Autophagy inhibition uncovers the neurotoxic action of the antipsychotic drug olanzapine

Abstract

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.

Introduction

Schizophrenia is a mental disorder that affects 1% of the world's population, and represents a leading cause of chronic disability among young adults.¹ Although antipsychotic medications efficiently reduce psychotic symptoms and relapse rates in schizophrenia patients, some recent studies on relatively large cohorts of schizophrenia patients,^2,3 as well as controlled studies in animals,^4-6 suggest that long-term antipsychotic treatment may contribute to brain tissue volume loss associated with the disease. Furthermore, the increased levels of apoptotic cell death marker tissue transglutaminase have been found in the cerebrospinal fluid of nonschizophrenic patients (including those with Alzheimer disease) receiving typical (flupentixol, haloperidol) or atypical (olanzapine, melperone, zotepine) antipsychotics.⁷ The levels of cerebral apoptosis in nondemented patients on antipsychotic medications were apparently similar to those of Alzheimer disease patients not treated with antipsychotics,⁷ thus indicating that antipsychotic drugs may actually trigger some degenerative process. Accordingly, the exposure to neuroleptics (typical and atypical) is associated with a significant increase in neurofibrillary tangles and amyloid plaques in the frontal lobe cortex of patients suffering from dementia.⁸ Therefore, keeping in mind the rise in antipsychotic use for disorders other than schizophrenia,⁹ understanding the negative effects of antipsychotics on the brain has important clinical implications beyond schizophrenia treatment.

The physiological role of macroautophagy (referred to hereafter as autophagy) is to remove long-lived and damaged proteins, as well as dysfunctional organelles (e.g., mitochondria), through lysosomal machinery.^10,11 Autophagy also acts as a survival mechanism by providing energy and/or directly interfering with cell death pathways during nutrient deprivation, hypoxia, oxidative stress, and DNA damage.^12,13 On the other hand, if extensive or aberrant, autophagy can contribute to apoptosis (programmed cell death type I) or function as an alternative cell-death pathway (programmed cell death type II).^14,15 Accordingly, autophagy is involved in the maintenance of neuronal homeostasis, with either defective or excessive autophagy contributing to neurodegeneration associated with accumulation of misfolded proteins and damaged mitochondria.^16,17 Autophagy proceeds through hierarchically ordered activation of autophagy-related (ATG) proteins, controlled by the main autophagy repressor, mechanistic target of rapamycin (MTOR), and its principal upstream modulators AMP-activated protein kinase (AMPK) and AKT1/PKB.¹⁸ While a few recent gene array studies indicate that autophagy might be dysregulated in brains of schizophrenia patients,^19,20 the possible role of autophagy in schizophrenia has not been extensively investigated. Interestingly, though, it seems that the capacity for autophagy induction is shared by several antipsychotic drugs. This has been emphasized by the finding that 3 of 7 autophagy-inducing compounds detected in the screen of 480 bioactive molecules are the clinically approved antipsychotics fluspirilene, trifluoperazine, and pimozide.²¹ In the subsequent independent studies, antipsychotic agents haloperidol, clozapine, and sertindole have been found to trigger an autophagic response in neuronal cells,^22,23 while chlorpromazine and raclopride-induced autophagy in glioma cells and cardiac myocytes, respectively.^24,25 However, with the exception of the report demonstrating the in vitro neurotoxicity of sertindole-induced autophagy,²³ the role of autophagy induction by antipsychotics in neuronal survival and death has not been examined so far. Furthermore, it is unknown whether antipsychotic medications can trigger neuronal autophagy in vivo.

In the present study, we show that olanzapine, a widely used atypical antipsychotic with dopamine and serotonine receptor antagonist activity,²⁶ induces autophagic response in human neuronal cell line and mouse brain in vivo. Autophagy induction by olanzapine was mediated by oxidative stress in an AMPK-AKT1-MTOR-independent manner, leading to the clearance of dysfunctional mitochondria and prevention of overt neuronal damage.

Results

Olanzapine induces autophagy in SH-SY5Y cells

SH-SY5Y human neuroblastoma cells have been frequently used as an in vitro model of neurons.²⁷ Based on previous studies^28,29 and our preliminary results, we have chosen the concentration of olanzapine of ≤100 μM as nontoxic for SH-SY5Y cells. The flow cytometric analysis of the cells stained with acridine orange, which fluoresces red at acidic pH, demonstrated that olanzapine was more potent than risperidone (atypical antipsychotic) and haloperidol (typical antipsychotic) in increasing the numbers of acidic vesicles in SH-SY5Y cells (Fig. 1A, up; the representative histogram is presented in Fig. 1B). The observed effect of olanzapine was also time dependent throughout 72 h of incubation (Fig. 1A, down). The fluorescent microscopy analysis confirmed the increase in numbers of acridine orange-stained red fluorescent acidic vesicles (Fig. 1C), which is consistent with the induction of autophagy. Accordingly, immunoblotting demonstrated that olanzapine treatment increased the conversion of microtubule-associated protein 1 light-chain 3B (MAP1LC3B/LC3B)-I to its lipidated, autophagosome-associated form LC3B-II, both in a time-dependent (Fig. 1D) and concentration-dependent manner (Fig. 1E). The increase in LC3B-II levels was also observed in the presence of proteolysis inhibitors bafilomycin A₁ and NH₄Cl (Fig. 1F), thus confirming that olanzapine-mediated LC3B-II accumulation was due to the increased LC3B conversion, rather than block in its proteolytic degradation. Ultrastructural analysis of olanzapine-treated cells by transmission electron microscopy revealed the presence of double-membrane autophagosomes (Fig. 2A, middle left panel) and single-membrane autolysosomes frequently containing membranous structures resembling mitochondrial remnants (Fig. 2A, middle right panel). The presence of relatively preserved mitochondria in autolysosomes was confirmed in olanzapine-treated cells in which intralysosomal digestion was inhibited by bafilomycin A₁ (Fig. 2A, right panel), a proton pump inhibitor that blocks the activity of acid hydrolases by raising lysosomal pH.³⁰ The analysis of electron micrographs demonstrated a time-dependent increase in the volume fraction of both early autophagic vesicles (autophagosomes and phagophores) and autolysosomes in olanzapine-treated compared to control cells (Fig. 2B). Accordingly, olanzapine increased the numbers of phagophores, autophagosomes, and autolysosomes in SH-SY5Y cells (data not shown). The fusion of autophagosomes with lysosomes and formation of autolysosomes upon olanzapine treatment was evaluated by confocal microscopy. The pattern of LC3B expression in control cells became more punctate upon olanzapine treatment (Fig. 2C, middle panel), which is consistent with the aggregation of LC3B-II in autophagosomes. Furthermore, a clear colocalization of autophagic LC3B puncta (green) and LysoTracker Red-stained lysosomes (red) was demonstrated in olanzapine-treated, but not control cells (Fig. 2C, right panel). Taken together, these data confirm the induction of autophagy in olanzapine-exposed SH-SY5Y cells.

