Targeted Ubiquitin-Proteasomal Proteolysis Pathway in Chronic

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Targeted Ubiquitin-Proteasomal Proteolysis Pathway in Chronic Social Defeat Stress Hsiao-Yun Lin, Yu-Shu Liu, Yu-Ching Liu, Chao-Jung Chen, and Dah-Yuu Lu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00519 • Publication Date (Web): 23 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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Journal of Proteome Research

Targeted Ubiquitin-Proteasomal Proteolysis Pathway in Chronic Social Defeat Stress Hsiao-Yun Lin1,2, Yu-Shu Liu1, Yu-Ching Liu3, Chao-Jung Chen3,4, Dah-Yuu Lu1,5*

1Department

of Pharmacology, School of Medicine, China Medical University, Taichung,

Taiwan; 2Fishberg Department of Neuroscience and the Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, USA; 3Proteomics Core Laboratory, Department of Medical Research, China Medical University Hospital, Taichung, Taiwan; 4Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan; 5Brain Disease Research Center, China Medical University Hospital, Taichung, Taiwan.

*Corresponding author: Dah-Yuu Lu, School of Medicine, Department of Pharmacology, China Medical University, No.91 Hsueh-Shih Road, Taichung, Taiwan. Tel: (886) 4-22053366 ext. 2253. ([email protected])

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Abstract Stressful events promote psychopathogenic changes that might contribute to the development of mental illnesses. There are some individuals tend to recover from the stress response or some are not. However, the molecular mechanisms of stress resilience during stress are not well characterized. Here, we identify proteomic changes in the hippocampus by using proteomic technique to examine mice following chronic social defeat stress. We showed that small ubiquitin-like modifier (SUMO)-1 expression was significantly decreased in susceptible mice following chronic social defeat stress. We also examined protein inhibitor of activated signal transducer of transcription (PIAS)1 levels, an E3 SUMO-protein ligase protein inhibitor of activated STAT1, which is known to interact with SUMO-1. PIAS1 were shown to be profoundly decreased and monoamine oxidase (MAO)-A increased in the hippocampus of susceptible mice following chronic social defeat stress. Furthermore, manipulated PIAS1 expression in hippocampus also have an influence on glucocorticoid receptor (GR) translocation. We also found that knockdown of PIAS1 expression in hippocampus then subject to submaximal stress increased GR to GRE-binding site on the MAO-A promoter. The present study raises the possibility that differs level of PIAS1 between individuals in response to chronic social defeat stress, and that such differences may contribute to the susceptiblity to stress.

Keywords: chronic social defeat stress; PIAS1; MAO-A; GR;


susceptibility

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Introduction Social stress is most common in modern society and largely increase the risk of psychopathologies (1). Various models of chronic stress have been used to induce symptoms in mice that are relevant to depression (2-4), while the animal models of depression enable research that cannot be performed on human subjects. Social defeat, a paradigm with accepted ethological validity, has been applied to imitate the social-stress-related disorders of human society (5). In animal model, chronic social defeat stress showed a decrease in food intake and inhibition of body weight gain (6), and most animal that have undergone social defeat exhibit social-avoidance behaviors toward the stressors. However, not all mice subjected to such repeated stress develop these symptoms, which has allowed development of the social defeat paradigm to study the resilient individuals encountering chronic psychosocial stress minimize pathophysiological outcomes (7). Studies in rodents have suggested that social defeat may alter glucocorticoid receptor sensitivity (8), monoamine activity (9), brain-derived neurotrophic factor (BDNF) (10), within the relevance of prefrontal cortex (11, 12), nucleus accumbens (13), hippocampal (14) and basolateral amygdala (15) brain regions to regulate stress susceptibility. Thus, to use the stress resiliency paradigms in animals will facilitate discovery of effective medicines against the onset of social defeat-related diseases. Physiological

and

psychological

stresses

have

both

been

found

to

change

hypothalamic-pituitary-adrenal (HPA)-axis reactivity (16). Glucocorticoids are thought to play a prominent role in the regulation of HPA-axis activation. The glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) are the two main receptors that mediate the stressful effects of glucocorticoids (17). Impaired GR function, reduced GR-mediated negative feedback on the HPA-axis, and increased secretion of corticotropin-releasing factor (CRF) in various brain



