Effect of point mutations in Aβ aggregation

The major goal of the proposed work is to use a combination of ex situ and in situ atomic force microscopy (AFM) to characterize the structures and toxic biological properties of nanoscale aggregates formed by β-amyloid (Aβ) peptides containing various point mutations, which are involved implicated in variants of Alzheimer’s disease (AD).

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AD is a fatal neurodegenerative disorder that is the most common cause of dementia, and it is part of a growing group of diseases commonly classified as ‘protein misfolding’ or ‘conformational’ diseases. These diseases are defined by the rearrangement of a specific protein to a non-native conformation that promotes the formation and deposition of toxic, nanoscale aggregates within tissues or cellular compartments. In addition to AD, many other neurodegenerative diseases are associated with the accumulation of nanoscale protein aggregates, including Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and the prion encephalopathies. For the vast majority of these diseases, there are no widely effective preventative measures or therapeutic treatments.

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The precise mechanisms by which protein aggregates are toxic and cause neurodegeneration remains unclear. Deposited protein aggregates of each disease (generically called amyloid) typically are comprised of 2-6 protofilaments (diameter ~ 2-5 nm) that twist together into rope-like fibrillar structures (average diameter ~5-13 nm). Common biochemical characteristics of these fibrils such as detergent-insolubility, a cross-b sheet structure, protease resistance and the ability to bind lipophilic dyes such as congo red indicate a conserved mechanism of pathogenesis might be shared among these diverse diseases; however, increasing evidence suggests that fibrillar aggregates may be inert or even protective. Substantial effort to identify and characterize soluble oligomeric precursors to fibril formation has lead to an alternative hypothesis whereby small, potentially diffusible assemblies, such as spherical oligomers (~5-10 nm diameter), protofibrils (~8-12 nm diameter) and pore-like annular structures (~50-500 nm diameter), are the structural entities that mediate neurodegeneration. The ultimate objective of the amyloidogenic peptide AFM studies is to elucidate the physicochemical aspects and molecular mechanisms of pathological self-assembly of biological macromolecules that lead to toxicity.

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The role that cellular environment, and in particular surfaces, may play in dictating and stabilizing early oligomeric structures and the eventual formation of amyloid is poorly understood. In our preliminary data, we show that surface chemistries, ranging from hydrophilic and hydrophobic model surfaces to supported lipid membranes, influence Aβ aggregation both kinetically and morphologically. We further show that aggregation of an Aβ peptide containing the Arctic point mutation (E22E) significantly differs (both kinetically and morphologically) on model surfaces in comparison to Aβ-WT. Based on our studies, published high resolution structural studies of Aβ fibrils, and the identification of several point mutation clustered around position 22 of Aβ, we hypothesize that these point mutations alter the Aβ aggregation pathway and its interaction with cellular surfaces, resulting in different disease progression and phenotypes. Understanding the alterations in the Aβ aggregation pathway will provide insights into potential modes of toxicity.

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To begin to test this hypothesis we propose to use a combination of ex situ and in situAFM. This is currently the best available technique to examine the physicochemical properties, structures and dynamic assembly of aggregation-prone proteins, especially near surfaces. While ex situ AFM provides for the study of protein aggregation if free solution conditions, in situ AFM allows for real time observations of the aggregation of mutant Aβ peptides under near physiological conditions with nanoscale resolution on surfaces. Our long-term goal is to relate a nanoscale understanding of the structures and biological properties of different aggregates formed by mutant Aβ peptides to the process of neurodegeneration, and we anticipate that such studies will elicit the design of potential therapeutics to alter and/or manipulate mutant Aβ assemblies.

Figure: Schematic representation of amyloid precursor protein processing and point mutations in Aβ.  

Figure: Different chemical preparations of Aβ result in different aggregation behavior in solution and on lipid bilayers.