Figure 1.

Olanzapine increases the numbers of acidic vesicles and autophagic flux in SH-SY5Y cells. (A to C) SH-SY5Y cells were incubated for 24 h or for the indicated time periods with or without different concentrations of olanzapine (OLA), risperidone, and haloperidol (A), or with 100 μM of olanzapine (B, C). After staining with acridine orange (AO), the number of acidic vesicles was determined as an increase in red vs. green (FL3/FL1) fluorescence by flow cytometry (A, B) or fluorescent microscopy (C). The data in (A) are presented as mean ± SD values of 3 independent experiments (*P < 0.05), while the representative histograms and micrographs are shown in (B) and (C), respectively. (D to F) SH-SY5Y cells were incubated with or without olanzapine (100 μM) for the indicated time periods (D), 16 h (E) or 8 h (F), in the presence or absence of proteolysis inhibitors bafilomycin A₁ (BAF; 20 nM) or ammonium chloride (AC; 20 mM) (F). LC3B conversion was assessed by immunoblotting, and the densitometry data are presented mean ± SD values of 3 independent experiments (*P < 0.05)

Figure 2.

Olanzapine increases the formation of autophagic structures in SH-SY5Y cells. (A, B) SH-SY5Y were incubated with or without olanzapine (100 μM) for 16 h (A) or the indicated time periods (B), and electron microscopy analysis was performed. The representative micrographs from 3 independent experiments are presented (A), showing untreated cell with intact mitochondria (left panel, arrow), olanzapine-treated cell with an autophagosome (middle left panel, arrow), and olanzapine-treated cell with an autolysosome containing membranous structures resembling mitochondrial remnants (middle right panel, arrow). The relatively preserved mitochondria (right panel, arrow) could be observed in olanzapine-exposed cells simultaneously treated with proteolysis inhibitor bafilomycin A₁ (10 nM, 16 h). (B) The volume fraction (VF) values of early autophagic structures (autophagosomes and phagophores) and autolysosomes were determined in at least 60 cells per sample, and the results are presented as mean ± SD values of 3 independent experiments (*P < 0.05). (C) SH-SY5Y cells were incubated with or without olanzapine (100 μM) for 24 h, and autophagosome-lysosome fusion was assessed by staining with anti-LC3B-Alexa Fluor 488 and LysoTracker Red. The representative micrographs of 3 independent experiments are presented.

Olanzapine-triggered autophagy is MTOR-independent and mediated by oxidative stress

We next assessed the mechanisms responsible for olanzapine-mediated autophagy induction in SH-SY5Y cells. A real-time RT-PCR analysis demonstrated that olanzapine at various time-points increased the expression of the key autophagy-related genes ATG4B, ATG5, and ATG7, while the increase in ATG12 did not reach statistical significance (Fig. 3A). The levels of the proautophagic protein BECN1/Beclin 1 were also slightly increased early upon incubation with olanzapine (Fig. 3B). On the other hand, olanzapine failed to modulate the activation (phosphorylation) of the main autophagy inhibitor MTOR and its direct target RPS6KB1 (Fig. 3B). Accordingly, the phosphorylation of the MTOR inhibitor AMPK and MTOR activator AKT1 was not significantly affected by olanzapine treatment (Fig. 3B). Olanzapine stimulated reactive oxygen species (ROS) generation in SH-SY5Y cells, as demonstrated by the increase in fluorescence of the redox-sensitive dye dihydrorhodamine 123 (DHR) (Fig. 3C). The levels of malondialdehyde, an indicator of lipid peroxidation, were also increased by olanzapine (Fig. 3D), thus further suggesting the induction of oxidative stress by the drug. The observed increase in ROS production was required for olanzapine-mediated autophagy, as the antioxidant agents N-acetylcysteine and α-tocopherol reduced olanzapine-induced LC3 conversion in SH-SY5Y cells (Fig. 3E). It therefore appears that olanzapine-triggered expression of autophagy genes and subsequent induction of autophagy were MTOR-independent and mediated by oxidative stress.

Figure 3.

Olanzapine-triggered autophagy is oxidative stress-mediated and MTOR-independent. (A-D) SH-SY5Y cells were incubated for the indicated time periods with or without olanzapine (100 μM). The expression of mRNA encoding different ATG proteins was analyzed by real-time RT-PCR (A), the levels and phosphorylation status of molecules involved in autophagy regulation were determined by immunoblotting (B), the intracellular ROS production was assessed by flow cytometric analysis of DHR-stained cells (C), while the lipid peroxidation was examined by colorimetric detection of malondialdehyde (MDA) (D). The results in (A, D) are presented as mean ± SD values of triplicates from a representative of 3 independent experiments (*P < 0.05), while the representative blots or flow cytometry histograms from 3 independent experiments are shown in (B, C). (E) SH-SY5Y cells were incubated for 8 h with or without olanzapine (100 μM), in the presence or absence of N-acetylcysteine (NAC; 2 mM) or α-tocopherol (αT; 0.5 mM). The LC3 conversion was assessed by immunoblotting, and the densitometry data are presented as mean ± SD values of 3 independent experiments (*P < 0.05).

