Photo-Induced Oxidative Stress Impairs Mitochondrial Metabolism in Neurons and Astrocytes

Photodynamic therapy is selective destruction of cells stained with a photosensitizer upon irradiation with light at a specific wavelength in the presence of oxygen. Cell death upon photodynamic treatment is known to occur mainly due to free radical production and subsequent development of oxidative stress. During photodynamic therapy of brain tumors, healthy cells are also damaged; considering this, it is important to investigate the effect of the treatment on normal neurons and glia. We employed live-cell imaging technique to investigate the cellular mechanism of photodynamic action of radachlorin (200 nM) on neurons and astrocytes in primary rat cell culture. We found that the photodynamic effect of radachlorin increases production of reactive oxygen species measured by dihydroethidium and significantly decrease mitochondrial membrane potential. Mitochondrial depolarization was independent of opening of mitochondrial permeability transition pore and was insensitive to blocker of this pore cyclosporine A. However, irradiation of cells with radachlorin dramatically decreased NADH autofluorescence and also reduced mitochondrial NADH pool suggesting inhibition of mitochondrial respiration by limitation of substrate. This effect could be prevented by inhibition of poly (ADP-ribose) polymerase (PARP) with DPQ. Thus, irradiation of neurons and astrocytes in the presence of radachlorin leads to activation of PARP and decrease in NADH that leads to mitochondrial dysfunction.     

Reactive Oxygen Species Produced by a Photodynamic Effect Induced Calcium Signal in Neurons and Astrocytes

Photodynamic therapy (PDT) leads to production of reactive oxygen species (ROS) and cell destruction due to oxidative stress. We used photodynamic effect of photosensitizer radachlorin to unravel the effect of photo-induced oxidative stress on the calcium signal and lipid peroxidation in primary culture of cortical neurons and astrocytes using live cell imaging. We have found that irradiation in presence of 200 nM of radachlorin induces calcium signal in primary neurons and astrocytes. Photo-induced neuronal calcium signal depends on internal calcium stores as it was still observed in calcium-free medium and could be blocked by depletion of endoplasmic reticulum (ER) stores with inhibitor of sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA) thapsigargin. Both inhibitors of phospholipase C activity U73122 and water-soluble analogue of vitamin E Trolox suppressed calcium response activated by PDT. We have also observed that the photodynamic effect of radachlorin induces lipid peroxidation in neurons and astrocytes. This data demonstrate that lipid peroxidation induced by PDT in neurons and astrocytes leads to activation of phospholipase C that results in production of inositol 1,4,5-trisphosphate (IP3).     

具有自噬抑制作用的卟啉金属有机框架的制备及其光动力癌症治疗的研究*

光动力疗法(PDT)具有微创、可控、低毒、可重复治疗等优点,已成为临床医学中不可缺少的治疗手段。但由于肿瘤细胞的自我保护机制,大大降低了PDT疗效。使用PDT治疗方法的同时实施药理自噬抑制策略,切断因光动力治疗下严重氧化损伤下的保护性自噬。通过油浴加热法合成卟啉金属有机框架PCN-224,并在PCN-224上负载自噬抑制剂硫酸羟氯喹(HCQ),通过扫描电子显微镜(SEM)、粒径测试(DLS)、紫外可见光谱测试等方法检测,结果表明成功地合成了该材料,增强了卟啉光敏剂的水溶性,并且光照后对4T1小鼠乳腺癌细胞毒性明显增强,且装载了HCQ后进一步提高了肿瘤杀伤能力。     

Responses of crayfish neurons and glial cells to photodynamic impact: Intracellular signaling,ultrastructural changes,and neuroglial interactions

