Depression-like behaviors are accompanied by disrupted mitochondrial energy metabolism in chronic corticosterone-induced mice

Journal Pre-proof Depression-like behaviors are accompanied by disrupted mitochondrial energy metabolism in chronic corticosterone-induced mice Xiaoxian Xie, Qichen Shen, Chunan Yu, Qingfeng Xiao, Jiafeng Zhou, Ze Xiong, Zezhi Li, Zhengwei Fu

PII:

S0960-0760(19)30544-8

DOI:

https://doi.org/10.1016/j.jsbmb.2020.105607

Reference:

SBMB 105607

To appear in:

Journal of Steroid Biochemistry and Molecular Biology

Received Date:

12 September 2019

Revised Date:

19 January 2020

Accepted Date:

24 January 2020

Please cite this article as: Xie X, Shen Q, Yu C, Xiao Q, Zhou J, Xiong Z, Li Z, Fu Z, Depression-like behaviors are accompanied by disrupted mitochondrial energy metabolism in chronic corticosterone-induced mice, Journal of Steroid Biochemistry and Molecular Biology (2020), doi: https://doi.org/10.1016/j.jsbmb.2020.105607

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Depression-like behaviors are accompanied by disrupted mitochondrial energy metabolism in chronic corticosterone-induced mice

Xiaoxian Xie1#, Qichen Shen1#, Chunan Yu1, Qingfeng Xiao1, Jiafeng Zhou1, Ze Xiong1, Zezhi Li2*, Zhengwei Fu1* 1College of Biotechnology and Bioengineering, Zhejiang University of Technology,

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Hangzhou 310032, China

Department of Neurology, Ren Ji Hospital, Shanghai Jiao Tong University School of

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Medicine, Shanghai 200025, China

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Short title: CORT links depression and mitochondrial energy metabolism These authors contributed equally to this work

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Corresponding author:

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Prof. Zhengwei Fu, Dr. Zezhi Li

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College of Biotechnology and Bioengineering, Zhejiang University of Technology, No.6 District, Zhaohui, Hangzhou, Zhejiang, 310032, China E-mail: [email protected], [email protected] Tel: 86-0571-8832-0599 Fax: 86-0571-8832-0599

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Highlights 1. CORT alters mitochondria energy-related transcriptome and metabolomics profiles 2. CORT injection disrupts mitochondrial energy metabolism in depressive mice 3. ATP production is inhibited via NAD+ synthesis and SIRT3 signaling pathway

Abstract Stress exerts its negative effects by interference with mitochondrial energy production

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in rodents, and is able to impair mitochondrial bioenergetics. However, the underlying mechanism that stress hormone impacts depression-like behaviors and mitochondrial

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energy metabolism is still not well understood. Here, we investigated the changes of depression-like behaviors and mitochondrial energy metabolism induced by chronic

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corticosterone (CORT). The results showed that after treatment with CORT for 6

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weeks, mice displayed depression-like behaviors, which were identified by tail suspension test, forced swimming test and open field test. Then, the livers were

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isolated and tested by RNA sequencing and metabolome analysis. RNA sequencing showed 354 up-regulated genes and 284 down-regulated genes, and metabolome

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analysis revealed 280 metabolites with increased abundances and 193 metabolites

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with reduced abundances in the liver of mice after CORT, which were closely associated with lipid metabolism and oxidative phosphorylation in mitochondria. Based on these findings, the changes of mitochondrial energy metabolism were investigated, and we revealed that CORT condition inhibited glycolysis and fatty acid degradation pathway, and activated synthesis of triacylglycerol, leading to the reduced

levels of acetyl-CoA and attenuated TCA cycle. Also, the pathways of NAD+ synthesis were inhibited, resulting in the reduced activity of sirtuin 3 (SIRT3). Thus, all of these observations disrupted the function of mitochondria, and led to the decrease of ATP production. Our findings uncover a novel mechanism of stress on depression-like behaviors and mitochondrial energy metabolism in rodents.

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Keywords: depression; mitochondrial function; energy metabolism; NAD+; SIRT3

1. Introduction Depression is a common but serious mood disorder linked to negative effects on daily activities and decreasing quality of life for millions of people worldwide [1]. It is projected to be one of three leading causes of disease burden in 2030 [2], and becomes an important cause of disability and suicidal behavior [3]. The previous studies demonstrated that there was a high incidence of suicide (10–15%) in

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depressive patients [4]. Depression is also known to exert its negative effects by, at

one hand, interference with mitochondrial energy production in chronic mild stress- [5]

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and corticosterone (CORT) -induced depressive rats [6]. Mitochondria are the cellular sites of aerobic respiration and produce majority of cellular ATP through oxidative

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phosphorylatioin [7]. Nicotinamide adenine dinucleotide (NAD+)-dependent enzymes

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are important in oxidative metabolism, and nicotinamidephospho-ribosyltransferase (NAMPT) is the rate-limiting enzyme in NAD+ synthesis, thus the increase of NAD+

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is associated with enforced mitochondrial function [8]. The dysfuncction of mitochondria was observed in depressive animals [9]; the reduced adenosine

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triphosphate (ATP) production was also found in the depressed patients [10],

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suggesting the association of depression with mitochondrial energy metabolism. Excessive circulating CORT level leads to the dysfunction of hypothalamic

pituitary adrenal (HPA) axis through negative feedback inhibitory mediation in depressed patients [11], which was also tested in the depressed animals [12]. Accumulated studies have demonstrated that chronic CORT injection can increase the

probability of depression-like behaviors in sucrose preference [13], and forced swimming test (FST) in rats [14]. The decrease in sucrose intake and elevation in immobility time in FST may be caused by dysregulation of the HPA axis due to the injection of CORT [15]. Also, CORT injection was corrected with the eukaryotic electron transport process via induction of oxidase activity in mitochondria, and impairs mitochondrial bioenergetics [9, 16], thus, it results in the reduction of ATP

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production [15]. Patients with mitochondrial diseases often display symptoms

representing mood disorders [17]. The alterations in the processes of mitochondria

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including oxidative phosphorylation and membrane polarity can increase oxidative stress and apoptosis, preceding the development of depressive symptoms [18].

