Figure 3

Oxidized cross-linking of synthetic Aβ _1-42 generates a dimer that potently induces rods. (A) Western blot showing SDS-stable species of Aβ in different preparations. Two gel filtration fractions from medium of 7PA2 cells are combined to give Aβd/t. Last three lanes are synthetic human Aβ_1-42. Lane 1: traditional oligomers prepared in DMSO/F12 [44, 45]; Lane 2: peptide incubated (37°C, 3 d) in PBS containing 250 μM H₂O₂; Lane 3: peptide incubated (5 d) in PBS containing 250 μM peroxide plus 25 μM CuCl₂. Dimer is absent in traditional oligomer preparations but forms in peroxide alone. Dimer, trimer and tetramer are generated with Cu²⁺/peroxide. (B) Comparison of rod-inducing activity between untreated (Untr) dissociated neurons and neurons treated with: Cu²⁺/peroxide (veh cont), 1 μM traditional synthetic human Aβ oligomers (HAβsyn), 1 μM synthetic rodent Aβ (RAβsyn) treated identically to HAβsyn, 1 μM Cu²⁺/peroxide-treated RAβsyn, 10 nM (45 ng/mL) and 1 nM peroxide-oxidized HAβsyn, and 5 nM each of high-n and low-n oligomers-containing fractions of oxidized HAβsyn (see Additional file 3). (C) The rod-inducing activity of different Aβ preparations. The effective concentration for a 50% maximal response (EC₅₀; arrows) was calculated from dose-response curves for traditional Aβ oligomers (Aβsyn; ED₅₀ = 3100 ng/mL), Cu²⁺/peroxide oxidized synthetic Aβ (OxAβsyn; ED₅₀ = 5 ng/mL) and Aβd/t (ED₅₀ = 0.4 ng/mL). Dashed line is control (untreated). Bars are standard deviations. (D) Human and rodent Aβ_1-42 sequences differ in three residues (bold), but only tyrosine at position 10 is likely to generate the peroxide-induced SDS-stable species.