Activation of Hippocampal Neuronal NADPH Oxidase NOX2 Promotes Depressive-Like Behaviour and Cognition Deficits in Chronic Restraint Stress Mouse Model

• Abstract
• Introduction
• Materials and Methods
• Animals
• Chronic restraint stress
• Drug administration
• Tail suspension test (TST)
• Forced swimming test (FST)
• Open field test (OFT)
• Morris water maze (MWM) test
• Immunofluorescence staining
• Western blot analysis
• Measurement of corticosterone levels
• Real-time-polymerase chain reaction (RT-PCR)
• Statistical analyses
• Results
• CRS-induced depression-like behaviours and impaired learning and memory in mice
• Chronic resistant stress increased NOX2 expression in the hippocampus but had no effect on NOX3 or NOX4 expression
• NOX2 is expressed mainly in hippocampal neurons rather than in microglia or astrocytes in mice with chronic resistant stress
• Gp91ds-Tat treatment rescued chronic resistant stress-induced depression-like behaviours and spatial cognitive impairments
• Blockade of NOX2 signalling restores the expression of brain-derived neurotrophic factor in hippocampal region of chronic resistant stress mice
• Discussion
• Conclusion
• Ethics approval and consent to participate
• Availability of data and materials
• Authorsʼ contributions
• References

Abstract

Background Nicotinamide adenosine dinucleotide phosphate oxidases (NOX) play important roles in mediating stress-induced depression. Three NOX isotypes are expressed mainly in the brain: NOX2, NOX3 and NOX4. In this study, the expression and cellular sources of these NOX isoforms was investigated in the context of stress-induced depression.

Methods Chronic restraint stress (CRS)-induced depressive-like behaviour and cognitive deficits were evaluated by tail suspension tests, forced swimming tests and the Morris water maze test. Hippocampal NOX expression was determined by immunofluorescence staining and western blotting. The hippocampal levels of the brain-derived neurotrophic factor (BDNF) mRNA were determined via quantitative real-time –polymerase chain reaction. Glucocorticoid levels in the hippocampus were measured using ELISA kits.

Results In the mouse CRS model, a significant increase in NOX2 expression was observed in the hippocampus, whereas no significant changes in NOX3 and NOX4 expression were detected. Next, NOX2 expression was primarily localised to neurons (NeuN⁺) but not microglia (Iba-1⁺) or astrocytes (GFAP⁺). Treatment with gp91ds-tat, a specific NOX2 inhibitor, effectively mitigated the behavioural deficits induced by CRS. The decreased expression of the BDNF mRNA in the hippocampus of CRS mice was restored upon gp91ds-tat treatment. A positive correlation was identified between neuronal NOX2 expression and serum glucocorticoid levels.

Conclusions Our study indicated that neuronal NOX2 may be a critical mediator of depression-like behaviours and spatial cognitive deficits in mice subjected to CRS. Blockade of NOX2 signalling may be a promising therapeutic strategy for depression.

Introduction

Depression is a common mental disorder and a leading cause of disability that significantly contributes to the global disease burden [1]. Stress, a primary adverse life event, can trigger depressive episodes in humans. Rodents subjected to chronic restraint stress (CRS) have been widely used as depression models [2] [3] [4] [5]. Recent studies using this model have shown that chronic stress increases the expression of nicotinamide adenosine dinucleotide phosphate (NADPH) oxidases (NOX) in a glucocorticoid-dependent manner, eventually promoting depression-like behavior [6].

NOX, a superoxide-generating enzyme family consisting of seven members (NOX1–5, DUOX1/2), primarily regulates the production of reactive oxygen species (ROS) in various tissues, especially in the central nervous system [7] [8] [9] [10] [11] [12]. Studies have documented elevated ROS levels due to other NOX isoforms, including NOX4 and NOX3 [8] [13] [14]. The expression of NOX has also been confirmed to be associated with increased oxidative stress in numerous brain disorders [6] [15] [16]. For example, increasing evidence suggests that elevated NOX2 levels are involved in memory and cognitive impairments in Alzheimer’s disease, sepsis, and aging [17] [18]. However, the specific member and cellular sources of NOX that mediate chronic stress-induced depressive-like behaviour, along with the underlying mechanisms, are poorly understood.

