Chelation of Lysosomal Iron Protects Dopaminergic SH-SY5Y Neuroblastoma Cells from Hydrogen Peroxide Toxicity by Precluding Autophagy and Akt Dephosphorylation

In human neuroblastoma SH-SY5Y cells, hydrogen peroxide (H2O2, 200 mu M) rapidly (< 5 min) induced autophagy, as shown by processing and vacuolar relocation of light chain 3(LC3). Accumulation of autophagosome peaked at 30 min of H2O2 exposure. The continuous presence of H2O2 eventually (at > 60 min) caused autophagy-dependent annexin V-positive cell death. However, the cells exposed to H2O2 for 30 min and then cultivated in fresh medium could recover and grow, despite ongoing autophagy. H2O2 rapidly (5 min) triggered the formation of dichlorofluorescein-sensitive HO center dot-free radicals within mitochondria, whereas the mitochondria-associated oxidoradicals revealed by MitoSox (O-2(center dot-)) became apparent after 30 min of exposure to H2O2. 3-Methyladenine inhibited autophagy and cell death, but not the generation of HO center dot. Genetic silencing of beclin-1 prevented bax- and annexin V-positive cell death induced by H2O2, confirming the involvement of canonical autophagy in peroxide toxicity. The lysosomotropic iron chelator deferoxamine (DFO) prevented the mitochondrial generation of both HO. and O-2(center dot-) and suppressed the induction of autophagy and of cell death by H2O2. Upon exposure to H2O2, Akt was intensely phosphorylated in the first 30 min, concurrently with mammalian target of rapamycin inactivation and autophagy, and it was dephosphorylated at 2 h, when > 50% of the cells were dead. DFO did not impede Akt phosphorylation, which therefore was independent of reactive oxygen species (ROS) generation but inhibited Akt dephosphorylation. In conclusion, exogenous H2O2 triggers two parallel independent pathways, one leading to autophagy and autophagy-dependent apoptosis, the other to transient Akt phosphorylation, and both are inhibited by DFO. The present work establishes HO center dot as the autophagy-inducing ROS and highlights the need for free lysosomal iron for its production within mitochondria in response to hydrogen peroxide.