N-acetylcysteine prevents olanzapine-induced oxidative stress in mHypoA-59 hypothalamic neurons

Olanzapine is a second-generation antipsychotic (AP) drug commonly prescribed for the treatment of schizophrenia. Recently, olanzapine has been found to cause brain tissue volume loss in rodent and primate studies; however, the underlying mechanism remains unknown. Abnormal autophagy and oxidative stress have been implicated to have a role in AP-induced neurodegeneration, while N-acetylcysteine (NAC) is a potent antioxidant, shown to be beneficial in the treatment of schizophrenia. Here, we investigate the role of olanzapine and NAC on cell viability, oxidative stress, mitochondrial mass and mitophagy in hypothalamic cells. Firstly, cell viability was assessed in mHypoA-59 and mHypoA NPY/GFP cells using an MTS assay and flow cytometric analyses. Olanzapine treated mHypoA-59 cells were then assessed for mitophagy markers and oxidative stress; including quantification of lysosomes, autophagosomes, LC3B-II, p62, superoxide anion (O2–) and mitochondrial mass. NAC (10 mM) was used to reverse the effects of olanzapine (100 µM) on O2−, mitochondrial mass and LC3B-II. We found that olanzapine significantly impacted cell viability in mHypoA-59 hypothalamic cells in a dose and time-dependent manner. Olanzapine inhibited mitophagy, instigated oxidative stress and prompted mitochondrial abnormalities. NAC was able to mitigate olanzapine-induced effects. These findings suggest that high doses of olanzapine may cause neurotoxicity of hypothalamic neurons via increased production of reactive oxygen species (ROS), mitochondrial damage and mitophagy inhibition. This could in part explain data suggesting that APs may reduce brain volume.

www.nature.com/scientificreports/ containing membrane proteins LC3B and p62 will engulf all "recyclable" units and carry them to a lysosome. The autophagosome and lysosome will fuse to form an autophagolysosome. Here, the damaged cellular units and p62 are degraded by acidic vesicles in the lysosome and are used as an energy source. It has previously been noted that schizophrenia patients have a marked increase in oxidative stress markers. For example, thiobarbituric acid-reactive substances (TBARS) and malondiadehyde (MDA) levels in patients are significantly increased 14,15 . Chronic olanzapine treatment also increases ROS production and MDA in SHSY5Y cells 9,[16][17][18] . Furthermore, autophagy has a key role in the pathophysiology of schizophrenia. For example, Beclin1 (an essential autophagy protein) has been found to be decreased by 40% in post-mortem hippocampal tissue of schizophrenia patients 19 . Moreover, post-mortem analysis of brain tissue confirm increased mitophagy in schizophrenia patients 20,21 . Interestingly, clinically approved olanzapine, haloperidol, clozapine and sertindole have been found to induce autophagy in neuronal cells [22][23][24] . Therefore, it is possible that the oxidative stress and autophagy observed in patients is caused by AP medication. Most literature supporting AP-induced neurodegeneration focuses on the frontal and temporal lobes, however, a 10% reduction in whole brain volume has been reported in animal models with a strong focus on grey matter reductions 2 . The primary function of the hypothalamus is metabolic regulation, yet, the hypothalamus is also grey matter 25 . Olanzapine is notorious for causing weight-gain and other metabolic side-effects 26 . It is possible this is related to the neurodegeneration of grey matter in the hypothalamus. Previous studies investigating the role of APs on the autophagy pathway have used SH-SY5Y cells or primary neurons 24 . Therefore our study is novel in using hypothalamic neuronal cells to investigate the role of mitophagy in AP-induced neurodegeneration.
N-acetylcysteine (NAC) is a potent antioxidant derived from the amino acid L-cysteine and is used for the treatment of various conditions such as schizophrenia, amyotrophic lateral sclerosis, bipolar disorder, Alzheimer's disease and various cancers 27 . We hypothesise that NAC may alleviate olanzapine-induced effects on neurodegeneration via countering oxidative stress.
In this study we aimed to investigate the underlying mechanism of olanzapine-induced neurodegeneration, primarily focusing on the role of oxidative stress, mitochondrial damage, and mitophagy in mHypoA-59 hypothalamic neurons. We also tested the hypothesis that antioxidant NAC would alleviate any observed effects.