Recent studies have reported that the hippocampal dysfunction induced by chronic stress is closely related to the pathology of depression [19] [20] [21]. Furthermore, the enrichment and increased expression of glucocorticoid receptors, pivotal mediators of stress-induced neurotoxicity, have been observed in the hippocampus of depressed individuals [22]. Therefore, these findings suggest that the hippocampus is an important region for the progression of depression [23].

Here, we evaluated behavioural abnormalities associated with depression and spatial cognition in CRS mice and further demonstrated that NOX2 expressed by hippocampal neurons plays a key role in stress-induced behavioural deficits. We also investigated whether the specific NOX2 inhibitor Gp91ds-tat rescued the behavioural deficits and the expression of brain-derived neurotrophic factor (BDNF) induced by CRS.

Materials and Methods

Animals

All experiments were performed according to the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Zhongshan School of Medicine on Laboratory Animal Care. Male C57BL/6 J mice (7 weeks old) obtained from the Laboratory Animal Centre of Sun Yat-Sen University (Guangzhou, China) were housed in pairs under specific pathogen-free conditions. The animals were bred at a controlled temperature (22±1°C) with a 12-h light/12-h dark artificial light cycle and specific pathogen-free food and water were available ad libitum.

Chronic restraint stress

The restraint treatment in the present study was performed as previously described [24] [25]. Briefly, the mice were individually placed headfirst into well-ventilated 50 mL polypropylene conical tubes, which were then plugged with a 3-cm-long middle tube and finally tied with a cap of the 50 mL tube to limit their autonomous activities for 6 h/day (from 9:00 a.m. to 3:00 p.m.) for 28 consecutive days. The animals were returned to their home cages after each session of restraint. Food and water were provided for 24 h during the chronic treatment regimen.

Drug administration

The mice were intraperitoneally injected with the NOX2 inhibitor, Gp91ds-tat (5 mg/kg/d) and the NADPH oxidase inhibitor, apocynin (15 mg/kg/d) [6]. The NOX4 inhibitor GKT137831 (60 mg/kg/d) was administered subcutaneously [13]. All drugs were purchased from Sigma-Aldrich and dissolved in a normal saline solution. The mice were administered the drugs 1 h before the start of each 6 h restraint in the cotreatment paradigm.

Tail suspension test (TST)

As previously described [26], each mouse was suspended upside down by its tail for 6 min in a soundproof room. The total duration of immobility during the last 5 min was measured using the SuperTst high-throughput TST analysis system (Shanghai Xinruan Information Technology Co., Ltd., China).

Forced swimming test (FST)

Three days after the TST, individual animals were placed in an open cylindrical container (diameter=10 cm) containing water at 22±1°C and forced to swim for 6 min, as previously described [26]. During the last 5 min, the total duration of immobility was recorded and analyzed using the SuperFst high-throughput FST system (Shanghai Xinruan Information Technology Co., Ltd., China).

Open field test (OFT)

Each mouse was individually placed in a 40 cm×40 cm×40 cm Plexiglas cubicle and allowed to explore for 5 min. The total distance traveled and the average speed of each mouse during the task were calculated using the TopScan^TM 2.0 system (Clever Sys. Inc.). After each trial, 70% ethanol was used to clean the apparatus thoroughly.

Morris water maze (MWM) test

In a separate experiment, the MWM test was used to assess the effects of CRS/drug treatment on spatial learning and memory. As previously described [27], in this hippocampus-dependent task, the mouse must learn to use surrounding environmental cues to locate the hidden platform (9 cm in diameter), which was 1.0 cm below the water surface in a circular pool filled with opacified water made with nontoxic white dye (80 cm in diameter, 22–24°C). The pool was divided into four equal quadrants. The mice were placed on the platform for 30 s before each testing phase. Each mouse performed four trials per day and was allowed to swim freely for a maximum of 60 s or until the platform was found for 5 consecutive days. Mice that failed to find the platform within 60 s were guided to the platform and allowed to stay on it for 10 s before being dried. For the probe trial on day 6, the platform was removed, and each mouse was allowed to swim freely for 60 s to assess their spatial memory of the platform location. The latency of finding the platform and swimming distance were recorded with a video camera and a computerised tracking system (MT-200, Chengdu, China).

