Introduction
Schizophrenia is a chronic mental illness affecting approximately 1% of the global population, often beginning undetected in childhood and leading to psychosis in early adulthood Research indicates that Phencyclidine (PCP), an NMDA antagonist, can induce schizophrenia-like symptoms in humans, with effects lasting weeks after cessation of use Studies have shown that repeated PCP treatment in mice leads to significant behavioral abnormalities, including increased immobility, social deficits, and impairments in learning Consequently, the PCP mouse model serves as a valuable tool for assessing new therapeutic options and understanding the underlying mechanisms of schizophrenia.
Recent studies have highlighted the role of oxidative stress in the disease's pathophysiology, with glutathione (GSH) identified as the primary cellular antioxidant that defends against this stress GSH is crucial for protecting nervous tissue from reactive oxygen species and for modulating redox-sensitive sites, such as NMDA receptors.
Glutathione (GSH) functions as a neuromodulator or neurotransmitter, binding to NMDA receptors through its γ-glutamyl moiety to influence neuronal responses Studies on GSH-deficient models have shown morphological, electrophysiological, and behavioral abnormalities akin to those seen in patients with schizophrenia.
Mice lacking the glutamate-cysteine ligase modifier subunit (GCLm) exhibit schizophrenia-like behaviors linked to NMDA receptor hypofunction, disrupted dopamine neurotransmission, abnormal neuronal synchronization, and impaired myelination N-acetylcysteine, a pro-drug of cysteine and a precursor to glutathione (GSH), has been recognized for its potential therapeutic effects.
2 exhibit positive effects against neurochemical changes induced by GCLm gene knockout [das Neves Duarte et al., 2012]
Nuclear factor erythroid-2-related factor 2 (Nrf2) is a crucial transcription factor that operates at the antioxidant response element, a specific DNA sequence in the promoter region of genes responsible for encoding antioxidant and phase II detoxifying enzymes Normally, Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein 1 (KEAP1) However, during oxidative stress, the Nrf2/KEAP1 complex is disrupted, allowing Nrf2 to translocate into the nucleus, where it promotes the transcription of genes regulated by the antioxidant response element.
The genes responsible for encoding heme oxygenase (HO)-1 and the catalytic and modifier subunits of γ-glutamylcysteine ligase play a crucial role in the synthesis of glutathione (GSH), as highlighted by Numazawa et al (2004) Research indicates that Nrf2-null mice have significantly reduced GSH levels, underscoring the importance of Nrf2 in GSH synthesis Furthermore, Nrf2-dependent GSH synthesis is vital for providing neuroprotection, as demonstrated by Shih et al (2003).
Recent studies indicate that nuclear factor kappa-B (NF-κB) acts as a redox-sensitive transcription factor, with its activation observed under various experimental conditions Antioxidants like N-acetylcysteine and pyrrolidine dithiocarbamate can inhibit NF-κB activation triggered by cytokines, viruses, and lipopolysaccharides Additionally, NF-κB activation has been linked to animal models of Alzheimer’s and Parkinson's diseases, which are characterized by heightened oxidative stress Research also shows that dietary antioxidants can prevent the nuclear translocation of activated NF-κB, highlighting its crucial role in protecting the brain from oxidative stress.
Glutathione (GSH) serves as a crucial cofactor for various enzymes, particularly glutathione peroxidase (GPx), which facilitates the reduction of hydrogen peroxide (H2O2) and other peroxides by utilizing electrons from GSH Research indicates that GPx levels are significantly lower in the erythrocytes and plasma of individuals with schizophrenia Moreover, studies have shown that unmedicated schizophrenic patients exhibit decreased GPx activity compared to control groups.
GPx-1 is a selenium-dependent isoenzyme that is widely expressed in various tissues Numerous studies have explored the relationship between different variants of the GPx-1 gene and schizophrenia, highlighting its potential significance in understanding this mental health disorder.
This study investigates the role of GPx-1 in abnormal behaviors induced by PCP using homozygous GPx-1 depletion and transgenic overexpression in mice It also examines the efficacy of antipsychotics, clozapine and haloperidol, in relation to the GPx-1 gene for treating these symptoms The findings suggest that GPx-1 and Nrf2-dependent GSH systems are essential in mediating PCP-induced abnormal behaviors Additionally, the neuroprotective effects of clozapine are linked to the positive modulation of the GPx-1 gene and Nrf2-dependent GSH systems.
