Fig. 8. Involvement of H₂O₂ in Aβ1–42- and superoxide-induced activation of N-SMase in human primary neurons.

A, cells were treated with 1 μm Aβ1–42 in the presence or absence of 5 μm hypoxanthine (H) and 0.5 milliunits/ml xanthine oxidase (XO). At different intervals (measured in minutes), activity of N-SMase was assayed. B, in one set, cells were treated with 1 μm Aβ1–42 and in the other, 1 μm Aβ1–42 was also added to media only. At different intervals (measured in minutes), the level of H₂O₂ was measured in supernatant, as described under “Materials and Methods.” Control group served as 100%, and data from other groups were expressed as percent of control. C, cells received either ASO or ScO at 1 μm against p22phox, and after 40 h of incubation, the cells were treated with 1 μm fibrillar Aβ1–42. After 15 min of stimulation, H₂O₂ production was assayed in supernatants. Results are mean ± S.D. of three different experiments. a, p < 0.001 versus Aβ1–42 + vehicle. Cells pretreated with different amounts of either superoxide dismutase (SOD) or catalase (Cat) for 15 min were stimulated with either 1 μm fibrillar Aβ1–42 (D) or the combination of H (5 μm) and XO (0.5 milliunits/ml) (E). After 30 min of incubation, activity of N-SMase was assayed in total cell extracts. Control group served as 100%, and data from other groups were expressed as percent of control. Results are mean ± S.D. of three different experiments.