Olanzapine causes mitochondrial damage and mitophagy in SH-SY5Y cells

The induction of oxidative stress in neurons is frequently associated with mitochondrial dysfunction.³¹ Indeed, the increase in ROS generation in olanzapine-treated SH-SY5Y cells was accompanied by a loss of mitochondrial membrane potential, as demonstrated by the increase in green-to-red (FL1/FL2) fluorescence ratio of the mitochondria binding fluorochrome JC-1 (Fig. 4A). The time-kinetics analysis revealed that the rise in DHR fluorescence preceded the increase in JC-1 FL1/FL2 ratio (Fig. 4B), thus indicating that ROS production occurs before mitochondrial depolarization during olanzapine treatment. The treatment with α-tocopherol, a potent and relatively selective antioxidant,³² significantly inhibited both the increase in DHR fluorescence and JC-1 FL2/FL1 conversion in olanzapine-exposed cells (Fig. 4C, D), suggesting that ROS production was involved in the drug-mediated mitochondrial depolarization. In contrast to untreated cells with intact mitochondria (Fig. 2A, left panel), the electron microscopy analysis of olanzapine-treated cells demonstrated the presence of mitochondria with electron dense matrix and ballooned cristae (Fig. 5A left and middle panel), or swollen mitochondria with hypodense matrix and loss of cristae (Fig. 5A left and right panel). Some of the swollen mitochondria with hypodense matrix contained myelin figures (Fig. 5A, right panel), possibly as a result of concentric whorling of the inner mitochondrial membrane.³³ The quantification of damaged mitochondria revealed that their proportion significantly increased during olanzapine treatment, reaching the maximum levels after 8 to 16 h of exposure, and then slightly declining after 24 h (Fig. 5B). While the similar pattern of increase was observed for both types of mitochondrial damage, the mitochondria with electron dense matrix were markedly more frequent (Fig. 5B). The levels of SQSTM1, a cargo receptor that labels intracellular targets, including damaged mitochondria, for selective autophagic degradation,^34-36 was markedly increased in mitochondrial, but not cytoplasmic fraction of olanzapine-treated cells (Fig. 5C). Moreover, confocal microscopy revealed a clear colocalization of autophagic LC3B puncta (green) and MitoTracker Red-labeled mitochondria (red) in olanzapine-treated, but not control cells (Fig. 5D). These data indicate that olanzapine-induced damage to mitochondria is accompanied by their autophagic clearance.

Figure 4.

Olanzapine causes ROS-dependent mitochondrial depolarization in SH-SY5Y cells. (A-D) SH-SY5Y cells were incubated for 16 h (A) or for the indicated time periods (B to D) with or without olanzapine (100 μM), in the absence (A and B) or presence (C, D) of α-tocopherol (αT; 0.5 mM). Mitochondrial depolarization (A, B, D) and ROS production (B, C) were determined by flow cytometric analysis of JC-1 green/red (FL1/FL2) fluorescence ratio or DHR green fluorescence, respectively. The representative JC-1 histograms are shown in (A), while the data in (B to D) are presented as mean ± SD values of 3 independent experiments [*P < 0.05 compared to untreated cells (B) or all other treatments (C, D)]. The results are expressed relative to untreated cells at corresponding time points (control values arbitrarily set to 1–the dashed lines).

Figure 5.

Olanzapine induces mitochondrial damage and colocalization with SQSTM1 and LC3B. (A to D) SH-SY5Y cells were incubated for 16 h (A, C), 24 h (D) or for the indicated time periods (B) with or without olanzapine (100 μM). (A, B) The presence of mitochondrial damage in olanzapine-treated cells was assessed by electron microscopy (the arrow shows a myelin figures), while the percentage of damaged mitochondria (B) is presented as mean ± SD values (*P < 0.05; n = 60 cells). (C) The levels of SQSTM1 in mitochondrial fraction (COX4I1-rich, actin-low) and cytoplasmic fraction (actin-rich, COX4I1-low) were analyzed by immunoblotting, and the blots from a representative of 2 independent experiments are shown. (D) The presence of LC3B/mitochondria colocalization was assessed by confocal fluorescent microscopy using LC3-Alexa Fluor 488 and MitoTracker Red. The representative micrographs from 3 independent experiments are presented.

Autophagy protects SH-SY5Y cells from olanzapine toxicity

Treatment with olanzapine (up to 100 μM) did not markedly compromise the viability of SH-SY5Y cells, as only a minor reduction in cell number (crystal violet staining) in the absence of cell membrane damage (no increase in lactate dehydrogenase [LDH] release) was observed after 72 h of incubation (Fig. 6A). Moreover, we did not observe a significant ATP loss throughout this period (data not shown), possibly due to the reliance of SH-SY5Y cells on glycolysis for energy production.³⁷ However, the agents that block class III phosphatidylinositol 3-kinase-dependent autophagosome formation (3-methyladenine)³⁸ or autophagosome acidification and subsequent proteolytic digestion (bafilomycin A₁),³⁰ significantly reduced the cell numbers and caused cell membrane damage in olanzapine-treated, but not control cells (Fig. 6B). As both inhibitors could have autophagy inhibition-independent effects,³⁹ and 3-methyladenine can even induce autophagy,³⁸ we assessed the role of autophagy in olanzapine-exposed SH-SY5Y cells by using RNA interference-mediated knockdown of LC3B and BECN1. Both crystal violet and LDH release assay demonstrated that the cells transfected with LC3B short hairpin (sh)RNA or BECN1 small-interfering (si)RNA were more sensitive to olanzapine treatment than their control RNA-transfected counterparts (Fig. 6C), thus confirming that the induction of autophagy protected SH-SY5Y cells from the cytotoxic effects of the drug. Combined treatment with olanzapine and bafilomycin A₁, but not with each agent alone, significantly increased caspase activation and DNA fragmentation (Fig. 6D), indicating the induction of apoptosis.

Figure 6.