Materials and methods
All mice used in the study were treated in accordance with the NIH Guide for the Humane Care and Use of Laboratory Animals The subjects included eight-week-old male C57BL/6 wild-type mice, male glutathione peroxidase-1 knockout (GPx-1 KO) mice, male GPx-1 overexpressing transgenic (GPx-1 TG) mice, and non-transgenic (non-TG) mice, all weighing approximately 25 ± 3 g They were kept on a 12:12 h light:dark cycle and had unrestricted access to food, with a two-week adaptation period prior to experimentation The GPx-1 KO mice, derived from 129/SVJ x C57BL/6 strains, exhibited undetectable GPx-1 mRNA levels and a significant reduction in total GPx activity (80-99%) in various tissues compared to wild-type mice Breeding pairs of GPx-1 KO mice were generously provided by Professor Ye-Shih.
The GPx-1 transgenic (TG) mice, which possess three copies of the transgene, were developed from (C57BL/6 × C3H) hybrid mice, as reported by Cheng et al in 1998 Breeding pairs of these GPx-1 TG mice were generously supplied by Professor Xin Gen Lei from Cornell University For genotyping, polymerase chain reaction (PCR) primers were utilized, including specific sequences for detecting knockout mutants and overexpressing transgenic variants, provided by Bioneer Corporation in South Korea.
2.2 Drug treatment and experimental design
Phencyclidine hydrochloride (PCP) was prepared in 0.9% sterile saline, while clozapine was dissolved in 0.1N HCl at a concentration of 10 mg/ml and diluted with saline Haloperidol hydrochloride was prepared in distilled water at 1 mg/ml and also diluted with saline All solutions were freshly prepared prior to administration Mice received a daily subcutaneous dose of PCP (10 mg/kg) for 14 days To assess the impact of antipsychotics on behavioral activity and GSH-dependent antioxidant systems, clozapine (10 mg/kg/day, orally) or haloperidol (1 mg/kg/day, orally) was administered once daily for seven days following the final PCP treatment.
1, mice were sacrificed 4h, 1d, 2d, 4d and 7d after the final PCP treatment for examining antioxidant
Following the final PCP treatment, behaviors, transcription factors, and the GSH-dependent antioxidant system were assessed The prefrontal cortices and hippocampi of the mice were rapidly dissected post-sacrifice by decapitation and subsequently preserved in liquid nitrogen for neurochemical analyses.
Western blotting analysis was conducted following established protocols [Shin et al., 2005a] Tissues were homogenized using a lysis buffer containing 200 mM Tris–HCl (pH 6.8), 10% sodium dodecyl sulfate (SDS), 5 mM ethylene glycol tetraacetic acid (EGTA), 5 mM ethylenediaminetetraacetic acid (EDTA), and 10% glycerol The lysate was centrifuged at 13,000 × g for 30 minutes, and the supernatant was utilized for Western blot analysis Proteins (20 μg/lane) were separated via 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes, which were then blocked in phosphate buffered saline (PBS) with 3% skim milk for 30 minutes Blots were incubated overnight at 4 °C with primary antibodies against β-actin (1:300000), GPx-1 (1:1000), glutathione reductase (GR; 1:2000), cytosolic Cu/Zn-superoxide dismutase (SOD-1; 1:15000), and mitochondrial Mn-superoxide dismutase (SOD-2; 1:30000).
In a study conducted by Kim et al (2003), membranes were washed in PBS and then incubated with horseradish peroxidase (HRP)-conjugated secondary anti-rabbit immunoglobulin (IgG) at a dilution of 1:5000 (Thermo Scientific, Rockford, IL, USA) or anti-mouse IgG at the same dilution (Sigma-Aldrich) for two hours Visualization of the bands was achieved using an enhanced chemiluminescence system (GenDEPOT, Houston, TX, USA), and the relative intensities of the bands were quantified using PhotoCapt MW software (version 10.01 for Windows) from Vilber Lourmat, Marne la Vallée, France.