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Moreover, CORT injection is involved in the metabolic perturbations taking place in

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mitochondria, and these metabolites, such as pyruvate and creatine, were closely associated with energy metabolism [19]. In the present study, identification of the

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mitochondrial energy metabolism disruption produced by CORT administration suggests a new implication for understanding the mechanisms of stress

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pathophysiology.

2 Material and method For detailed procedures, see the Supplemental Experimental Procedures 2.1 Animals and corticosterone administration Male C57BL/6J mice were housed in groups of 4 per cage. The body weight was

recorded every week and food intake was measured every day. After 1 week of acclimatization, mice were administered subcutaneously daily with CORT suspension liquid (20mg/kg) or a vehicle (n=8) for 6 weeks. The experimental design was shown in Figure S1. 2.2 Behavioral assessment and locomotor analysis After CORT administration for 6 weeks, depression-like behaviors were evaluated by

protocols were performed as a previous study [20].

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2.3 Library preparation for RNA sequencing

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tail suspension test (TST), forced swimming test (FST) and open field test (OFT). The

Total RNA was isolated and purified, and then the mRNA was randomly segmented

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using ragmentation buffer (Illumina) at 94 °C. The first strand cDNA and second

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strand cDNA were successively synthesized and amplificated using Phusion High-Fidelity DNA polymerase. Analysis of sequencing data was performed using

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BMKCloud (www.biocloud.net).

2.4 Metabolome sample preparation and GC-TOF-MS Analysis

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Metabolites were isolated from liver and transferred into a GC/MS glass vial. Gas

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chromatograph system coupled with a Pegasus HT time-of-flight mass spectrometer (GC-TOF-MS) was performed using an Agilent 7890 gas chromatograph system (Agilent Technologies, Palo Alto, Calif.) coupled with a Pegasus HT time-of-flight mass spectrometer (AB Sciex, Foster City, CA, USA). Metabolome data analysis was performed using BMKCloud (www.biocloud.net).

2.5 Real-time quantitative polymerase chain reaction (RT-qPCR) The cDNA was synthesized using reverse transcriptase kit (Toyobo, Japan). The cDNA was quantified using a Master Cycler RealPlex4 system (Eppendorf Vertrieb Deutschland GmbH, Wesseling-Berzdorf, Germany), and all primers were listed in Table S2. 2.6 Activities of enzymes, and enzyme linked immunosorbent assay (ELISA)

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The activities of sirtuin 3 (SIRT3), superoxide dismutase 2 (SOD2), glutathione

s-transferase (GST) and Acetyl-CoA were determined using kits purchased from

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Cusabio (Cusabio Biotech CO., LTD., Wuhan, China). GSH levels were determined using ELISA kit (Mlbio, Shanghai, China) according to the manufacturer’s

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instructions. ROS levels were detected as a previous description [21].

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2.7 Transmission electron microscopy (TEM)

Liver samples of mice were cut into about 1 cubic mm, and TEM was performed as a

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previous study [22]. Samples were imaged using a transmission electron microscope (TEM; FEI Tecnai G2 20; Hillsboro, OR, USA) equipped with a charge-coupled

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device camera.

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2.8 Statistical analysis

All measurements of figures were presented as the mean ± standard error of the mean (SEM). Differences between normally distributed data were analyzed by student’s t-test. The p value < 0.05 was considered statistically significant.

3 Results 3.1 Corticosterone affects physiological parameters in mice expressing depression-like behaviors After 3 weeks of CORT injection, mice displayed depressive behaviors as evidenced by increased duration of immobility in both TST (p < 0.01, Fig. 1A) and FST (p < 0.01, Fig. 1B), along with reduced time in center zone and number of entries in the

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OFT (p < 0.01, Fig. 1C, D), when compared to non-stressed control animals. As

expected, CORT significantly decreased the body weight of mice after one week (p <

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0.01, Fig. 1E), but it significantly enhanced the average food intake per day as

compared to controls (p < 0.05, Fig. 1F), whereas it almost did not change the total

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locomotor activity per day (p > 0.05, Fig. 1G). In addition, CORT increased the liver

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weight and percentage of liver to body weight (p < 0.001, Fig. 1H, I). The contents of triglyceride were calculated by ELISA and oil O red staining, and then we found lipid

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accumulation in the liver of mice after CORT (p < 0.05, Fig. 1J, K, L). 3.2 Transcriptome analysis and experimental identification by RT-qPCR in the liver

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To identify the expression changes of gene clusters in mice after CORT adminstration,

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the libraries were established, and three repeated animals were prepared in each group. The RNA-Seq of six samples generated grossly 272.9 million of clean reads, which ranged from 40.3-52.7 million reads per sample. Subsequently, 86.41%-90.56% of the reads were mapped to the gene bank of mus musculus (Table 1). Principal component analysis (PCA) analysis revealed that the transcriptional levels of mice after CORT

were significantly differed from controls (Fig. 2A). The volcano plots showed that totally 638 transcripts were differently expressed as compared to control group, including that 354 genes were up-regulated and 284 genes were down-regulated (Fig. 2B). We listed the top 10 most enriched GO terms of each category (Fig. 2C). Apparently, in category of molecular function, DEGs were concentrated on “ATP binding”, “protein binding” and “protein homodimerization activity”. The most

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enriched groups in cellular component were mainly “cytoplasm”, “mitochondrion”,

and “cytosol”. The most enriched groups “positive regulation of apoptotic process”,

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“response to drug”, “RNA polymerase Ⅱ promoter”, “response to hypoxia” and

“aging” were significant enrichments. More details of GO enrichment analysis were

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listed in Table S1. To evaluate the reliability of transcriptome sequencing data, 16

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genes with various degrees of expression levels were selected and identified by RT-qPCR. Our results were consistent with the DEGs analysis (Fig. 2D, E), and the

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liner regression equation Y = 1.16X - 0.4888 with high correlation (R2 = 0.9332, p < 0.0001; Fig. 2D), indicative of high accuracy and quality of RNA-Seq analysis.

