Mutations Enhance the Aggregation Propensity of the Alzheimer’s Aβ Peptide

Abstract

Aggregation of the amyloid β (Aβ) peptide plays a key role in the molecular etiology of Alzheimer’s disease. Despite the importance of this process, the relationship between the sequence of Aβ and the propensity of the peptide to aggregate has not been fully elucidated. The sequence determinants of aggregation can be revealed by probing the ability of amino acid substitutions (mutations) to increase or decrease aggregation. Numerous mutations that decrease aggregation have been isolated by laboratory-based studies. In contrast, very few mutations that increase aggregation have been reported, and most of these were isolated from rare individuals with early-onset familial Alzheimer’s disease. To augment the limited data set of clinically derived mutations, we developed an artificial genetic screen to isolate novel mutations that increase aggregation propensity. The screen relies on the expression of Aβ–green fluorescent protein fusion in Escherichia coli. In this fusion, the ability of the green fluorescent protein reporter to fold and fluoresce is inversely correlated with the aggregation propensity of the Aβ sequence. Implementation of this screen enabled the isolation of 20 mutant versions of Aβ with amino acid substitutions at 17 positions in the 42-residue sequence of Aβ. Biophysical studies of synthetic peptides corresponding to sequences isolated by the screen confirm the increased aggregation propensity and amyloidogenic behavior of the mutants. The mutations were isolated using an unbiased screen that makes no assumptions about the sequence determinants of aggregation. Nonetheless, all 16 of the most aggregating mutants contain substitutions that reduce charge and/or increase hydrophobicity. These findings provide compelling evidence supporting the hypothesis that sequence hydrophobicity is a major determinant of Aβ aggregation.

Introduction

Postmortem studies of the brains of patients with Alzheimer’s disease (AD) reveal significant quantities of senile plaque. Biochemical analyses of amyloid fibrils in these plaques indicate that amyloid β (Aβ) peptides are the primary components of the fibrils.^1,2 These Aβ peptides are produced by proteolytic cleavage of the amyloid precursor protein (APP). Because cleavage of APP can occur at several sites, Aβ peptides occur in several different lengths, with the 40-residue Aβ40 and the 42-residue Aβ42 being the most abundant. Although Aβ40 is produced in larger amounts, Aβ42 aggregates more readily, and increased ratios of Aβ42/Aβ40 have been observed in the brains of AD patients.^3,4

The molecular details of Aβ aggregation and the mechanism through which this aggregation causes AD are not fully understood. Nonetheless, a large number of studies support the “amyloid cascade” hypothesis,⁵ which posits that accumulation of aggregated Aβ initiates a multistep cascade that ultimately leads to AD. Several lines of evidence support this hypothesis. First, genetic studies show that several forms of familial Alzheimer’s disease (FAD) are caused by mutations either in APP or in enzymes that process APP. Both classes of mutations increase the production and/or aggregation of Aβ42 and lead to the early onset of AD.6, 7, 8 Second, early-onset AD is also observed in Down syndrome, wherein trisomy of chromosome 21, which encodes APP, leads to increased production of Aβ42.9, 10, 11, 12 Third, construction of transgenic animals, including nematodes, fruit flies, and mice, has demonstrated that introduction of APP and/or Aβ produces cognitive and behavioral impairments.13, 14, 15 Finally, studies of enzymes that metabolize Aβ confirm the relationship between Aβ accumulation and AD. For example, decreased expression of insulin-degrading enzyme or neprilysin, both of which are known to degrade Aβ, leads to increased accumulation of Aβ and, ultimately, to AD. In contrast, overexpression of these enzymes reduces Aβ levels and attenuates Aβ-related memory deficit.16, 17, 18, 19, 20 Together, these studies provide a compelling case for the role of Aβ aggregation in the pathogenesis of AD.

Although Aβ accumulation and aggregation clearly play a role in AD, recent studies indicate that the insoluble fibrils themselves may not be the toxic species. Instead, it now appears that oligomers or intermediates in the aggregation process are the major toxic species in AD. For example, Lesne et al. demonstrated that extracellular accumulation of a 56-kDa soluble oligomer of Aβ42 (presumably a dodecamer) causes memory deficits in transgenic mice.²¹ Similarly, Walsh et al. demonstrated that small oligomers of Aβ inhibit long-term potentiation of neurons, resulting in memory deficits, whereas monomers or fibrils of Aβ show no effect.^22,23

To enhance understanding of the molecular etiology of AD, we and others have probed the amino acid sequence determinants of Aβ aggregation.24, 25, 26, 27, 28, 29 Previously, our laboratory developed an artificial genetic system to screen for mutations in the sequence of Aβ42 that prevent aggregation.²⁴ By using this system to screen randomly generated libraries of mutations, we demonstrated that replacement of nonpolar residues with polar residues inhibited aggregation and caused dramatic increases in the solubility of Aβ42. More recently, we also showed that at many positions in the Aβ42 sequence, random mutations of nonpolar residues to other nonpolar residues had little or no effect, thereby demonstrating that “generic” hydrophobic residues—rather than particular nonpolar side chains—are sufficient to promote the aggregation of Aβ42.

Complementary studies by both Williams et al. and Morimoto et al. used proline-scanning mutagenesis to demonstrate that disruption of the β-sheet regions of Aβ decreases aggregation propensity.^27,28 Thus, mutagenesis experiments have shown that both sequence hydrophobicity and β-sheet propensity are key determinants of aggregation. Experimental and bioinformatics approaches by Chiti et al. support these findings, both for Aβ42 and for other amyloidogenic proteins.²⁹

In addition to the laboratory-generated mutations described above, naturally occurring mutants in the human population provide insights into the sequence determinants of Aβ aggregation. Several examples of familial early-onset AD are caused by mutations in Aβ that increase its aggregation propensity. For example, the Dutch mutant, Glu22 → Gln, increases Aβ aggregation and leads to early-onset AD.³⁰

Laboratory-based studies of the sequence determinants of Aβ aggregation have focused primarily on mutations that decrease aggregation. In contrast, genetic studies of early-onset FAD in the human population have discovered mutations that increase aggregation propensity. In this latter class, however, only a few mutants are known, presumably because those mutations that cause the most dramatic increase in aggregation are lethal and do not survive in the population. To augment the clinically isolated collection of aggregation-prone mutants in Aβ, we have developed an unbiased screen for mutations that increase aggregation. Here we describe the implementation of this screen to isolate a collection of mutations that increase aggregation propensity beyond that of wild-type Aβ.