regions are thought to be involved in the development of depressive behaviors (18). A post-mortem brain study showed reduced GR gene expression in the frontal cortex and hippocampus, and a reduction in the expression of GRs and MRs in the frontal cortex in patients with schizophrenia, major depression, and bipolar disorder (18). It has been proposed that an imbalance between the GR and the MR underlies HPA-axis dysregulation, and is associated with the susceptibility to psychopathology (19). These changes might account for the delayed recovery from or compromised adaptation to stressors, and are thought to increase the vulnerability to stress-related psychiatric disorders (20). There are four distinct members of the PIAS (protein inhibitor of activated STAT) family, namely PIAS1, PIASx (PIAS2), PIAS3, and PIASy (PIAS4). PIAS1 was initially identified as an inhibitor of STAT1, blocks the DNA-binding activity of STAT1 and inhibits the transcriptional activity of STAT1 in response to cytokine stimulation (21). PIAS1 also acts as a SUMO (small ubiquitin-like modifier) E3 ligase, modulates cellular processes including, cell proliferation, DNA damage responses, and cytokine signaling. Our latest study also provided the function of PIAS1 as a regulator of the inflammatory process in microglia (22). It has been demonstrated that PIAS1 regulates transcription through several mechanisms, including promoting the sumoylation of a transcription factor or blocking the DNA binding activity of a transcription factor (23). Previous study showed that PIAS1 inhibit MR- and GR-mediated transcription (24, 25). Moreover, PIAS1 has an impact on SUMO-modified MeCP2 in rat hippocampus rescued the social behavior and memory performance (26). Proteomic profiling by iTRAQ provides a high-throughput approach to analyze differently expressed proteins in control, resilient and susceptible mice. In this findings, we found that PIAS1 protein express differently between divide susceptibility of chronic social defeat stress,


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and decreased the expression of PIAS1 have an influence on GR translocation. Furthermore, modulation of PIAS1 expression alters the social behavior in mice and modulates the MAO-A expression. In present study, we used the proteome analyses as a tool to elucidate the pathophysiological mechanisms of animal model of depression as well as discovery the potential diagnostic, therapeutic biomarkers.



Materials and methods Animals Male 8-week-old C57BL/6 mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Under standard laboratory conditions (21 ± 2 °C, 12-h L/D cycle, with food and water available ad libitum), the mice were group-housed before the social defeat experiments and then housed alone after the social defeat experiments and during behavioral testing. Male 20-40-week-old CD-1 mice (National Laboratory Animal Center) were used as aggressors in the social defeat paradigm. All animal procedures were performed in accordance with the Animal Care and Use Guidelines of China Medical University (Taichung, Taiwan). Chronic social defeat stress The social defeat stress paradigm was performed according to a standard social defeat stress protocol but with some modified (5). CD-1 mice were screened for aggressive characteristics before the start of the social defeat experiments. The experimental C57BL/6J mice were subjected to a new CD-1 aggressor mouse for 10 min once per day for 14 consecutive days. After 10 min of physical contact, the experimental mice were separated by a transparent polycarbonate perforated divider into opposite compartments and housed during the next 24 h. The experimental C57BL/6J mice were housed alone after the final session of social defeat stress. The social avoidance tests were performed 24 h after the last defeat episode. For the tissue collection after the social interaction test, the mice were euthanized, and the brain tissues were dissected and then stored at −80 °C. Submaximal social defeat To examine whether changes in PIAS1 potentiated the animals’ susceptibility to psychosocial stress that was induced by submaximal repeated defeat, C57BL/6J mice were subjected to three


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social-defeat episodes in one day, with each defeat episode separated by 15 min of rest. After the third defeat of the submaximal social defeat paradigm, the C57BL/6J mice were singly housed overnight. Twenty-four h later, the mice were subjected to the social avoidance, Elevated plus-maze test and Light-dark box tests. Social interaction test In the first 2.5-min session (empty), the C57BL/6J mouse was introduced to the arena in an empty mesh cage. In the second 2.5-min session (target), a mesh cage containing an unfamiliar CD-1 mouse was introduced to the arena. The mesh cages allowed visual and olfactory interactions between the test and target mice. The area (16 cm × 35 cm) was defined as the interaction zone. The time spent in the interaction zone and corner zones were measured with the Noldus EthoVision behavioral tracking system (Noldus Information Technology, Wageningen, Netherlands). The social interaction (SI) ratio was calculated as the ratio of the time spent in the interaction zone near the novel animal divided by the time spent in the same area near the empty cage. Mice with a ratio over 1 spent more time near the novel animal than the empty cage and were classified as resilient. Mice with a ratio below 1 spent less time near the novel animal than the empty cage and were classified as susceptible. Isobaric tags for relative and absolute quantification (iTRAQ) analysis iTRAQ analysis was performed by Proteomics Core Laboratory, China Medical University Hospital. For iTRAQ experiment, the each group of proteins isolated from 6 mice hippocampus. 50 μg of each sample was denatured and the cysteines blocked as described in the iTRAQ protocol (Applied Biosystems, Foster City, CA). Each sample was then digested with 2μg trypsin (Promega) solution at 37°C overnight and labeled with the iTRAQ tags as follows: control (114), resilient (115), and susceptible (116). The labeled samples were pooled and subjected to