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3.3 Metabolome analysis in the liver

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To further isolate the impacts of stress CORT conditioning on physiological response to the liver, we performed metabolite profiling using GC-MS. We detected a total of 1823 metabolites, and found 233 target metabolites. According to these identified metabolites, the correlations among biological replicates were assessed using Spearman Rank Correlation (Fig. 3A). We found significant differences between mice

after CORT and control animals, which was also proved by the Orthogonal Projections to Latent Structures-Discriminant Analysis (OPLS-DA) (Fig. 3B), and inspected reliability (Fig. S3A). Then, 453 metabolites were screened, and mice after CORT showed the increased abundances of 280 metabolites, and the reduced abundances of 193 metabolites (Fig. 3C). Also, the score plot (S-plot) of 233 target metabolites showed that the significant changes of metabolites in the abundances

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were mainly lipid-related (Fig.3D), such as oleic acid and palmitic acid. Strikingly, we found the metabolites with significant change were rich in energy metabolism, such as

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fatty acid, amino acid and oxidative phosphorylation-related metabolites in mice after CORT (Fig. S3B-D).

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3.4 Alterations in energy metabolism in mice after corticosterone

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Accordingly, our analysis of transcriptome and metabolome revealed the link of CORT condition with mitochondrial energy metabolism, and we further tested the

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expression of the key genes and metabolites associated with lipid-related energy metabolism. Our data showed that that CORT reduced the mRNA expression of

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phosphofructokinase (PFKL) in glycolysis pathway (p < 0.05, Fig. 4A), and increased

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the mRNA expression of pyruvate carboxylase (PCx) in gluconeogenesis pathway (p < 0.05, Fig. 4B), when compared to controls. In addition, mice after CORT displayed increased mRNA expression of phosphatidate phosphatase Lpin1 and 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT, p < 0.05, Fig. 4C, S4A) in TG synthesis. Moreover, CORT condition decreased the expression of fatty

acid-binding protein (Fabp1; p < 0.05, Fig. 4D), peroxisome proliferator-activated receptor α (PPARα; p < 0.05, Fig. 4E), and increased the expression of fatty acid synthase (Fasn) in fatty acid synthesis (p < 0.05, Fig. S4B) as compared to controls. Thus, these changes contributed to the accumulation of fatty acid (p < 0.05, Fig. 5F, S2B). Furthermore, we found that exposure to CORT decreased the acetyl-CoA level (p < 0.05, Fig. 4G) and increased the level of oxaloacetate (p < 0.05, Fig. S4C), and

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significantly reduced the nicotinamide adenine dinucleotide (NAD+) and oxidized

form of nicotinamide adenine dinucleotide phosphate (NADP+) (p < 0.05, Fig. 4H, I).

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3.5 Mediations of NAD+ synthesis and its effect on SIRT3 activity in mice after corticosterone

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Given the decreased levels of coenzyme NAD+ resulted in the reduction of catalytic

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ability of SIRT3, we tested the alteration of NAD+ synthesis in the liver of mice. Our result demonstrated that CORT condition reduced the mRNA levels of nicotinamide

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phosphoribosyl transferase (NAMPT) and icotinamide mononucleotide adenylyl transferase (NMNAT) as compared to controls (p < 0.05, Fig. 5A, B), whereas it had

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no effect on the expression of nicotinamide mononucleotide adenylyl transferase

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(NMRK; p > 0.05, Fig. 5C). Moreover, the reduced level of tryptophan was found in mice after CORT (p < 0.05, Fig. 5D), which inhibited the de novo pathway (Fig. S4D-F) that converts tryptophan to finally produce NAD+ [23]. Thus, we investigated the changes of SIRT3 activity, and our results showed that CORT condition significantly reduced the mRNA levels and activity of SIRT3 (p < 0.05, Fig. 5E, F),

which was coupled with the decreased mRNA levels and activities of superoxide dismutase (SOD) 2, glutathione reductase (GSR) and GST (p < 0.05, Fig. 5G-L). Also, the corresponding increase of reactive oxygen species (ROS) and the reduction of glutathione (GSH) were found in the latter group (p < 0.05, Fig. 5M, N). 3.6 Corticosterone affects mitochondrial production of ATP To further test whether CORT condition affects the production of ATP in mitochondria,

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we measured the mRNA levels of mitochondrial genes. Our results showed that the expression levels of genes associated with electron transport chains were decreased

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compared to controls, including cytochrome c oxidase 1 (COX 1), and COX2 (p <

0.05, Fig. 6A, B), NADH-ubiquinone oxidoreductase 1 (ND1), ND2, ND4, ND6 (p <

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0.05, Fig. 6C-F), ATP synthase 5 (ATP5), ATP synthase F0 subunit 6 (ATP6) (Fig. 6G,

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H), cytochrome b (Cytb) (p < 0.05, Fig. 6I), cytochrome b-c1 complex subunit 1 (Uqcrc1) and Uqcrc2 (p < 0.05, Fig. 6J, K). Moreover, the changes of mitochondria

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were further analyzed by detecting the ultrastructure of the liver. Our results demonstrated that unrecognizable mitochondria-like material instead of normal

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mitochondria, as well as the mitochondria with cristae lost, were detected in mice

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after CORT in comparison to controls (Fig. 6L). Collectively, mice exposure to CORT exhibited the corresponding decrease of ATP production (p < 0.05, Fig. 6M).

4 Discussion This study is to combine transcriptome sequencing and metabolomics analysis to

investigate the pathophysiological mechanisms association with depression-like behaviors and the disruption of energy metabolism in CORT-induced individuals. Stress is a critical environmental trigger for the production of clinical depression, and accumulated animal models have been developed in an attempt to determine the impacts of stress on the physiological metabolism [24]. Chronic administration of hormone CORT produces reliable and robust changes in a variety of behaviors that

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can be considered symptomatic of depression [12]. Specifically, in our current

experiments, mice treated by CORT shows a decrease of latency to immobilize in the

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forced swimming test, which is a measure of despair with good predictive value for

antidepressant effects, together with an increase in the duration of the tail suspension

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test, all measures typically used to infer a depressive phenotype [12, 25, 26]. This

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phenotype was also shown by a reduction in the time into the center zone and the number of entries into a new zone in the open field test, which assesses anxiety in

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novel environments[27], being consistent with a previous description [28]. Moreover, the depression-like behavior induced by CORT can be rescued by antidepressant

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treatment, which also supported the predictive validity of this depression model [19].