2.4 Determination of superoxide dismutase (SOD) activity
Homogenates of tissues were centrifuged at 25,000 × g for 15 min at 4 °C and the supernatant was dialyzed in 50 mM PBS (pH 7.8) containing 1 mM EDTA SOD activity was determined based on
The inhibition of superoxide-dependent reactions was studied using a reaction mixture consisting of 70 mM potassium phosphate buffer (pH 7.8), 30 μM cytochrome c, 150 μM xanthine, and a tissue extract diluted 10 times with PBS, totaling 3 ml The reaction commenced with the addition of 10 μl of 50 units of xanthine oxidase, and the absorbance change at 550 nm was monitored One unit of superoxide dismutase (SOD) is defined as the amount needed to reduce the rate of cytochrome c reduction by 50% To estimate total SOD, 10 μM potassium cyanide (KCN) was introduced to inhibit cytochrome oxidase activity For Mn-SOD (SOD-2) activity assessment, 1 mM KCN was added to inhibit Cu,Zn-SOD (SOD-1) activity, allowing SOD-1 activity to be calculated by subtracting SOD-2 activity from total SOD activity [Shin et al., 2008].
2.5 Determination of glutathione peroxidase (GPx) activity
GPx activity was assessed using a method outlined by Shin et al (2008), involving an incubation mixture composed of 1 mM glutathione, 0.2 mM NADPH, and 1.4 IU of glutathione reductase in a potassium phosphate buffer at pH 7.0 The reaction commenced with the addition of supernatant (0.3–0.8 mg protein) and 0.25 mM H2O2, with absorbance changes measured at 340 nm over 4.5 minutes One unit of GPx activity is defined as the amount needed to oxidize 1 μM NADPH per minute, calculated using the molar absorptivity of 6.22 × 10 −6 for NADPH.
2.6 Determination of glutathione reductase (GR) activity
The measurement of GR activities was conducted using a modified method based on previous studies The reaction mixture consisted of 1 mM oxidized glutathione and 100 μl of a sample in a phosphate buffer at pH 7.0, supplemented with 1 mM EDTA The reaction was initiated by the addition of NADPH to achieve a final concentration of 0.11 mM, and the decrease in NADPH absorbance at 340 nm was recorded One unit of activity is defined as the oxidation of 1 nM of NADPH.
2.7 Determination of reactive oxygen species (ROS) formation
ROS formation in the brain tissue of mice was assessed by measuring the conversion from 2’,7’- dichlorofluorescin diacetate (DCFH-DA) to dichlorofluorescein (DCF), as previously described
A solution of 7 μM DCFH-DA was prepared in methanol and incubated at 37 °C for 3 hours Following incubation, fluorescence was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm, using DCF as a standard reference [Shin et al., 2016].
The extent of protein oxidation in brain tissue was evaluated by measuring protein carbonyl groups using a spectrophotometric method with the 2,4-dinitrophenylhydrazine (DNPH)-labeling procedure, as outlined by Oliver et al (1987) and modified by Shin et al (2012) Results are reported as nanomoles of DNPH incorporated per milligram of protein, calculated using an extinction coefficient for aliphatic hydrazones of 21 mM −1 cm −1.
2.9 Determinations of malondialdehyde (MDA) by high-performance liquid chromatography (HPLC)
Lipid peroxidation levels were assessed by measuring the accumulation of thiobarbituric acid-reactive substances in tissue homogenates, with results expressed as MDA content The MDA levels in tissue homogenates were quantified using the methodology established by Jareno et al.
In a modified procedure based on Kim et al (1999), 0.1 ml of a homogenate was diluted with phosphate buffered saline (PBS) and mixed with a working solution of thiobarbituric acid and perchloric acid, then heated to 95 °C for one hour After cooling in an ice water bath for 10 minutes, the flocculent precipitate was removed by centrifugation at 3200 × g for 10 minutes The resulting supernatant was neutralized, filtered, and injected onto a 5-μm octadecylsilane column The mobile phase comprised 50 mM PBS (pH 6.0) and methanol in a 58:42 ratio, with isocratic separation at a flow rate of 1.0 ml/min and detection at 532 nm using a UV-VIS high-performance liquid chromatography detector, as detailed by Shin et al (2012).
