Postmortem studies of the brains of Alzheimer’s disease (AD) patients reveal significant quantities of senile plaque. Biochemical analyses of the amyloid fibrils in these plaques indicate that Aβ peptides are the primary components of the fibrils1,2
. 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” hypothesis5
, which posits that accumulation of aggregated Aβ initiates a multi-step cascade that ultimately leads to Alzheimer’s disease. 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 the enzymes that process APP. Both classes of mutations increase the production and/or aggregation of Aβ42, and lead to the early onset of AD6–8
. Second, early onset AD is also observed in Down syndrome, wherein trisomy of chromosome 21, which encodes APP, leads to increased production of Aβ429–12
. Third, construction of transgenic animals, including nematodes, fruit flies, and mice, has demonstrated that introduction of APP and/or Aβ produces cognitive and behavioral impairment13–15
. Finally, studies of enzymes that metabolize Aβ confirm the relationship between Aβ accumulation and AD. For example, decreased expression of insulin degrading enzyme (IDE) 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 16–20
. Together, these studies provide a compelling case for the role of Aβ aggregation in the pathogenesis of Alzheimer’s disease.
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 mice21
. 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 showed no effect22,23
To enhance understanding of the molecular etiology of AD, we and others have probed the amino acid sequence determinants of Aβ aggregation 24–29
. Previously, our lab developed an artificial genetic system to screen for mutations in the sequence of Aβ42 that prevent aggregation24
. 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 Wetzel’s group and Shirasawa’s group 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 proteins29
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 AD30
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 later 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.