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Thus, these studies suggest that exogenous injection of CORT is useful for inducing depression-like behavior in rodents. Of note, weight loss is one of the important behavioral indicative of depressive syndromes in rats [29]. In our current study, the observed decrease in body weight at the beginning of CORT injection was agreement with the previous reports, which, to some extent, is an important physiological

manifestation of depression in CORT-induced animals [29, 30]. Surprisingly, the increase of food intake and almost no changes of locomotor activity were observed in depression-like mice in our present study, which was similar to several previous studies [31]. As we knew, glucose is the major source of energy for brain biochemical processes in some disease states such as depression, and its utilization may be useful in detecting the alterations in brain biochemical activity [32].

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Depression can enhance brain glucose utilization in depressed rats [33], indicative of elevated energy expenditure in the brain of depressive rats. These reports help to

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eliminate our results that mice after CORT exhibited increased food intake, whereas

these animals exhibited reduced body weight. In addition, almost no differences were

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observed in range of serum markers in depression-like mice in comparison to controls

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(Fig. S2), suggesting that CORT condition did not affect the blood composition of mice. Thus, depression-like mice were identified to not exhibit any gross pathological

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abnormalities or illness.

Accumulated proteins involved in glycolysis were significantly altered in

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depressed individuals, such as pyruvate kinase that catalyzes the rate-determining step

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in glycolysis, responding for the transfer of a phosphate group from phosphoenolpyruvate to ADP to yield one molecule of pyruvate and one molecule of ATP [34]. As an essential product of glycolysis, pyruvate is the major source of acetyl-CoA in the mitochondrion as a result of pyruvate degydrogenase activity [35]. As we know, the acetyl-CoA is a central metabolite in carbon and energy metabolism

[36]. These findings explain our current results of a disrupted energy metabolism in mice after CORT. Furthermore, the reduced acetyl-CoA may attenuate the processes of glycolysis and fatty acid degradation pathway [37], as well as the levels of oxaloacetate, leading to the decrease of coenzyme levels, such as NADH and the increase of glucose in serum and liver, as observed in our current study (Fig. S4G, H). In addition, the mRNA expression of PCX was activated to promote

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glyceroneogenesis pathway [38, 39], and to produce a mass of dihydroxyacetone

phosphate (DHAP) to result in a significant enforce of TG synthesis pathway from

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DHAP. Meanwhile, the excessive glucose and the attenuated processes of glycolysis

inhibited the degeneration pathway of fatty acid and stimulated its synthesis, leading

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to the accumulation of TG, thus this increased TG conversely impact these two

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processes [40]. These findings demonstrated that CORT condition involved in disrupting the processes of energy metabolism.

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Mitochondria produce cellular energy via the tricarboxylic-acid cycle and oxidative phosphorylation [7]. Because 2%-4% of the oxygen consumed by

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mitochondria is converted to superoxide anions, mitochondria are susceptible to

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oxidative damage [41]. Thus, many functional properties of mitochondria are substantially disrupted after exposure to a variety of ROS [42]. Excessive ROS is scavenged through SIRT3, a member of the deacetylase family [43] due to that SIRT3 can deacetylate SOD2 to catalyze the conversion of superoxide to hydrogen peroxide in mitochondria and to mediate superoxide radical formation in response to stress [44],

as well as GSR to stimulate GSH production [45]. These reports well explain our results of the reduced SOD2 and GSR activities in mice after CORT, leading to increased ROS and decreased GSH levels, which may be resulted from downregulation of SIRT3, as reported by a previous report [46]. In addition, SIRT3 is associated with multiple dehydrogenase activation, such as pyruvate dehydrogenase in pyruvic oxidation and long-chain acetyl-CoA dehydrogenase in fatty acid degradation

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[47]. Also, the reduced NAD+ due to the inhibition of synthesis from the tryptophan

and niacin [23], as well as the accumulation of NAM in the liver (Fig. S3D) [48], led

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to the reduced activity of SIRT3 [46], supporting our findings that the reduced levels of NAD+ along with attenuating SIRT3 activity was found in depressive mice. Thus,

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alteration of NAD+ affected the β-oxidation, pyruvic oxidation and succinate

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oxidation in mitochondria [48]. In addition, we found glutamate accumulation in the liver (Fig. S3C), and this increase may activate the N-methyl-D-aspartate receptor, a

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subtype of glutamate receptors [49]. As a result, elevated glutamate increases the level of intracellular calcium (Ca2+) level in mitochondrial matrix, contributing to the

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increase of ROS [50]. Consistent with our hypothesis, the reduction of SIRT3 activity

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in mice after CORT impacts mitochondrial energy metabolism. Increasing evidences have indicated essential roles of nicotinamide adenine

dinucleotide, oxidized form (NAD+) in various biological functions. NAD+ deficiency has been observed in models of a number of diseases such as depressive syndromes [48], consistent with our current study, as also evidenced by our previous description

[5]. NAD+ produces its beneficial effects via targeting at multiple pathological pathways, including attenuating mitochondrial alterations and oxidative stress, by modulating such enzymes as sirtuins [48]. As we know, NAD+ can be synthesized from simple building-blocks (de novo) from the tryptophan, or be synthesized via a salvage pathway from niacin [23]. The NAD+ deficiency in the diseases results from the reduced nicotinamide phosphoribosyltransferase (NAMPT) activity, nicotinamide

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mononucleotide adenylyl transferase (NMNAT) and nicotinamide/nicotinate riboside kinase (NMRK) [48]. These reports well explained our observations of reduced

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expression of NAMPT, NMNAT and NMRK in mice after CORT, leading to the accumulated NAM, a precursor of NAD+ in salvage pathway, and the reduced

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contents of NAD+ in the liver. The reduced production of NAD+ is possibly resulted

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from the decreased tryptophan in the novo pathway [23], consistent with our observation in our present study.

