Alzheimer Disease Amyloid Beta Peptides: AFM and Voltammetric Characterization
Authors: Oliveira-Brett Ana Maria , Chiorcea-Paquim Ana-Maria , Enache Teodor Adrian 
Published: February 2020

Alzheimer’s disease (AD) is the most widespread form of dementia and was recently estimated to affect 44.4 million people worldwide. The main clinical manifestations of AD are loss of memory, cognitive skills, speech, and normative behaviour; however, accumulation of fibrillary amyloid beta (Aβ) peptides in plaques can occur and be observed decades before the AD symptoms.
Native human amyloid beta (Aβ) peptides exist as short isoforms, soluble monomers, varying from 39 to 43 amino acids. In AD, dysregulation and overproduction of Aβ peptides occurs, leading to a cascade mechanism in which the peptides undergo morphological changes, from soluble, monomeric random coil or α-helix conformations, into aggregated β-sheet structures, a fibrillization process that involves numerous intermediate structures. While at the beginning of ’90 it was considered that the neurodegeneration in AD is caused by the deposition of Aβ peptide plaques (large Aβ aggregates) in the brain tissue, nowadays, what appeared to be primarily responsible for neurodegeneration are these transient heterogeneous small soluble oligomers and protofibrils, which are difficult to be investigated due to the fast aggregation rate of Aβ peptide.
The time dependent structural modifications undergone by the synthetic human Aβ isoform peptides containing 40 and 42 amino acids, Aβ1-40 and Aβ1-42 peptides, i.e.the intermediary structures which appear during fibrilization, were studied by atomic force microscopy (AFM) at highly oriented pyrolytic graphite and differential pulse (DP) voltammetry at a glassy carbon electrode. The Aβ peptide solutions were incubated at room temperature, and in free chloride media, to limit the effect of temperature and salt concentration on the aggregation rate,.
In order to fully understand how the electron transfer reaction was influenced by both the specific amino acid side-chain position and the fibrilization process, the Aβ1-40 and Aβ1-42 peptides aggregation was compared with the aggregation of four Aβ peptides control sequences that: (i) do not aggregate (inverse Aβ40-1 and Aβ42-1 and rat Aβ1-40Rat peptides), and (ii) lack specific electroactive amino acids (mutants Aβ1-40Phe10 and Aβ1-40Nle35peptides).
The fibrilization process was investigated by AFM, via changes in the adsorption morphology, from initially random coiled structures, corresponding to the Aβ peptide monomers in random coil or in α-helix conformations, to aggregates, protofibrils and two types of fibrils, corresponding to the Aβ peptide in a β-sheet configuration. The hydrophobic carbon surface induced rapid changes in the Aβ peptides conformation, and differences between the adsorbed fibrils, formed at the carbon surface or in solution, were detected.
The Aβ peptides fibrilization was electrochemically detected via the decrease of the Aβ peptide electroactive amino acids oxidation peak currents, in the DP voltammograms, that occurred in a time dependent manner. Structurally, the Aβ peptide sequences contained five electroactive amino acid residues, one tyrosine (Tyr10), three histidines (His6, His13 and His14) and one methionine (Met35). DP voltammograms showed that, according to their primary structure, the Aβ peptides undergo oxidation in one or two steps, the first electron transfer reaction corresponding to the Tyr10 residue oxidation and the second to His6, His13, His14 and Met35 residues. The highest contribution to the second oxidation peak current was from the His13 residue, followed by His14 and His6 residues, while the Met35residue presented the lowest oxidation peak current. The Aβ peptides electron transfer reaction depended on the sequence hydrophobicity, on the position of the electroactive amino acid residues in the sequence (the residues close to N-termini giving the highest oxidation peak currents), being also strongly influenced by the Aβ peptide secondary structure.
The AFM and voltammetric results were consistent with the in vitro fibrilization mechanism, observed for different Aβ peptide sequences. The Aβ peptide monomers followed a nucleation process, leading to the formation of small nuclei. Once the nucleation process started, Aβ free monomers in solution were added to the nuclei and their dimensions grown forming various aggregate types, such as small oligomers and long fibrils with different morphologies. This polymerization reaction followed first-order kinetics, and is thermodynamically favored by the hydrophobic regions of Aβ peptides, as well as their β-sheet secondary structure. As fibrilization proceeded, the electroactive residues became more protected, and the transfer of electrons became more difficult.