Non-Esterified Fatty Acids Generate Distinct Low-Molecular Weight Amyloid-β (Aβ42) Oligomers along Pathway Different from Fibril Formation

Abstract

Amyloid-β (Aβ) peptide aggregation is known to play a central role in the etiology of Alzheimer’s disease (AD). Among various aggregates, low-molecular weight soluble oligomers of Aβ are increasingly believed to be the primary neurotoxic agents responsible for memory impairment. Anionic interfaces are known to influence the Aβ aggregation process significantly. Here, we report the effects of interfaces formed by medium-chain (C9–C12), saturated non-esterified fatty acids (NEFAs) on Aβ42 aggregation. NEFAs uniquely affected Aβ42 aggregation rates that depended on both the ratio of Aβ:NEFA as well the critical micelle concentration (CMC) of the NEFAs. More importantly, irrespective of the kind of NEFA used, we observed that two distinct oligomers, 12–18 mers and 4–5 mers were formed via different pathway of aggregation under specific experimental conditions: (i) 12–18 mers were generated near the CMC in which NEFAs augment the rate of Aβ42 aggregation towards fibril formation, and, (ii) 4–5 mers were formed above the CMC, where NEFAs inhibit fibril formation. The data indicated that both 12–18 mers and 4–5 mers are formed along an alternate pathway called ‘off-pathway’ that did not result in fibril formation and yet have subtle structural and morphological differences that distinguish their bulk molecular behavior. These observations, (i) reflect the possible mechanism of Aβ aggregation in physiological lipid-rich environments, and (ii) reiterate the fact that all oligomeric forms of Aβ need not be obligatory intermediates of the fibril formation pathway.

Specific Soluble Oligomers of Amyloid-β Peptide Undergo Replication and Form Non-fibrillar Aggregates in Interfacial Environments

“…This property has been recently demonstrated by Kayed et al (28), who reported that specific soluble oligomers called PFOs were able to seed their own replication upon interacting with monomeric A␤. The property of replication could be a manifestation of the unique structure of the oligomeric assembly and more importantly may complement our previously reported hypothesis that such oligomers are formed along a pathway different from fibril formation (1,19). To explore this, a 20 M sample of freshly purified, "seedfree" A␤42 was incubated with 0.4 M isolated LFAOs as seeds (2% molar ratio) at room temperature.…”

“…Briefly, 1.5-2 mg of peptide was dissolved in 0.5 ml of 30 mM NaOH and stored for 15 min at room temperature prior to size exclusion chromatography (SEC) onto a 1 ϫ 30-cm Superdex-75 HR 10/30 column (GE Healthcare) attached to an ÄKTA FPLC system (GE Healthcare) to remove any preformed aggregates as reported previously (1). The column was preequilibrated in 20 mM Tris-HCl (pH 8.0) at 25°C and run at a flow rate of 0.5 ml/min.…”

“…Preparation and Isolation of LFAOs-LFAOs were prepared and isolated as described previously (1). Briefly, the freshly purified A␤42 (50 M) was incubated with 50 mM NaCl and 5 mM C12:0 fatty acid at 37°C for 48 h. After 48 h, the sample was subjected to SEC, and the fractions corresponding to the peak near the void volume (V 0 ) were collected.…”

“…Gels were electroblotted onto 0.45-m nitrocellulose membranes (BioTrace TM NT, Pall Life Sciences). Blots were boiled in a microwave oven in PBS for 2 min and blocked overnight with 1ϫ PBS containing 5% nonfat dry milk and 0.1% Tween 20 before probing (1-2 h) with 1:1000 -1:2500 dilutions of Ab9 monoclonal antibody, which detects amino acid residues of A␤ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Blots were then incubated with anti-mouse horseradish peroxide (HRP) conjugate and developed with ECL reagent (Thermo Scientific).…”

“…Unfortunately, there is a paucity of structural, mechanistic, and pathological understanding of LMW oligomers in general and more so with the oligomers that are not formed along the fibril formation pathway. In our previous work, we reported that non-esterified fatty acid (NEFA) interfaces can induce multiple pathways of A␤ aggregation in carefully controlled conditions of NEFA concentrations and A␤-to-NEFA molar ratios (1). More importantly, we reported that two distinct off-pathway oligomers of A␤42 can be generated: 4 -5-mers (small fatty acid-derived oligomers) and 12-18-mers (large fatty acid-derived oligomers (LFAOs)).…”