Molecular engineering of a secreted, highly homogeneous, and neurotoxic aβ dimer

Aβ oligomers play a key role in the pathophysiology of Alzheimer's disease. Research into structure-function relationships of Aβ oligomers has been hampered by the lack of large amounts of homogeneous and stable material. Using computational chemistry, we designed conservative cysteine substitutions in Aβ aiming at accelerating and stabilizing assembly of Aβ dimers by an intermolecular disulfide bond without changing its folding. Molecular dynamics simulations suggested that mutants AβS8C and AβM35C exhibited structural properties similar to those of Aβ wildtype dimers. Full length, mutant APP was stably expressed in transfected cell lines to study assembly of Aβ oligomers in the physiological, secretory pathway and to avoid artifacts resulting from simultaneous in vitro oxidation and aggregation. Biochemical and neurophysiological analysis of supernatants indicated that AβS8C generated an exclusive, homogeneous, and neurotoxic dimer, whereas AβM35C assembled into dimers, tetramers, and higher oligomers. Thus, molecular engineering enabled generation of bioactive, homogeneous, and correctly processed Aβ dimers in vivo.