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Similar to a previous study [8], the significant decrease in ATP content was found in mice after exposure to CORT in our experiments, indicating that CORT

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condition affected mitochondrial ATP production, as evidenced by other reports [8,

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51]. ATP production was associated with altered ultrastructure of mitochondria, and an elevation in oxidative stress was likely the cause of the observed morphological changes [52]; these reports were consistent with the previous description in a mouse [53] and rat [54] model of stress-induced depression, which were similar to the observation in the current research. Moreover, mitochondrion is assembled from the

genes encoded by nDNA and mtDNA, and seven polypeptides (ND1-6) in oxidative phosphorylation complex Ι, as well as APT6, COXI and COXII, are encoded by mtDNA [55]. The mitochondrial respiratory chain is coupled to the synthesis of ATP, and oxidative phosphorylation is one of mitochondrial ATP production [7], and the significant reduction of genes including COX1, COX2, ND1, ND2, ND4, ND6, ATP6 genes in mitochondria and ATP5 gene in nucleus were found in the liver of depressive

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mice in our experiment, suggesting CORT condition may affect the expression of

genes in oxidative phosphorylation, leading to the decrease of ATP production. Our

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results were similar to a previous description that the abnormal expression of

mitochondrial respiratory chain complex proteins was found in a stress rat model [56] .

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Collectively, CORT condition can drive the changes of mitochondrial function via

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mediating NAD+ synthesis and gene expression encoded by mtDNA. Based on the combination of transcriptome sequencing and metabolomics sources,

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a summary of the present study was depicted in Figure 7. Our results demonstrated that CORT condition induces depression-like behaviors and disturbed energy

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metabolism in the liver of mice as indicated by perturbation in NAD+ synthesis,

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glycolysis, TCA cycle, leading to the dysfunction of mitochondria for reduced ATP production, all of which were possibly mediated by SIRT3. More details are presented in Figure S5. These findings provide unique opportunities to further dissect the mechanism underlying energy metabolism and perceived threat in conditions of chronic stress.

Author contributions Conceived and designed the experiments: Zhengwei Fu and Zezhi Li. Performed the experiments: Qichen Shen, Xiaoxian Xie, Chunan Yu, Jiafeng Zhou, and Qingfeng Xiao. Analyzed the data: Qichen Shen and Ze Xiong. Wrote the paper: Xiaoxian Xie

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and Qichen Shen.

Author statement

Conceived and designed the experiments: Zhengwei Fu and Zezhi Li. Performed the

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experiments: Qichen Shen, Xiaoxian Xie, Chunan Yu, Jiafeng Zhou, and Qingfeng Xiao. Analyzed the data: Qichen Shen and Ze Xiong. Wrote the paper: Xiaoxian Xie

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and Qichen Shen.

This work was supported by a grant from the National Natural Science Foundation of

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China (No. 31701028), Program for Changjiang Scholars and Innovative Research Team in University (IRT_17R97) and the National Key Research and Development

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Program of China (2017YFD0200503).

Declaration of Competing Interest

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The authors declare no competing financial interests.

Author statement Conceived and designed the experiments: Zhengwei Fu and Zezhi Li. Performed the experiments: Qichen Shen, Xiaoxian Xie, Chunan Yu, Jiafeng Zhou, and Qingfeng Xiao. Analyzed the data: Qichen Shen and Ze Xiong. Wrote the paper: Xiaoxian Xie

and Qichen Shen. This work was supported by a grant from the National Natural Science Foundation of China (No. 31701028), Program for Changjiang Scholars and Innovative Research Team in University (IRT_17R97) and the National Key Research and Development Program of China (2017YFD0200503).

Funding

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This work was supported by a grant from the National Natural Science Foundation of China (No. 31701028), Program for Changjiang Scholars and Innovative Research

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Team in University (IRT_17R97) and the National Key Research and Development

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Program of China (2017YFD0200503).

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References

[1] T. Tokuda, J. Yoshimoto, Y. Shimizu, G. Okada, M. Takamura, Y. Okamoto, S. Yamawaki, K. Doya, Identification of depression subtypes and relevant brain regions

na

using a data-driven approach, Sci. Rep. 8 (2018) 14082. [2] B.M. Mathers, L. Degenhardt, B. Phillips, L. Wiessing, M. Hickman, S.A.

ur

Strathdee, A. Wodak, S. Panda, M. Tyndall, A. Toufik, Global epidemiology of injecting drug use and HIV among people who inject drugs: a systematic review,

Jo

Lancet 372 (2008) 1733-1745. [3] D. Chisholm, K. Sweeny, P. Sheehan, B. Rasmussen, F. Smit, P. Cuijpers, S. Saxena, Scaling-up treatment of depression and anxiety: a global return on investment analysis, Lancet Psychiatry 3 (2016) 415-424. [4] H.J. Möller, Suicide, suicidality and suicide prevention in affective disorders, Acta Psychiatr. Scand. Suppl. 108(s418) (2010) 73-80. [5] X. Xie, Y. Chen, L. Ma, Q. Shen, L. Huang, B. Zhao, T. Wu, Z. Fu, Major

depressive disorder mediates accelerated aging in rats subjected to chronic mild stress, Behav. Brain Res. 329 (2017) 96-103. [6] L.T. Yi, J. Li, H.C. Li, Y. Zhou, B.F. Su, K.F. Yang, M. Jiang, Y.T. Zhang, Ethanol extracts from Hemerocallis citrina attenuate the decreases of brain-derived neurotrophic factor, TrkB levels in rat induced by corticosterone administration, J. Ethnopharmacol. 144 (2012) 328-334. [7] C.B. Peek, A.H. Affinati, K.M. Ramsey, H.Y. Kuo, W. Yu, L.A. Sena, O. Ilkayeva, B. Marcheva, Y. Kobayashi, C. Omura, Circadian clock NAD+ cycle drives

ro of

mitochondrial oxidative metabolism in mice, Science 342 (2013) 1243417.