2.10 Determination of GSH and glutathione disulfide (GSSG) by HPLC
GSH and GSSG levels were analyzed immediately after tissue dissection, following the method outlined by Shin et al (2015) In this process, a sample of the acidified supernatant was combined with an internal standard of 1 mM cysteic acid and 0.88 M iodoacetic acid, along with excess potassium hydrogen bicarbonate.
In the experiment, potassium perchlorate was precipitated by adding 8 to the reaction mixture, followed by the addition of 0.5 ml of a 1.5% (v/v) alcoholic solution of 2,4-dinitrofluorobenzene, which was incubated for 4 hours Afterward, 1.0 ml of diethyl ether was introduced, and the samples were shaken and centrifuged at 2000 g for 20 minutes at room temperature The aqueous phase containing derived glutathione was separated and analyzed using an HPLC–UV/Vis detection system (Model LC-20AT and SPD-20A, Shimadzu) The separation of 5-carboxymethyl glutathione was performed at room temperature with a flow rate of 1.2 ml/min, injecting 10 μl samples onto a Spherisorb NH2 5-μm analytical column Glutathione was detected at 365 nm, and the quantities of glutathione derivatives (GSH and GSSG) were determined relative to the internal standard, cysteic acid.
2.11 Analysis of nuclear translocation of Nrf2 or NF-κB p65
Result
3.1 Time-dependent changes in the expression and activity of cytosolic Cu,Zn superoxide dismutase (SOD-1), mitochondrial Mn superoxide dismutase (SOD-2), glutathione peroxidase (GPx), and glutathione reductase (GR) induced by repeated PCP treatment in the prefrontal cortex of wild type mice
This study investigates the effects of repeated PCP treatment on antioxidant defenses in an animal model, focusing on enzymatic antioxidants at various time points after withdrawal Significant increases in the expression and activity of SOD-1 were observed four hours post-PCP, while a significant decrease was noted seven days later In contrast, no significant changes in SOD-2 expression or activity were detected at any time point Additionally, GPx-1 expression significantly increased four hours post-PCP, remaining elevated at seven days, with the most pronounced changes at one and two days post-treatment GPx activity also rose significantly at four hours and one day post-PCP but decreased at seven days Furthermore, GR expression significantly increased at four hours and one day post-PCP, although its activity remained unchanged.
3.2 Time-dependent changes in reactive oxygen species (ROS), protein oxidation, lipid peroxidation, GSH and GSSG levels, and GSH/GSSG ratios induced by repeated PCP treatment in the prefrontal cortex of wild type mice
The exact cause of schizophrenia is still unclear, but research indicates a link between oxidative stress from free radicals and the disease's development Studies have consistently demonstrated an imbalance in the production and removal of free radicals in individuals with schizophrenia.
Our investigation into the effects of repeated PCP treatment revealed significant alterations in oxidative parameters and glutathione systems Notably, there was a marked increase in reactive oxygen species (ROS) and protein oxidation, indicated by protein carbonyl levels, at both 1 day (P < 0.01) and 2 days (P < 0.05) following PCP administration Additionally, lipid peroxidation, measured by malondialdehyde (MDA) levels, showed a significant rise (P < 0.05) at 1 day post-PCP.
Repeated PCP treatment led to a significant decrease in GSH levels (P < 0.05) at 7 days post-treatment, while GSSG levels significantly increased (P < 0.05) during the same period Additionally, notable reductions in the GSH/GSSG ratio were recorded at both 4 days (P < 0.05) and 7 days (P < 0.01) following PCP administration.
3.3 Repeated PCP treatment-induced changes in social interaction, novel object recognition, forced swimming, and behavior sensitization in glutathione peroxidase-1 knockout (GPx-1 KO) and GPx-1 overexpressing transgenic (GPx-1 TG) mice
We explored the effects of GPx-1 gene manipulation in mice on abnormal behaviors caused by repeated PCP treatment, which disrupts GSH-dependent systems Our findings revealed that repeated PCP administration significantly reduced interaction times in the social interaction test (P < 0.01) and the recognition index in the novel object recognition test (P < 0.05) Additionally, it led to increased immobility time in the forced swimming test (P < 0.05) and heightened behavioral sensitization (P < 0.05) in both wild type and GPx-1 non-TG mice.
