Suppression of Oligomer Formation and Formation of Non‐Toxic Fibrils upon Addition of Mirror‐Image Aβ42 to the Natural l‐Enantiomer

Abstract

Racemates often have lower solubility than enantiopure compounds, and mixing of enantiomers can enhance aggregation propensity of peptides. Amyloid β (Aβ) 42 is an aggregation-prone peptide, believed to play a key role in Alzheimer’s Disease. Soluble Aβ42 aggregation intermediates (oligomers) have emerged as particularly neurotoxic. We hypothesized that addition of mirror image (D-) Aβ42 should reduce the concentration of toxic oligomers formed by natural (L-) Aβ42. We synthesized L- and D-Aβ42 and found their equimolar mixing to lead to accelerated fibril formation. Confocal microscopy with fluorescently labeled analogs of the enantiomers showed their co-localization in racemic fibrils. Reflecting enhanced fibril formation propensity, racemic Aβ42 was less prone to form soluble oligomers. This resulted in protection of cells from toxicity of L-Aβ42 at concentrations ranging up to 50 µM. In summary, mixing of Aβ42 enantiomers induces accelerated formation of non-toxic fibrils.

New insights into differential aggregation of enantiomerically pure and racemic Aβ40 systems

“…To probe further the acceleration of fibril formation upon enantiomer mixing, 1 H NMR experiments were conducted. It was anticipated that the relatively high solubility of Aβ40, as compared with Aβ42, which had been the subject of the majority of our experiments in the past, would be an asset for these experiments. An enantiopure aqueous solution of L‐Aβ40 displayed a well‐defined 1 H NMR spectrum, and showed no evidence of precipitation (298 K; 160 μM L‐Aβ40; 9:1 H 2 O/D 2 O mixture, phosphate‐buffered to pH 7.4; Figure A).…”

“…Taken together, findings presented here corroborate our initial observations made with the Aβ42 system, further supporting the notion that the racemic Aβ system is fundamentally different from the enantiopure one. The morphological differences determined for the Aβ40 isoform using TEM were more pronounced and well defined than those observed with Aβ42 . This allowed for a quantitative fibril analysis, revealing an approximately 2‐fold narrowing of rac ‐Aβ40 fibrils along with the disappearance of the helical twisting that was often present with enantiopure Aβ40.…”

“…In continuation of our research efforts on the racemic aggregating Aβ system, we performed experiments with the two enantiopure Aβ40 peptides and their racemic mixture. The two Aβ40 enantiomers were synthesized and purified, using our previously published protocols and procedures . The two peptides displayed reciprocal CD bands of comparable intensity, as expected for mirror‐image peptides, and mixing of the enantiomers in a 1:1 ratio led to disappearance of that band (Figure A).…”

“…Mimicking conditions of ThT fibril formation assays, L‐, D‐, or rac ‐Aβ40 was incubated for 24 h with shaking at 37 °C. Samples were subsequently transferred onto TEM grids, stained with uranyl acetate and imaged, following previously published protocols and procedures . Fibrils obtained from L‐ or D‐Aβ40 had an appearance that was distinct from those derived from racemic Aβ40.…”

“…Peptides were synthesized by Fmoc HOBt//DIC/Piperidine chemistry on a Liberty One CEM peptide synthesizer equipped with a CEM discovery microwave, as previously described . All syntheses were performed at a 0.1‐mmol scale.…”