscale aggregates within tissues or cellular compartments. A pathological hallmark of Alzheimer's disease (AD), an age-related neurodegenerative disease, is the formation of neuritic amyloid plaques. These plaques consist predominantly of extracellular masses of filamentous aggregates of the β-amyloid peptide (Aβ) as well as other plaque-associated proteins (e.g., apoE, apoJ, inflammatory molecules), which are associated with dystrophic dendrites and axons, activated microglia, and reactive astrocytes.1 Aβ is a monomeric, amphipathic, 39- to 43-amino-acid residue cleavage product of the transmembrane amyloid precursor protein.2

Like many other amyloid-forming peptides, Aβ can form a variety of aggregate structures on and off pathway to fibril formation, including distinct oligomers and protofibrils.[2], [3] Beyond this heterogeneity of smaller intermediate aggregate structures, Aβ also has the ability to form numerous morphologically distinct fibril structures, often referred to as polymorphs.[4], [5], [6], [7] A variety of environmental factors can influence the emergence of different polymorphic fibrils. For example, varying sample preparation of Aβ1–40 results in five structurally distinct fibrillar aggregates in vitro.6 Furthermore, Aβ can bind metal ions, and their presence in solution can facilitate aggregation leading to neurotoxicity.[8], [9], [10] As such, several small molecules that inhibit metal-induced Aβ aggregation for potential therapeutic purposes have been developed.[11], [12], [13] Another contributing factor to the emergence of distinct polymorphic aggregates is the presence of surfaces. Aβ aggregates into distinct forms in the presence of mica and graphite.[14], [15], [16] The addition of disease-related point mutations can directly lead to distinct polymorphic aggregates of Aβ in the presence of surfaces,17 suggesting that electrostatic and hydrophobic interactions between the peptide and surface strongly influence the aggregation process. The role of surfaces may underlie the ability of different synthetic nanoparticles, which have high surface-to-volume ratios, to either promote18 or inhibit19 Aβ aggregation, and thus could prove useful in understanding their potential therapeutic use. The formation of specific polymorphs may play an important role in disease pathology, as two structurally distinct polymorphic fibrils of Aβ1–40 were associated with significantly different levels of toxicity to neuronal cell cultures.20

With respect to surfaces, a potentially relevant environmental factor regulating Aβ aggregation is lipid bilayers. The fluid surfaces provided by lipid bilayers are well known to influence protein structure and dynamics, which can nucleate the aggregation process. Importantly for AD, lipid bilayer properties alter protein conformation and exert enormous influence on the aggregation state, as substantial enhancement of Aβ aggregation is observed in the presence of lipid membranes.[21], [22], [23], [24], [25] While cellular membranes may act to aid protein aggregation,[26], [27], [28] these same membranes may be damaged by the aggregation process, leading to membrane dysfunction caused by membrane permeabilization by Aβ either perturbing bilayer structure[29], [30] or forming unregulated pores.[31], [32], [33], [34] While several physicochemical aspects of membranes (such as phase state, curvature, charge, and elasticity) associated with lipid composition play an important role in specific peptide/lipid interactions, these interactions are also dependent on protein properties. Understanding the basic interaction between Aβ and lipid membranes could lead to a better understanding of Aβ aggregation associated with cellular membranes.

As Aβ is a cleavage product of amyloid precursor protein, it contains a hydrophobic transmembrane domain and a predominately hydrophilic extracellular domain, imparting amphiphilic character to Aβ (Fig. 1). Furthermore, based on a variety of structural and computational studies, several domains have been identified in Aβ. The N-terminal region of Aβ has been shown to form α-helical or β-sheet structure dependent on solution conditions, such as pH.[35], [36] The hydrophobic C-terminal end of Aβ has a high propensity to aggregate into β-sheet-rich structures independent of solvent conditions.[35], [36] Despite the appearance of various polymorphs, Aβ fibrils are composed of bundled β-sheets with backbones orthogonal to the fiber axis creating a cross-β structure.37 Site-directed spin labeling electron paramagnetic resonance studies of Aβ fibrils identified two β-strand-forming domains (residues 11–21 and 29–39, respectively) separated by turn/bend region (around residues 23–26).38 NMR studies on a variety of Aβ fragments support the notion of two β-strand regions separated by a β-turn in different fibril structures.[20], [39], [40] The appearance of a β-turn between two β-strands is further supported by computational studies of Aβ fibrils.41 The central region of Aβ (residues 16–21) has been shown to form a hydrophobic core with enhanced amyloidogenic properties42 and is contained within one of the β-strand-forming regions. A variety of NMR studies of Aβ in solution indicate that the monomer is predominately unstructured with fluctuating residual structure.[43], [44] A more recent NMR study though has demonstrated that this central hydrophobic domain of Aβ can form a 310 helix, resulting in a compact structure as other hydrophobic residues cluster against the helix.45 The variations in Aβ monomer and aggregate structure associated with these different studies may be partially attributed to the variation in preparation protocols that lead to polymorphic aggregates.