Immunofluorescence staining

As previously described [27], the animals were deeply anesthetised and transcardially perfused with phosphate buffer solution (PBS), followed by cold 4% paraformaldehyde (PFA). After excision, the brains were immediately postfixed with 4% PFA overnight at 4°C and then sequentially dehydrated by incubating with 10%, 20% and 30% sucrose solutions for 24 h at 4°C. Serial coronal sections (40 μm) were obtained using a freezing microtome (Leica SM2000R) and stored in PBS at 4°C prior to immunostaining. Free-floating sections were washed with PBS and then blocked with PBS containing 1% bovine serum albumin (BSA) and 0.25% Triton X-100 (Sigma-Aldrich) for 1 h at 37°C. The slices were then incubated with the following primary antibodies: rabbit anti-NOX2 (ab80508; 1:400; Abcam), rabbit anti-NOX3 (PA5–38036; 1:400; Invitrogen), rabbit anti-NOX4 (ab216654; 1:400; Abcam), mouse anti-NeuN (MAB377; 1:1000; Sigma Aldrich), mouse anti-Iba-1 (019–19741; 1:1000; Wako Chemical), and mouse anti-GFAP (AMAB91033; 1:10,000; Sigma-Aldrich). The primary antibodies were diluted in PBS containing 1% BSA and 0.25% Triton X-100. The sections were then incubated overnight at 4°C. The sections were washed three times before being incubated with the following secondary antibodies for 2 h at 37°C: Alexa Fluor 555-conjugated donkey anti-rabbit and Alexa Fluor 488-conjugated donkey anti-mouse. The secondary antibodies were diluted 1:400. A Zeiss LSM780 confocal laser-scanning microscope was used to capture representative confocal micrographs. The fluorescence intensity of the acquired images was quantified using NIH ImageJ software.

Western blot analysis

As previously described, the mice were deeply anesthetised and transcardially perfused with cold saline, and hippocampal tissue was collected immediately and stored frozen (−70°C) until use. Lysates of hippocampal tissues were generated using protein lysis buffer (Beyotime Institute of Biotechnology, P0013C). The protein concentration was measured using a BCA protein assay kit (Beyotime Institute of Biotechnology, P0012). A proper volume of an equivalent amount of protein was separated through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (Bio-Rad, L1620177). The membrane was blocked with 5% (w/v) BSA for 2 h at room temperature. The membrane was incubated with primary antibodies against NOX2 (Abcam, ab80508, 1:1000), NOX3 (Invitrogen, PA5–38036, 1:1000), NOX4 (Abcam, ab216654, 1:1000) and β-actin (Cell Signaling Technology, 4967, 1:1000) overnight at 4°C, followed by incubation with HRP-conjugated anti-mouse (KPL, 5110–0011, 1:2000) or anti-rabbit (KPL, 5110–0010, 1:2000) antibodies at room temperature for 0.5 h. The bands were visualised using an ECL western blot detection kit (FD BioScience, FD8030). Images were acquired with a GE AI600 instrument (General-Electrics Healthcare) and Fiji (NIH, Bethesda, Maryland, USA) was used for the band pixel density analysis. The data were normalised to the WT controls and used for statistical analysis.

Measurement of corticosterone levels

Corticosterone levels in the hippocampus were measured using enzyme-linked immunosorbent assay kits (Corticosterone ELISA Kit; EIAab Science Co, Ltd., Wuhan, China) according to the manufacturerʼs instructions. The reaction was measured at 450 nm using a spectrofluorometer (Biotek Synergy HTX, Biotek).

Real-time-polymerase chain reaction (RT-PCR)

Total RNA was extracted from the hippocampus using the SE Total RNA Kit I (Omega) according to the manufacturerʼs instructions. Messenger RNA (mRNA) (2 μg) was reverse transcribed into cDNA using a Prime-Script RT Reagent Kit (Takara, Dalian, Liao Ning, China) according to the manufacturerʼs instructions. Quantitative PCRs were performed in triplicate with SYBR Premix Ex Taq (Takara, Dalian, Liao Ning, China). β-Actin was chosen as the reference gene. All the results of the quantitative real-time PCRs were analysed using a Light Cycler 96 system (Roche) following the standard curve method and normalised to the expression of β-actin. The gene-specific PCR primers for BDNF are as follows: 5′-TGTGACAGTATTAGCGAGTGGGT-3ʼ (forward) and 5′-ACGATTGGGTAGTTCGGCATT-3ʼ (reverse).

