X.-M. Li, Department of Psychiatry, Faculty of Medicine, University of Manitoba, PZ432-771 Bannatyne Avenue, Winnipeg, Manitoba, Canada Fax: +1 204 789 3929 Tel: +1 204 787 7432 E-mail: firstname.lastname@example.org X. Li, Laboratory of Neuropharmacology, School of Pharmaceutical Science, Wenzhou Medical College, Zhenjiang, China Fax: +86 577 86689983 Tel: +86 577 8669350 E-mail: email@example.com
We have shown that quetiapine, a new antipsychotic drug, protects cultured cells against oxidative stress-related cytotoxicities induced by amyloid β (Aβ)25-35, and that quetiapine prevents memory impairment and decreases Aβ plaques in the brains of amyloid precursor protein (APP)/presenilin-1 (PS-1) double-mutant mice. The aim of this study was to understand why quetiapine has these protective effects. Because the cytotoxicity of both Aβ(25-35) and Aβ(1-40) requires fibril formation, our first experiments determined the effect of quetiapine on Aβ(25-35) aggregation. Quetiapine inhibited Aβ(25-35) aggregation in cell-free aqueous solutions and blocked the fibrillar aggregation of Aβ(25-35), as observed under an electron microscope. We then investigated why quetiapine inhibits Aβ(25-35) aggregation. During the aggregation of Aβ(25-35), a hydroxyl radical (OH•) was released, which in turn amplified Aβ(25-35) aggregation. Quetiapine blocked OH•-induced Aβ(25-35) aggregation and scavenged the OH• produced in the Fenton system and in the Aβ(25-35) solution, as analyzed using electron paramagnetic resonance spectroscopy. Furthermore, new compounds formed by quetiapine and OH• were observed in MS analysis. Finally, we applied Aβ(25-35) to PC12 cells to observe the effect of quetiapine on living cells. Aβ(25-35) increased levels of intracellular reactive oxygen species and calcium in PC12 cells and caused cell death, but these toxic effects were prevented by quetiapine. These results demonstrate an anti-oxidative stress mechanism of quetiapine, which contributes to its protective effects observed in our previous studies and explains the effectiveness of this drug for Alzheimer’s disease patients with psychiatric and behavioral complications.
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Quetiapine is an atypical antipsychotic drug (APD) for the treatment of symptoms in patients with schizophrenia. This drug effectively alleviates positive and negative symptoms as well as cognitive impairment [1–3]. In addition, quetiapine is well tolerated and causes the lowest incidence of extra-pyramidal symptoms [1,4]. Moreover, quetiapine ameliorates psychotic symptoms in patients with Alzheimer’s disease (AD) and Parkinson’s disease (PD), and improves cognitive function in patients with these neurodegenerative diseases [5–8]. The therapeutic effects observed in AD and PD patients suggest an unexplored mechanism beyond the antagonistic actions of this drug on dopamine and serotonin receptors in the brain.
We conducted extensive studies using various techniques and models to understand why quetiapine has unique therapeutic effects in schizophrenia as well as AD and PD patients. Our previous in vitro data have shown that quetiapine protects PC12 cells against cytotoxicities induced by serum withdrawal , N-methyl-4-phenylpyridinium ions  and amyloid β (Aβ)25-35 [11,12]. These insults have the common property of producing oxidative stress in cells, suggesting that quetiapine may function as a scavenger of reactive oxygen species (ROS). In a recent animal study using an amyloid precursor protein (APP)/presenilin-1 (PS-1) double transgenic mouse model of AD , administration of quetiapine for 4 and 7 months prevented memory impairment and diminished the number of Aβ plaques in the cortex and hippocampus of the transgenic mice. In addition, quetiapine lowered levels of brain Aβ peptides and decreased β-secretase activity and expression. These results suggest that quetiapine may delay the progress of AD while improving patients’ cognitive functions.
