P1-391: Monomeric Aβ peptides bearing the Flemish A21G mutation are less readily degraded by neprilysin than wild type Aβ

Abstract

Mutations within the Aβ sequence of the APP gene are associated with hereditary disorders which are similar or identical to Alzheimer's disease and encode the A21G (Flemish), E22K (Italian), E22G (Arctic), E22Q (Dutch) and the D23N (Iowa) amino acid substitutions. Although substantial data exist on the effects of these mutations on APP processing, whether or not peptides bearing intra–Aβ mutations show alter degradation and/or aggregation remains unclear. We examined the ability of neprilysin (NEP), insulin degrading enzyme (IDE) and plasmin to degrade cell–derived and synthetic wild type (wt) and mutant Aβ peptides. Media from HEK cells transiently transfected with WT huAPP695 or APP bearing one of the disease–associated point mutations was incubated for 24h ± protease. The reaction was stopped by addition of protease inhibitors and Aβ content assessed by immunoprecipitation/western blotting. Synthetic peptides (40μM) were dissolved in 50mM Tris, pH7.4, aggregates removed by using Micron 3 filters and peptide concentrations adjusted to 20μM. Aggregation of Aβ peptide was achieved by incubation at 37°C for 1 week and was confirmed by Congo Red binding and electron microscopy. Peptides were incubated for varying periods of time with proteases and digestion arrested by the addition of an inhibitor cocktail. The extent of degradation was assessed by HPLC analysis and/or densitometric analysis of peptides separated by SDS–PAGE. Each protease was capable of degrading both wt and mutant (aggregate–free) synthetic and cell–derived Aβ peptides. Moreover, experiments investigating the rates of cleavage of synthetic Aβ peptides revealed that all peptides are degraded similarly by IDE and plasmin, but that the Flemish peptide was degraded significantly more slowly by NEP than wt or other mutant peptides. Importantly, we show that the ability of these proteases to degrade Aβ is strongly retarded by aggregation. However, the decreased proteolysis of the G21 peptide by NEP cannot be attributed to its aggregation state as we also find that the G21 peptide aggregates less readily than the WT peptide. The resistance of the G21 peptide to proteolysis by NEP may represent one mechanism by which this mutation causes increased intracerebral accumulation of Aβ.