MTS assay & cell viability. Cell viability was assessed using the CellTiter 96 Aqueous One Solution Cell
Proliferation Assay and absorbance was measured at 490 nm. Cell viability was confirmed using flow cytometric analysis to outline live cell population while a live-cell imager was used to illustrate these results. Acridine orange staining. Acidic vesicles were quantified using acridine orange (AO) (Sigma-Aldrich, 318337); a pH sensitive dye used to detect lysosomal activity. We used flow cytometry or fluorescent microscopy to detect FL1/orange-red vs. FL3/green fluorescence.
Cyto-ID staining. Pre-autophagosomes, autophagosomes and autophagolysosomes were measured by staining with the Cyto-ID Autophagy detection kit (ENZO, 51031) followed by flow cytometric analysis.
ROS and mitochondrial stress. MitoSOX Red Mitochondrial Superoxide Indicator (ThermoFisher Scientific, M36008) was used to detect mitochondrial ROS, while, MitoTracker Green FM (ThermoFisher Scientific, M7514) was used to determine mitochondrial mass. Cells were stained with the given dyes followed by flow cytometric analyses and fluorescent imaging. Immunoblot analysis. Cells were treated for 24hrs, harvested with lysis buffer and protein concentration was determined using DC Protein Assay Kit (Bio-Rad, 5000111). Samples were heated at 95 °C in Laemmli buffer, loaded for fractionation to 10% SDS-PAGE gels (Bio-Rad, 4561033), and then transferred into Immuno-Blot PVDF membranes (Bio-Rad, 1620177). 5% skim milk in TBST was used as blocking buffer. Membranes were incubated overnight at 4•C with the following antibodies; LC3B (Cell Signalling Technology, #2775), and p62 (Cell Signalling Technology, 5114) in TBST containing 1% milk. Anti-rabbit IgG conjugated with horseradish peroxidase was used as the secondary antibody (Santa Cruz Biotechnology). We used ECL detection reagents (Sigma-Aldrich, RPN3004) and captured high resolution images with Amersham Gel Imager (GE Healthcare life Sciences).

Statistical analysis.
Statistical analysis was performed with the SPSS program (version 21; Chicago, IL, United States). One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used for multiple comparisons for all data as there were only two factors at any given time. This includes data for cell viability, www.nature.com/scientificreports/ AO, MitoSOX and MitoTracker flow cytometric analysis as well as LC3B-II and p62 immunoblotting. Data were expressed as mean ± SEM, and p < 0.05 was considered statistically significant.
Significance statement. Olanzapine is one of the most commonly prescribed second-generation antipsychotic drugs. Notoriously known for metabolic side-effects such as weight gain and diabetes, olanzapine has also been demonstrated to cause neurodegeneration. However, there is limited mechanistic knowledge surrounding this area. Here, for the first time, we demonstrate that olanzapine causes cell death in mHypoA-59 hypothalamic neurons via increased oxidative stress and mitochondrial damage. Furthermore, the antioxidant N-acetylcysteine is able to reverse these effects. This study provides a base-line in understanding the mechanism underlying olanzapine-induced neurodegeneration and proposes a potential treatment outlet for these severe side-effects.

Results
Olanzapine may cause neuronal toxicity in a dose and time-dependent manner in hypothalamic neurons. Hypothalamic neurons have been used in vitro to investigate central nervous system disorders such as obesity, stress and metabolic conditions 28,29 . An MTS assay was used to determine cell viability and optimum working concentrations for both mHypoA-59 neurons and mHypoA NPY/GFP after 24 h of olanzapine treatment. mHypoA-59 neurons were susceptible to cell toxicity at concentrations ≥ 100 µM after 24 h (  Cyto-ID stains autophagic vacuoles; which includes, pre-autophagosomes, autophagosomes and autophagolysosomes. Hence, we used Cyto-ID to determine the level of autophagic components found after olanzapine exposure in mHypoA-59 neurons. Flow cytometric analysis of Cyto-ID stained mHypoA-59 neurons demonstrated that olanzapine increases the amount of autophagic vacuoles in serum-starved conditions (Fig. 2D, the representative histogram is illustrated in Fig. 2E, p < 0.001, F (3, 11) = 148.547). www.nature.com/scientificreports/ LC3B-II and p62 are key proteins in the autophagy process and are commonly used as markers for autophagy inhibition or completion. The LC3B and p62 proteins are required to form the membrane of the autophagosome. While, LC3B is accumulated with autophagy upregulation, p62 is degraded upon completion of autophagy. Interestingly, western blot analysis revealed a significant increase in both LC3B-II and p62 proteins indicating an aberrant autophagy process (Fig. 2F). LC3B-II showed a significant increase at both 25 µM (p < 0.001, F (2,17) = 28.449) and 100 µM (p < 0.001). P62 also showed a significant increase at both 25 µM (p = 0.002, F (2, 17) = 34.778) and 100 µM (p < 0.001).