Photodynamic therapy (PDT), an inducer of oxidative stress, is used for treatment of cancer, including brain tumors. To study the mechanisms of photodynamic injury of neurons and glial cells (GC), we used a simple model object — isolated crayfish mechanoreceptor consisting of a single sensory neuron surrounded by a multilayered glial envelope. PDT caused inhibition and elimination of neuronal activity, impairment of intracellular organelles involved in the biosynthetic, bioenergetic, and transport processes and neuroglial interactions, necrosis of neurons and glial cells, and in glial apoptosis. PDT-induced death of a neuron and GC was mediated by intercellular molecular messengers and intracellular signaling cascades. PDT-induced inhibition and elimination of neuronal activity was associated with opening of mitochondrial permeability transition pores, Ca²⁺ release into cytosol, protein kinase C and NO synthase activities. Necrosis of neurons was mediated by protein kinases B/Akt, GSK-3β and mTOR, opening of mitochondrial permeability transition pores and Ca²⁺/calmodulin/CaMKII pathway. NO and GDNF reduced neuronal necrosis. Multiple signal pathways, such as phospholipase C/Ca²⁺, Ca²⁺/calmodulin/CaMKII, Ca²⁺/PKC, Akt/mTOR, MEK/p38, and protein kinase G mediated PDT-induced necrosis both in glial cells and in neurons. NOS/NO and neurotrophic factors NGF and GDNF protected glial cells and demonstrated antinecrotic activity. Glial apoptosis was reduced by neurotrophic factors NGF and GDNF, protein kinase C, and MAP kinase JNK. In contrast, mitochondrial permeability transition pores and phospholipase C, which mobilize intracellular Ca²⁺, NOS/NO/protein kinase G, proteins GSK-3β and mTOR, stimulated apoptosis of glial cells. The schemes of involvement of various inter- and intracellular signaling processes in the responses of neurons and GC to PDT are developed.     

The outcome of 5-ALA-mediated photodynamic treatment in melanoma cells is influenced by vitamin C and heme oxygenase-1

Photodynamic therapy (PDT) is an important clinical approach for cancer treatment. It involves the administration of a photosensitizer, followed by its activation with light and induction of cell death. The underlying mechanism is an increased production of reactive oxygen species (ROS) leading to oxidative stress, which is followed by cell death. However, effectiveness of PDT is limited due to an initiation of endogenous rescue response systems like heme oxygenase-1 (HO-1) in tumor cells. In recent years, consuming of antioxidant supplements has become widespread, but the effect of exogenously applied antioxidants on cancer therapy outcome remains unclear. Thus, this study was aimed to investigate if exogenous antioxidants might decrease ROS-induced cytotoxicity in photodynamic treatment. Lycopene, β-carotene, vitamin C, N-acetylcysteine, trolox, and N-tert-butyl-α-phenylnitrone in different doses were administered to human melanoma cells prior exposure to photodynamic treatment. Supplementation with vitamin C resulted in a significant decrease of the cell death rate, whereas the other tested antioxidants had no effect on cell viability and oxidative stress markers. The simultaneous application of vitamin C with the HO-1 activity inhibitor zinc protoporphyrine IX (ZnPPIX) caused a considerable decrease of photodynamic treatment-induced cytotoxicity compared to ZnPPIX alone. It can be summarized that exogenously applied antioxidants do not have a leading role in the protective response against photodynamic treatment. However, further studies are necessary to investigate more antioxidants and other substances, which might affect the outcome of photodynamic treatment in cancer therapy.     

Tumor necrosis factor-alpha-associated lysosomal permeabilization is cathepsin B dependent

Cathepsin B (Cat B) is released from lysososomes during tumor necrosis factor-alpha (TNF-alpha) cytotoxic signaling in hepatocytes and contributes to cell death. Sphingosine has recently been implicated in lysosomal permeabilization and is increased in the liver by TNF-alpha. Thus the aims of this study were to examine the mechanisms involved in TNF-alpha-associated lysosomal permeabilization, especially the role of sphingosine. Confocal microscopy demonstrated Cat B-green fluorescent protein and LysoTracker Red were both released from lysosomes after treatment of McNtcp.24 cells with TNF-alpha/actinomycin D, a finding compatible with lysosomal destabilization. In contrast, endosomes labeled with Texas Red dextran remained intact, suggesting lysosomes were specifically targeted for permeabilization. LysoTracker Red was released from lysosomes in hepatocytes treated with TNF-alpha or sphingosine in Cat B(+/+) but not Cat B(-/-) hepatocytes, as assessed by a fluorescence-based assay. With the use of a calcein release assay in isolated lysosomes, sphingosine permeabilized liver lysosomes isolated from Cat B(+/+) but not Cat B(-/-) liver. C(6) ceramide did not permeabilize lysosomes. In conclusion, these data implicate a sphingosine-Cat B interaction inducing lysosomal destabilization during TNF-alpha cytotoxic signaling.     

