Aggregation and catabolism of disease-associated intra-Aβ mutations: reduced proteolysis of AβA21G by neprilysin

Abstract

Five point mutations within the amyloid β-protein (Aβ) sequence of the APP gene are associated with hereditary diseases which are similar or identical to Alzheimer's disease and encode: the A21G (Flemish), E22G (Arctic), E22K (Italian), E22Q (Dutch) and the D23N (Iowa) amino acid substitutions. Although a substantial body of data exists on the effects of these mutations on Aβ production, whether or not intra-Aβ mutations alter degradation and how this relates to their aggregation state remain unclear. Here we report that the E22G, E22Q and the D23N substitutions significantly increase fibril nucleation and extension, whereas the E22K substitution exhibits only an increased rate of extension and the A21G substitution actually causes a decrease in the extension rate. These substantial differences in aggregation together with our observation that aggregated wild type Aβ(1-40) was much less well degraded than monomeric wild type Aβ(1-40), prompted us to assess whether or not disease-associated intra-Aβ mutations alter proteolysis independent of their effects on aggregation. Neprilysin (NEP), insulin degrading enzyme (IDE) and plasmin play a major role in Aβ catabolism, therefore we compared the ability of these enzymes to degrade wild type and mutant monomeric Aβ peptides. Experiments investigating proteolysis revealed that all monomeric peptides are degraded similarly by IDE and plasmin, but that the Flemish peptide was degraded significantly more slowly by NEP than wild type Aβ or any of the other mutant peptides. This finding suggests that resistance to NEP-mediated proteolysis may underlie the pathogenicity associated with the A21G mutation.

Preparation and characterization of toxic Aβ aggregates for structural and functional studies in Alzheimer's disease research

“…2j and 4d). These observations are in accordance with previously published reports showing that Arctic (E22G) and Dutch (E22Q) mutations in Aβ significantly increase fibril nucleation and growth, whereas the Flemish (A21G) mutation decreases fibril growth 85 .…”

“…Figure 4 summarizes the results of these studies. Aβ40-Arctic (E22G) exhibits faster aggregation kinetics than does wt Aβ40 and has a higher tendency to form and accumulate protofibrils 18,36,85 . Accordingly, we found that dissolving synthetic Aβ40-Arctic in DMSO (Step 1B) resulted in a solution that is highly enriched with protofibrils and monomeric Aβ.…”

The Familial British Dementia Mutation Promotes Formation of Neurotoxic Cystine Cross-linked Amyloid Bri (ABri) Oligomers

“…Thioflavin T Binding Assay-Aggregation of Bri and ABri was monitored using a continuous thioflavin T (ThT) binding assay (21). Peptides were diluted to the desired concentration with the appropriate SEC elution buffer and ThT was added from a ϫ100 stock solution to a final concentration of 20 M. Aliquots (120 l) of the peptide solutions were then dispensed into the wells of an ice-cold 96-well black microtiter plate (Nunc, Roskilde, Denmark) and read immediately.…”

“…Electron Microscopy-Peptide samples were prepared as for the ThT binding assay, but in the absence of ThT, and analyzed by negative contrast microscopy as previously described (21 Circular Dichroism (CD) Spectroscopy-Peptide solutions in 20 mM NaP, pH 8.0, were analyzed at 22°C in a 1-mm quartz cuvette (Starna Scientific, Hainault, UK) using a J-810 JASCO spectropolarimeter (JASCO Corp., Tokyo, Japan). Spectra were collected from three data accumulations between 190 and 260 nm with 10 nm/min continuous scanning and 1 nm bandwidth.…”

Phosphorylation of Amyloid-β Peptide at Serine 8 Attenuates Its Clearance via Insulin-degrading and Angiotensin-converting Enzymes

“…In particular, NEP, ACE, and plasmin are considered to be physiologically and pathologically relevant in sporadic AD. NEP is a membrane-bound zinc metallopeptidase localized at the cell surface and in cytoplasmic vesicles preferentially hydrolyzing extracellular oligopeptides on the amino side of hydrophobic residues (53) and has been shown to be capable of degrading A␤ both in vivo (54,55) and in vitro (55)(56)(57). NEP is also reported to be expressed on neuronal pre-and postsynaptic membranes (52).…”