Olanzapine induces oxidative stress and mitochondrial dysfunction in mHypoA-59 cells.
Aberrant autophagy is commonly related to oxidative stress and mitochondrial damage. To investigate this pathway further, we used MitoSOX to measure ROS production. Flow cytometric analysis of MitoSOX stained mHy-poA-59 cells revealed a significant increase of mitochondrial ROS (O 2 − ) in a dose-dependent manner (Fig. 3A, representative histogram is illustrated in Fig. 3C, p = 0.001 at 100 µM and p < 0.001 at 150 µM, F (3, 11) = 26.887). This increase was also confirmed with live cell images (Fig. 3B).
Further examination of mitochondrial stress revealed an abnormality in mitochondrial mass. Flow cytometric analysis of MitoTracker stained mHypoA-59 cells showed that olanzapine significantly increased mitochondrial mass in a dose-dependent manner (Fig. 4A, representative histogram is illustrated in Fig. 4B, p < 0.001, F (3,11) = 213.787 for all concentrations). Live cell imaging with the incucyte ZOOM confirmed an increase in green fluorescent mitochondria (Fig. 4C). This increase in mitochondrial mass correlated with a decline in live cell population (Fig. 4A' , representative scatter plot is illustrated in Fig. 4D, p = 0.021 at 100 µM and p = 0.001 at 150 µM, F (3, 11) = 16.929). Given the autophagy pathway was inhibited; it is possible that increased mitochondrial mass indicates aberrant mitophagy and thereby causes cell death.