Statistical analyses

The data were statistically analysed using SPSS 23.0 statistical software for Windows (Chicago, IL, USA). The data are presented as the means±standard error of the mean. The data were analyzed using the Student’s t-test or one/two-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. Spearman’s correlation analysis was used to assess the correlation between hippocampal glucocorticoid (GC) levels and NOX2 expression. P<0.05 was considered statistically significant.

Results

CRS-induced depression-like behaviours and impaired learning and memory in mice

We restrained the mice for 6 h daily for 28 days to induce a depression model ([Fig. 1a]). Compared with control mice, CRS mice exhibited increased immobility in the TST ([Fig. 1b], t=3.868, df=22, p=0.0008) and the FST ([Fig. 1c], t=4.454, df=22, p=0.0002). These findings suggest that the adopted CRS procedure is sufficient for the induction of depression-like behaviours, consistent with previous reports [24].

Dysfunction of the hippocampus has been proposed to be involved in the neuropathology of major depressive disorder and may play a critical role in spatial learning and memory [19] [20]. The MWM test was used to assess spatial learning and memory abilities and to test whether the CRS treatment affected hippocampal-dependent learning and memory. In the acquisition trial, the CRS mice spent more time locating the hidden platform than the control mice on days 3, 4 and 5 ([Fig. 1d], day 3: t=2.646, df=22, p=0.0148; day 4: t=4.043, df=22, p=0.0005; day 5: t=3.346, df=22, p=0.0029). Moreover, compared with control mice, CRS mice spent significantly less time in the target quadrant (Q3) in the probe trial ([Fig. 1e], t=3.63, df=22, p=0.0015), suggesting impaired spatial learning and memory. The swimming speeds were comparable between the two groups (data not shown). We observed similar distance ([Fig. 1f]) and speed ([Fig. 1g]) of locomotion between the CRS and the control mice in the OFT, excluding locomotion disorders induced by CRS. These findings showed that CRS not only induces depression-like behaviours but also impairs the hippocampus-dependent spatial memory in mice.

Chronic resistant stress increased NOX2 expression in the hippocampus but had no effect on NOX3 or NOX4 expression

NOX is an enzyme that mainly regulates ROS generation in the brain [8] [9] [10] [12]; however, the NOX isoform in the brain that is responsible for CRS-induced behavioural symptoms remains unclear. NOX2 was first detected via immunofluorescence staining in the hippocampus of the CRS group and control group to address this question ([Fig. 2a–c]). Our data revealed a significant increase in NOX2 expression in the hippocampus, including the CA1, CA3 and DG regions of CRS model mice compared with CON model mice ([Fig. 2c], CA1: t=3.513, df=10, p=0.0056; CA3: t=4.545, df=10, p=0.0011; DG: t=4.835, df=10, p=0.0007). Similar results were further confirmed by western blot analysis ([Fig. 2d and e], t=10.09, df=10, p<0.0001). Interestingly, immunofluorescence staining and western blot analysis did not show any significant difference in NOX3 or NOX4 levels in either the cortex or hippocampus between the two groups ([Fig. 3a–c]; [Fig. 3d–f]). These findings suggest that CRS promotes hippocampal NOX2 expression but does not affect the expression of NOX3 and NOX4 in the brain.

