The Ratio of Monomeric to Aggregated Forms of Aβ40 and Aβ42 Is an Important Determinant of Amyloid-β Aggregation, Fibrillogenesis, and Toxicity

Abstract

Aggregation and fibril formation of amyloid-␤ (A␤) peptides A␤40 and A␤42 are central events in the pathogenesis of Alzheimer disease. Previous studies have established the ratio of A␤40 to A␤42 as an important factor in determining the fibrillogenesis, toxicity, and pathological distribution of A␤. To better understand the molecular basis underlying the pathologic consequences associated with alterations in the ratio of A␤40 to A␤42, we probed the concentration-and ratio-dependent interactions between well defined states of the two peptides at different stages of aggregation along the amyloid formation pathway. We report that monomeric A␤40 alters the kinetic stability, solubility, and morphological properties of A␤42 aggregates and prevents their conversion into mature fibrils. A␤40, at approximately equimolar ratios (A␤40/A␤42 ϳ 0.5-1), inhibits (>50%) fibril formation by monomeric A␤42, whereas inhibition of protofibrillar A␤42 fibrillogenesis is achieved at lower, substoichiometric ratios (A␤40/A␤42 ϳ 0.1). The inhibitory effect of A␤40 on A␤42 fibrillogenesis is reversed by the introduction of excess A␤42 monomer. Additionally, monomeric A␤42 and A␤40 are constantly recycled and compete for binding to the ends of protofibrillar and fibrillar A␤ aggregates. Whereas the fibrillogenesis of both monomeric species can be seeded by fibrils composed of either peptide, A␤42 protofibrils selectively seed the fibrillogenesis of monomeric A␤42 but not monomeric A␤40. Finally, we also show that the amyloidogenic propensities of different individual and mixed A␤ species correlates with their relative neuronal toxicities. These findings, which highlight specific points in the amyloid peptide equilibrium that are highly sensitive to the ratio of A␤40 to A␤42, carry important implications for the pathogenesis and current therapeutic strategies of Alzheimer disease.Alzheimer disease is a progressive neurodegenerative disorder characterized by age-related accumulation of amyloid-␤ (A␤) 2 proteins in the form of diffuse and neuritic plaques in regions of the brain that are affected by the disease (1-4). The discovery of A␤ fibrils as principal constituents of amyloid plaques led to the emergence of the amyloid hypothesis, which implicates the aggregation of A␤ as the primary trigger for a cascade of pathogenic events culminating in neurodegeneration and development of AD (1, 5-7). A␤ proteins are produced in neuronal and non-neuronal cells as a result of sequential proteolytic cleavage of the type I transmembrane amyloid precursor protein (APP) by ␤-and ␥-secretases (8 -12). Depending on the site of APP cleavage by ␥-secretase, A␤ proteins of various chain lengths are generated (13-16). The predominant A␤ species in human plasma and CSF, as well as in conditioned media of APP-expressing cells, is A␤40 (ϳ90%) followed by A␤42 (ϳ10%). Despite the preponderance of A␤40, in vivo studies reveal that A␤42 is a major constituent of amyloid plaques and suggest that A␤42 aggregation plays a critical role in the initiation of plaque f...

Potent amyloidogenicity and pathogenicity of Aβ43

“…In agreement with Aβ42 having higher hydrophobicity and higher toxicity than Aβ40 in vitro and in vivo, a large number of reports also show that Aβ42 contributes to synaptic dysfunctions [31][32][33][34] . Herein, we compared the toxicity of Aβ40, Aβ42 and Aβ43.…”

Biometals and Their Therapeutic Implications in Alzheimer's Disease

“…Traditionally, it was believed that Aβ1-42 is predominately responsible for toxicity observed in AD. Two key observations support this theory: 1) familial AD mutations in APP elevate the expression of Aβ1-42 [24,25]; and 2) Aβ1-42 is more prone to aggregation and is more toxic than Aβ1-40 [26,27]. However, it was reported that pyroglutamate-Aβ, which is also presented in AD plaques, could also be the toxic isoform of Aβ [28][29][30][31].…”

Preparation and characterization of toxic Aβ aggregates for structural and functional studies in Alzheimer's disease research

“…Whether some of these species represent off-or on-pathway intermediates to amyloid fibril formation remains unknown. Some of the key distinguishing features of protofibrillar aggregates are that they (i) contain a mixture of different secondary structure elements with a predominance of β-sheet 20 ; (ii) bind amyloid-specific dyes, such as Congo red and Thioflavin-T (ThT), although less strongly than mature fibrils [20][21][22] ; and (iii) are less stable than mature amyloid fibrils and exist in equilibrium with monomeric Aβ 20,21,23 . In the presence of monomeric Aβ, all these species convert to mature amyloid fibrils by monomer addition.…”

“…Treatment of lyophilized Aβ with strong acids and bases to disrupt preformed aggregates and enhance solubility 52 , filtration through LMW cutoff filters 53 , photo-induced crosslinking of unmodified proteins 17 , density gradient centrifugation 54 and size exclusion chromatography (SEC) 13,14,17 have all been used to prepare soluble aggregates of Aβ and yield preparations that vary in size and morphology distribution. SEC, in particular, offers several advantages: (i) a variety of column matrices with different separation capacities are readily available and can be used in isolation or in combination with other columns to obtain high-resolution separation and fractions containing Aβ aggregates of defined size distribution; (ii) generally, the columns are equipped with filters at the top that allow for the removal of fibrillar (or insoluble) material from the injected sample, thus ensuring that the Aβ fractions are free of fibrillar seeds; (iii) if SEC is coupled to a light-scattering detector, accurate determination of Aβ aggregates' size distribution becomes possible 20 ; (iv) in analytical mode, SEC is a valuable tool to monitor early events in amyloid formation and quantification of monomer and/or protofibril loss during the time course of fibril formation 21 ; and (v) by choosing proper running conditions (i.e., buffer pH and contents), fractions are obtained in solution conditions suitable for biological systems, free of harmful or undesired substances (e.g., organic solvents and so on). SEC has also been successfully used to isolate Aβ oligomers (such as dimers and trimers) and high-molecular-weight aggregates (including protofibrils and amorphous aggregates) from complex cell culture media 55 or from postmortem AD brain extracts 56 .…”

“…Although it is one of the most commonly used methods for isolating and purifying Aβ oligomers 13,21,23 , SEC suffers from certain drawbacks that should be taken into consideration when using this technique: (i) some aggregates, because of their variable stability, may dissociate on interacting with column matrix and elute as LMW species 21 ; (ii) there may be a low resolution of separation if the sample is composed of a continuum of interconverting aggregates of related sizes; (iii) the samples are diluted in concentration after SEC fractionation and may not be suitable for use in certain experiments requiring high Aβ concentration; (iv) long duration of separation may allow interconversion and mask changes taking place within the time scale of minutes or less; (v) interactions with column matrices and separation may also be affected by factors other than size, such as aggregate conformation and tertiary structure; and (vi) nonspecific adsorption of hydrophobic peptides (such as Aβ) to the column matrix might result in a low yield of purified material.…”