The aim of this study was to demonstrate the cellular and molecular mechanisms underlying the protective effects of quetiapine observed in our previous studies [9–13] and the therapeutic profiles of this drug in treating schizophrenia or AD patients. Our first objective was to determine if quetiapine affects Aβ aggregation as the cytotoxicity of both Aβ(25-35) and Aβ(1-40) requires fibril formation [14–16]. The second objective was to see how quetiapine affects Aβ aggregation. We focused on the effects of quetiapine on ROS for the following reasons: Aβ peptides produce superoxide, hydrogen peroxide (H2O2) and hydroxyl radicals (OH•) [16–18]; these ROS in turn induce Aβ production ; this cycle of oxidative stress and Aβ accumulation is thought to contribute to neurodegeneration in AD . The third objective was to simulate real episodes in living cells exposed to Aβ and to confirm the protective effects of quetiapine on living cells. To achieve these objectives, we chose Aβ(25-35) for our experiments from among the available peptides (Aβ(1-40), Aβ(1-42), Aβ31-35 and Aβ(25-35)) on the basis that: (a) Aβ(25-35) is the core toxic fragment and a proposed biologically active fragment of full-length Aβ(1-40) [19,20]; (b) Aβ(25-35) induces oxidative stress when it aggregates  and has been commonly used in in vitro studies simulating the oxidative stress mechanism of AD [21–23]; and (c) APP and PS-1 mice show oxidative stress in their brains .
Quetiapine inhibits fibrillar aggregation of Aβ(25-35)
A thioflavin T (ThT) fluorescence assay was performed to assess the aggregation of Aβ(25-35). As expected, the fluorescence intensity of an Aβ(25-35) solution (100 μm) increased when the incubation period was prolonged, and the intensity reached its peak when incubated for 72 h (Fig. 1A), indicating that Aβ(25-35) aggregation occurred spontaneously in the solution as a function of incubation time. Based on this result, Aβ(25-35) solutions of the same concentration (100 μm) were incubated at 37 °C for 72 h in the presence or absence of various concentrations of quetiapine. Aggregation of Aβ(25-35) was completely blocked in the presence of 50 or 100 μm quetiapine (Fig. 1B). To confirm this effect, a Congo red binding assay was performed. Again, the aggregation of Aβ(25-35) was inhibited, dependent upon the concentration of quetiapine, and was completely blocked by 100 μm quetiapine (Fig. 1C).
To obtain more compelling evidence for the inhibition of Aβ(25-35) aggregation by quetiapine, an electronic microscopy observation was made on prepared samples following the incubation of cell-free aqueous Aβ(25-35) (100 μm) solutions at 37 °C for 72 h in the presence or absence of various concentrations of quetiapine. Typical fibrillar aggregates were observed in the samples containing only Aβ(25-35) (100 μm) solution (Fig. 2A). These fibrillar aggregates decreased when an Aβ(25-35) solution was co-incubated with 12.5 or 25 μm quetiapine (Fig. 2B,C), and were not observed in an Aβ(25-35) solution co-incubated with 50 or 100 μm quetiapine (Fig. 2D,E).
Quetiapine scavenges hydroxyl radicals and blocks radical-induced Aβ(25-35) aggregation
Aβ(1-40), Aβ(1-42) and Aβ(25-35) produce free radicals in their incubation solutions [16–18], and free radicals produced by the Fenton reaction amplify the aggregation of Aβ . To explore the chemical mechanisms by which quetiapine inhibits Aβ(25-35) aggregation, we tested the ability of quetiapine to scavenge free radicals and to block the amplification of Aβ(25-35) aggregation by free radicals. In electron paramagnetic resonance (EPR) spectroscopy experiments, we detected a typical four-line spectrum of OH• after incubating 1 mm Aβ(25-35) for 24 h (Fig. 3A). In the presence of quetiapine (0.5 or 1.0 mm), the intensity of OH• spectra decreased in a concentration-dependent manner (Fig. 3B,C), suggesting that quetiapine has the capacity to scavenge OH• generated in the Aβ(25-35) solution. To further confirm this capacity, the same EPR analysis was performed for the Fenton reaction solution, in which OH• was produced from H2O2 in the presence of the ferrous ion (Fe2+). Again, quetiapine reduced the EPR signal of OH• in a concentration-dependent manner (Fig. 3D–F).