(See previous page). Inhibition of autophagy protects SH-SY5Y cells from olanzapine toxicity. (A, B) SH-SY5Y cells were incubated for 24 to 72 h with different concentrations of olanzapine (A) or for 48 h with or without olanzapine (100 μM), in the presence or absence of autophagy inhibitors bafilomycin A₁ (BAF; 10 nM) or 3-methyladenine (3MA; 2.5 mM) (B). The cell viability was determined by crystal violet staining or LDH release assay, and the results are presented as mean ± SD values of triplicates from a representative of 3 independent experiments (*P < 0.05 compared to olanzapine alone). (C) SH-SY5Y cells transfected with LC3B shRNA or BECN1 siRNA (the inset shows immunoblot confirmation of knockdown) and appropriate control RNA-transfected cells were incubated for 48 h with or without olanzapine (100 μM). The cell viability was determined by crystal violet staining or LDH release assay, and the results are presented as mean ± SD values of triplicates from a representative of 3 independent experiments (*P < 0.05 compared to all other treatments). (D) SH-SY5Y cells were incubated for 48 h with or without olanzapine (100 μM), in the presence or absence of the autophagy inhibitor bafilomycin A₁ (BAF; 10 nM), and the caspase activation and DNA fragmentation were analyzed by flow cytometry. The representative histograms are shown, while the data are presented as mean ± SD values of 3 independent experiments (*P < 0.05 compared to all other treatments).

The toxicity of nitric oxide (NO) to olanzapine-treated cells is associated with autophagy inhibition

To demonstrate the autophagy-mediated protection from olanzapine in a biologically more relevant setting, we used NO, a neurotoxic-free radical recently found to inhibit autophagic response.^40,41 Chemical NO donor diethylamine NONOate (DEA-NONOate) readily inhibited olanzapine-triggered autophagy, as confirmed by a significant reduction of the olanzapine-mediated increase in LC3 conversion and the numbers of acidic vesicles (Fig. 7A, B). While displaying a dose-dependent toxicity toward SH-SY5Y cells, DEA-NONOate potently synergized with olanzapine in the induction of cell death, as demonstrated by the reduction of crystal violet staining and the increase in LDH release (Fig. 7C). A similar synergistic cytotoxic effect was observed when olanzapine was combined with another NO donor, propylamine propylamine NONOate (PAPA-NONOate) (Fig. 7D). Therefore, the inhibition of autophagy by NO is associated with its ability to synergistically enhance the in vitro neurotoxicity of olanzapine.

Figure 7.

The toxicity of NO to olanzapine-treated cells is associated with autophagy inhibition. (A) SH-SY5Y cells were incubated for 8 h with 100 μM olanzapine in the presence or absence of NO donor DEA-NONOate (0.25 mM), and LC3B conversion was analyzed by immunoblotting. The representative blot from 3 independent experiments is shown. (B) SH-SY5Y cells were incubated for 24 h with olanzapine (50 or 100 μM) and/or different concentrations of DEA-NONOate, and the presence of acidic vesicles was assessed by flow cytometric analysis of acridine orange (AO) red/green fluorescence (FL3/FL1) ratio. The flow cytometry data are presented as mean ± SD values of triplicates from a representative of 2 experiments (*P < 0.05 compared to olanzapine-treated cells without NO donors). (C, D) SH-SY5Y cells were incubated for 48 h with or without olanzapine (100 μM), in the presence or absence of different concentrations of NO donors DEA-NONOate (C) or PAPA-NONOate (D). The cell viability was determined by crystal violet staining or LDH release assay, and the data are presented as mean ± SD values of triplicates from a representative of 3 experiments (*P < 0.05 compared to untreated cells and cells treated with olanzapine or NO alone).

Autophagy inhibitor chloroquine induces proapoptotic changes in brains of olanzapine-treated mice

We next examined the ability of olanzapine to trigger neuronal autophagy in vivo, using the dose of 2 mg/kg/d. According to the formula: animal dose (mg/kg) × animal Km/human Km, where Km is a correction factor reflecting the relationship between body weight and body surface area (Km = 37 for an adult human of 60 kg, and Km = 3 for a mouse of 20 g), the human equivalent dose is 9.7 mg. This dose is equal to the target dose (10 mg/d) and lower than the maximum dose (20 mg/d) in the treatment of schizophrenia, according to the Physicians' Desk Reference. Mice were treated with olanzapine for 2 wk, and the induction of autophagy in the brain was assessed by immunoblot detection of LC3B conversion, as well as by RT-PCR analysis of Atg gene expression. In comparison with saline-treated controls, frontal brain region and hippocampus of olanzapine-treated mice displayed higher levels of LC3B-II (Fig. 8A). Furthermore, the levels of mRNA encoding Atg4B, Atg5, Atg7, Atg12, Becn1, and Gabarap were all significantly increased in the frontal brain region of olanzapine-treated mice (Fig. 8B), thus indicating the induction of autophagy. We then used chloroquine, a well-known inhibitor of autophagic proteolysis^42,43 to assess the role of olanzapine-mediated autophagy in modulating the expression of various molecules that regulate apoptotic cell death. The treatment with olanzapine increased the mRNA levels of proapoptotic Cdkn1a and antiapoptotic Xiap (Fig. 8C), while other proapoptotic/antiapoptotic factors were not significantly affected either by olanzapine or chloroquine alone (Fig. 8C). On the other hand, their combination significantly increased the mRNA levels of proapoptotic molecules Cdkn1a, Cdkn1b, Pmaip1, Bax, Bak1, Bcl2l11, and Trp53 in comparison to untreated animals (Fig. 8C). The expression of antiapoptotic Bcl2, Bcl2l1, and Xiap was also increased, while the levels of mRNA encoding the proapoptotic Bbc3, Pten, Bad, and Apaf1, as well as antiapoptotic Birc5, were not significantly altered by olanzapine/chloroquine cotreatment. Finally, the mice receiving olanzapine/chloroquine combination, compared to the sham controls, displayed an increase in neuronal DNA fragmentation in the frontal brain region, as revealed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Fig. 8D). We found no or few TUNEL-positive cells in the brains of animals administered olanzapine or chloroquine alone (Fig. 8D). These data indicate that olanzapine at a clinically relevant dose can trigger autophagic response in the brain, and that inhibition of autophagy could increase brain expression of proapoptotic genes and neuronal DNA fragmentation.