OFFGEL-IEF fractionation. Peptide OFFGEL-IEF Fractionation For pI-based peptide separation, the 3100 OFFGEL Fractionator (Agilent Technologies) was used with a 24-well setup. Sample was resolubilized in 3.6 mL of 1﹪(v/v) IPG buffer, at pH 3-10 (GE Healthcare). The 24 cm-long IPG gel strips (GE Healthcare), with a 3-10 linear pH range, were rehydrated for 15 minutes according to the manufacturer’s manual. Then, sample was loaded in each of the 24 wells. Electrofocusing of the peptides was performed at 20℃ until a level of 50 kVh was reached. After focusing, the 24 peptide fractions were withdrawn and the wells were rinsed with 200 L of a solution of H2O/methanol/formic acid (49/50/1). After 15 minutes, each of the rinsing solutions was pooled with its corresponding peptide fraction. All of the fractions were evaporated by centrifugation under vacuum. Just prior to nanoLC-MS/MS analysis, the fractions were resuspended in 15 L of 0.1%formic acid. NanoLC–MS/MS Analysis Identification of phosphopeptides was performed with a nanoflow UPLC system (UltiMate 300 RSLCnano system, Dionex, Ameterdam) coupled with a captive spray ion source and a Q-TOF mass spectrometer (maXis impact, Bruker). Samples were injected into a homemade tunnel-frit trap column (C18, 5 m, 180 m × 20 mm) with a flow rate of 10 L/min for duration of 4 min. The trapped pep-tides were separated by a commercial analytical column (Acclaim PepMap C18, 2 m, 100Å, 75 m × 250 mm, Thermo Scientific) with the ACN/water gradient of 1–40% at a flow rate of 300 nL/min. For MS detection, peptides with a charge identification of phosphopeptides were performed with a nanoflow UPLC system (UltiMate 300 RSLCnano system, Dionex, Ameterdam) coupled with a captive spray ion source and a Q-TOF mass spectrometer (maXis impact, Bruker). Samples were injected into a homemade tunnel-frit trap 8 ACS Paragon Plus Environment

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column (C18, 5 m, 180 m × 20 mm) with a flow rate of 10 L/min for duration of 4 min. The trapped peptides were separated by a commercial analytical column (Acclaim PepMap C18, 2 m, 100 Å, 75 m × 250 mm, Thermo Scientific) with the ACN/water gradient of 1–40% at a flow rate of 300 nL/min. For MS detection, peptides with a charge, the peptide fractions were identified with a nanoflow UPLC system (UltiMate 3000 RSLCnano system, Dionex, Thermo Scientific) coupled with a captive spray ion source and a Q-TOF mass spectrometer (maXis impact, Bruker). Samples were injected into trap column (Acclaim PepMap C18, 5 m, 100Å 100 m × 20 mm, Thermo Scientific) with a flow rate of 10 μL/min for a duration of 4 min. The trapped peptides were separated by analytical column (Acclaim PepMap C18, 2 m, 100Å 75 m × 250 mm, Thermo Scientific) with the ACN/water gradient of 1–40 % at a flow rate of 300 nL/min. For MS detection, peptides with a charge of 2+ and 3+, intensity above 50 counts were chosen for data-dependent acquisition, set to 1 full MS scan (400-2000 m/z) with 1 Hz, and switched to 16 product ion scans (50–2000 m/z) with 10 Hz. Protein identification NanoLC-MS/MS spectra were deisotoped, centroided, and converted to .mgf files by DataAnalysis software (version 4.1, Bruker Daltonics). To identify proteins, mass spectra obtained were compared to SwissPort database (release 51.0) via MASCOT algorithm (version 2.2.07) through the Bruker ProteinScape interface (version 3.1). The search parameters of peptide MS and MS/MS mass tolerance set at 70 ppm and 0.07 Da, respectively. The taxonomy was set mouse, enzyme as trypsin, maximum of two missed cleavages allowed, fixed modification - methylthio (Cys), iTRAQ 4-plex (N-term), and iTRAQ 4-plex (Lys) and a variable modification was oxidation (Met). The protein identifications using automatically generated decoy databases to determine the false discovery rate for each search (FDR 1.2 or

Targeted Ubiquitin-Proteasomal Proteolysis Pathway in Chronic

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