We then sought to determine whether quetiapine inhibits Aβ aggregation by scavenging OH• in solution. A ThT fluorescence assay was performed for various solutions and the results showed that OH• amplified Aβ(25-35) aggregation and quetiapine blocked this amplification (Fig. 4). Considering these results, and those shown in Fig. 3, we conclude that: (a) Aβ(25-35) aggregates spontaneously and during the aggregation process produces OH•, which in turn amplifies Aβ(25-35) aggregation; and (b) quetiapine inhibits Aβ aggregation by scavenging OH•.
New compounds formed by quetiapine and hydroxyl radicals
We next conducted experiments to show new compounds made of quetiapine and OH•, which would be the strongest evidence of OH• scavenging by quetiapine. It was also important to determine any possible interaction between quetiapine and Aβ(25-35), as this interaction could also contribute to the observed inhibition of Aβ aggregation. Therefore, we performed MS analyses for the following aqueous cell-free solutions: quetiapine (100 μm), quetiapine (100 μm) in the Fenton system, Aβ(25-35) (1 mm) and Aβ(25-35) (1 mm) plus quetiapine (1 mm). In the quetiapine (C21H25N3O2S, M1 = 383) solution, a peak was detected at m/z 384, which corresponds to the [M1 +H]+ ion of the drug (Fig. 5A). In the Fenton solution with quetiapine, two new peaks (in addition to the peak at m/z 384) were detected at m/z 400 (384 + 16) and m/z 416 (384 + 32) (Fig. 5B), demonstrating that one quetiapine molecule captured one or two OH•. In the Aβ(25-35) (C45H81N13O14S, M2 = 1060) solution, a main peak was detected at m/z 530.8, which corresponds to the [M2 + 2H]2+ ion of the peptide (Fig. 5C). In the solution with quetiapine and Aβ(25-35), in addition to the peaks at m/z 384 and m/z 530.8, another was detected at m/z 400 (Fig. 5D, insert), demonstrating that one quetiapine molecule captured one OH• produced in the peptide solution. However, no peak represented the combination of quetiapine and Aβ(25-35).
Quetiapine prevents downstream cellular events induced by Aβ(25-35)
Similar to previous studies [11,12,21–23], PC12 cells treated with an aged Aβ(25-35) solution showed a reduced viability. However, this decrease was effectively prevented in cultures treated with Aβ(25-35) that had been co-incubated with quetiapine for 72 h at 37 °C before addition to the culture medium (Fig. 6A). We then examined molecular signals leading to the death of PC12 cells exposed to the aged Aβ(25-35) solution. We found that: (a) exposure to an aged Aβ(25-35) solution for 6 h increased intracellular ROS levels in PC12 cells; (b) this increase did not occur when the Aβ(25-35) solution had been co-incubated with quetiapine, although quetiapine per se did not alter intracellular levels of ROS (Fig. 6B); (c) aged Aβ(25-35) solution increased intracellular levels of calcium in PC12 cells; and (d) this increase in calcium did not occur when the Aβ(25-35) solution had been co-incubated with quetiapine (Fig. 7).
In cell-free systems Aβ(25-35) aggregates spontaneously (Figs 1 and 2) and the aggregation process produces OH• (Fig. 3), which, in turn, amplifies the aggregation of Aβ(25-35) (Fig. 4). Via this mechanism, Aβ(25-35) causes oxidative stress, indicated by high levels of intracellular ROS (Fig. 6), and disturbs the calcium homeostasis of PC12 cells (Fig. 7). High levels of both ROS and calcium are fatal to living cells. Quetiapine captures OH• in the Aβ(25-35) solution (Figs 3 and 5) and thus inhibits the aggregation of Aβ(25-35) (Figs 1 and 4). Via this mechanism, quetiapine can decrease Aβ aggregation seen in senile plaques (extracellular), as shown in our previous animal study using the APP/PS-1 double transgenic mouse model of AD . Using the same mechanism, quetiapine can prevent increases in intracellular ROS and calcium and thus allow cells to survive in the presence of Aβ(25-35), as demonstrated in this study (Figs 6 and 7), because aggregates of Aβ can indirectly and directly induce oxidative stress in cells in vitro [22,25,26]. Quetaipine prevents or alleviates oxidative stress by capturing OH• that may be produced in the cytoplasm for calcium dyshomeostasis (for example) and/or from extracellular sites such as senile plaques where Aβ aggregates and OH• is produced. With this anti-oxidative stress mechanism we can explain why quetiapine exerted protective effects on PC12 cells experiencing serum withdrawal  or exposed to N-methyl-4-phenylpyridinium ion  as these two insults increase intracellular ROS [27,28]. We also understand why this drug decreased Aβ plaques in the brains of APP/PS-1 double-mutant mice .
