Fig. 2.

Effect of iAβ on cholesterol and phosphatidylcholine release from neurons in culture. Neuron-rich cultures were labeled with [¹⁴C]acetate for 48 hr as described in Materials and Methods. Cells were then washed three times with DMEM and incubated with iAβ at various concentrations for 4 hr. Synthetic Aβ1–40 dissolved at high concentration and incubated at 37°C for 24 hr, followed by filtration, was used. The released lipids in the media and the cellular lipids were extracted and analyzed as described in Materials and Methods. The iAβ-mediated release of cholesterol and phosphatidylcholine (PC) (a) was significantly increased in a dose-dependent manner. Each data point represents mean ± SE for three samples. For the time course study of iAβ-mediated lipid release from neurons, cultured neurons were labeled with [¹⁴C]acetate for 48 hr and then washed three times with DMEM and incubated with iAβ at a final concentration of 8 μm. The iAβ-mediated release of cholesterol and PC (b) increased with incubation time. Each data point represents mean ± SE for three samples. Effect of Congo red on iAβ-mediated lipid release and effect of fresh Aβ on lipid release from neurons were investigated using labeled neurons with [¹⁴C]acetate for 48 hr. c, Cells were washed three times with DMEM and then incubated with iAβ (10 μm), iAβ (10 μm) with Congo red (CR) (10 μm), CR alone (10 μm), freshly dissolved Aβ (frAβ) (10 μm), and frAβ plus CR (10 μm) in serum-free N₂ medium for 24 hr. The release of cholesterol and PC in iAβ-treated culture medium was abolished by concurrent treatment with Congo red. Freshly dissolved Aβ1–40 did not promote lipid release from these cells. Each data point represents mean ± SE for four samples. *p < 0.005 versus CONT, iAβ + CR, frAβ, and frAβ + CR. CONT, Control cultures;iAβ, incubated Aβ1–40; CR, Congo red; frAβ, fresh Aβ1–40. d, Cells were washed three times with DMEM and then incubated with none (CONT), iAβ (5 μm), iAβ (5 μm) + NAC (1 mm), NAC (1 mm), H₂O₂ (2 mm), and H₂O₂ (2 mm) + NAC (1 mm). *p < 0.001 versus CONT and NAC; **p < 0.0001 versus H₂O₂ + NAC; ^#p < 0.06 versus CONT and NAC.NAC, N-acetyl-l-cysteine.e, The cultures were washed three times with DMEM and then incubated with none (CONT), iAβ (5 μm), and iAβ (5 μm) + H7 (30 nm) for 16 hr at 37°C, and the lipids in the medium and the cells were quantified as described in Materials and Methods. *p < 0.004 versus CONT and iAβ + H7.