Synthesis, aggregation, neurotoxicity, and secondary structure of various Aβ1–42 mutants of familial Alzheimer's disease at positions 21–23

Murakami, Kazuma; Irie, Kazuhiro; Morimoto, Akira; Ohigashi, Hajime; Shindo, Mayumi; Nagao, Masaya; Shimizu, Takahiko; Shirasawa, Takuji

doi:10.1016/s0006-291x(02)00430-8

Cited by 136 publications

(167 citation statements)

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“…Because proline has a propensity to form a ␤-turn structure as a Pro-X corner (13), the data strongly suggest that turn at positions 22 and 23 is a critical secondary structure in the A␤42 fibrils. This finding demonstrates very well the high aggregative ability of the A␤42 mutants in cerebral amyloid angiopathy (21,22), E22Q-A␤42 (Dutch), and E22K-A␤42 (Italian), because Gln-Asp (Dutch) and Lys-Asp (Italian) sequences at positions 22 and 23 are more frequently found in the two-residue ␤-turn (13) than in Glu-Asp (wild type). Recent investigations using solid-state NMR (7-9) have indicated a parallel organization of ␤-sheets in A␤40 fibrils, because the ␤-carbons of Ala-21 and Ala-30 are located within 5.5 Å, respectively.…”

Section: Discussionsupporting

confidence: 61%

“…Sedimentation Assay for Fibril Formation-Each A␤ derivative was dissolved in 0.02% NH 4 OH at 250 M. After a 10-fold dilution by 50 mM sodium phosphate containing 100 mM NaCl at pH 7.4, the resultant peptide solution (25 M) was incubated at 37°C for 4,8,16,24, or 48 h. After centrifugation at 15,000 rpm in an Eppendorf microcentrifuge at 4°C for 10 min, 25 l of the supernatant was then analyzed by HPLC as reported previously (15,21,22). The area of the absorption at 220 nm was integrated and expressed as a percentage of the control.…”

Section: Methodsmentioning

confidence: 99%

“…Cell Culture of PC12 Cells-Rat pheochromocytoma PC12 cells were obtained from Riken Cell Bank and were cultured as reported previously (15,21,22). For experimental purposes, near-confluent cultures of the cells were plated at ϳ10 4 cells/100 l/well fresh culture medium in a 96-well tissue culture plate coated with collagen and incubated at 37°C under 5% CO 2 overnight before the experiments.…”

Section: Methodsmentioning

confidence: 99%

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Analysis of the Secondary Structure of β-Amyloid (Aβ42) Fibrils by Systematic Proline Replacement

Morimoto

Irie

Murakami

et al. 2004

Journal of Biological Chemistry

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Amyloid fibrils in Alzheimer's disease mainly consist of 40-and 42-mer ␤-amyloid peptides (A␤40 and A␤42) that exhibit aggregative ability and neurotoxicity. Although the aggregates of A␤ peptides are rich in intermolecular ␤-sheet, the precise secondary structure of A␤ in the aggregates remains unclear. To identify the amino acid residues involved in the ␤-sheet formation, 34 proline-substituted mutants of A␤42 were synthesized and their aggregative ability and neurotoxicity on PC12 cells were examined. Prolines are rarely present in ␤-sheet, whereas they are easily accommodated in ␤-turn as a Pro-X corner. Among the mutants at positions 15-32, only E22P-A␤42 extensively aggregated with stronger neurotoxicity than wild-type A␤42, suggesting that the residues at positions 15-21 and 24 -32 are involved in the ␤-sheet and that the turn at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of A␤42. The C-terminal proline mutants (A42P-, I41P-, and V40P-A␤42) hardly aggregated with extremely weak cytotoxicity, whereas the C-terminal threonine mutants (A42T-and I41T-A␤42) aggregated potently with significant cytotoxicity. These results indicate that the hydrophobicity of the C-terminal two residues of A␤42 is not related to its aggregative ability and neurotoxicity, rather the C-terminal three residues adopt the ␤-sheet. These results demonstrate well the large difference in aggregative ability and neurotoxicity between A␤42 and A␤40. In contrast, the proline mutants at the N-terminal 13 residues showed potent aggregative ability and neurotoxicity similar to those of wild-type A␤42. The identification of the ␤-sheet region of A␤42 is a basis for designing new aggregation inhibitors of A␤ peptides. Alzheimer's disease (AD)1 is neuropathologically characterized by the progressive deposition of amyloid fibrils in the brain parenchyma and cortical blood vessels (1). This deposition mainly consists of 40-and 42-mer peptides (A␤40 and A␤42) generated from amyloid precursor protein by two proteases, ␤-and ␥-secretase (2, 3). A␤42 plays a pivotal role in the pathogenesis of AD, because the aggregative ability and neurotoxicity of A␤42 are considerably higher than those of A␤40 (4). Because the aggregative ability of A␤ peptides is closely related to the neurotoxicity, precise structural information for amyloid fibrils is indispensable for understanding the molecular mechanisms of AD and related folding diseases and for developing new medicinal leads using the inhibitory activity of amyloid fibril formation.Previous studies on A␤ fibrils showed that A␤ aggregates mainly consist of intermolecular parallel ␤-sheet (5-10

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Section: Discussionsupporting

confidence: 61%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Analysis of the Secondary Structure of β-Amyloid (Aβ42) Fibrils by Systematic Proline Replacement

Morimoto

Irie

Murakami

et al. 2004

Journal of Biological Chemistry

Self Cite

167

202

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“…Numerous in vitro experiments have shown that A␤ peptides with the Dutch mutation aggregate more readily than their WT A␤ counterparts, in addition to exhibiting increased cytotoxic activity (1,3,22). These observations have led to the hypothesis that increased propensity for aggregation in A␤E22Q underlies the main mechanism of pathology of the Dutch form of AD (3,23).…”

Section: Discussionmentioning

confidence: 99%

Role of the familial Dutch mutation E22Q in the folding and aggregation of the 15–28 fragment of the Alzheimer amyloid-β protein

Baumketner

Krone

Shea³

2008

Proc. Natl. Acad. Sci. U.S.A.

