Chelation of lysosomal iron protects dopaminergic SH-SY5Y neuroblastoma cells from hydrogen peroxide toxicity by precluding autophagy and Akt dephosphorylation

Abstract

In human neuroblastoma SH-SY5Y cells, hydrogen peroxide (H 2O 2, 200μM) 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 H 2O 2 exposure. The continuous presence of H 2O 2 eventually (at > 60 min) caused autophagy-dependent annexin V-positive cell death. However, the cells exposed to H 2O 2 for 30 min and then cultivated in fresh medium could recover and grow, despite ongoing autophagy. H 2O 2 rapidly (5 min) triggered the formation of dichlorofluorescein-sensitive HO ·-free radicals within mitochondria, whereas the mitochondria-associated oxidoradicals revealed by MitoSox (O 2·-) became apparent after 30 min of exposure to H 2O 2. 3-Methyladenine inhibited autophagy and cell death, but not the generation of HO ·. Genetic silencing of beclin-1 prevented bax- and annexin V-positive cell death induced by H 2O 2, 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·- and suppressed the induction of autophagy and of cell death by H 2O 2. Upon exposure to H 2O 2, 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 H 2O 2 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 · as the autophagy-inducing ROS and highlights the need for free lysosomal iron for its production within mitochondria in response to hydrogen peroxide. © The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.