NOX2 is expressed mainly in hippocampal neurons rather than in microglia or astrocytes in mice with chronic resistant stress

Brain slices from CRS mice or controls were double-stained for NOX2/Iba-1, NOX2/GFAP, and NOX2/NeuN to investigate the cellular source of increased NOX2 in the hippocampus of CRS mice. Only a few NOX2/Iba-1⁺cells and NOX2/GFAP⁺cells were observed near the blood vessels rather than in the parenchyma of the brain ([Fig. 4a and b]). Almost all NOX2 signals were colocalised with those in hippocampal neurons (NeuN⁺cells) ([Fig. 4c]). Our quantification results revealed that the relative expression of NOX2 in hippocampal neurons was significantly increased after CRS-induced depression ([Fig. 4c], t=3.206, df=10, p=0.0094). These data showed that hippocampal neurons are the primary source of NOX2 production, suggesting their pivotal role in depressive-like behaviour and cognitive deficits induced by CRS.

Gp91ds-Tat treatment rescued chronic resistant stress-induced depression-like behaviours and spatial cognitive impairments

We used Gp91ds-tat (a specific inhibitor of NOX2), GKT137831 (a specific inhibitor of NOX4) and apocynin (a nonspecific NOX inhibitor) to verify the role of NOX2, NOX4 and NADPH oxidase signalling in behavioural deficits and to further determine the specific type of NADPH oxidase that serves as the main mediator of behavioural impairments. The results showed that depression-like behaviours in the TST ([Fig. 5a], one way ANOVA F (4, 55)=9.458, p<0.0001) and FST ([Fig. 5b], one way ANOVA F (4, 55)=8.656, p<0.0001) and spatial cognitive deficits in the MWM- test ([Fig. 5c], one way ANOVA day 3: F (4, 55)=8.88, p<0.0001; day 4: F (4, 55)=9.663, p<0.0001; day 5: F (4, 55)=10.46, p<0.0001, and D, one way ANOVA F (4, 55)=7.032, p=0.0001) were reversed by Gp91ds-tat and apocynin treatments, but not by GKT137831 treatment in CRS mice. These findings provide strong evidence for the hypothesis that activated NOX2 signalling induced by CRS primarily mediates depression-like behaviours and spatial cognitive deficits in mice.

Blockade of NOX2 signalling restores the expression of brain-derived neurotrophic factor in hippocampal region of chronic resistant stress mice

According to the evidence described previously, BDNF plays an important role in the pathogenesis of depression and spatial cognitive deficits by regulating neural plasticity processes [21] [28]. Increased NOX2 levels attenuate BDNF expression in the hippocampus; therefore, we quantified the expression of the BDNF gene via RT-qPCR in control and CRS mice following Gp91ds-tat treatment to determine the downstream effect of BDNF on NOX2-induced behavioural deficits after CRS. We found reduced hippocampal BDNF expression in the CRS mice compared with the controls, whereas the expression of BDNF was significantly rescued in the hippocampus of Gp91ds-tat-treated CRS mice ([Fig. 6a], one way ANOVA F (3, 28)=16.95, p<0.0001). These findings showed that hippocampal NOX2 signalling may contribute to depression-like behaviours and spatial cognitive impairments, partly by decreasing BDNF expression.

A previous study verified that NOX2 expression can be induced in a GC-dependent manner [29]. Therefore, we explored the GC level in the hippocampus and detected an increase in the GC levels in CRS mice ([Fig. 6b], t=14.33, df=10, p<0.0001). Furthermore, a significant positive correlation was observed between GC levels and NOX2 expression ([Fig. 6c], Pearson r=0.9933, p<0.0001). These findings suggested that the GC might act as an upstream signal to promote NOX2-associated depression-like behaviours and spatial cognitive impairments in the CRS mouse model.

Discussion

In the present study, we observed a significant increase in the levels of NOX2, expressed predominantly by neurons in the hippocampus of a mouse model of depression induced by CRS. Furthermore, treatment with Gp91ds-tat, a NOX2 inhibitor, completely abrogated CRS-induced behavioural deficits, accompanied by the rescue of BDNF mRNA expression in the hippocampus. Our study revealed that CRS elicits depression-like behavioural and spatial cognitive deficits partly through the activation of the neuronal NOX2-BDNF pathway in the hippocampus, suggesting a potential therapeutic intervention for depression.