Figure 8.

Autophagy inhibitor chloroquine induces apoptotic changes in brains of olanzapine-treated mice. (A to C) Mice (6 per group) were treated for 14 d with PBS (control) or olanzapine (2 mg/kg) (A, B) or with PBS (control), olanzapine (2 mg/kg), chloroquine (20 mg/kg), or olanzapine + chloroquine (C), as described in Materials and Methods. The levels of LC3B-II in the frontal brain region and hippocampus were assessed by immunoblotting (A), while the expression of mRNA for Atg genes (B) and proapoptotic or antiapoptotic genes (C) in the frontal brain was analyzed by real-time RT-PCR. The results are presented as mean ± SEM values (*P < 0.05). (D) Mice were treated as in (C) and DNA fragmentation in the frontal brain was assessed by TUNEL assay. The arrows point at the representative cells with red-stained TUNEL-positive nuclei in mice treated with olanzapine + chloroquine.

Discussion

The present study shows for the first time the ability of olanzapine to induce an autophagic response in human neuronal cells in vitro and in the mouse brain in vivo. The induction of autophagy by other antipsychotic drugs has recently been reported,^21-25 but its contribution to their therapeutic or side effects has not been elucidated. By demonstrating that the inhibition of autophagy unmasks the neurotoxic action of olanzapine both in vitro and in vivo, our study implicates autophagy as an important protective mechanism against the olanzapine-mediated neurotoxicity.

Having in mind the difficulties in monitoring autophagy,⁴⁴ we have examined an array of autophagic markers in olanzapine-exposed SH-SY5Y cells. The LC3B conversion and punctuation, increase in autophagic flux and numbers of acidic vesicles, presence of autophagosomes and autolysosomes according to morphological criteria, autophagosome-lysosome fusion, and increased expression of several ATG genes, confirmed the ability of olanzapine to induce autophagic response in SH-SY5Y neurons. Olanzapine also increased LC3B conversion in the mouse brain, indicating that autophagy could be induced by antipsychotics in vivo. In addition, the colocalization of autophagosome-associated LC3B and mitochondria, as well as mitochondrial association with the autophagic cargo receptor SQSTM1, strongly suggests that olanzapine-triggered autophagic response involved mitophagy. While a direct role of SQSTM1 in triggering mitophagy is controversial, its accumulation in the damaged mitochondria seems to be required for their clustering.^36,45,46 This is consistent with the here-reported ability of olanzapine to induce ROS production and subsequent loss of the mitochondrial membrane potential, as mitochondrial depolarization seems to be a crucial event in triggering autophagic clearance of oxidative stress-damaged mitochondria.^47-50 Although olanzapine has been shown to inhibit mitochondrial succinate dehydrogenase and electron transport complexes I and IV,^51,52 our data suggest that the drug could initially increase neuronal ROS generation independently of, and prior to mitochondrial dysfunction (Fig. 9). Nevertheless, the ROS produced by dysfunctional mitochondria can lead to further mitochondrial damage and oxidative stress through a positive feedback circle,⁵³ eventually leading to autophagic removal of damaged mitochondria (Fig. 9). Interestingly, the patterns of mitochondrial damage observed in our study have also been reported to occur in mitoptosis, a phenomenon defined as a sort of mitochondrial death program associated with both apoptosis and autophagy.⁵⁴ Mitochondria with a hypodense matrix and an electron dense matrix found in olanzapine-exposed SH-SY5Y neurons bear a close resemblance to those seen in so-called inner membrane mitoptosis and outer membrane mitoptosis, respectively.⁵⁴ However, the relevance of this apparent similarity remains to be explored. It should also be noted that autophagy induction by clozapine and haloperidol in rat primary neurons, in contrast to our findings, was associated with the block of autophagosome-lysosome fusion.²² Therefore, the progression of antipsychotic-induced autophagy to the degradative stage might be regulated in a drug- and/or cell type-dependent manner.

Figure 9.

A hypothetical representation of the mechanisms and role of autophagy in olanzapine neurotoxicity. Olanzapine triggers a positive feedback circle of cell-damaging ROS production and mitochondrial dysfunction, but the overt neurotoxicity is prevented by a concomitant induction of cytoprotective autophagy and mitophagy.

Enhanced ROS production by mitochondria induces general autophagy,^55,56 and autophagy induction by the antipsychotic drug sertindole was mediated by oxidative stress,²³ as confirmed in the present study by the ability of the antioxidant treatment to prevent olanzapine-triggered LC3B conversion and increase in the number of acidic vesicles. The main mechanism apparently responsible for the oxidative stress-mediated autophagy in various cell types, including neurons, is the inhibition of the autophagy repressor MTOR via AMPK activation and/or AKT1 blockade.^55,57 Interestingly, it has recently been shown that olanzapine increases both AMPK and MTOR activation in mouse liver in vivo, but autophagy has not been assessed.⁵⁸ In the present study, olanzapine-mediated increase in ATG gene expression was not associated with modulation of AMPK, AKT1 or MTOR activity, suggesting that transcriptional induction of autophagy by the drug was MTOR-independent. Indeed, oxidative stress can trigger transcription of crucial ATG genes by activating FOXO transcription factors,^59,60 which control transcriptional activation of autophagy independently of MTOR.⁵⁵ Accordingly, the expression of ATG4B, ATG12, BECN1, and GABARAP, which was upregulated by olanzapine in SH-SY5Y neurons and/or mouse brain, is under direct control of FOXO3.⁵⁵ The MTOR-independent mechanisms involved in oxidative stress-triggered autophagic response also include a direct interaction of cytosolic FOXO1 with ATG7,⁶¹ as well as activation of mitogen-activated protein kinase pathway⁶² or endoplasmic reticulum stress-dependent phosphorylation of PRKC/protein kinase C.⁶³ The possibility that some of the above mechanisms may function as an autophagy-inducing signal downstream of ROS production in olanzapine-exposed neurons is currently being tested in our laboratory. As for the upstream events responsible for the autophagy induction by olanzapine, the involvement of dopaminergic receptor blockade seems rather unlikely, as dopamine actually induces autophagy in SH-SY5Y cells.^64,65 However, having in mind that several dopamine/serotonine receptor-blocking drugs were able to trigger autophagic response,^21-25 the role of dopamine and/or serotonine receptor antagonism in the observed effect clearly requires further exploration.

