Targeted Metabolomic Pathway Analysis and Validation Revealed

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Targeted Metabolomic Pathway Analysis and Validation Revealed Glutamatergic Disorder in the Prefrontal Cortex among Chronic Social Defeat Stress Mice Model of Depression Wei Wang, Hua Guo, Shu-Xiao Zhang, Juan Li, Ke Cheng, Shun-Jie Bai, DeYu Yang, Hai-Yang Wang, Zi-Hong Liang, Li Liao, Lin Sun, and Peng Xie J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00577 • Publication Date (Web): 06 Sep 2016 Downloaded from http://pubs.acs.org on September 6, 2016

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Journal of Proteome Research

Targeted Metabolomic Pathway Analysis and Validation Revealed Glutamatergic Disorder in the Prefrontal Cortex among Chronic Social Defeat Stress Mice Model of Depression †, #, ‡, §

Wei Wang

, Hua Guo

†, #, ‡, §

, Shu-Xiao Zhang#, ‡, §, Juan Li #, ‡, €, §, Ke Cheng #, ‡, €,§,

†

†, #, ‡

Shun-Jie Bai#, ‡, De-Yu Yang , Hai-Yang Wang Lin Sun€, Peng Xie

†

, Zi-Hong Liang #, ‡, €, Li Liao #, ‡,

†, #, ‡, €,*

Department of Neurology, Yongchuan Hospital of Chongqing Medical University,

Chongqing 402460, China #

Institute of Neuroscience and Collaborative Innovation Center for Brain Science,

Chongqing Medical University, Chongqing 400016, China ‡

Chongqing Key Laboratory of Neurobiology, Chongqing 400016, China

€

Department of Neurology, the First Affiliated Hospital of Chongqing Medical

University, Chongqing 400016, China §

These authors made equal contribution to the manuscript.

*

Correspondence to: Peng Xie, Yongchuan Hospital, Chongqing Medical University,

China Tel: +86-23-68485490 E-mail: [email protected]

ABSTRACT Major depressive disorder is a severe psychiatric disease that has critically affected life quality for millions of people. Chronic stress is gradually recognized as a primary 1

ACS Paragon Plus Environment


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pathogenesis risk factor of MDD. Despite the remarkable progress in mechanism research, the pathogenesis mechanism of MDD is still not well understood. Therefore, we conducted a liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection

of

25

major

metabolites

of

tryptophanic,

GABAergic

and

catecholaminergic pathways in the prefontal cortex (PFC) of mice in chronic social defeat stress (CSDS). The depressed mice exhibit significant reduction of glutamate in GABAergic pathway and an increase of L-DOPA and vanillylmandelic acid in catecholaminergic pathways. The data of real-time-quantitative polymerase chain reaction (RT-qPCR) and western blotting analysis revealed altered level of glutamatergic circuitry. The metabolomic and molecular data reveal that the glutamatergic disorder in mice shed lights to reveal a mechanism on depression-like and stress resilient phenotype.

KEYWORDS: depression, metabolomics, glutamate, CSDS, LC-MS/MS INTRODUCTION MDD is a severe complex psychiatric disorder along with high morbidity and mortality rates1. Up to now, MDD has been reported to be involving with complex etiopathogenesis, including alterations of the HPA axis, hypofunction of monoaminergic and GABAergic neurotransmission, as well as abnormal expression of BDNF2-5. Nevertheless, the etiology of MDD is complex and still unknown. Among aforementioned hypothesis, scientists have been focusing on turbulent neurotransmitters that yielded promising insights into the neurobiology of MDD6-9. The glutamatergic system is well studied in relation to many neuropsychiatric 2


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diseases and deserves consideration when unveiling novel pathogenic and therapeutic mechanisms of MDD10-14. PFC, a crucial neural area for functions of mood regulating, memory and cognitive, has been involved in depression pathogenesis study for decades15. Our group has previously conducted non-targeted metabolomics research using GC-MS and 1H NMR-based metabolic profiling on the rodent model of depression, monkeys (Macaca fascicularis), and patients with MDD16-21. Previous metabolomics studies have examined the PFC in chronic unpredictable mild stress (CUMS) and lipopolysaccharide (LPS) model22-24. While our early researches measured the metabolites of neurotransmitter, the non-targeted profiling approach above can not accurately quantify the variation during the metabolic procedure. To overcome this issue, we quantified 25 neurotransmitters and relevant metabolites in a targeted approach of LC-MS/MS. This approach can precisely detect metabolites in the tryptophanic, GABAergic and catecholaminergic systems. Chronic social defeat stress (CSDS) model of rodents has been widely applied in MDD research, which allows mimicking the characteristics of depression patients with depressive-like phenotypes25. According to different sensitivity of the paradigm, the defeated mice can be divided into depressed and resilient categories26. In the present