Mutations in APP and PS-1 are thought to cause familial AD and increase the production of Aβ42 [29,30]. APP and PS-1 mice show oxidative stress . In a previous study , quetiapine prevented memory impairment, decreased the number of Aβ plaques in the brains of APP/PS-1 mice, diminished levels of brain Aβ peptides and reduced the activity of β-secretase. These results show that quetiapine is able to delay the disease process of AD by acting on β-secretase, which mediates the initial step of Aβ production by β-cleavage of APP . This is consistent with data from previous studies showing that Aβ load is correlated with increased β-secretase expression and enzymatic activity in sporadic AD patients [32,33]. By demonstrating the anti-oxidative properties of quetiapine, this study offers a reasonable explanation of the effect of this drug on β-secretase activity in APP/PS-1 mice, as observed previously . That is, by capturing OH•, quetiapine attenuates oxidative stress and thus decreases β-secretase activity. In line with this explanation, oxidative stress has been shown to facilitate β-secretase activity and Aβ generation .
This study demonstrates a vicious cycle of Aβ aggregation → OH• → Aβ aggregation through bench experiments. Although we do not know if the same cycle happens in the brains of APP/PS-1 mice, previous studies have shown that Aβ peptides produce H2O2, which in the presence of Fe2+ generates OH• (Fenton reaction) and causes oxidative stress, which in turn induces Aβ production. This cycle of oxidative stress and Aβ accumulation eventually leads to neurodegeneration in AD . With these advances and our discovery that quetiapine captures OH•, inhibits the aggregation of Aβ(25-35), decreases ROS levels and protects PC12 cells exposed to Aβ(25-35), we hypothesize that by eliminating OH• from the microenvironment where Aβ localizes, quetiapine interrupts the cycle of Aβ aggregation → OH• → Aβ aggregation in the brain and thus delays disease progression in AD.
The anti-oxidative stress mechanism of quetiapine demonstrated in this study may also help explain the effects of this drug on cognitive deficits in patients with schizophrenia and PD. Similar to AD, the pathogenesis of PD involves a mechanism whereby oxidative stress causes damage to brain neurons thus leading to cognitive deficits [35,36]. Oxidative stress is also an important factor in the pathophysiology of schizophrenia, as indicated by recent reports showing alterations in the genes related to oxidative stress, increased oxidative stress and diminished enzymatic anti-oxidants in patients with schizophrenia compared with control subjects [37–40]. Therefore, we reason that quetiapine, by eliminating OH•, attenuates oxidative stress thus protecting brain cells against oxidative-stress-related damage and improving cognitive function in patients with schizophrenia and PD. In support of this explanation is the detection of the 7-hydroxy-metabolite of quetiapine (a compound indicated by the peak at m/z 400 in Fig. 5B,D) in the plasma of patients who received quetiapine treatment , suggesting that this drug effectively captures OH• in the human body. In future, efforts should be made to detect similar compounds in the brain tissues of patients with schizophrenia.
Lastly, the anti-oxidative stress mechanism of quetiapine demonstrated in this study may contribute to the low incidence of extra-pyramidal symptoms and movement disorders caused by this drug in patients with schizophrenia . APD-induced movement disorders, including tardive dyskinesia, are thought to result from neuronal cell damage caused by free radicals . In animal studies, oxidative stress and elevated levels of lipid peroxidation have been implicated in haloperidol (a typical APD) toxicity . Psychotic patients treated with typical APDs show elevated levels of lipid peroxidation [44,45]. In this study, we demonstrated that quetiapine is an anti-oxidant, as indicated by its ability to eliminate OH•, therefore this drug shows the lowest incidence of extra-pyramidal symptoms and movement disorders. In line with this, recent clinical reports indicate that quetiapine is effective in treating tardive dyskinesia caused by other APDs [46–48]. It should be noted that quetiapine is not accepted as a medicine to treat tardive dyskinesia in patients with schizophrenia, despite the foregoing clinical reports and the anti-oxidative stress mechanism demonstrated here.
