FIG.4.
Reactive oxygen species (ROS)-dependent changes in the amyloid beta peptide (Aβ) response. (A) Glial cells were exposed to the indicated compounds for 8 h, and intracellular ROS determined by FACS using DCF. The single-cell fluorescence intensity is plotted vs. cell number (counts) for 10 000 cells. Aβ1–42, 1 µm, desferoxamine (DFO), 50 µm. (B) The ROS production shown in (A) presented as a histogram, plus ROS data under the same conditions and time for 1 µm rotenone (Rot), 1 µm diphenyliodonium (DPI), 100 µm propyl gallate (PG), 30 mm N-acetyl-l-cysteine (NAC) and 100 µm apocynin (APO), both individually and in combination with Aβ. *P < 0.001 one-way ANOVA vs. Aβ alone; **P < 0.0001, two-tailed P-value, unpaired t-test (n = 3) vs. vehicle alone. (C) Hypoxia-inducible factor (HIF)-1α protein was determined by Western blotting and quantified relative to actin as a function of time in the presence of 1 µm Rot or 1 µm Aβ1–42. *P < 0.05; **P < 0.01; ***P < 0.001 anova vs. 0 time (n = 4). (D) NAC does not inhibit HIF-1α expression. Cells were exposed to vehicle (cont), 30 mm NAC alone (NAC), 1 µm Aβ1–42 (Aβ) or both together (NAC + Aβ) for 4 h, and the HIF-1α amount determined by Western blotting. *P < 0.001, unpaired t-test (n = 4). (E) Glial activation was determined at 8 h by morphological criteria or by NO release. Rot, 1 µm, 100 µm H2O2, 100 µm PG, 30 mm NAC, 1 µm Aβ1–42, 1 µm DPI, 100 µm APO or 30 nm lactacystin (LACTO). *P < 0.01, significantly different from controls of vehicle alone in the morphology assay (n = 5) and **P < 0.001 from Aβ-induced NO release (n = 3) using ANOVA and Tukey’s post hoc test.