[8] A.P. Gomes, N.L. Price, A.J. Ling, J.J. Moslehi, M.K. Montgomery, L. Rajman, J.P. White, J.S. Teodoro, C.D. Wrann, B.P. Hubbard, Declining NAD+ induces a

-p

pseudohypoxic state disrupting nuclear-mitochondrial communication during aging, Cell 155 (2013) 1624-1638.

re

[9] X. Xie, Q. Shen, L. Ma, Y. Chen, B. Zhao, Z. Fu, Chronic corticosterone-induced depression mediates premature aging in rats, J. Affect. Disord. 229 (2018) 254-261.

lP

[10] D. Martins-de-Souza, L.W. Harris, P.C. Guest, S. Bahn, The Role of Energy Metabolism Dysfunction and Oxidative Stress in Schizophrenia Revealed by

na

Proteomics, Antioxid. Redox Signal. 15 (2010) 2067-2079. [11] B. Peeters, E. Boonen, L. Langouche, G. Van den Berghe, The HPA axis response to critical illness: New study results with diagnostic and therapeutic implications, Mol.

ur

Cell. Endocrinol. 408 (2015) 235-240. [12] S.H. Ali, R.M. Madhana, K. Athira, E.R. Kasala, L.N. Bodduluru, S. Pitta, J.R.

Jo

Mahareddy, M. Lahkar, Resveratrol ameliorates depressive-like behavior in repeated corticosterone-induced depression in mice, Steroids 101 (2015) 37-42. [13] B. Lee, B. Sur, I. Shim, H. Lee, D.H. Hahm, Angelica gigas ameliorate depression-like symptoms in rats following chronic corticosterone injection, BMC Complement. Altern. Med. 15 (2015) 1-13. [14] L.E. Kalynchuk, A. Gregus, D. Boudreau, T.S. Perrotsinal, Corticosterone

increases depression-like behavior, with some effects on predator odor-induced defensive behavior, in male and female rats, Behav. Neurosci. 118 (2004) 1365-1377. [15] L. Zhang, J. Zhang, H. Sun, H. Liu, Y. Yang, Z. Yao, Exposure to enriched environment restores the mRNA expression of mineralocorticoid and glucocorticoid receptors in the hippocampus and ameliorates depressive-like symptoms in chronically stressed rats, Curr. Neurovasc. Res. 8 (2011) 286-293. [16] X. Xie, C. Yu, J. Zhou, Q. Xiao, Q. Shen, Z. Xiong, Z. Li, Z. Fu, Nicotinamide mononucleotide ameliorates the depression-like behaviors and is associated with

ro of

attenuating the disruption of mitochondrial bioenergetics in depressed mice, J. Affect. Disord. 263 (2020) 166-174.

[17] E. Morava, T. Gardeitchik, T. Kozicz, L. de Boer, S. Koene, M.C. de Vries, R.

-p

McFarland, T. Roobol, R.J.T. Rodenburg, C.M. Verhaak, Depressive behaviour in

children diagnosed with a mitochondrial disorder, Mitochondrion 10 (2010) 528-533.

re

[18] J. Allen, R. Romay-Tallon, K.J. Brymer, H.J. Caruncho, L.E. Kalynchuk, Mitochondria and mood: mitochondrial dysfunction as a key player in the

lP

manifestation of depression, Front. Neurosci. 12 (2018).

[19] M.-j. Gong, B. Han, S.-m. Wang, S.-w. Liang, Z.-j. Zou, Icariin reverses

na

corticosterone-induced depression-like behavior, decrease in hippocampal brain-derived neurotrophic factor (BDNF) and metabolic network disturbances

63-73.

ur

revealed by NMR-based metabonomics in rats, J. Pharm. Biomed. Anal. 123 (2016)

[20] L. Zhang, A. Hirano, P.K. Hsu, C.R. Jones, N. Sakai, M. Okuro, T. Mcmahon, M.

Jo

Yamazaki, Y. Xu, N. Saigoh, A PERIOD3 variant causes a circadian phenotype and is associated with a seasonal mood trait, Proc. Natl. Acad. Sci. U S A 113 (2016) 1-9. [21] Q. Xiao, Z. Xiong, X. Xie, C. Yu, Q. Shen, J. Zhou, Z. Fu, Increased Oxidative Damage Contributes to Mitochondrial Dysfunction in Muscle of Depressed Rats Induced by Chronic Mild Stress Probably Mediated by SIRT3 Pathway, Biol. Bull. 46 (2020) 615-625.

[22]H. Nojima, C.M. Freeman, R.M. Schuster, L. Japtok, B. Kleuser, M.J. Edwards, E. Gulbins, A.B. Lentsch, Hepatocyte exosomes mediate liver repair and regeneration via sphingosine-1-phosphate, J. Hepatol. 64 (2016) 60-68. [23] G. Oxenkrug, Insulin resistance and dysregulation of tryptophan-kynurenine and kynurenine-nicotinamide adenine dinucleotide metabolic pathways, Mol. Neurobiol. 48(2) (2013) 294-301. [24] M. Schieber, N.S. Chandel, ROS function in redox signaling and oxidative stress, Cur. Biol. 24 (2014) R453-R462.

ro of

[25] S.A. Johnson, N.M. Fournier, L.E. Kalynchuk, Johnson SA, Fournier NM,

Kalynchuk LE. Effect of different doses of corticosterone on depression-like behavior and HPA axis responses to a novel stressor. Behav Brain Res 168: 280-288, Behav.

-p

Brain Res. 168(2) (2006) 280.

[26] S. Hong, J.M. Moreno-Navarrete, X. Wei, Y. Kikukawa, I. Tzameli, D. Prasad, Y.

re

Lee, J.M. Asara, J.M. Fernandez-Real, E. Maratos-Flier, P. Pissios, Nicotinamide N-methyltransferase regulates hepatic nutrient metabolism through Sirt1 protein

lP

stabilization, Nat. Med. 21 (2015) 887.