Discussion
Here, using cellular techniques, we have demonstrated that (i) olanzapine induces oxidative stress, inhibits mitophagy and causes apoptosis in mHypoA-59 neuronal cells; (ii) NAC is able to reverse olanzapine-induced oxidative stress and cellular damage.
Olanzapine is generally prescribed at a range between 5-20 mg/day and dosage is usually increased in 5 mg intervals on a fortnightly basis. However, more and more frequently case reports confirm prescriptions above the recommended range. Between 1997 and 2006, the percentage of patients prescribed olanzapine above the licensed range of 20 mg/day increased from 16.2% to over 50% 38 . This is particularly the case for treatment resistant patients and those presenting with acute or high levels of psychopathology 39 . A study conducted between www.nature.com/scientificreports/ nine psychiatric centres in Denmark, concluded that 80% of patients were administered between 50 and 80 mg/ day, the maximum dose being 160 mg/day 40 . Another study examined 58 post-mortem blood specimens and found a mean olanzapine concentration of 358 ng/ml with a range of 10 to 5200 ng/ml 41 . Since serum levels should not exceed 100 ng/ml, it is clear that patients are taking more than what have previously been reported 42 . Given these accounts from clinical practice and post-mortem blood serum levels it is not uncommon that doses of olanzapine higher than recommended are prescribed to patients. Therapeutic serum levels of olanzapine are reported to be between 0.1-0.3 µM 43,44 ; however, these serum levels are based on patients receiving only 5-20 mg/day. Interestingly, post-mortem samples reveal an average of 1.1 µM olanzapine blood concentration, ranging up to 16 µM 41 . It is known that antipsychotics accumulate in brain tissue at 25-30 folds higher than serum levels 45,46 . Clinical studies predict a micro molar concentration in the brain 47 . Although in vitro doses are not exactly comparable to doses in vivo, the olanzapine concentration in this study ranging between 25 and 150 µM used for neuronal culture may mimic the micro molar concentration of olanzapine predicted in the CNS of patients.
This present study demonstrates that olanzapine induces cell death in mHypoA-59 hypothalamic neurons in a dose and time-dependent manner, but not mHypoA NPY/GFP cells. Interestingly, previous studies extensively report the neuroprotective nature of olanzapine against various agents. For example, olanzapine (1 mg/ kg) prevents methamphetamine-induced apoptosis in the medial prefrontal cortex of rats injected daily for 5 days 48 . Another study reports olanzapine (80-120 µM) neuroprotection of glutamine and serum deprived PC12 cells after 72 h 49 . Similarly, the neuroprotective nature of olanzapine (100-200 µM) is reported against hydrogen peroxide toxicity in PC12 cells after 24hrs of treatment [48][49][50] . Olanzapine is also reported to increase cell survival in PC12 cell cultures, SH-SY5Y cells, and 3T3 preadipocytes 49,51 . Conversely, the neurotoxicity of olanzapine is not well documented in cell culture studies, particularly hypothalamic neurons. However, one study reports the neurotoxic nature of olanzapine (100 µM) in SH-SY5Y cells 9 . Interestingly, haloperidol and clozapine (10-25 µM) treated primary rat neurons, significantly decreases cell viability of neurons in a dose and time-dependent manner 22 .
Comparable to the results found in mHypoA NPY/GFP cells here, Kowalchuk et. al. report that neither 24 h olanzapine treatment at 20 µM or 100 µM effected cell viability in rHypoE-19 and rHypoE-46 hypothalamic cells 29 . The variance in cell viability observed between hypothalamic cell lines could be due to higher olanzapine tolerability of mHypoA NPY/GFP cells compared to mHypoA-59 cells. Previously, differential responses have been observed in similar cell lines in reaction to hormones and other compounds 52 . This can be attributed to the differing origin of the cell lines, differential receptor profiles or other physical properties such as the addition of a GFP as seen here.
Nonetheless, in vivo, there are substantial reports to suggest the neurotoxic nature of olanzapine as well as other antipsychotics. For example, Vernon et al. 7 report brain volume reductions of 10%, primarily of grey matter, in rats infused with clinical levels of either olanzapine or haloperidol for 8 weeks compared to untreated controls. Another study in monkeys also reveals an 8-11% loss of brain volume after oral administration of olanzapine or haloperidol for a period of 17-27 months 53,54 . Here we show that olanzapine can cause hypothalamic toxicity in neurons, which form the bases of hypothalamic grey matter. Olanzapine causes severe metabolic side effects and it is possible that these are related to hypothalamic toxicity and neurodegeneration. However, a future study confirming this hypothesis in vivo is necessary for a better understanding of the effects of APs on grey matter. Taken together, it is possible that olanzapine elicits neuroprotective properties in neuroblastoma and PC12 cell lines against certain stressors, while exhibiting neurotoxic qualities in a dose and time-dependent manner in certain hypothalamic neurons and causes grey matter reductions in primates and rodents.
Vucicevic et al. 9 report that olanzapine initiates autophagy in SH-SY5Y blastoma cells. This present study demonstrates that olanzapine causes an increase in autophagosomes, lysosomes, LC3B-II and p62 proteins. Routinely, p62 is degraded upon autophagy completion; increased LC3B-II and p62, as found in this study, imply interference in the autophagy process. Haloperidol and clozapine are also reported to cause an increase of autophagosomes, lysosomes, LC3B-II and p62 proteins in primary rat neurons 22 . These APs are suggested to block the fusion between autophagosomes and lysosomes and thereby hinder the autophagy process. It is well known that aberrant autophagy may cause programmed cell death or apoptosis. Here, we confirm that olanzapine also inhibits the formation of autophagolysosomes in mHypoA-59 hypothalamic neurons and thereby causes cell death.
We demonstrate that olanzapine induces oxidative stress via increasing O 2 − ; the most renowned free radical primarily produced in the mitochondria. Further analysis also reveals a substantial increase in mitochondrial mass, implying a defect in the mitophagy process. Vucicevic et al. 9 also report that olanzapine (100 µM) causes oxidative stress and mitochondrial damage in SH-SY5Y cells after 24hrs. Another study treating human blood samples with 80 µM and 160 µM olanzapine for 1 hr found an increase in total oxidative stress at 160 µM. Although 100 µM was not measured, no oxidative stress was detected at concentrations lower than 80 µM 55 . Dietrich-Muszalska et al. 56 conducted a similar study where blood samples treated with 20 and 40 µM olanzapine for 1 and 24hrs exhibit no significance in TBARS-an oxidative stress marker. These reports are consistent with our findings being that a concentration of ≥ 100 µM olanzapine causes oxidative stress while lower concentrations do not.
In the clinic olanzapine is reported to have antioxidant properties. For example, Al-Chalabi et al. 15 report that administration of olanzapine 10-20 mg/day for 2 months significantly increases total antioxidant status and decreases MDA levels in the serum of schizophrenia patients. On the other hand, an animal study observing the effect of olanzapine (6 mM) on rat hepatocytes report cytotoxicity, increased ROS production, lipid peroxidation, mitochondrial and lysosomal damage after a mere 2 hr exposure period 57 . As previously suggested, it is possible that low doses of olanzapine (< 100 µM) have positive implications while, high doses (≥ 100 µM) www.nature.com/scientificreports/ may cause cellular damage via the ROS pathway. Also, ROS may be used as a biomarker to monitor possible olanzapine-induced toxic effects. N-acetylcysteine is commonly used in the study of ROS and is now more often considered in psychiatric therapy. The aetiologies of psychiatric illnesses are multifactorial; this involves inflammatory pathways, oxidative stress, mitochondrial function and apoptotic pathways among others 27 . NAC is able to intervene in all of these pathways which is why it has gained popularity in the clinical setting. For example, in a double-blind, placebocontrolled, randomized trial of 140 participants, NAC was given in addition to regular medication for 6 months and an improvement in the Positive and Negative Symptoms Scale (PANSS) was observed 58 . In another case study, a treatment-resistant patient directed to hospital with a PANSS of 143 was treated with NAC for 7 days and her PANSS score decreased to 59 59 . In this current study we found that NAC reverses olanzapine-induced oxidative stress, mitochondrial damage and LC3B-II. Vucicevic et al. 9 also report that NAC reversed olanzapine-induced LC3B conversion in SH-SY5Y cells. Given that NAC has beneficial effects in schizophrenia patients, it is possible that NAC administered in conjunction with olanzapine may alleviate olanzapine-induced effects via regulating ROS and mitophagy and reinforcing the stability of hypothalamic neurons.