Suppression of REDD1 attenuates oxygen glucose deprivation/reoxygenation-evoked ischemic injury in neuron by suppressing mTOR-mediated excessive autophagy

Cerebral ischemia/reperfusion (I/R) typically occurs after mechanical thrombectomy to treat ischemic stroke, generation of reactive oxygen species (ROS) after reperfusion may result in neuronal insult, ultimately leading to disability and death. Regulated in development and DNA damage responses 1 (REDD1) is a conserved stress response protein under various pathogenic conditions. Recent research confirms the controversial role of REDD1 in injury processes. Nevertheless, the role of REDD1 in cerebral I/R remains poorly defined. In the current study, increased expression of REDD1 was observed in neurons exposed to simulated I/R via oxygen glucose deprivation/reoxygenation (OGD/R) treatment. Knockdown of REDD1 enhanced OGD/R-inhibited cell viability, but suppressed lactate dehydrogenase (LDH) release in neurons upon OGD/R. Simultaneously, suppression of REDD1 also antagonized OGD/R-evoked cell apoptosis, Bax expression, and caspase-3 activity. Intriguingly, REDD1 depression abrogated neuronal oxidative stress under OGD/R condition by suppressing ROS, MDA generation, and increasing antioxidant SOD levels. Further mechanism analysis corroborated the excessive activation of autophagy in neurons upon OGD/R with increased expression of autophagy-related LC3 and Beclin-1, but decreased autophagy substrate p62 expression. Notably, REDD1 inhibition reversed OGD/R-triggered excessive neuronal autophagy. More importantly, depression of REDD1 also elevated the expression of p-mTOR. Preconditioning with mTOR inhibitor rapamycin engendered not only a reduction in mTOR activation, but also a reactivation of autophagy in REDD1 knockdown-neurons upon OGD/R. In addition, blocking the mTOR pathway muted the protective roles of REDD1 inhibition against OGD/R-induced neuron injury and oxidative stress. Together these data suggested that REDD1 may regulate I/R-induced oxidative stress injury in neurons by mediating mTOR-autophagy signaling, supporting a promising therapeutic strategy against brain ischemic diseases.     

Photosensibilization with Endogenous Riboflavin of the Isolated Mechanoreceptor Neuron and Satellite Glial Cells of the Crayfish <Emphasis Type="Italic">Astacus leptodactilus</Emphasis>

The photodynamic effect of the exogenous riboflavin on the mechanoreceptor neuron and satellite glial cells was studied in the isolated stretch receptor of the river crayfish. It was shown that the photodynamic action of exogenous riboflavin produced lesion of the cytoplasmic membrane and irreversible cessation of the neuron impulse activity, disturbance of integrity of the plasma membrane of glial cells, and development of necrotic processes in them. It also induces apoptosis of the glial cells. A disturbance of bioenergetic processes in the neuron and development of apoptosis in the glial cells was observed with a 4-h delay after the photoinduced cessation of the neuron impulsation. Riboflavin is known to be a photosensitizer of the first kind, which generates superoxide-anion during illumination. Its photodynamic effect on the neuron was essentially lower than the photodynamic effect of the earlier studied photosensitizers of the second kind—porphyrines, chlorines, and phthalocyanines. They produced the cell lesions that did not developed cessation of impulsation, as this took place in the case of photosensitizers of the first kind.__________Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 3, 2005, pp. 259–265.Original Russian Text Copyright © 2005 by Dergacheva, Kolosov, Uzdensky.     

