We investigated the role of autophagy, a controlled cellular self-digestion process, in regulating survival of neurons exposed to atypical antipsychotic olanzapine. Olanzapine induced autophagy in human SH-SY5Y neuronal cell line, as confirmed by the increase in autophagic flux and presence of autophagic vesicles, fusion of autophagosomes with lysosomes, and increase in the expression of autophagy-related (ATG) genes ATG4B, ATG5, and ATG7. The production of reactive oxygen species, but not modulation of the main autophagy repressor MTOR or its upstream regulators AMP-activated protein kinase and AKT1, was responsible for olanzapine-triggered autophagy. Olanzapine-mediated oxidative stress also induced mitochondrial depolarization and damage, and the autophagic clearance of dysfunctional mitochondria was confirmed by electron microscopy, colocalization of autophagosome-associated MAP1LC3B (LC3B henceforth) and mitochondria, and mitochondrial association with the autophagic cargo receptor SQSTM1/p62. While olanzapine-triggered mitochondrial damage was not overtly toxic to SH-SY5Y cells, their death was readily initiated upon the inhibition of autophagy with pharmacological inhibitors, RNA interference knockdown of BECN1 and LC3B, or biological free radical nitric oxide. The treatment of mice with olanzapine for 14 d increased the brain levels of autophagosome-associated LC3B-II and mRNA encoding Atg4b, Atg5, Atg7, Atg12, Gabarap, and Becn1. The administration of the autophagy inhibitor chloroquine significantly increased the expression of proapoptotic genes (Trp53, Bax, Bak1, Pmaip1, Bcl2l11, Cdkn1a, and Cdkn1b) and DNA fragmentation in the frontal brain region of olanzapine-exposed animals. These data indicate that olanzapine-triggered autophagy protects neurons from otherwise fatal mitochondrial damage, and that inhibition of autophagy might unmask the neurotoxic action of the drug.
Keywords: AKT1, v-akt murine thymoma viral oncogene homolog 1; AMPK, AMP-activated protein kinase; APAF1, apoptotic protease activating factor 1; ATG, autophagy-related; BAD, BCL2-associated agonist of cell death; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BBC3, BCL2 binding component 3; BCL2, B-cell CLL/lymphoma 2; BCL2L1, BCL2-like 1; BCL2L11, BCL2-like 11 (apoptosis facilitator); BECN1, Beclin 1, autophagy-related; BIRC5, baculoviral IAP repeat containing 5; CDKN1A, cyclin-dependent kinase inhibitor 1A (p21, Cip1); CDKN1B, cyclin-dependent kinase inhibitor 1B (p27, Kip1); CFLAR/FLIP, CASP8 and FADD-like apoptosis regulator; COX4I1/COX IV, cytochrome c oxidase IV isoform 1; DEA-NONOate, diethylamine NONOate; DHR, dihydrorhodamine 123; FOXO, forkhead box O; GABARAP, GABA(A) receptor-associated protein; LDH, lactate dehydrogenase; MAP1LC3B, microtubule-associated protein 1 light chain 3 β; MTOR, mechanistic target of rapamycin; PAPA-NONOate, propylamine propylamine NONOate; PMAIP1, phorbol-12-myristate-13-acetate-induced protein 1; PTEN, phosphatase and tensin homolog; ROS, reactive oxygen species; RPS6KB1/S6K1, ribosomal protein S6 kinase, 70kDa, polypeptide 1; SQSTM1; SQSTM1/p62, sequestosome 1; TRP53, transformation related protein 53 (mouse ortholog of human TP53, tumor protein p53); TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; XIAP, X-linked inhibitor of apoptosis; antipsychotic; apoptosis; autophagy; mitophagy; neurotoxicity; nitric oxide, NO; olanzapine; oxidative stress.