Elizabeth A. Yates

Biophysical insights into how surfaces, including lipid membranes, modulate protein aggregation related to neurodegeneration.

There are a vast number of neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD), associated with the rearrangement of specific proteins to non-native conformations that promotes aggregation and deposition within tissues and/or cellular compartments. These diseases are commonly classified as protein-misfolding or amyloid diseases. The interaction of these proteins with liquid/surface interfaces is a fundamental phenomenon with potential implications for protein-misfolding diseases. Kinetic and thermodynamic studies indicate that significant conformational changes can be induced in proteins encountering surfaces, which can play a critical role in nucleating aggregate formation or stabilizing specific aggregation states. Surfaces of particular interest in neurodegenerative diseases are cellular and subcellular membranes that are predominately comprised of lipid components. The two-dimensional liquid environments provided by lipid bilayers can profoundly alter protein structure and dynamics by both specific and non-specific interactions. Importantly for misfolding diseases, these bilayer properties can not only modulate protein conformation, but also exert influence on aggregation state. A detailed understanding of the influence of (sub)cellular surfaces in driving protein aggregation and/or stabilizing specific aggregate forms could provide new insights into toxic mechanisms associated with these diseases. Here, we review the influence of surfaces in driving and stabilizing protein aggregation with a specific emphasis on lipid membranes.

Amyloid-forming proteins alter the local mechanical properties of lipid membranes.

A diverse number of diseases, including Alzheimers disease, Huntingtons disease, and type 2 diabetes, are characterized by the formation of fibrillar protein aggregates termed amyloids. The precise mechanism by which aggregates are toxic remains unclear; however, these proteins have been shown to interact strongly with lipid membranes. We investigated morphological and mechanical changes in model lipid bilayers exposed to amyloid-forming proteins by reconstructing the tapping forces associated with atomic force microscopy (AFM) imaging in solution. Tip/sample tapping forces contain information regarding mechanical properties of surfaces. Interpretation of the mechanical changes in the bilayers was aided by numerical simulations of the entire AFM experiment. Amyloid-forming proteins disrupted distinct regions of the bilayer morphology, and these regions were associated with decreased Youngs modulus and adhesive properties. These changes in bilayer mechanical properties upon exposure to amyloid-forming proteins may represent a common mechanism leading to membrane dysfunction in amyloid diseases.

Point Mutations in Aβ Result in the Formation of Distinct Polymorphic Aggregates in the Presence of Lipid Bilayers

A hallmark of Alzheimers disease (AD) is the rearrangement of the β-amyloid (Aβ) peptide to a non-native conformation that promotes the formation of toxic, nanoscale aggregates. Recent studies have pointed to the role of sample preparation in creating polymorphic fibrillar species. One of many potential pathways for Aβ toxicity may be modulation of lipid membrane function on cellular surfaces. There are several mutations clustered around the central hydrophobic core of Aβ near the α-secretase cleavage site (E22G Arctic mutation, E22K Italian mutation, D23N Iowa mutation, and A21G Flemish mutation). These point mutations are associated with hereditary diseases ranging from almost pure cerebral amyloid angiopathy (CAA) to typical Alzheimers disease pathology with plaques and tangles. We investigated how these point mutations alter Aβ aggregation in the presence of supported lipid membranes comprised of total brain lipid extract. Brain lipid extract bilayers were used as a physiologically relevant model of a neuronal cell surface. Intact lipid bilayers were exposed to predominantly monomeric preparations of Wild Type or different mutant forms of Aβ, and atomic force microscopy was used to monitor aggregate formation and morphology as well as bilayer integrity over a 12 hour period. The goal of this study was to determine how point mutations in Aβ, which alter peptide charge and hydrophobic character, influence interactions between Aβ and the lipid surface. While fibril morphology did not appear to be significantly altered when mutants were prepped similarly and incubated under free solution conditions, aggregation in the lipid membranes resulted in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also had a variable ability to disrupt bilayer integrity.

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

A hallmark of Alzheimers 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.

Point Mutations in Aβ Induce Polymorphic Aggregates at Liquid/Solid Interfaces

A pathological hallmark of Alzheimers disease (AD), a late onset neurodegenerative disease, is the development of neuritic amyloid plaques, composed predominantly of aggregates of the β-amyloid (Aβ) peptide. It has been demonstrated that Aβ can aggregate into a variety of polymorphic aggregate structures under different chemical environments, and a potentially important environmental factor in dictating aggregate structure is the presence of surfaces. There are also several mutations clustered around the central hydrophobic core of Aβ (E22G Arctic mutation, E22K Italian mutation, D23N Iowa mutation, and A21G Flemish mutation). These mutations are associated with hereditary diseases ranging from almost pure cerebral amyloid angiopathy (CAA) to typical Alzheimers disease pathology. The goal of this study was to determine how these mutations influence the morphology of Aβ aggregates under free solution conditions and at an anionic surface/liquid interface. While the rate of formation of specific aggregates was altered by mutations in Aβ under free solution conditions, the respective aggregate morphologies were similar. However, aggregation occurring directly on a negatively charged mica surface resulted in distinct aggregate morphologies formed by different mutant forms of Aβ. These studies provide insight into the potential role anionic surfaces play in dictating the formation of Aβ polymorphic aggregate structures.

