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.

Introduction

Convergent evidence suggests that accumulation of the amyloid β-protein (Aβ) and its subsequent aggregation initiates a complex cascade that culminates in Alzheimer's disease (AD) (LaFerla and Oddo, 2005, Walsh and Selkoe, 2004). Five point mutations within the Aβ sequence that are associated with hereditary diseases similar or identical to AD are clustered around the central hydrophobic core of Aβ and include: the A21G Flemish mutation, E22K Italian mutation, E22G Arctic mutation, E22Q Dutch mutation and the D23N Iowa mutation (Fig. 1). These mutations have the potential to impact upon all factors known to regulate Aβ monomer levels, namely production, degradation and aggregation. To date, no simple correlation between Aβ production or aggregation and disease phenotype has emerged, but it, was suggested that the pathogenic effect of the Dutch, Flemish, Italian and Arctic mutations arise as a consequence of their resistance to proteolysis (Tsubuki et al., 2003).

A large number of proteases have been implicated in the catabolism of Aβ, but of these neprilysin (NEP, EC 3.4.24.11) and insulin-degrading enzyme (IDE, EC 3.4.22.11) are the most studied (Eckman and Eckman, 2005, Selkoe, 2001, Turner et al., 2004). NEP is a membrane-bound zinc-metallopeptidase that exists as an ectoenzyme preferentially hydrolysing extracellular oligopeptides on the amino side of hydrophobic residues (Carson and Turner, 2002). NEP has been shown by numerous investigators to be capable of degrading Aβ both in vivo (Iwata et al., 2004) and in vitro (Howell et al., 1995, Kanemitsu et al., 2003, Liu et al., 2007, Shirotani et al., 2001), and its physiologic role has been demonstrated by the finding that genetic ablation of NEP causes elevation of endogenous Aβ (Iwata et al., 2001), whereas transgenic or viral expression of NEP causes a lowering of cerebral Aβ (El-Amouri et al., 2007, Hemming et al., 2007, Iwata et al., 2004, Leissring et al., 2003, Marr et al., 2003). IDE is a zinc-metalloprotease which shows no obvious primary amino acid sequence specificity but has been proposed to recognize a conformation that is prone to conversion to β-sheet structure (Kurochkin, 1998). As with NEP, support for the physiological importance of IDE in regulating Aβ levels comes from the findings that genetic deletion of IDE in mice (Farris et al., 2003, Miller et al., 2003) leads to elevated levels of cerebral Aβ, whereas transgenic over-expression of IDE causes a decrease in brain Aβ levels (Leissring et al., 2003). Unlike NEP and IDE, plasmin (EC 3.4.21.7) does not appear to contribute significantly to the normal catabolism of Aβ (Tucker et al., 2004), but rather seems to play a role in the diseased brain. Plasmin is a serine protease that is produced from its inactive precursor, plasminogen, by the action of tissue-type plasminogen activator (tPA), and mice lacking either tPA or plasminogen clear injected Aβ much less well than wild type mice (Melchor et al., 2003). Moreover, recent studies indicate that high levels of Aβ cause a decrease in tPA activity suggesting that excessive accumulation of Aβ results, in part, due to a loss of plasmin (Cacquevel et al., 2007).

In order to investigate the possibility that intra-Aβ mutations mediate their effect through a common pathogenic mechanism, namely resistance to proteolysis, we examined the ability of NEP, IDE and plasmin to degrade both wild type (wt) Aβ(1–40) and Aβ(1–40) bearing disease-associated single amino acid substitutions (Fig. 1). Here we show that the ability of these proteases to degrade Aβ is strongly retarded by aggregation but that when Aβ peptides are presented in their unaggregated, monomeric state, they are efficiently degraded by NEP, IDE and plasmin, with one exception. The Aβ(1–40)A21G peptide is degraded more slowly by NEP than wt Aβ(1–40) or any of the other mutant peptides studied. This resistance to NEP-mediated proteolysis may represent one mechanism by which the A21G mutation causes increased cerebral accumulation of Aβ. On the other hand, our data suggest that resistance to proteolysis by the major known Aβ-degrading enzymes cannot explain the effects of the other four disease-associated intra-Aβ mutations.