13,15-N-cycloimide derivatives of chlorin p6 with isonicotinyl substituent are photosensitizers targeted to lysosomes.

Four monocationic cycloimide derivatives of chlorin p(6) (CICD) were studied as photosensitizers and compared to a structurally similar neutral derivative. Cationic CICD are highly photostable (quantum yield of photobleaching is about 1 x 10(-5), generate singlet oxygen under irradiation (quantum yields are 0.3-0.45), can be involved in a photo-induced substrate-dependent generation of superoxide radicals, but do not produce OH . 17,18-delta-lacton 13(2)-(N-methylisonicotinylamido)-13,15-cycloimide mesochlorin p(6) () and 13(2)-(N-methylisonicotinylamido)-13,15-cycloimide mesochlorin p(6) methyl ester () possess high cancer cell killing photodynamic activity, but they provide no photoinduced bactericidal effect. Substitution of an ethyl group with a hydroxyethyl or acetyl group at position 3 of the macrocycle results in a decrease in extinction and intracellular accumulation that finally leads to the reduced photocytotoxicity. Cationic CICD are targeted to lysosomes, and their intracellular penetration occurs most probably via caveolae-dependent endocytosis. Photodynamic treatment with cationic CICD results in the cell death via necrosis at both sub-phototoxic (40-70% of dead cells) and phototoxic (90-100% of dead cells) regimes of cell treatment. Irradiation induces lysosome damage, leakage of CICD from lysosomes and development of protease activity in cytoplasm, whereas mitochondria are not affected with irradiation. A positive charge of cationic CICD modified drastically an internalization pathway, sites of intracellular localization and mechanisms of photoinduced cytotoxicity as compared to previously studied neutral and anionic CICD. Our experiments with different CICD show that varying charge and structure of substituents it is possible to modulate many cellular properties of CICD in order to find the best molecular template of the advanced near-IR photosensitizer for photodynamic therapy.     

Receptor-mediated endocytosis of phosphodiester oligonucleotides in the HepG2 cell line: evidence for non-conventional intracellular trafficking

Having identified an oligonucleotide (ON) receptor in the HepG2 cell line, we have re-examined here the kinetics of ON uptake, subcellular distribution and intracellular localisation in these cells, at concentrations relevant for the study of a receptor-dependent process. Kinetic parameters of ON endocytosis were comparable with those of the receptor-mediated endocytosis tracer, transferrin (uptake equilibrium, saturation with concentration, specific competition and rapid efflux) and were clearly distinct from those of fluid-phase endocytosis. By analytical subcellular fractionation, particulate ON showed a bimodal distribution after 2 h of uptake, with a low-density peak superimposed on the distribution of endosomes, and a high-density peak overlapping lysosomes. After an overnight chase, only the high-density peak remained, but it could be dissociated from lysosomes, based on its refractoriness to displacement upon chloroquine-induced swelling. After 2 h of uptake at 300 nM ON-Alexa, a punctate pattern was resolved, by confocal microscopy, from those of transferrin, of a fluid-phase tracer, and of vital staining of lysosomes by LysoTracker. At 3 µM ON-Alexa, its pattern largely overlapped with the fluid-phase tracer and LysoTracker. Taken together, these data suggest that ON may be internalised at low concentrations by receptor-mediated endocytosis into unique endosomes, then to dense structures that are distinct from lysosomes. The nature of these two compartments and their significance for ON effect deserve further investigation.     

Cell death in HIV dementia

Many patients infected with human immunodeficiency virus type-1 (HIV-1) suffer cognitive impairment ranging from mild to severe (HIV dementia), which may result from neuronal death in the basal ganglia, cerebral cortex and hippocampus. HIV-1 does not kill neurons by infecting them. Instead, viral proteins released from infected glial cells, macrophages and/or stem cells may directly kill neurons or may increase their vulnerability to other cell death stimuli. By binding to and/or indirectly activating cell surface receptors such as CXCR4 and the N-methyl-D-aspartate receptor, the HIV-1 proteins gp120 and Tat may trigger neuronal apoptosis and excitotoxicity as a result of oxidative stress, perturbed cellular calcium homeostasis and mitochondrial alterations. Membrane lipid metabolism and inflammation may also play important roles in determining whether neurons live or die in HIV-1-infected patients. Drugs and diets that target oxidative stress, excitotoxicity, inflammation and lipid metabolism are in development for the treatment of HIV-1 patients.     