Preparation Protocols of Aβ(1–40) Promote the Formation of Polymorphic Aggregates and Altered Interactions with Lipid Bilayers

The appearance of neuritic amyloid plaques comprised of β-amyloid peptide (Aβ) in the brain is a predominant feature in Alzheimers disease (AD). In the aggregation process, Aβ samples a variety of potentially toxic aggregate species, ranging from small oligomers to fibrils. Aβ has the ability to form a variety of morphologically distinct and stable amyloid fibrils. Commonly termed polymorphs, such distinct aggregate species may play a role in variations of AD pathology. It has been well documented that polymorphic aggregates of Aβ can be produced by changes in the chemical environment and peptide preparations. As Aβ and several of its aggregated forms are known to interact directly with lipid membranes and this interaction may play a role in a variety of potential toxic mechanisms associated with AD, we determine how different Aβ(1-40) preparation protocols that lead to distinct polymorphic fibril aggregates influence the interaction of Aβ(1-40) with model lipid membranes. Using three distinct protocols for preparing Aβ(1-40), the aggregate species formed in the absence and presence of a lipid bilayers were investigated using a variety of scanning probe microscopy techniques. The three preparations of Aβ(1-40) promoted distinct oligomeric and fibrillar aggregates in the absence of bilayers that formed at different rates. Despite these differences in aggregation properties, all Aβ(1-40) preparations were able to disrupt supported total brain lipid extract bilayers, altering the bilayers morphological and mechanical properties.

Insertion of Aβ alters the local mechanical properties of lipid membranes

Background: Background:Compelling data suggest that the aggregation of the b-amyloid peptide plays a central role in AD. Importantly for AD and other amyloid diseases, lipid bilayer properties canmodulate protein conformation and exert influence on the protein’s aggregation state. Cell membranes may also be a direct target of amyloid-forming peptides, resulting in cell death. This may be due to the ability of amyloid-forming peptides to induce membrane permeabilization by altering bilayer structure or by forming unregulated pore-like structures. We performed a series of studies to dynamically assess the impact that Ab and other amyloid-forming proteins have on the mechanical properties of lipid membranes. Methods: We used an atomic-force microscopy technique to measure with nanoscale spatial resolution, the mechanical properties of brain-lipid extract bilayers exposed to Ab and other amyloid-forming proteins.Results:When exposed to monomeric Ab, regions of increased surface roughness formed in the lipid membranes. These regions were often associated with the formation of aggregates. These regions of membrane disruption were associated with a softening of the membrane locally and decreased adhesion to the AFM probe.Conclusions: These results suggest that Ab decreases the order of lipid components comprising a lipid bilayer, which could potentially lead to membrane dysfunction.

Specific Sequences within Beta-Amyloid Mediate Aggregation Associated with Lipid Membranes

A hallmark of Alzheimers disease (AD), a late onset neurodegenerative disease, is the presence of neuritic amyloid plaques deposited within the brain comprised of beta-amyloid (As) peptide aggregates. As forms a variety of nanoscale, toxic aggregates which have been shown to strongly interact with supported lipid bilayers, which may represent a key step in potential toxic mechanisms. Understanding how specific regions of As regulate its aggregation in the absence and presence of surfaces can provide insight into the fundamental interaction of As with cellular surfaces. We investigated the interaction of specific fragments of As (As1-11, As1-28, Abeta10-26, As12-24, As16-22, As22-35, and As1-40) with lipid membranes. These sequences represent a variety of chemically unique regions along As, i.e., the extracellular domain, the central hydrophobic core, and transmembrane domain. We determined how these As sequences alter aggregate morphology and induce mechanical changes of lipid bilayers using various scanning probe microscopic techniques, and compared these aggregates with those formed under free solution conditions. In free solution, oligomer and fibrillar aggregate species were formed with varied rate of formation and morphology, i.e. smaller fragments (As1-11, As12-24, As16-22, and As22-35) formed smaller oligomers, and shorter, less rigid fibrils. Interaction with model lipid bilayers resulted in distinct aggregates and changes in bilayer stability dependent on the As fragment. As10-26, As16-22, As22-35, and As1-40 caused disruption of the lipid bilayer structure upon exposure and resulted in a variety of distinct fibrillar aggregates. These interactions were associated with altered mechanical properties of the lipid bilayer. Conversely, As1-11, As1-28, and As12-24 had minimal interaction with a lipid membrane, forming only oligomers. These studies illustrate the potential role of specific amino acid sequences within As on aggregation and interactions with lipid membranes.

Investigation of Protein/Lipid Interactions via Scanning Probe Acceleration Microscopy: Theory and Experiment

There is great interest in the application of proximal probe techniques to simultaneously image and measure mechancial properties of surfaces with nanoscale spatial resolution. There have been several innovations in generating time-resolved force interaction between the tip and surface while acquiring a tapping mode AFM image. These tip/sample forces contain information regarding mechanical properties of surfaces in an analogous fashion to a force curve experiment. Here, we demonstrate, via simulation, that the maximum and minimum tapping forces change with respect to the Young’s modulus and adhesiveness of a surface, but the roughness of the surfaces has no effect on the tapping forces. Using these changes in tapping forces, we determine the mechanical changes of a lipid membrane after exposure to a huntingtin exon1 (htt exon1) protein with an expanded polyglutamine (polyQ) domain. Expanded polyQ domains in htt is associated with Huntington’s disease, a genetic neurodegenerative disorder. The htt exon1 protein caused regions of increased surface roughness to appear in the lipid membrane, and these areas were associated with decreased elasticity and adhesion to the AFM probe.© 2012 ASME