Effects of the English (H6R) and Tottori (D7N) Familial Alzheimer Disease Mutations on Amyloid β-Protein Assembly and Toxicity

Abstract

Mutations in the amyloid ␤-protein (A␤) precursor gene cause autosomal dominant Alzheimer disease in a number of kindreds. In two such kindreds, the English and the Tottori, the mutations produce amyloid ␤-proteins containing amino acid substitutions, H6R and D7N, respectively, at the peptide N terminus. To elucidate the structural and biological effects of the mutations, we began by examining monomer conformational dynamics and oligomerization. Relative to their wild type homologues, and in both the A␤40 and A␤42 systems, the English and Tottori substitutions accelerated the kinetics of secondary structure change from statistical coil 3 ␣/␤ 3 ␤ and produced oligomer size distributions skewed to higher order. This skewing was reflected in increases in average oligomer size, as measured using electron microscopy and atomic force microscopy. Stabilization of peptide oligomers using in situ chemical crosslinking allowed detailed study of their properties. Each substitution produced an oligomer that displayed substantial ␤-strand (H6R) or ␣/␤ (D7N) structure, in contrast to the predominately statistical coil structure of wild type A␤ oligomers. Mutant oligomers functioned as fibril seeds, and with efficiencies significantly higher than those of their wild type homologues. Importantly, the mutant forms of both native and chemically stabilized oligomers were significantly more toxic in assays of cell physiology and death. The results show that the English and Tottori mutations alter A␤ assembly at its earliest stages, monomer folding and oligomerization, and produce oligomers that are more toxic to cultured neuronal cells than are wild type oligomers. Alzheimer disease (AD)2 is characterized by the accumulation of intraneuronal filaments formed by the microtubuleassociated protein Tau and by extracellular parenchymal and vascular amyloid deposits largely comprising the amyloid ␤-protein (A␤) (1). A␤ is produced by sequential proteolytic cleavage of the amyloid ␤-protein precursor (APP) by ␤-and ␥-secretase (2). In some kindreds, AD occurs in an autosomal dominant manner because of mutations in the genes encoding APP or ␥-secretase (3). These mutations alter the absolute or relative amounts of A␤40 or A␤42 that are produced or they alter peptide primary structure (4 -6).The first intra-A␤ missense mutations that were observed all clustered within the APP gene region encoding amino acids Ala 21 -Asp 23 of A␤. These mutations included the Flemish (A21G), Dutch (E22Q), Italian (E22K), Arctic (E22G), and Iowa (D23N) (7-11). Each of these mutations alters peptide assembly or metabolism. For example, the Flemish mutation causes a decrease in the fibril extension rate (12). The Dutch, Italian, and Iowa mutations cause disease with fulminant vascular pathology (8, 9, 11). The Arctic mutation causes early onset AD that involves enhanced protofibril formation (13). Recently, a new mutation in the Ala 21 -Asp 23 region, causing a deletion of Glu 22 (⌬E22), was reported (14). This deletion causes enhanced peptide oligomerization b...

High-speed atomic force microscopy reveals structural dynamics of amyloid β 1–42 aggregates

“…In preparation for HS-AFM observation, we separated Aβ 1-42 peptides into low-and high-molecularweight populations (LMW and HMW, respectively) and confirmed that their structure and assembly properties were consistent with previous studies (Supporting Information and Figs. S1 and S2) (28,(42)(43)(44)(45)(46)(47)(48)(49). The LMW population comprised monomeric and low-order oligomeric Aβ (28).…”

A comparative analysis of the aggregation behavior of amyloid‐β peptide variants

“…Alternatively, the denser peptide networks can be more prone to precipitation in the test tube which would lead to a similar observation. Although the effect of FAD-related mutations of Ab 1-42 on aggregation has been investigated in the past [23][24][25][26], no comprehensive study has been reported that directly compares the majority of the currently known mutations. Different Ab preparation methods and experimental conditions have led to considerable variation in reported effects of these mutations.…”

Effect of the Tottori Familial Disease Mutation (D7N) on the Monomers and Dimers of Aβ40and Aβ42

“…44,56,62 Second, Ono et al by following the secondary structures of Aβ as a function of time showed that D7N accelerates the conversion from random coil to β-sheet by 10-fold in Aβ 40 and 5-fold in Aβ 42 system. 30 Our monomer simulations reveal a 5% and 14% reduction of coil in Aβ 40 and Aβ 42 upon mutation. In contrast, our dimer simulations show that either the coil content remains constant in Aβ 40 or increases by 16% in Aβ 42 upon D7N mutation.…”

“…29 A more recent experimental study showed that the Tottori mutation alters Aβ assembly at its earliest stages, monomer folding and oligomerization. 30 While the effects of mutations at positions 21−23 on the Aβ monomer and oligomers have been extensively studied by computational means, 31−36 the impact of mutations at the Nterminus has not been investigated theoretically. In this work, we study the effect of the Tottori mutation on the monomers and dimers of Aβ 40 and Aβ 42 using all-atom molecular dynamics (MD) simulations at 300 K in explicit solvent within the microsecond time scale.…”