Genetic deletion of GPx-1 significantly worsened PCP-induced sociability deficits, impaired visual recognition memory, increased immobility time, and enhanced behavioral sensitization compared to wild type mice, with all results showing statistical significance (P < 0.05) Conversely, genetic overexpression of GPx-1 notably reduced these PCP-induced behaviors, indicating a protective effect against sociability deficits, memory impairment, increased immobility, and behavioral sensitization in GPx-1 non-TG mice, also with statistical significance (P < 0.05).
3.4 Repeated PCP treatment-induced changes in nuclear translocation and DNA binding activity of Nrf2 and NF-κB p65 in the prefrontal cortex of glutathione peroxidase-1 knockout (GPx-1 KO) and GPx-1 overexpressing transgenic (GPx-1 TG) mice
In our investigation of repeated PCP treatment, we found that it significantly decreased cytosolic Nrf2 expression in both wild type and GPx-1 non-TG mice (P < 0.01) Conversely, PCP treatment led to a significant increase in nuclear Nrf2 expression and DNA binding activity in these mice (P < 0.01 for nuclear expression and P < 0.05 for DNA binding) Notably, the knockout of glutathione peroxidase-1 (GPx-1) significantly reduced nuclear translocation and DNA binding activity of Nrf2 compared to wild type (P < 0.01 and P < 0.05, respectively) In contrast, genetic overexpression of GPx-1 enhanced both nuclear translocation and DNA binding activity of Nrf2 (P < 0.05).
Repeated PCP treatment did not significantly affect cytosolic NF-κB p65 levels but notably increased its nuclear translocation in both WT and GPx-1 non-TG mice (P < 0.01) Additionally, PCP treatment enhanced the DNA binding activity of NF-κB in wild type (P < 0.05) and GPx-1 non-TG mice (P < 0.01) The absence of GPx-1 (KO) further amplified these increases in nuclear translocation (P < 0.05 vs WT) and DNA binding activity of NF-κB (P < 0.05 vs WT) Conversely, genetic overexpression of GPx-1 significantly reduced the nuclear translocation (P < 0.05 vs GPx-1 non-TG) and DNA binding activity of NF-κB (P < 0.05 vs GPx-1 non-TG).
3.5 Repeated PCP treatment-induced changes in GCLm and GCLc mRNA expressions, GSH and GSSG levels, GSH/GSSG ratios, GPx-1 expression, and GPx activity in the prefrontal cortex of glutathione peroxidase-1 knockout (GPx-1 KO) and GPx-1 overexpressing transgenic (GPx-1 TG) mice
Fifth, we investigated whether repeated PCP treatment is shown to alter GSH-dependent systems and whether GPx-1 gene manipulation affects PCP-induced changes in these systems, given that both
Repeated treatment with PCP significantly increased GCLm mRNA expression in wild-type (WT) and GPx-1 non-transgenic (non-TG) mice (P < 0.01) Additionally, PCP treatment induced GCLc mRNA expression in both WT and GPx-1 non-TG mice (P < 0.05) However, glutathione peroxidase-1 knockout notably reduced these increases in GCLm and GCLc mRNA expressions compared to WT (P < 0.05) Conversely, genetic overexpression of GPx-1 led to a significant rise in both GCLm and GCLc mRNA expressions compared to GPx-1 non-TG mice (P < 0.05).
Repeated PCP treatment significantly decreased GSH levels (P < 0.05) (Fig 6c) and GSH/GSSG ratios (P < 0.01) (Fig 6e), while it significantly increased GSSG (P < 0.05) (Fig 6d) of
In a study comparing wild-type (WT) and glutathione peroxidase-1 (GPx-1) knockout (TG) mice, it was found that the knockout significantly reduced glutathione (GSH) levels and GSH/GSSG ratios (P < 0.01), while increasing GSSG levels (P < 0.05 vs WT) Conversely, genetic overexpression of GPx-1 provided substantial protection against decreases in GSH levels induced by PCP (P < 0.05 vs GPx-1).