The anti-oxidative stress property of quetiapine demonstrated in this study is beyond the realm of neurotransmitters and neurotransmitter receptors relevant to antipsychotics. Therefore, we do not expect anti-oxidative stress to be a mechanism common to all APDs; even so, in future we will test other atypical APDs using similar experimental strategies. However, anti-oxidants should be recommended to improve cognitive function in schizophrenia and AD patients because oxidative stress is involved in the pathophysiology of these two brain diseases. Quetiapine has both antipsychotic actions and anti-oxidative stress effects, and therefore is beneficial not only for patients with schizophrenia, but also for AD patients with psychiatric and behavioral complications [6,7,49–51].
Quetiapine was supplied by AstraZeneca Pharmaceuticals (Macclesfield, UK). The PC12 cell line was obtained from American Type Culture Collection (CRL-1721; ATCC, Rockville, MD, USA). Dulbecco’s modified Eagle’s medium was from Gibco BRL (Grand Island, NY, USA) and Aβ(25-35) from Oncogene Research (La Jolla, CA, USA). ThT, Congo red, 2′,7′–dicholorofluorescin diacetate, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetra-zolium bromide, N-tert-butyl-α-phenylnitrone and 5,5-dimethyl-1-pyrroline-N-oxide were from Sigma Chemical Company (St Louis, MO, USA).
ThT fluorescence assay
Following a method described by LeVine , we performed a ThT fluorescence assay. Briefly, 140 μL sample solutions of Aβ(25-35) (100 or 25 μm in 0.1 m NaCl/Pi) were incubated at 37 °C for the indicated periods in the presence or absence of various reagents. Forty microliters of ThT (16 μm) was then added to the sample solutions. Measurements were carried out in triplicate on a microplate fluorometer using softmax pro software, 1 min after mixing. ThT specifically binds to sites on Aβ fibrils. The ThT–Aβ mixture has an excitation at 450 nm and emission at 482 nm, whereas ThT alone has no fluorescence signal at this wavelength. All data were scaled using Aβ(25-35) alone as 100%. The fluorescence of non-aggregated Aβ(25-35) is ≤ 50 fluorescence units at the wavelengths tested, while results after aggregation were ≥ 350 fluorescence units.
Congo red binding
Aβ(25-35) fibril formation was also monitored by Congo red binding. A 10 μL sample solution was added to 240 μL Congo red (25 μm) in 0.1 mm potassium phosphate buffer with 150 mm NaCl (pH 7.4). Thirty minutes later, the absorbance of the mixture was read at 540 and 480 nm in triplicate on a microplate spectrophotometer using softmax pro software and the amount of Congo red bound (Cb) was calculated.
After the indicated incubation periods, sample solutions of Aβ(25-35) in the presence or absence of quetiapine were spread on carbon-coated grids for 2 min, blotted and stained with 1% phosphotungstic acid for 30 s. Specimens were visualized under a Zeiss 10C TEM at 80 kV.
EPR spectroscopy and spin trapping
Spin-trapping experiments were conducted as described by Varadarajan et al. . Aβ(25-35) (1 mm) solutions containing 50 mmN-tert-butyl-α-phenylnitrone were incubated in the presence or absence of 0.5 or 1 mm quetiapine at 37 °C for 24 h. Twenty microliters of the sample solution was then placed in a capillary tube set in a quartz tube. The EPR spectra were determined using a Bruker (Billerica, MA, USA) EMX EPR spectrometer. Instrument parameters were as follows: microwave power of 2.0 mW, modulation amplitude of 1.0 G, gain at 2 × 105, conversion time of 10.24 ms and spectrum accumulation over 48 scans. To evaluate the radical-quenching ability of quetiapine, we took advantage of the Fenton reaction: H2O2 + Fe2+ → OH• + OH− + Fe3+. This reaction is recognized as one of the most powerful oxidizing reactions. Various concentrations of quetiapine were added to the reaction mixture consisting of 10 μL 0.08% H2O2, 16 μL 0.5 m 5,5-dimethyl-1-pyrroline-N-oxide, 10 μL H2O, 34 μL H2O and 10 μL 0.1 mm ferrous sulfate. Three minutes after the addition of ferrous sulfate, EPR spectra were determined using a Bruker EMX EPR spectrometer.