It is well established that oxidative stress-mediated mitochondrial dysfunction can lead to apoptotic or necrotic neuronal death.⁶⁶ However, despite the ability to block important mitochondrial enzymes,^51,52 and cause mitochondrial depolarization (the present study), olanzapine was not overtly toxic to cultured neurons, as demonstrated previously^29,67-69 and confirmed here, thus indicating some protective mechanism(s). We here identify an important role of autophagy in this protection by showing that olanzapine neurotoxicity is readily revealed if the autophagic response is inhibited pharmacologically or by genetic knockdown of autophagy-essential LC3B and BECN1. This finding is consistent with the proposed role of autophagy in removal of ROS-producing depolarized mitochondria, which reduces oxidative stress and allows residual, undamaged mitochondria to repopulate the mitochondrial pool and rescue the cell.^70,71 Accordingly, the failure of autophagy-mediated mitochondrial quality control could contribute to neuronal damage in neurodegenerative diseases.⁷² In addition to mitophagy, general autophagy itself may promote cell survival under oxidative stress not only by providing ATP and basic building blocks for the synthesis of antiapoptotic molecules, but also by eliminating toxic protein aggregates, modulating cell cycle progression, and releasing antiapoptotic molecules like BCL2 and the CASP8/caspase 8 inhibitor CFLAR/FLIP from the inhibitory interaction with BECN1 and ATG3, respectively.⁷³ Therefore, it is conceivable that both olanzapine-triggered mitophagy and general autophagy protected SH-SY5Y cells from otherwise fatal mitochondrial injury (Fig. 9). A similar mechanism might be operative in vivo, as the concomitant administration of clinically relevant doses of olanzapine and autophagy inhibitor chloroquine in our study caused a significant increase in brain expression of several proapoptotic genes (Trp53, Pmaip1, Bax, Bak1, Bcl2l11, Cdkn1a, and Cdkn1b), associated with fragmentation of neuronal DNA. This suggests that autophagy/mitophagy-mediated blockade of oxidative stress in olanzapine-treated neurons might inhibit the expression of proapoptotic genes and subsequent apoptotic cell death, which is consistent with the findings that ROS-mediated control of apoptotic machinery in neuronal cells involves transcription initiation of several genes, including TRP53, PMAIP1, BAX, BAK1, BCL2L11, CDKN1A, and CDKN1B.^74-76 It should be noted, however, that autophagy was apparently responsible for the SH-SY5Y cell death induced by sertindole,²³ thus indicating that the role of autophagy in modulation of neuronal death might depend on the type of antipsychotic drug.

Finally, to provide additional support for biological and clinical relevance of our findings, we have demonstrated that NO, an intracellularly produced biologically active free radical with autophagy-blocking capacity,^40,41 potently synergized with olanzapine in killing SH-SY5Y neurons. NO-mediated S-nitrosylation impairs autophagy in various cell types, including neurons, mainly by inhibiting MAPK8/JNK1-mediated phosphorylation of BCL2 and increasing its interaction with autophagy-essential BECN1.⁴¹ Moreover, the inhibition of NO-producing NO synthase family of enzymes enhanced the clearance of autophagic substrates and reduced neurodegeneration in models of Huntington disease.⁴¹ Consistent with these findings, the synergistic neurotoxicity of olanzapine and NO in our study was associated with NO-mediated interference with olanzapine-triggered autophagic response. It should be noted, however, that the death of olanzapine-treated cells in our study was more efficiently induced with NO than with selective autophagy inhibition. While this is probably due to the ability of NO to directly modulate various cell death pathways independently of autophagy,⁷⁷ it is still very likely that autophagy inhibition contributed to the observed neurotoxicity in the presence of olanzapine.

In conclusion, our data indicate that autophagy/mitophagy could protect olanzapine-exposed neurons from oxidative stress-mediated mitochondrial damage and subsequent apoptotic death (Fig. 9). As some studies indicate that the production of NO in certain brain regions is increased in schizophrenia,⁷⁸ it is tempting to speculate that autophagy inhibition by NO might unmask the neurodamaging effects of antipsychotic medications. It would also seem reasonable to consider caution when using olanzapine to treat patients who receive drugs with autophagy-inhibiting potential (e.g., chloroquine, intravenous proton-pump inhibitors), or suffer from neuroinflammatory/neurodegenerative disorders associated with increased NO production and/or impaired autophagic clearance, such as multiple sclerosis and Alzheimer and Parkinson diseases.^79,80 The future studies should investigate if the concept proposed here also applies to other antipsychotic medications and glial cells, as well as to explore its possible significance for the side effects of antipsychotic treatment.

Materials and Methods

Cell culture

The human neuroblastoma cell line SH-SY5Y (ATCC CRL-2266) was grown at 37 °C in a humidified atmosphere with 5% CO₂, in Dulbecco's Modified Eagle Medium/Ham's Nutrient Mixture F12 (Sigma-Aldrich, 51445C) supplemented with 10% fetal calf serum (Sigma-Aldrich, F4135) and 10 μl/ml of penicillin/streptomycin (Sigma-Aldrich, P0781). The trypsinized cells were incubated in 96-well flat-bottom cell culture plates (1.5 × 10⁴ cells/well) for the cell viability assessment, 24-well plates (1.2 × 10⁵ cells/well) for the flow cytometric analysis, or 60 mm cell culture plates (1.5 × 10⁶ cells) for the immunoblotting, electron microscopy, and real-time RT-PCR. Cells were rested for 24 h, and then treated with olanzapine (Sigma-Aldrich, O1141) in the absence or presence of autophagy inhibitors bafilomycin A₁ (Sigma-Aldrich, 11711) and 3-methyladenine (Sigma-Aldrich, M9281), antioxidants N-acetylcysteine (Sigma-Aldrich, A8199) and α-tocopherol (Sigma-Aldrich, T3251), or NO donors DEA-NONOate (Sigma-Aldrich, D5431), and PAPA-NONOate (Sigma-Aldrich, P8102), as described in Results and in Figure Legends.