study,

metabolites

involved

in

tryptophanic,

GABAergic

and

catecholaminergic metabolism in the PFC of depressed, resilient and control mice were all monitored via LC-MS/MS. Additionally, RT-qPCR and western blotting were applied in monitoring the key genes and enzymes of glutamatergic pathway. As 3



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revealed in our study clearly that metabolomic alterations of glutamatergic pathway exist in the PFC of defeated mice.

MATERIALS AND METHOD Animals All animals were obtained from experimental animal center of the Chongqing Medical University. C57BL/6 mice (age, two months; weight, 18-20g) were composed of two groups (control: defeat=15:30) for LC-MS/MS (control: depressed: resilient=10:10:10), while another batch of mice with a similar phenotype were used for the molecular studies (control: depressed: resilient=8:8:8). All mice were maintained under stable conditions (12 hours light dark cycle, 23 ± 2 °C, relative humidity conditions and freely to food and water). The procedures were granted by the Ethics Committee of Chongqing Medical University. Intruder Mice We applied two-month-old C57BL/6 male mice as “intruder” mice that were defeated by resident CD1 in the CSDS model. Aggressive Resident Mice Before the stress, 80 male CD1 mice (18-20 weeks old) were screened for aggressive behavior as previously described26. There are two criteria used to evaluate the aggressive behavior of CD-1 mice as below: (1) the CD-1 must have attacked the C57BL/6 mice in at least two continuous days in the period of 3 days of screening (180 s each). (2) The time before initial attack must be less than 60 s. Ultimately 38 CD1 mice were qualified and 30 of them were selected as residents to habituate in the 4


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defeat cage for two weeks before the experiments. Chronic Social Defeat Stress Paradigm The stress paradigm similar to a protocol described26 was employed with slight modifications. The physical stress was last 5 min everyday during the defeat procedure. Immediately after the defeat, the intruder mice were transferred to the opposite compartment for the remainder 24h. The C57BL/6 mice were turned daily while the CD1 mice were not in the period of 10-day defeat procedure. Social Interaction Test In order to assess the social aversion of C57BL/6 intruders, the 5-min social interaction was recorded 6 h after the last attack of defeat stress. Social interaction was proceeded in an open field (40 cm × 40 cm × 18 cm) with a perforated plastic box (10 cm × 7 cm × 18 cm) was placed on one side of it. Procedure was carried out under red-light conditions. Test consisted of two parts that with and without the target CD-1 mouse in the perforated plastic box. The movements of C57BL/6 mice were recorded by the video recorder. The social interaction ratio (SI ratio) is a ratio calculated by the time when CD1 is present and absent in the interaction zone. Sucrose Preference Test The SPT is proceeded as previously described27. Mice were adapted to 1% sucrose solution for 3 days prior to the test28. After 12 h water- and food-deprival, the water and sucrose consumption were weighed at 8:00 PM and 12h later by skilled experimenters. The preference for sucrose (%) = (sucrose amount/total amount) × 100. 5



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Open Field Test The OFT was performed after the SPT was accomplished. Each mouse was placed in the open field (50 cm × 50 cm × 40 cm) center for 5 min movement29. Distance traveled, rears , numbers of entrances and percentage of time spent in the center (50% of the field) were adopted30. Sample Preparation After the tests, all mice were sacrificed simultaneously and the prefrontal tissues were separated quickly then stored in the liquid nitrogen. All tissues were frozen at -80 °C before biochemical analysis. After being accurately weighed, frozen brain tissue samples were placed into EP tube (EP tube and ball in the first precooling -80 °C), with 400µL 75ng/mL of L-Phe (2Cl)-OH as an internal standard methanol-water mixed solution (4/1, v/v), and the tubes were then placed into a low-temperature homogenizer (Tissuelyser-48, Shanghai Jingxin Technology). After 10-min ice-water bath ultrasonic extraction, the reagent was centrifuged at 14000g, 4 °C for 10 min. Then the 350µL upper solution was transferred to a new EP tube. 200µL of methanol-water (4/1, v/v) was added to the residue EP tube and vortex-mixed for 30s then ultrasonically extracted after 5 min in an ice water. The mixture was centrifuged at 14000g, 4 °C for 10min and the upper 200µL solution was extracted. The two extracts were mixed to total of 550µL and for 10 min centrifugation at 14000g, 4 °C. Then the 150µL supernatant was used for the LC-MS/MS. LC-MS/MS We used the AB Sciex Triple QuadTM6500 mass spectrometry (MS) system with 6


