David Teplow

Aberrant protein oligomerization is an important pathogenetic process in vivo. In Alzheimer's disease, the amyloid β-protein forms neurotoxic oligomers. The predominant in vivo Aβ alloforms, Aβ40 and Aβ42, have distinct oligomerization pathways. Aβ42 monomers oligomerize into pentamer/hexamer units which self-associate to form larger oligomers. Aβ40 does not form these paranuclei, a fact which may explain the particularly strong linkage of Aβ42 with AD. Here, we sought to determine the structural elements controlling paranucleus formation as a first step toward the development of strategies for treating AD. Because oxidation of Met35 is associated with altered Aβ assembly, we examined the role of Met35 in controlling Aβ oligomerization. Oxidation of Met35 in Aβ42 blocked paranucleus formation and produced oligomers indistinguishable in size and morphology from those produced by Aβ40. Systematic structural alterations of the Cγ 35-substituent group revealed that its electronic nature, rather than its size, was the factor controlling oligomerization pathway choice. Preventing assembly of toxic Aβ42 paranuclei through selective oxidation of Met35 thus represents a potential therapeutic approach for AD.

Hereditary cerebral haemorrhage with amyloidosis, Dutch type, is a cerebral amyloidosis characterized by prominent vascular deposits and fatal haemorrhages. The disorder is caused by a point mutation in codon 693 of the gene encoding the amyloid precursor protein, resulting in a Glu→Gln amino acid substitution at position 22 of the amyloid β-protein region. The pathogenetic mechanisms of HCHWA-D are unknown but could involve alterations in the proteolytic processing of APP and in amyloid fibril formation. We examined Aβ production and stability by using cultured human embryonic kidney 293 cells stably expressing wild-type or 'Dutch' APP. Radiosequencing and quantitative immunoprecipitation experiments showed that cells expressing Dutch APP secreted increased quantities of Aβ peptides beginning at Asp1, and of truncated peptides beginning at Val18 and Phe19. The ratio of levels of 4 kDa to 3 kDa peptides remained constant due to co-ordinate decreases in other peptide species. Novel truncated or elongated peptides were not observed. Pulse-chase experiments showed that the Dutch mutation did not affect the stability of the Aβ or p3 populations. These results are consistent with a disease process in which the Dutch mutation results in the production of Aβ peptides with enhanced propensities for fibrillogenesis, leading to accelerated vascular deposition and disease.

In a Flemish kindred, an Ala692 → Gly amino acid substitution in the amyloid β-protein precursor causes a form of early-onset Alzheimer's disease which displays prominent amyloid angiopathy and unusually large senile plaque cores. The mechanistic basis of this Flemish form of AD is unknown. Previous in vitro studies of amyloid β-protein production in HEK-293 cells transfected with cDNA encoding Flemish AβPP have shown that full-length [Aβ]and truncated [Aβ and Aβ] forms of Aβ are produced. In an effort to determine how these peptides might contribute to the pathogenesis of the Flemish disease, comparative biophysical and neurotoxicity studies were performed on wild-type and Flemish Aβ, Aβ and Aβ. The results revealed that the Flemish amino acid substitution increased the solubility of each form of peptide, decreased the rate of formation of thioflavin-T-positive assemblies, and increased the SDS-stability of peptide oligomers. Although the kinetics of peptide assembly were altered by the Ala21 → Gly substitution, all three Flemish variants formed fibrils, as did the wild-type peptides. Importantly, toxicity studies using cultured primary rat cortical cells showed that the Flemish assemblies were as potent a neurotoxin as were the wild-type assemblies. Our results are consistent with a pathogenetic process in which conformational changes in Aβ induced by the Ala21 → Gly substitution would facilitate peptide adherence to the vascular endothelium, creating nidi for amyloid growth. Increased peptide solubility and assembly stability would favour formation of larger deposits and inhibit their elimination. In addition, increased concentrations of neurotoxic assemblies would accelerate neuronal injury and death.