Depression and spatial cognitive deficits have been found to be closely linked to aberrant neural plasticity in the hippocampus [30] [31], which is particularly sensitive to pathological changes in response to chronic stress in both humans and rodents [32] [33] [34]. Given that the brain consumes a large amount of oxygen and is rich in lipids, it is vulnerable to oxidative stress caused by an imbalance between ROS and antioxidants [35]. The effects of ROS on the regulation of neural plasticity in the brain and behaviour have been extensively documented [36]. Previous studies have revealed that NOX mediates depressive behaviour induced by chronic stress in mice [6]. However, the isoform of NADPH oxidase that is responsible for behaviour impairments remains unclear. Previous studies have suggested that NADPH oxidase is a major source of ROS production in various cell types and is central to the production of oxidative stress in response to stress [6] [37] [38]. Several isoforms of NADPH oxidase, especially NOX2, NOX3 and NOX4, are highly expressed in the central nervous system [14] [15]. Interestingly, we observed a significant increase in NOX2 expression in CRS mice, whereas NOX3 and NOX4 expression remained unaffected. Although differences in the distribution of these NOX isoforms have been confirmed, the mechanisms underlying the difference in NOX expression in response to CRS require further investigation.

NOX2 is involved in the development of multiple disorders of the central nervous system, such as Parkinsonʼs disease, Alzheimerʼs disease, bipolar disorder and brain injury resulting from a high-fat diet [39] [40] [41] [42] [43]. Thus, alteration in NOX2 expression in the CNS may be influenced by CRS to induce the pathological processes of depression. Following the discovery of elevated NOX2 levels in the hippocampus of CRS mice, we investigated the origin of this increase in NOX2 expression. The cellular expression of NOX2 in the brain under physiological conditions in rodent models remains unclear. While NOX2 has been reported to be expressed in both neurons and microglia but not in astrocytes in rats under physiological conditions, another study failed to detect NOX2 in the brain rats via RT-PCR [39]. In our present study, we observed that NOX2 is exclusively expressed by the hippocampal neurons of CRS mice using immunofluorescence staining. This inconsistency may be due to species differences. Notably, our findings on the cellular expression of NOX3 and NOX4 in the mouse brain are consistent with Cooney’s report [12], which revealed their exclusive neuronal expression without their presence in microglia or astrocytes. Our findings suggest that CRS induces depression-like behaviour and spatial cognitive impairments through a NOX2-dependent mechanism.

BDNF, a well-known neurotrophic factor, is involved in the regulation of activity-dependent changes in synapse structure and function and synaptogenesis [44]. Numerous studies support a strong association between BDNF expression and depression [45]. Synaptic plasticity is also recognised as a key mechanism supporting memory formation and retention. Numerous studies have documented the direct link between BDNF expression and learning and memory [46] [47]. Importantly, increased NOX2 expression has been shown to suppress hippocampal BDNF expression. Our data showed that CRS-exposed mice exhibit diminished BDNF expression in the hippocampus, which can be rescued by a specific NOX2 inhibitor. These findings suggest that the NOX2 signalling may contribute to behaviour deficits in CRS mice by regulating the downstream molecule BDNF.

Previous studies have shown that NADPH oxidase activity can be increased in a GC-dependent manner [6]. We also found a significantly increased concentration of GCs in the hippocampus of CRS mice. Additionally, our depression model exhibited a significant positive correlation between GC levels and NOX2 expression in the hippocampus, indicating a potential GC-dependent mechanism for NOX2 activation. However, whether the increase in the GC levels represents a compensatory response to the increase in NOX2 expression requires further investigation.

Conclusion

In summary, our study demonstrated that neuronal NOX2 may be a critical mediator of depression-like behaviour and spatial cognitive deficits in mice subjected to CRS. Our findings indicate that the specific blockade of NOX2 signalling may be a promising therapeutic strategy for depression and dementia.

Ethics approval and consent to participate

All experiments were performed under the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Zhongshan School of Medicine on Laboratory Animal Care.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.

Authorsʼ contributions

Z.Y., Z.Z. and J.Y.: study conceptualization; Z.Z., H.Z., Z.L. and H.H.: all animal work; H.Z. and Z.Z.: manuscript preparation and data analysis; Z.Y., J.Y., F.Q. and H.H.: assisted in manuscript revision.

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Correspondence

Publication History

Received: 25 June 2024

Accepted: 20 September 2024

Article published online:
15 November 2024

© 2024. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

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