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Amyloid fibrils, large ordered aggregates of amyloid ␤ proteins (A␤), are clinical hallmarks of Alzheimer's disease (AD). The aggregation properties of amyloid ␤ proteins can be strongly affected by singlepoint mutations at positions 22 and 23. The Dutch mutation involves a substitution at position 22 (E22Q) and leads to increased deposition rates of the A␤E22Q peptide onto preseeded fibrils. We investigate the effect of the E22Q mutation on two key regions involved in the folding and aggregation of the A␤ peptide through replica exchange molecular dynamics simulations of the 15-28 fragment of the A␤ peptide. The A␤15-28 peptide encompasses the 22-28 region that constitutes the most structured part of the A␤ peptide (the E22-K28 bend), as well as the central hydrophobic cluster (CHC) (segment 17-21), the primary docking site for A␤ monomers depositing onto fibrils. Our simulations show that the 22-28 bend is preserved in the A␤(15-28) peptide and that the CHC, which is mostly unstructured, interacts with this bend region. The E22Q mutation does not affect the structure of the bend but weakens the interactions between the CHC and the bend. This leads to an increased population of ␤-structure in the CHC. Our analysis of the fibril elongation reaction reveals that the CHC adopts a ␤-strand conformation in the transition state ensemble, and that the E22Q mutation increases aggregation rates by lowering the barrier for A␤ monomer deposition onto a fibril. Thermodynamic signatures of this enhanced fibrillization process from our simulations are in good agreement with experimental observations. Alzheimer's disease ͉ explicit water ͉ familial type ͉ molecular dynamics simulations ͉ replica exchange T he presence of amyloid fibrils in the brain is a clinical hallmark of Alzheimer's disease (AD). The fibrils consist of large ordered aggregates of amyloid-␤ (A␤) peptides, proteolytic by-products of the enzymatic cleavage of the Alzheimer amyloid precursor protein (APP). AD can be sporadic (occurring in elderly patients and characterized by a slow progression) or familial (a hereditary form characterized by early onset of the disease and aggravated severity). The majority of familial forms of AD involve single-point mutations in the 22-23 segment of the A␤ peptide. The focus of this work is on the Dutch form of AD, which involves a mutation encoded by a point substitution G to C at codon 693 of the amyloid precursor protein (APP). The result is the production of an A␤ peptide in which residue E22 is mutated to Q (E22Q mutation). Patients who have this mutation are at risk of developing cerebral amyloid angiopathy that typically leads to cerebral hemorrhage and stroke. Many in vitro experiments show that the A␤E22Q peptide aggregates much more readily than its wild-type (WT) counterpart (1-3). As a free monomer in solution, the WT A␤ peptide is for the most part unstructured with residual structure observed locally in a few regions of the primary sequence (4). The A␤ peptide can populate a variety of oligomeric species, some...

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“…Others have suggested that the peptide's unique aggregation properties are responsible for its disease phenotype. The Flemish substitution leads to decreased β-sheet formation 16 and decreased fibril extension 18 compared with wild-type. Despite these negative effects on fibril formation, the A21G substitution enhances formation of protofibrils.…”

mentioning

confidence: 99%

Familial Alzheimer’s Disease Mutations Differentially Alter Amyloid β-Protein Oligomerization

Gessel

Bernstein

Kemper

et al. 2012

ACS Chem. Neurosci.

109

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Although most cases of Alzheimer's disease (AD) are sporadic, ∼5% of cases are genetic in origin. These cases, known as familial Alzheimer's disease (FAD), are caused by mutations that alter the rate of production or the primary structure of the amyloid β-protein (Aβ). Changes in the primary structure of Aβ alter the peptide's assembly and toxic activity. Recently, a primary working hypothesis for AD has evolved where causation has been attributed to early, soluble peptide oligomer states. Here we posit that both experimental and pathological differences between FAD-related mutants and wild-type Aβ could be reflected in the early oligomer distributions of these peptides. We use ion mobility-based mass spectrometry to probe the structure and early aggregation states of three mutant forms of Aβ40 and Aβ42: Tottori (D7N), Flemish (A21G), and Arctic (E22G). Our results indicate that the FAD-related amino acid substitutions have no noticeable effect on Aβ monomer cross section, indicating there are no major structural changes in the monomers. However, we observe significant changes to the aggregation states populated by the various Aβ mutants, indicating that structural changes present in the monomers are reflected in the oligomers. Moreover, the early oligomer distributions differ for each mutant, suggesting a possible structural basis for the varied pathogenesis of different forms of FAD.

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Synthesis, aggregation, neurotoxicity, and secondary structure of various Aβ1–42 mutants of familial Alzheimer's disease at positions 21–23

Cited by 136 publications

References 23 publications

Analysis of the Secondary Structure of β-Amyloid (Aβ42) Fibrils by Systematic Proline Replacement

Analysis of the Secondary Structure of β-Amyloid (Aβ42) Fibrils by Systematic Proline Replacement

Role of the familial Dutch mutation E22Q in the folding and aggregation of the 15–28 fragment of the Alzheimer amyloid-β protein

Familial Alzheimer’s Disease Mutations Differentially Alter Amyloid β-Protein Oligomerization

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