Cell viability

Crystal violet staining of adherent, viable cells, and the release of intracellular enzyme LDH as a marker of cell membrane damage, were used to determine cell viability/cytotoxicity exactly as previously described.⁸¹ The results are presented as percent of the crystal violet absorbance obtained in untreated cells (100%), or fold increase in the absorbance measured in LDH release assay (set to 1 in untreated cells).

ROS production and mitochondrial depolarization

The production of ROS and mitochondrial depolarization were measured by flow cytometry, using a FACSCalibur flow cytometer (BD, Heidelberg, Germany) and CellQuest Pro software. Intracellular ROS production was determined by measuring the intensity of green fluorescence emitted by a redox-sensitive dye DHR (Life Technologies, D-23806). After incubated with DHR (2 μM) for the whole duration of treatment, cells were detached by trypsinization and washed in phosphate buffered saline (PBS). The green fluorescence (FL1), corresponding to total ROS levels, was measured, and the results are presented as the fold change in mean fluorescence intensity in comparison to the untreated control cells. The mitochondrial depolarization was assessed using JC-1(Life Technologies, T-3168), a lipophilic cation that forms orange-red fluorescent aggregates upon binding to polarized mitochondria. If the mitochondrial membrane potential is disturbed, the dye cannot access the transmembrane space and remains or reverts to its green monomeric form. The cells were stained as described by the manufacturer, and the results are presented as a green/red fluorescence ratio (mean FL1/FL2), the increase of which reflects mitochondrial depolarization.

Lipid peroxidation

Malondialdehyde, an indicator of lipid peroxidation, was measured using a colorimetric thiobarbituric acid assay. The binding of thiobarbituric acid to malondialdehyde-bis-(dimethylacetal)1,1,3,3-tetramethoxypropan formed during lipid peroxidation results in a chromogenic complex. The homogenate obtained by lysing 5 × 10⁶ cells with 10% ice-cold trichloroacetic acid (Sigma-Aldrich, T6399) was centrifuged at 800 g for 10 min, and the supernatant was mixed (1:1) with 0.6% 2-thiobarbituric acid (Sigma-Aldrich, T5500) and heated in a boiling water for 10 min. The absorbance was measured at 535 nm, and the results are expressed as the fold change of absorbance intensity compared to control, untreated cells.

Caspase activation and DNA fragmentation

Activation of caspases was measured by flow cytometry after labeling the cells with a cell-permeable, FITC-conjugated pancaspase inhibitor (ApoStat; R&D Systems, FMK012) according to the manufacturer's instructions. The increase in the green fluorescence (FL1) as a measure of caspase activity was determined using FACSCalibur flow cytometer, and the results are presented as the fold change in mean fluorescence intensity in comparison to the untreated control cells. DNA fragmentation as a marker of apoptosis was assessed by flow cytometric analysis of cells stained with DNA-binding dye propidium iodide as previously described.⁸² The TUNEL assay (Roche, 11684809910) was used to identify fragmented DNA in the brain cryosections. After fixation in 4% paraformaldehyde and permeabilization with 0.2% Triton X-100 (Sigma-Aldrich, X100), cells were treated with 3% bovine serum albumin (Sigma-Aldrich, A2153) and TUNEL reaction mixture. Converter for alkaline phosphatase (Roche, 11684809910) was applied and Fast Red (Roche, 11496549001) was used to visualize the signal for light microscopy. TUNEL-stained sections were counterstained with Mayer's hematoxylin (Sigma-Aldrich, MHS1).

Quantification of acidic vesicles by acridine orange staining

The number of intracellular acidic vesicles was assessed by staining with a pH-sensitive dye acridine orange (Sigma-Aldrich, 318337) and subsequent flow cytometric and fluorescent microscopy analysis as previously described.⁸²

Transmission electron microscopy analysis of damaged mitochondria and autophagy

The trypsinized cells were fixed with 3% glutaraldehyde in cacodylate buffer and postfixed in 1% OsO₄. After dehydration in graded alcohols, cells were embedded in Epoxy medium (Sigma-Aldrich, 45345). Thin sections were mounted on copper grids (Sigma-Aldrich, G4901), and stained with uranyl acetate and lead citrate for examination on an electron microscope (Morgagni 268D, FEI, Hillsboro, OR). The sections and micrographs for the analysis were selected by using Systematic Uniform Random Sampling,⁸³ and the numbers of intact/damaged mitochondria and autophagic structures (phagophores, autophagosomes, and autolysosomes) were determined in 60 cells for each treatment. The phagophores and autophagosomes were counted together, as they cannot be clearly differentiated unless the open ends of the phagophores appear in the plane of section (both are surrounded by the double membrane). Autophagic structures were also quantified by fractional volume analysis as previously described,⁸³ using the following formula: volume fraction = ΣPAP/ ΣPcyt × ρ, where ΣPAP is the number of points of a dense grid counted on autophagic structures, ΣPcyt the number of points of a second grid counted within the cytoplasm, and ρ is the number of points on the dense grid that represent each point of the grid used for the cytoplasm (in this case 25). At least 60 cells per sample were analyzed in each experiment.

Confocal microscopy

Confocal fluorescence microscopy was used to analyze colocalization of LC3B with lysosomes or mitochondria. Cells grown in chamber slides were incubated with 100 nM LysoTracker Red (Life Technologies, L7528) or MitoTracker Red (Life Technologies, M7512) at 37°C for 60 or 15 min, respectively. After washing, cells were fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with ice-cold methanol for 10 min, and blocked with 5% normal goat serum and 0.3% Triton X-100 for 1 h. Cells were washed and incubated with rabbit anti-LC3B (Cell Signaling Technology, 2775) (1:200 in PBS [Sigma-Aldrich, P4417] with 1% bovine serum albumin and 0.3% Triton X-100 for 1.5 h at room temperature), followed by 1 h incubation with Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies, A-11034) as a secondary antibody. Cells were then washed with PBS and mounted on microscope slides with Flouromount-G (SouthernBiotech, 0100–01). Images were acquired with Leica Confocal Software on Confocal Scanner microscope Leica TCS SP2 (Leica Microsystems, Wetzlar, Germany) using a PL APO 63 × 1.3 Glycerol HCX CS objective with 2.54 digital zoom and FITC-TRITC laser combination in a single scan mode.