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Waters ACQUITY UPLC chromatographic instrument control. Analyst 1.5.2 software was applied to the data collection and analysis. The metabolites were isolated using waters UPLC amino column (2.1mm x 100mm, 1.7 µm) with the maintained column temperature of 40°C. Then the mobile phase comprised 0.15% (v/v) formic acid aqueous solution (A) and acetonitrile (B). The rate of flow was set at 0.25mL/min while the sample volume of5µL. MS operating parameters were conducted as follows: Ionspray voltage, (±) 4500 V; temperature, 550 ° C; Curtain gas, 20; CAD gas, 8; GS1, 50; GS2, 50; EP, 10; CXP, 10. The Multiple Reaction Monitoring (MRM) condition is shown in the following Table 1. The Analyst software (AB Sciex, version number: 1.5.2) uses the default parameters for automatic identification and integration of the MRM transition. The preparation of quantitative standard curve was as follows: the initial mixture of 25 analytes was gradually diluted 3 times with 8 gradients and repeated 3 times for LC-MS analysis. For each analysis, we chose 5 linear range concentrations from the 8 gradients to generate the standard curve. Standard linear regression curves were drawn with the mass of the analyte-peak area on the vertical axis, and analyte concentration on the horizontal axis. RT- qPCR analysis RT-qPCR was used to detect the gene expression in the glutamatergic pathway. 1µg RNA, isolated from the PFC brain tissue in Trizol (Invitrogen), was used for cDNA synthesis by PrimeScript™ RT reagent Kit (TAKARA). For RT-qPCR, a SYBR green detection system (Roche, Germany) was used in reactions. β-actin a the 7



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housekeeping gene was used to normalize to the data. 2-∆∆ CT method was applied for the data analysis and the specific primers are exhibited in Table 2. Western Blotting Validation The PFC brain tissue was lysed in RIPA buffer with protease inhibitor cocktail (Roche, Germany). The protein was transferred from 10%SDS-PAGE to a polyvinylidene fluoride membrane (Millipore, USA) for blocking. Then the plots were reacted with rabbit monoclonal anti-GluA1 (Abcam, 1:2000), rabbit monoclonal anti-EAAT2 (Abcam, 1:5000) and mouse monoclonal anti-Glul (Abcam, 1:2000) overnight at 4 °C and then incubated with secondary antibodies for 2 hours. Signals were visualized using an ECL kit (Millipore, USA). Statistical Analysis The behavior and molecular data were expressed as means ± SEM. Here we applied the SPSS 21.0 (Chicago, USA) to conduct the analysis of the data by one-way analysis of variance (ANOVA). Differences among three groups were identified using post-hoc Student t-test, correcting for multiple comparisons using Fisher’s LSD correction. Significance was considered at a corrected P = 0.05.

RESULTS The Social Interaction Test The test was applied to evaluate the depressed and resilient phenotype of the defeated mice. Historically, an SI ratio of 1, which mice spent equal time in the interaction zone when CD1 was present and absence, has been applied as the standard for dividing defeated mice into the depressed and resilient phenotype. As indicated in 8


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Figure 1A, 13 (46.4%) out of a total of 28 defeated mice showed resilient behavior according to their SI ratio. The SI ratio of depressed mice were lower than control and resilient groups (F (2, 40) =16.61, P < 0.05) (Figure 1B). Sucrose Preference Test After 10 days of the experiment, the CSDS procedure significantly decreased the sucrose preference of susceptible mice (control mice 75.23±2.0%; depressed 69.15±1.6%, P

Targeted Metabolomic Pathway Analysis and Validation Revealed

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