1 non-TG) (Fig 6c) and GSH/GSSG ratios (P < 0.05 vs GPx-1 non-TG) (Fig 6e), as well as attenuating PCP-induced increases in GSSG levels (P < 0.05 vs GPx-1 non-TG) (Fig 6d)
Repeated treatment with PCP led to a notable increase in GPx-1 expression in both wild-type (WT) and GPx-1 non-transgenic (non-TG) mice, with statistical significance at P < 0.05 Additionally, genetic overexpression of GPx-1, in the absence of PCP, resulted in a significant increase in GPx-1 levels compared to GPx-1 non-TG mice, with a P value of less than 0.01.
Discussion
This study reveals that the GPx-1 gene has significant antipsychotic potential against PCP-induced abnormal behaviors in mice, with effects comparable to the antipsychotic drug clozapine Notably, clozapine's antipsychotic effects are linked to the activation of the GPx-1 gene and the Nrf2-dependent GSH synthetic systems Additionally, our findings highlight the crucial role of the Nrf2-dependent system in the prefrontal cortex for neuromodulation mediated by the GPx-1 gene, which helps mitigate PCP-induced abnormal behaviors in mice.
Research indicates that dysfunction in the prefrontal cortex plays a significant role in schizophrenia, with various studies highlighting anatomical and metabolic anomalies in the frontal lobe of affected individuals Cognitive impairments in schizophrenia are largely attributed to this frontal cortex dysfunction Previous findings have demonstrated that repeated PCP treatment leads to glutamatergic hypofunction in the prefrontal cortex, evidenced by reduced extracellular glutamate release Additionally, prenatal exposure to PCP may disrupt glutamatergic neurodevelopment due to NMDA receptor blockage, resulting in similar hypofunction Furthermore, prenatal PCP exposure may also cause GABAergic dysfunction in the prefrontal cortex, contributing to behavioral deficits and underscoring the critical importance of the prefrontal cortex in the development of PCP-induced abnormal behaviors.
Numerous studies have indicated alterations in superoxide dismutase (SOD) activity among schizophrenia patients Specifically, untreated first-episode schizophrenic patients exhibit reduced SOD activity in their red blood cells Additionally, decreased SOD levels are observed in the cerebrospinal fluid and erythrocytes of patients with tardive dyskinesia Recent research has also shown diminished SOD activity in the prefrontal cortex of mice subjected to perinatal PCP treatment Conversely, some studies report increased SOD levels in the post-mortem brains and serum of schizophrenic patients However, there are also findings that suggest unchanged SOD activity when compared to healthy controls These conflicting results may be attributed to the induction of enzymatic antioxidants in response to various factors.
32 suppressed in different phases of the disease Thus more studies might be required for a better understanding of this issue
Research indicates that oxidative stress linked to dysfunctional GSH-dependent systems is crucial in the development of schizophrenia Notably, the activity of GCL, the key enzyme for GSH synthesis, is significantly reduced in individuals with schizophrenia compared to healthy controls Additionally, drug-free patients with schizophrenia exhibit markedly lower GSH levels in their cerebrospinal fluid Postmortem analyses of brain samples from schizophrenia patients reveal decreased GSH, GPx, and GR levels in the caudate region, highlighting the impaired ability to synthesize GSH as a potential vulnerability in schizophrenia.
Research indicates that glutathione peroxidase (GPx) activity is diminished in the blood of schizophrenia patients, with plasma GPx levels showing a significant positive correlation to psychosis rating scores In a recent study, a reduction in glutathione (GSH) levels resulted in decreased GPx activity seven days after PCP treatment, despite an elevated expression of GPx-1 at that time This discrepancy between GPx activity and GPx-1 expression suggests potential dysfunction in the neurochemical system, which may contribute to abnormal behaviors, although further investigation is needed to fully understand this relationship.
Nrf2 plays a crucial role in the cellular response to oxidative stress by activating genes involved in glutathione (GSH) synthesis, leading to a compensatory mechanism in response to GSH depletion caused by PCP exposure This mechanism involves Nrf2 translocating to the nucleus and enhancing the expression of GCLm and GCLc, which are vital for antioxidant defense Our findings suggest that PCP treatment initiates this compensatory response, further amplified by the overexpression of the GPx-1 gene Conversely, the knockout of the GPx-1 gene does not trigger this compensatory induction, highlighting the essential role of endogenous GPx-1 in protecting against PCP toxicity.