To detect whether quetiapine can react with hydroxyl radicals derived from Aβ(25-35) solution or the Fenton reaction solution, MS experiments were performed as previously described . Fifty microliters of Aβ(25-35) solution (1 mm) were incubated at 37 °C for 24 h in the presence or absence of 1 mm quetiapine. The Fenton reaction solution was incubated at 22 °C for 2 h in the presence or absence of 1 mm quetiapine. The solutions mentioned above were diluted 100–fold and the samples directly injected into a stream of water (containing 0.1% formic acid)/acetonitrile (1 : 1 v/v) at 5 μL·min−1 and introduced into the electrospray source of the mass spectrometer. Mass spectra were obtained on the QSTAR XL SYSTEM using an ESI source. The parameters applied were: source temperature of 50 °C, spray voltage of 5 kV, source house voltage of 70 V and N2 as the sheath gas.
Cell culture and cell viability assay
PC12 cells were cultured at 37 °C in a humidified atmosphere of 5% CO2/95% air in collagen-coated flasks containing Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum, 10% heat-inactivated horse serum, 50 U·mL−1 penicillin and 50 μg·mL−1 streptomycin. During the logarithmic growth phase, cells were re-suspended in the medium and seeded at 2 × 104·well−1 on poly-(l-lysine)-coated 96-well plates. After 24 h, the old medium was replaced with a fresh medium containing 10 μm Aβ(25-35), which had been incubated originally at 100 μm with the indicated concentrations of quetiapine in sterilized 0.1 m NaCl/Pi (pH 7.4) for 3 days. Following an additional 24 h incubation, the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetra-zolium bromide assay was performed to measure the viability of PC12 cells, as described previously . Briefly, the medium was removed from each well and replaced with 50 μL Dulbecco’s modified Eagle’s medium/high-glucose medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetra-zolium bromide at a concentration of 0.5 mg·mL−1. The cultures were incubated at 37 °C for an additional 4 h, and then 100 μL of a solubilization solution (33 μL HCl plus 67 μL isopropyl alcohol) was added to each well. After 3 h, the attenuance (D) of each well was determined with a micro-ELISA reader at a wavelength of 570 nm. The D values were normalized as percentages of the A values of the controls, which were exposed to neither quetiapine nor Aβ(25-35).
Measurement of intracellular ROS
Aβ(25-35) (100 μm) was incubated at 37 °C for 72 h in the presence or absence of 50 μm quetiapine, and the solution then added to PC12 cell culture medium (the final concentrations of Aβ(25-35) and quetiapine were 10 and 5 μm, respectively). After an additional 6 h incubation, the intracellular ROS was measured using the fluorescent probe 2′,7′-dicholorofluorescin diacetate as described previously , with an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
Measurement of intracellular calcium
Aβ(25-35) (100 μm) was incubated at 37 °C for 72 h in the presence or absence of 50 μm quetiapine, and the solution then added to PC12 cell culture medium (the final concentrations of Aβ(25-35) and quetiapine were 10 and 5 μm, respectively). After an additional 6 h incubation, cells were exposed to 2 μm fluo-3/AM at 22 °C for 30 min in Hanks’ balanced salt solution (HBSS) buffer. The cells were then rinsed with HBSS twice, left in 0.5 mL HBSS buffer, and the cell fluorescence intensity determined using a confocal laser-scanning microscope (Olympus, Tokyo, Japan) with an excitation wavelength of 488 nm and an emission wavelength of 510 nm. Calcium concentrations were calculated according to Grynkiewicz et al. .
This study was supported by the Canadian Psychiatric Research Foundation, the Schizophrenia Society of Saskatchewan and the Saskatchewan Health Research Foundation. The authors thank Ramaswami Sammynaiken and Kenneth Thoms for their excellent technical assistance with the EPR and MS studies, respectively. The authors are also grateful to Dr Ronald Browning who read the manuscript.