[27] T. Karl, R. Pabst, S.V.J.E. Hörsten, T. Pathology, Behavioral phenotyping of mice

na

in pharmacological and toxicological research, Exp. Toxicol. Pathol. 55 (2004) 69-83. [28] A. Gregus, A.J. Wintink, A.C. Davis, L.E.J.B.B.R. Kalynchuk, Effect of repeated corticosterone injections and restraint stress on anxiety and depression-like behavior

ur

in male rats, Behav. Brain Res. 156 (2005) 105-114. [29] E.Y. Sterner, L.E. Kalynchuk, Behavioral and neurobiological consequences of

Jo

prolonged glucocorticoid exposure in rats: relevance to depression, Prog. Neuropsychopharmacol Biol. Psychiatry 34(5) (2010) 777-790. [30] Y. Zhao, R. Ma, J. Shen, H. Su, D. Xing, L. Du, A mouse model of depression induced by repeated corticosterone injections, Eur. J. Pharmacol. 581 (2008) 113-120. [31] T. Ge, Z. Zhang, J. Lv, Y. Song, J. Fan, W. Liu, X. Wang, F.S. Hall, B. Li, R. Cui, The role of 5-HT2c receptor on corticosterone-mediated food intake, J. Biochem. Mol.

Toxicol. 31(6) (2017) e21890. [32] S.H. Kennedy, K.R. Evans, S. Krüger, ., H.S. Mayberg, J.H. Meyer, S. Mccann, ., A.I. Arifuzzman, S. Houle, ., F.J. Vaccarino, Changes in regional brain glucose metabolism measured with positron emission tomography after paroxetine treatment of major depression, Am. J. Psychiatry 158 (2001) 899-905. [33] K. Kanemaru, M. Diksic, The Flinders Sensitive Line of rats, a rat model of depression, has elevated brain glucose utilization when compared to normal rats and the Flinders Resistant Line of rats, Neurochem. Int. 55 (2009) 655-661.

ro of

[34] W.-H. Shao, J.-J. Chen, S.-H. Fan, Y. Lei, H.-B. Xu, J. Zhou, P.-F. Cheng, Y.-T.

Yang, C.-L. Rao, B. Wu, H.-P. Liu, P. Xie, Combined metabolomics and proteomics

analysis of major depression in an animal model: perturbed energy metabolism in the

-p

chronic mild stressed rat cerebellum, OMICS 19 (2015) 383-392.

[35] Q. Nie, H. Chen, J. Hu, H. Gao, L. Fan, Z. Long, S. Nie, Arabinoxylan Attenuates

re

type 2 diabetes by improvement of carbohydrate, lipid, and amino acid metabolism, Mol. Nutr. Food Res. 62 (2018) 1800222.

lP

[36] M.J. Hynes, S.L. Murray, ATP-citrate lyase is required for production of cytosolic acetyl coenzyme A and development in aspergillus nidulans, Eukaryot. Cell 9 (2010)

na

1039.

[37] H. Cui, C. Zhang, C. Li, L. Lin, Antimicrobial mechanism of clove oil on Listeria monocytogenes, Food Control 94 (2018) 140-146.

ur

[38] C.W. Hsieh, C. Huang, I. Bederman, J. Yang, M. Beidelschies, M. Hatzoglou, M. Puchowicz, C.M. Croniger, Function of phosphoenolpyruvate carboxykinase in

Jo

mammary gland epithelial cells, J. Lipid Res. 52 (2011) 1352-1362. [39] X. Xie, Q. Xiao, Q. Shen, Z. Xiong, C. Yu, J. Zhou, Z. Fu, Crocin-I ameliorates the disruption of lipid metabolism and dysbiosis of gut microbiota induced by chronic stress corticosterone in mice, Food Funct (2019). (dio: org/10.1039/C9FO00444K) [40] A. Adina-Zada, T.N. Zeczycki, M.M. St, S. Jitrapakdee, W.W. Cleland, P.V. Attwood, Allosteric regulation of the biotin-dependent enzyme pyruvate carboxylase

by acetyl-CoA, Biochem. Soc. Trans. 40(3) (2012) 567. [41] M.D. Williams, H. Van Remmen, C.C. Conrad, T.T. Huang, C.J. Epstein, A. Richardson, Increased oxidative damage is correlated to altered mitochondrial function in heterozygous manganese superoxide dismutase knockout mice, J. Biol. Chem. 273 (1998) 28510-28515. [42] J. Du, Y. Wang, R. Hunter, Y. Wei, R. Blumenthal, C. Falke, R. Khairova, R. Zhou, P. Yuan, R. Machado-Vieira, Dynamic regulation of mitochondrial function by glucocorticoids, Proc. Natl. Acad. Sci. U S A 106 (2009) 3543-3548.

ro of

[43] M.C. Haigis, L.P. Guarente, Mammalian sirtuins--emerging roles in physiology, aging, and calorie restriction, Genes Dev. 20 (2006) 2913-2921.

[44]R. Tao, M.C. Coleman, J.D. Pennington, O. Ozden, S.H. Park, H. Jiang, H.S. Kim,

-p

C.R. Flynn, S. Hill, M.W. Hayes, Sirt3-mediated deacetylation of evolutionarily

conserved lysine 122 regulates MnSOD activity in response to stress, Mol. Cell 40

re

(2010) 893-904.

[45] C. Sebastian, R. Mostoslavsky, SIRT3 in calorie restriction: can you hear me

lP

now?, Cell 143 (2010) 667-668.

[46] X. Qiu, K. Brown, M.D. Hirschey, E. Verdin, D. Chen, Calorie restriction reduces

na

oxidative stress by SIRT3-mediated SOD2 activation, Cell Metab. 12 (2010) 662-667. [47] A.E. Ringel, S.A. Tucker, M.C. Haigis, Chemical and Physiological Features of Mitochondrial Acylation, Mol. Cell 72 (2018) 610-624.

ur

[48] M. Zhang, W. Ying, NAD+ deficiency is a common central pathological factor of a number of diseases and aging: mechanisms and therapeutic implications, Antioxid.