Real-time RT-PCR

A real-time RT-PCR was used to determine the expression of genes involved in autophagy and apoptosis. Total RNA from cells or brain tissue was extracted using TRIZOL reagent (Life Technologies, 15596-026) according to the manufacturer's instructions. Approximately 1 μg of RNA was used in the reverse transcription reaction using MuLV reverse transcriptase (Life Technologies, N8080018) with oligo(dt) primers (Life Technologies, 18418012) according to the manufacturer's instructions. Real-time RT-PCR was performed in a Realplex² Mastercycler (Eppendorf, Hamburg, Germany) using 96-well reaction plates (Applied Biosystems, 4306737), TaqMan Universal PCR Master Mix (Applied Biosystems, 4364340), and TaqMan primers/probes (Applied Biosystems) for human ATG4B (Hs00367088_m1), ATG5 (Hs00169468_m1), ATG7 (Hs00197348_m1), ATG12 (Hs00740818_m1), SQSTM1 (Hs00177654_m1), and B2M (Hs00984230_m1), or mouse Atg4b (00558047_m1), Atg5 (00504340_m1), Atg7 (00512209_m1), Atg12 (00503201_m1), Gabarap (00490678_m1), Becn1 (01265461_m1), Sqstm1 (00448091_m1), Cdkn1a (00432448_m1), Cdkn1b (00438168_m1), Bbc3 (00519268_m1), Pmaip1 (00451763_m1), Bax (00432051_m1), Bad (00432042_m1), Bak1 (00432045_m1), Bcl2l11 (00437796_m1), Trp53 (01731287_m1), Pten (00477208_m1), Apaf1 (01223702_m1), Bcl2 (00477631_m1), Bcl2l1 (00437783_m1), Xiap (00776505_m1), Birc5 (00599749_m1), and Rn18s (03928990_g1). The reaction conditions were as recommended by the manufacturer. All assays were performed in triplicates. The cycle of threshold (Ct) values of B2M and Rn18s as housekeeping genes were subtracted from Ct values of target genes to obtain ΔCt, and relative gene expression was determined as 2^−ΔCt. The results were presented relative to the control value, which was arbitrarily set to 1.

Immunoblotting

The immunoblotting was performed as previously described,⁸² using rabbit antibodies (Cell Signaling Technology) against LC3B (2775), BECN1/Beclin 1 (3495), PRKAA/AMPKA (2603), phospho-PRKAA/AMPKA (Thr172; 2535), AKT1 (9272), phospho-AKT1 (Ser473; 4058), MTOR (2983), phospho-MTOR (Ser2448; 2971), RPS6KB1/S6K1 (2708), phospho-RPS6KB1/S6K1 (Thr389; 9205), COX4I1/COX IV (4850), and actin (4968) as primary antibodies, and peroxidase-conjugated goat anti-rabbit IgG (Jackson IP Laboratories, 111-035-003) as a secondary antibody. The levels of LC3B-II were quantified by densitometry using ImageJ software and expressed relative to actin. The results are presented as the fold change in signal intensity compared to that of the untreated control, which was arbitrarily set to 1.

Mitochondrial fractionation

Mitochondria were obtained by using a mitochondrial purification protocol by Abcam (www.abcam.com).

RNA interference

SH-SY5Y cells were transfected with plasmids encoding shRNA against human LC3B (sc-43390-SH) and scrambled control shRNA (sc-108060), using shRNA Plasmid Transfection Reagent (sc-108061) and shRNA Plasmid Transfection Medium (sc-108062) according to the protocol by the manufacturer (Santa Cruz Biotechnology). The stably transfected cells were selected as recommended by manufacturer and maintained in selection medium containing 10 μg/ml puromycin (Santa Cruz Biotechnology, sc-108071). The transfection of SHSY5Y with siRNA against human BECN1 (Qiagen, NM003766) and negative control siRNA (Qiagen, SI03650318) was performed using Lipofectamine (Life Technologies, 11668-019) according to the manufacturer's instructions. After transfection, cells were allowed to grow 24 h before being used for experiments.

In vivo treatments

All animal experiments were approved by the Local Animal Care Committee and conformed to the ethical guidelines stated in stated in the “Principles of Laboratory Animal Care” (NIH publication #85-23, revised in 1985). The study was performed on 6-wk-old male NMRI Han mice (Institute of Biomedical Research, Galenika) kept under a 12:12 h light-dark cycle at 22 ± 2°C and accustomed to daily handling. In the first experiment, the mice (6 per group) received 200 μl of PBS (control) or olanzapine (2 mg/kg in 200 μl PBS). In the second experiment, the mice were divided into 4 groups (n = 6 per group): 1. control (200 μl PBS), 2. olanzapine (2 mg/kg in 200 μl PBS), 3. chloroquine (Sigma-Aldrich, C6628; 20 mg/kg in 200 μl PBS), and 4. olanzapine (2 mg/kg in 100 μl PBS) + chloroquine (20 mg/kg in 100 μl PBS). All injections were given intraperitoneally daily for 14 d. At the end of the experiments, the animals were sacrificed, and the frontal brain region and hippocampal tissues were collected for RT-PCR and immunoblot analysis.

Statistical analysis

The statistical significance of the differences was analyzed by ANOVA, Man-Whitney test or Kruskall-Wallis test (followed by Man-Whitney) were appropriate. A P value less than 0.05 was considered statistically significant.

Funding

The study was supported by the Ministry of Education, Science, and Technological Development of the Republic of Serbia (Grant No. 41025 to VT, 173053 to LHT, and 41029 to NM).