Impairment of lysosomal integrity by B10, a glycosylated derivative of betulinic acid, leads to lysosomal cell death and converts autophagy into a detrimental process

In this study, we report a novel mechanism of action for a cytotoxic derivative of betulinic acid (BA). B10 is a semi-synthetic glycosylated derivative of BA selected for its enhanced cytotoxic activity. Interestingly, although B10 induces apoptosis, caspase-3 downregulation incompletely prevents B10-induced cell death, Bcl-2 overexpression fails to protect cells and DNA fragmentation rates do not reflect cell death rates in contrast to cytoplasmic membrane permeabilization. These results implicate that apoptotic and non-apoptotic cell death coexist upon B10 treatment. Unexpectedly, we found that B10 induces autophagy and also abrogates the autophagic flux. B10 destabilizes lysosomes as shown by Lysotracker Red staining and by cathepsin Z and B release from lysosomes into the cytoplasm. Consistently, the cathepsin inhibitor Ca074Me significantly decreases B10-induced cell death, further supporting the fact that the release of lysosomal enzymes contributes to B10-triggered cell death. Downregulation of ATG7, ATG5 or BECN1 by RNAi significantly decreases caspase-3 activation, lysosomal permeabilization and cell death. Thus, by concomitant induction of autophagy and inhibition of the autophagic flux, B10 turns autophagy into a cell death mechanism. These findings have important implications for the therapeutic exploitation of BA derivatives, particularly in apoptosis-resistant cancers.     

Apoptosis, autophagy and cell cycle arrest following photodamage to mitochondrial interior

Cell death induced by oxidative insult targeted to mitochondrial interior of A431 cells was investigated. For stimulated production of ROS in the inner space of mitochondria, safranin-mediated photodynamic treatment (PDT) was employed. Another photosensitizer, mTHPC, which diffusely localizes to cellular membranes, was used for comparison. Cell response to the oxidative insult in mitochondrial interior was different from the response to the photodamage produced in cellular membranes. Autophagy and apoptotic features of cell death in response to mTHPC-PDT was observed in a wide range of PDT doses. Cell response to the oxidative stress in mitochondrial interior was dose-dependent. Damage up to CD50 did not reveal hallmarks of dead cells. At intermediate damage (CD50), cells manifested enhanced autophagy and reduced population of S-phase, but not apoptosis. Severe damage (beyond CD70) induced apoptosis following release of cytochrome c and caspase activation, in addition to autophagy and cell cycle arrest.     

Protective mechanism of FSH against oxidative damage in mouse ovarian granulosa cells by repressing autophagy

Oxidative stress-induced granulosa cell (GCs) death represents a common reason for follicular atresia. Follicle-stimulating hormone (FSH) has been shown to prevent GCs from oxidative injury, although the underlying mechanism remains to be elucidated. Here we first report that the suppression of autophagic cell death via some novel signaling effectors is engaged in FSH-mediated GCs protection against oxidative damage. The decline in GCs viability caused by oxidant injury was remarkably reduced following FSH treatment, along with impaired macroautophagic/autophagic flux under conditions of oxidative stress both in vivo and in vitro. Blocking of autophagy displayed similar levels of suppression in oxidant-induced cell death compared with FSH treatment, but FSH did not further improve survival of GCs pretreated with autophagy inhibitors. Further investigations revealed that activation of the phosphoinositide 3-kinase (PI3K)-AKT-MTOR (mechanistic target of rapamycin [serine/threonine kinase]) signaling pathway was required for FSH-mediated GCs survival from oxidative stress-induced autophagy. Additionally, the FSH-PI3K-AKT axis also downregulated the autophagic response by targeting FOXO1, whereas constitutive activation of FOXO1 in GCs not only abolished the protection from FSH, but also emancipated the autophagic process, from the protein level of MAP1LC3B-II to autophagic gene expression. Furthermore, FSH inhibited the production of acetylated FOXO1 and its interaction with Atg proteins, followed by a decreased level of autophagic cell death upon oxidative stress. Taken together, our findings suggest a new mechanism involving FSH-FOXO1 signaling in defense against oxidative damage to GCs by restraining autophagy, which may be a potential avenue for the clinical treatment of anovulatory disorders.     