Jo

Redox Signal. 30 (2018) 890-905. [49] B.S. Meldrum, Glutamate as a neurotransmitter in the brain: review of physiology and pathology, J. Nutr. 130(4S Suppl) (2000) 1007s-1015s. [50] X. Gao, X. Xu, J. Pang, C. Zhang, J. Ding, X. Peng, Y. Liu, Jm %J Physiological Research, NMDA receptor activation induces mitochondrial dysfunction, oxidative stress and apoptosis in cultured neonatal rat cardiomyocytes, Physiol. Res. 56 (2007)

559-569. [51] A. Gardner, A. Johansson, R. Wibom, I. Nennesmo, U.V. Döbeln, L. Hagenfeldt, T. Hällström, Alterations of mitochondrial function and correlations with personality traits in selected major depressive disorder patients, J. Affect. Disord. 76 (2003) 55-68. [52] S.M. Solon-Biet, A.C. Mcmahon, J.W. Ballard, K. Ruohonen, L.E. Wu, V.C. Cogger, A. Warren, X. Huang, N. Pichaud, R.G. Melvin, The ratio of macronutrients,

libitum-fed mice, Cell Metab. 19 (2014) 418-430.

ro of

not caloric intake, dictates cardiometabolic health, aging, and longevity in ad

[53] X. Xie, Q. Shen, L. Cao, Y. Chen, L. Ma, Q. Xiao, C. Yu, Z. Fu, Depression

caused by long-term stress regulates premature aging and is possibly associated with

-p

disruption of circadian rhythms in mice, Physiol. Behav.199 (2019) 100-110.

[54] G. Lucca, C.M. Comim, S.S. Valvassori, G.Z. Réus, F. Vuolo, F. Petronilho, E.C.

re

Gavioli, F. Dalpizzol, J. Quevedo, Increased oxidative stress in submitochondrial particles into the brain of rats submitted to the chronic mild stress paradigm, J.

lP

Psychiatr. Res. 43 (2009) 864-869.

[55] D.C. Wallace, W. Fan, V. Procaccio, Mitochondrial energetics and therapeutics,

na

Annu. Rev. Pathol. 5 (2010) 297-348.

[56] G.T. Rezin, M.R. Cardoso, C.L. Gonçalves, G. Scaini, D.B. Fraga, R.E. Riegel, C.M. Comim, J. Quevedo, E.L. Streck, Inhibition of mitochondrial respiratory chain

ur

in brain of rats subjected to an experimental model of depression, Neurochem. Int. 53

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(2008) 395-400.

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Figure legends

Figure 1 Corticosterone alters physiological parameters in mice expressing

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depression-like behaviors

A CORT protocol was used to induce depressive behaviors, which were shown by (A) tail suspension test, (B) forced swimming test, (C, D) open field test. Then the impacts of CORT condition on (E) body weight (n=8), (F) food intake per day, and (G) locomotor activity, as well as (H) the weight of liver and (I) the percentage of liver to

body weight were analyzed. Triglyceride in livers were evaluated using (J) ELISA analysis, and (K, L) oil O red stain (n=6). Values are presented as the mean ± SEM.

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*p < 0.05 vs. control mice.

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Figure 2 Transcriptome analysis and identification of gene mRNA expression. (A) Principal component analysis (PCA) analysis of mice after CORT and controls. (B)

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Volcano plot of DEGs between two groups (n=3). The red and green dots expressed the up- and down-regulated genes, respectively. The black dots indicated the genes without significantly different expression. (C) The most enriched Gene Ontology (GO) terms (Top 10) in DEGs of mice after CORT. The green, blue and red bars represented the terms of biological process, cellular

component and molecular function, respectively. (D-E) Gene mRNA expression of DEGs by RT-qPCR and correlation analysis between RT-qPCR and

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RNA-sequencing.

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Figure 3 Metabolome analysis in the liver. (A) The correlation analysis of mice after

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CORT and controls in metabolome analysis. (B) The OPLS-DA comparison between two groups. (C) Volcano plot of different metabolites between two groups (n=6). The red and green dots expressed the metabolite with increased abundances and reduced abundances, respectively. The black dots indicated the metabolites without significantly difference. (D) The score plot (S-plot) of 233 target metabolites.

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Figure 4 Corticosterone disrupts energy metabolism in the liver The mRNA expression of (A) PFKL, (B) PCX, (C) Lpin1, (D) Fabp1, (E) Pparα (n=8) was analyzed. The relative levels of (F) palmitic acid and (I) NADP+ (n=6) were

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measured using GC-MS, and (G) acetyl-COA, and (H) NAD+ using ELISA kit.

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Values are presented as the mean ± SEM. *p < 0.05 vs. control mice.

Figure 5 Corticosterone mediates NAD+ synthesis and its association with the activity of SIRT3 in the liver.

The mRNA expression of (A) NAMPT, (B) NMNAT, (C) NMRK, (E) SIRT3, (G) SOD2, (I) GSR, (K) GSTA1 (n=8) was analyzed by RT-qPCR. The relative levels of (D) tryptophan in the liver were measured using GC-MS, and the activities of (F) SIRT3, (H) SOD2, (J) GSR, (L) GST and the levels of (M) GSH were analyzed using ELISA kit. (N) ROS content in the liver. Values are presented as the mean ± SEM. *p

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< 0.05 vs. control mice.

Figure 6 Corticosterone recedes mitochondrial production of ATP. The mRNA expression of (A) COX1, (B) COX2, (C) ND1, (D) ND2, (E) ND4, (F) ND6, (G) ATP5,

(H) ATP6, (I) Cytb, (J) Uqcrc1 and (K) Uqcrc2 was analyzed by RT-qPCR. (L) Transmission electron micrographs of the liver (n =3 per group). Scale bars: 1μm. The mitochondria with unrecognizable mitochondria-like materials instead of normal mitochondria are marked by blue arrows. (M) The levels of ATP in the liver. Values

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are presented as the mean ± SEM. *p < 0.05, vs. control mice.

Figure 7. CORT condition reduced the activity of SIRT3 to mediate the processes of

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ETC, TCA and FAO in mitochondria, thus leading to the degeneration of

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mitochondrial function mainly shown by the decrease of ATP production.