Amyloid β-protein (Aβ42) oligomerization is an early event in Alzheimer’s disease (AD). Current diagnostic methods using sequence-specific antibodies against less toxic fibrillar and monomeric Aβ42 run the risk of overdiagnosis. Hence, conformation-specific antibodies against neurotoxic Aβ42 oligomers have garnered much attention for developing more accurate diagnostics. Antibody 24B3, highly specific for the toxic Aβ42 conformer that has a turn at Glu22 and Asp23, recognizes a putative Aβ42 dimer, which forms stable and neurotoxic oligomers more potently than the monomer. 24B3 significantly rescues Aβ42-induced neurotoxicity, whereas sequence-specific antibodies such as 4G8 and 82E1, which recognizes the N-terminus, do not. The ratio of toxic to total Aβ42 in the cerebrospinal fluid of AD patients is significantly higher than in control subjects as measured by sandwich ELISA using antibodies 24B3 and 82E1. Thus, 24B3 may be useful for AD diagnosis and therapy.

The formation of soluble oligomers of amyloid β42 and 40 (Aβ42, Aβ40) is the initial event in the pathogenesis of Alzheimer's disease (AD). Based on previous systematic proline replacement and solid-state NMR, we proposed a toxic dimer structure of Aβ42, a highly aggregative alloform, with a turn at positions 22 and 23, and a hydrophobic core in the C-terminal region. However, in addition to Aβ42, Aβ40 dimers can also contribute to AD progression because of the more abundance of Aβ40 monomer in biological fluids. Here, we describe the synthesis and characterization of three dimer models of the toxic-conformation constrained E22P-Aβ40 using l,l-2,6-diaminopimeric acid (DAP) or l,l-2,8-diaminoazelaic acid (DAZ) linker at position 30, which is incorporated into the intermolecular parallel β-sheet region, and DAP at position 38 in the C-terminal hydrophobic core. E22P-A30DAP-Aβ40 dimer (1) and E22P-A30DAZ-Aβ40 dimer (2) existed mainly in oligomeric states even after 2 weeks incubation without forming fibrils, unlike the corresponding monomer. Their neurotoxicity toward SH-SY5Y neuroblastoma cells was very weak. In contrast, E22P-G38DAP-Aβ40 dimer (3) formed β-sheet-rich oligomeric aggregates, and exhibited more potent neurotoxicity than the corresponding monomer. Ion mobility-mass spectrometry suggested that high molecular-weight oligomers (12-24-mer) of 3 form, but not for 1 and 2 after 4 h incubation. These findings indicate that formation of the hydrophobic core at the C-terminus, rather than intermolecular parallel β-sheet, triggers the formation of toxic Aβ oligomers. Compound 3 may be a suitable model for studying the etiology of Alzheimer's disease.

The only trimer model to exhibit weak but significant neurotoxicity against SH-SY5Y cells was the one which was linked at position 38.

Characterization of amyloid β (Aβ) oligomers, the transition species present prior to the formation of Aβ fibrils and that have cytotoxicity, has become one of the major topics in the investigations of Alzheimer’s disease (AD) pathogenesis. However, studying pathophysiological properties of Aβ oligomers is challenging due to the instability of these protein complexes in vitro. Here, we report that conformation-restricted Aβ42 with an intramolecular disulfide bond at positions 17 and 28 (SS-Aβ42) formed stable Aβ oligomers in vitro. Thioflavin T binding assays, nondenaturing gel electrophoresis, and morphological analyses revealed that SS-Aβ42 maintained oligomeric structure, whereas wild-type Aβ42 and the highly aggregative Aβ42 mutant with E22P substitution (E22P-Aβ42) formed Aβ fibrils. In agreement with these observations, SS-Aβ42 was more cytotoxic compared to the wild-type and E22P-Aβ42 in cell cultures. Furthermore, we developed a monoclonal antibody, designated TxCo-1, using the toxic conformation of SS-Aβ42 as immunogen. X-ray crystallography of the TxCo-1/SS-Aβ42 complex, enzyme immunoassay, and immunohistochemical studies confirmed the recognition site and specificity of TxCo-1 to SS-Aβ42. Immunohistochemistry with TxCo-1 antibody identified structures resembling senile plaques and vascular Aβ in brain samples of AD subjects. However, TxCo-1 immunoreactivity did not colocalize extensively with Aβ plaques identified with conventional Aβ antibodies. Together, these findings indicate that Aβ with a turn at positions 22 and 23, which is prone to form Aβ oligomers, could show strong cytotoxicity and accumulated in brains of AD subjects. The SS-Aβ42 and TxCo-1 antibody should facilitate understanding of the pathological role of Aβ with toxic conformation in AD.