Disruption of neuronal autophagy by infected microglia results in neurodegeneration

There is compelling evidence to support the idea that autophagy has a protective function in neurons and its disruption results in neurodegenerative disorders. Neuronal damage is well-documented in the brains of HIV-infected individuals, and evidence of inflammation, oxidative stress, damage to synaptic and dendritic structures, and neuronal loss are present in the brains of those with HIV-associated dementia. We investigated the role of autophagy in microglia-induced neurotoxicity in primary rodent neurons, primate and human models. We demonstrate here that products of simian immunodeficiency virus (SIV)-infected microglia inhibit neuronal autophagy, resulting in decreased neuronal survival. Quantitative analysis of autophagy vacuole numbers in rat primary neurons revealed a striking loss from the processes. Assessment of multiple biochemical markers of autophagic activity confirmed the inhibition of autophagy in neurons. Importantly, autophagy could be induced in neurons through rapamycin treatment, and such treatment conferred significant protection to neurons. Two major mediators of HIV-induced neurotoxicity, tumor necrosis factor-alpha and glutamate, had similar effects on reducing autophagy in neurons. The mRNA level of p62 was increased in the brain in SIV encephalitis and as well as in brains from individuals with HIV dementia, and abnormal neuronal p62 dot structures immunoreactivity was present and had a similar pattern with abnormal ubiquitinylated proteins. Taken together, these results identify that induction of deficits in autophagy is a significant mechanism for neurodegenerative processes that arise from glial, as opposed to neuronal, sources, and that the maintenance of autophagy may have a pivotal role in neuroprotection in the setting of HIV infection.     

Autophagy Constitutes a Protective Mechanism against Ethanol Toxicity in Mouse Astrocytes and Neurons

Ethanol induces brain damage and neurodegeneration by triggering inflammatory processes in glial cells through activation of Toll-like receptor 4 (TLR4) signaling. Recent evidence indicates the role of protein degradation pathways in neurodegeneration and alcoholic liver disease, but how these processes affect the brain remains elusive. We have demonstrated that chronic ethanol consumption impairs proteolytic pathways in mouse brain, and the immune response mediated by TLR4 receptors participates in these dysfunctions. We evaluate the in vitro effects of an acute ethanol dose on the autophagy-lysosome pathway (ALP) on WT and TLR4^-/- mouse astrocytes and neurons in primary culture, and how these changes affect cell survival. Our results show that ethanol induces overexpression of several autophagy markers (ATG12, LC3-II, CTSB), and increases the number of lysosomes in WT astrocytes, effects accompanied by a basification of lysosomal pH and by lowered phosphorylation levels of autophagy inhibitor mTOR, along with activation of complexes beclin-1 and ULK1. Notably, we found only minor changes between control and ethanol-treated TLR4^-/- mouse astroglial cells. Ethanol also triggers the expression of the inflammatory mediators iNOS and COX-2, but induces astroglial death only slightly. Blocking autophagy by using specific inhibitors increases both inflammation and cell death. Conversely, in neurons, ethanol down-regulates the autophagy pathway and triggers cell death, which is partially recovered by using autophagy enhancers. These results support the protective role of the ALP against ethanol-induced astroglial cell damage in a TLR4-dependent manner, and provide new insight into the mechanisms that underlie ethanol-induced brain damage and are neuronal sensitive to the ethanol effects.     

Autophagy inhibition uncovers the neurotoxic action of the antipsychotic drug olanzapine

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.