Under normal conditions, Nrf2 is quickly degraded by the ubiquitin-proteasome system However, during oxidative stress caused by reactive toxicants or ARE inducers, the interaction between Nrf2 and Keap1 is disrupted This disruption allows Nrf2 to translocate to the nucleus, where it can initiate protective responses.
ARE inducers can directly alter cysteine thiol groups in Keap1, leading to the release and increased activity of Nrf2 Additionally, the activation of various kinases may contribute to this process by phosphorylating Nrf2, which facilitates its translocation to the nucleus.
Our study suggests that altering the interaction between Keap1 and Nrf2, or genetically overexpressing Nrf2, can enhance the antioxidant capacity, specifically GPx-1, in the prefrontal cortex This modulation may provide significant protection against oxidative stress induced by PCP.
Clozapine has been shown to suppress pro-inflammatory cytokines and inhibit microglial overactivation both in vitro and in vivo It also reverses cortico-striatal oxidative stress, repairs abnormal kynurenine metabolism, and rescues mitochondrial dysfunction in rat models of schizophrenia Recent studies indicate that the antipsychotic effects of clozapine may arise from epigenetic modifications triggered by the activation of the dopamine D1 receptor in the prefrontal cortex.
This study investigates the impact of clinically available antipsychotics, specifically haloperidol and clozapine, on the GPx-1 gene and PCP-induced abnormal behaviors in mice Previous findings indicated that haloperidol primarily improves hyperlocomotion, while clozapine effectively addresses hyperlocomotion, social deficits, cognitive dysfunction, and reduced motivation (Qiao et al., 2001; Aoyama et al., 2014) Our current results support these earlier observations, confirming the differential effects of clozapine and haloperidol on schizophrenia-like symptoms Notably, we provide new evidence that clozapine's efficacy is linked to GSH-dependent systems, highlighting its role in restoring PCP-induced abnormal behaviors.
Clozapine's protective effects are linked to Nrf2-dependent pathways, suggesting that sulforaphane, known for up-regulating Nrf2-dependent phase II detoxification enzymes, may help mitigate PCP-induced abnormal behaviors in a mouse model of schizophrenia Additionally, the antipsychotic aripiprazole has been shown to enhance NAD(P)H-quinone oxidoreductase-1 and heme oxygenase-1 through Nrf2-dependent mechanisms in vitro Therefore, it is plausible to consider that Nrf2-dependent GSH synthetic systems play a significant role in these protective effects.
Clozapine demonstrates significant protective effects against abnormal behaviors induced by PCP, comparable to the genetic overexpression of GPx-1 This suggests that Nrf2-dependent GSH synthetic systems and GPx-1 gene induction in the prefrontal cortex play a crucial role in clozapine's protective mechanisms Previous research has shown that NF-κB activation and its mRNA expression are markedly elevated in schizophrenia patients compared to healthy individuals.
In 2009, research revealed that PCP treatment triggers the nuclear translocation and DNA binding activity of NF-κB p65, an effect enhanced by the knockout of the GPx-1 gene and diminished by its overexpression Interestingly, clozapine selectively modulates the Nrf2 transcription factor without affecting NF-κB, indicating a need for further investigation into this specific interaction.
Acute administration of typical antipsychotic haloperidol and atypical clozapine leads to distinct patterns of c-Fos expression in the forebrain Haloperidol increases c-Fos-positive neurons in the dorsolateral striatum, nucleus accumbens shell and core, and lateral septal nucleus, while clozapine primarily affects the nucleus accumbens shell and medial prefrontal cortex Additionally, Zhao et al (2012) found that both acute and repeated haloperidol pretreatment significantly enhances PCP-induced c-Fos-immunoreactivity in the nucleus accumbens shell and ventral tegmental area, but decreases it in the central amygdaloid nucleus In contrast, clozapine pretreatment further elevates PCP-induced c-Fos-immunoreactivity in the lateral septal nucleus and ventral tegmental area, while reducing it in the medial prefrontal cortex, highlighting the prefrontal cortex's role in clozapine's antipsychotic effects.