Structure Optimization of the Toxic Conformation Model of Amyloid β42 by Intramolecular Disulfide Bond Formation

Abstract

Amyloid β (Aβ) oligomers play a critical role in the pathology of Alzheimer's disease. Recently, we reported that a conformation‐restricted Aβ42 with an intramolecular disulfide bond through cysteine residues at positions 17/28 formed stable oligomers with potent cytotoxicity. To further optimize this compound as a toxic conformer model, we synthesized three analogues with a combination of cysteine and homocysteine at positions 17/28. The analogues with Cys‐Cys, Cys‐homoCys, or homoCys‐Cys, but not the homoCys‐homoCys analogue, exhibited potent cytotoxicity against SH‐SY5Y and THP‐1 cells even at 10 nM. In contrast, the cytotoxicity of conformation‐restricted analogues at positions 16/29 or 18/27 was significantly weaker than that of wild‐type Aβ42. Furthermore, thioflavin‐T assay, non‐denaturing gel electrophoresis, and morphological studies suggested that the majority of these conformation‐restricted analogues exists in an oligomeric state in cell culture medium, indicating that the toxic conformation of Aβ42, rather than the oligomeric state, is essential to induce cytotoxicity.

Optimization of the Linker Length in the Dimer Model of E22P-Aβ40 Tethered at Position 38

“…48 After incubation for 24 h in the presence of each dimer model or E22P-Aβ40, cell viability was evaluated by the ability to reduce 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as reported previously. 38 As shown in Figure 2A, E22P-Aβ40 as a positive control significantly decreased cell viability at 3.3 μM. Consistent with the previous report, 34 dimer model 2 with a DAP linker (n = 3) was more cytotoxic than E22P-Aβ40.…”

“…Consistent with this trend, the cytotoxicity of 1 with a DAA linker (n = 2) was very strong; it reached maximum neurotoxicity at 1 μM. As shown in Figure 2B, the cytotoxicity of 1 was almost equal to that of E22P-Aβ42, the most cytotoxic analogue of Aβ42 except for those with an intramolecular disulfide bond at positions 17/28, 36,38 and was ca. 30 times more potent compared with the E22P-Aβ40 monomer.…”

“…Each dimer model (10 μM) was incubated with Th-T (20 μM), and Th-T fluorescence was automatically monitored every 10 min at room temperature (23 °C) as reported previously. 38,52 WT-Aβ42 and E22P-Aβ42 aggregated rapidly to reach maximum fluorescence after 4 h. The difference in the maximum fluorescence intensity might reflect the fibril structure to which Th-T is bound. E22P-Aβ40 started to aggregate after 8 h and reached almost maximum fluorescence after 16 h, followed by an incremental increase.…”

“…Each sample was analyzed after a 48 h incubation at 37 °C as reported previously. 38 WT-Aβ42 and E22P-Aβ40 were used as references and formed typical amyloid fibrils. Among dimer models 1−5, only 1 formed typical amyloid fibrils similar to E22P-Aβ40 and WT-Aβ42.…”

Defensive reactions against intrinsic stresses

“…γ-secretase inhibitor impedes proteolysis of amyloid precursor protein to generate Aβ, the pathogenic factor of Alzheimer's disease [1]. However, nearly all such drugs failed in clinical trials.…”