Elucidating the molecular determinants of Aß aggregation with deep mutational scanning

Despite the importance of Aß aggregation in Alzheimer's disease etiology, our understanding of the sequence determinants of aggregation is sparse and largely derived from in vitro studies. For example, in vitro proline and alanine scanning mutagenesis of Aß40 proposed core regions important for aggregation. However, we lack even this limited mutagenesis data for the more disease-relevant Aß42. Thus, to better understand the molecular determinants of Aß42 aggregation in a cell-based system, we combined a yeast DHFR aggregation assay with deep mutational scanning. We measured the effect of 791 of the 798 possible single amino acid substitutions on the aggregation propensity of Aß42. We found that ~75% of substitutions, largely to hydrophobic residues, maintained or increased aggregation. We identified 11 positions at which substitutions, particularly to hydrophilic and charged amino acids, disrupted Aß aggregation. These critical positions were similar but not identical to critical positions identified in previous Aß mutagenesis studies. Finally, we analyzed our large-scale mutagenesis data in the context of different Aß aggregate structural models, finding that the mutagenesis data agreed best with models derived from fibrils seeded using brain-derived Aß aggregates. Overall design: Examination of Aß mutations in 3 replicates