Here, we sought to determine the role specific domains of Aβ play in its aggregation under free solution conditions and in the presence of total brain lipid extract (TBLE) bilayers. The aggregation of seven different Aβ fragments (Aβ1–11, Aβ1–28, Aβ10–26, Aβ12–24, Aβ16–22, Aβ22–35, and Aβ1–40) was investigated. These Aβ fragments represent a variety of chemically unique regions, that is, the extracellular domain, the central hydrophobic core, different β-strand-forming sequences, and the transmembrane domain (Fig. 1). Atomic force microscopy (AFM) was used to characterize the Aβ fragment aggregate morphology and to monitor the degree of interaction with a model lipid bilayer system. In addition, we determined the mechanical impact of exposure to the different Aβ fragments on the TBLE bilayers.

Section snippets

Aβ fragments form distinct oligomeric and fibrillar aggregates in free solution

To compare oligomers and fibrils formed by different Aβ fragments under free solution conditions (i.e., no surface present), we incubated 20-μM solutions of Aβ1–11, Aβ1–28, Aβ10–26, Aβ12–24, Aβ16–22, Aβ22–35, or Aβ1–40 at 37 °C and sampled them at different time points, deposited them on mica, and imaged them by AFM in air. Each Aβ fragment was prepared via the same protocol46 to ensure that observed differences in aggregate morphology were not attributable to sample preparation, which has been

Discussion

We have investigated a variety of Aβ fragments in an effort to understand how specific regions of Aβ regulate its interaction with lipid membranes. In particular, we investigated the interaction of Aβ1–11, Aβ1–28, Aβ10–26, Aβ12–24, Aβ16–22, Aβ22–35, and Aβ1–40 with TBLE bilayers. These Aβ fragments represent a variety of chemically unique regions along the peptide, that is, the extracellular domain, β-strand, β-turns, the central hydrophobic core, and a portion of the transmembrane domain.

Peptide preparation

Synthetic fragments of Aβ1–11, Aβ1–28, Aβ10–26, Aβ12–24, Aβ16–22, Aβ22–35, and Aβ1–40 (AnaSpec Inc., San Jose, CA) were prepared in the same manner according to published protocols.46 Briefly, peptides were treated with hexafluoroisopropanol to dissolve seeds and preexisting aggregates within the lyophilized stock. Hexafluoroisopropanol was evaporated off in a Vacufuge concentrator (Eppendorf), resulting in peptide films. These peptide films were dissolved in 10 μL of dimethyl sulfoxide to make

Author Contributions

E.A.Y and J.L. conceived the experiments. E.A.Y accomplished all peptide preparation, performed and analyzed all lipid experiments, and analyzed all ex situ experiments. S.L.O. and C.S.U. developed, performed, and analyzed the PDA experiments. M.F.L. and E.M.C performed ex situ experiments. E.M.C. and S.L.O. assisted with peptide preparation. E.A.Y and J.L. wrote and edited the paper.

Acknowledgements

The authors acknowledge Dr. Jonathan Boyd and Julie Vrana for access and assistance in using the TECAN plate reader. This work was funded by the Brodie Discovery and Innovation fund, the National Science Foundation (NSF#1054211), and the Alzheimer's Association (NIRG-11-203834). E.M.C. was supported by the Center for Neuroscience Summer Undergraduate Research Internship program (Robert C. Byrd Health Sciences Center, West Virginia University). M.F.L. was supported by the Honors Summer

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