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Effect of Prolonged Simulated Microgravity on Metabolic Proteins in Rat Hippocampus: Steps Toward Safe Space Travel Yun Wang, Iqbal Javed, Yahui Liu, Song Lu, Guang Peng, Yongqian Zhang, Hong Qing, and Yulin Deng J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00777 • Publication Date (Web): 02 Nov 2015 Downloaded from http://pubs.acs.org on November 7, 2015

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Journal of Proteome Research

Effect of Prolonged Simulated Microgravity on Metabolic Proteins in Rat Hippocampus: Steps Toward Safe Space Travel

Yun Wang, Iqbal Javed, Yahui Liu, Song Lu, Guang Peng, Yongqian Zhang, Hong Qing and Yulin Deng* School of Life Sciences, Beijing Institute of Technology, No.5 Zhongguancun South Street Beijing 100081P.R China

Corresponding author: Dr.Yulin Deng School of Life Sciences, Beijing Institute of Technology No.5 Zhongguancun South Street, Beijing 100081,P.R China Email: [email protected] Tel:+86-10-68914907;Fax:+86-10-68914907

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ABSTRACT Mitochondria are not only the main source of energy in cells but also produce reactive oxygen species (ROS), which result in oxidative stress when in space. This oxidative stress is responsible for energy imbalances and cellular damage. In this study, a rat tail suspension model was used in individual experiments for seven and twenty-one days to explore the effect of simulated microgravity (SM) on metabolic proteins in hippocampus, a vital brain region involved in learning, memory and navigation. A comparative

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O-labelled quantitative proteomic strategy was used to observe the

differential expression of metabolic proteins. Forty-two and sixty-seven mitochondrial metabolic proteins were differentially expressed after 21 and 7 days of SM, respectively. Mitochondrial Complex I, III, and IV, isocitrate dehydrogenase and malate dehydrogenase were down-regulated. Moreover, DJ-1 and peroxiredoxin 6, which defend against oxidative damage, were up-regulated in the hippocampus. Western blot analysis of proteins DJ-1 and COX 5A confirmed the mass spectrometry results. Despite these changes in mitochondrial protein expression, no obvious cell apoptosis was observed after twenty-one days of SM. The results of this study indicate that the oxidative stress induced by SM has profound effects on metabolic proteins.

KEYWORDS hippocampus, mitochondrial metabolic, oxidative stress, proteomics, simulated microgravity (SM)


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INTRODUCTION Microgravity is a great concern for human activities in space environments because it can cause the redistribution of fluids in the body during space travel. Microgravity can directly affect the body and has been shown to activate multiple signals in different regions of the brain in terrestrial models.(1) Previous studies have suggested that microgravity affects several aspects of brain functions, such as rat cortical circuit formation,(2) spatial learning,(3) cognitive demand of human sensorimotor performance,(4) and many other functions. Critical brain regions supervise functions involving learning and memory processes. One such regions is the hippocampus, and extensive evidence has shown that the hippocampus might be susceptible to microgravity. The hippocampus of male mice has been shown to exhibit oxidative stress under simulated microgravity (SM) with activation of nuclear transcription factor kappa B.(5) Furthermore, Stein and coworkers observed increased lipid peroxidation and oxidative stress in humans and rodents after space flights, and these effects were more pronounced after long-duration space flights.(1) More recently, proteomic analysis of the hippocampus(6) and hypothalamus,(7, 8) as well as a microarray analysis of gene expression in mouse brain,(9) was performed to elucidate the mechanisms responsible for body malfunctioning under SM. After 7 days of tail suspension, the levels of cytoskeletal and metabolic proteins in the hippocampus of adult mice were decreased,(6) whereas biomarkers of oxidative stress in the hypothalamus of mice were increased.(8) All of these studies suggest that changes in gravitational force may affect several aspects of brain function after 7 days



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of SM exposure. Strong evidence suggests that actual spaceflight conditions induce oxidative damage resulting in mitochondrial apoptosis in the retina.(10) Mitochondria are multifunctional organelles of eukaryotic cells that generate energy packets in the form of adenosine triphosphate (ATP). Brain energy expenditure accounts for approximately 80% of total body energy consumption. Most energy is used to support neuronal function and plasticity.(11) Moreover, previous studies have revealed that spaceflight conditions induce oxidative damage. Therefore, the susceptibility of brain regions to microgravity might be a result of certain changes in metabolic pathways in mitochondria.(6, 12) Furthermore, mitochondria are thought to be involved in aging via an accumulation of mitochondrial DNA (mtDNA) mutations, ultimately leading to the production of reactive oxygen species (ROS).(13) Because mitochondrial proteins are intrinsically related to energy metabolism, energy-related proteins may also be affected under SM. This present study was designed to investigate the effects of long-term exposure (21 days) to microgravity environments on the rat brain, especially mitochondrial metabolic functions. This study represents a milestone in understanding the mechanisms involved in energy imbalances during long-term exposure to microgravity and will aid in the design of proper preventive measures to cope with microgravity losses, ultimately ensuring the safety of life during long-term stays in space.

EXPEIMENTAL PROCEDURES Animals


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Adult male Sprague-Dawley (SD) rats (240±20 g, 10 weeks old) were used in the study. Animals were housed individually in cages, kept on a 12 h light/dark cycle, and food and water were freely available. All animal procedures were conducted in accordance with the Chinese Academy of Medical Sciences, Institute of Laboratory Animal.

Tail suspension model In the laboratory, the tail suspension model has been widely used to induce SM in small animals, such as rats.(7, 14-16) A total of 40 male SD rats were used in two independent experiments, i.e., 7- and 21-day tail suspension. For each experiment, 20 rats were divided into SM and control groups consisting of 10 rats each. Control rats were kept in normal cages and had free access to drinking water and food. For SM, rats were kept for 7 and 21 days after suspending their bodies by their tail in such a position that only their front paws could touch the floor. All SM rats had free access to water and food. After completing the experimental protocol, the rats were sacrificed by cardio perfusion. The hippocampus tissues were removed from the brains of both groups of sacrificed rats and preserved at -80ºC for further experimentation.

Sample extraction The hippocampal proteins were extracted using pre-described methods with slight modifications.(17, 18) The rat hippocampus was put into an ice-cold buffer (250 mM sucrose, 5 mM MgCl2, 1.0 mM DTT, 25 µg/mL spermine, 25 µg/mL spermidine, 50 mM Tris-HCl, pH 7.4), and a protease inhibitor mixture tablet (Roche Diagnostics, Germany). The mixture was then homogenized with a Teflon-glass dunce



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homogenizer (ten strokes in total) followed by a low-speed centrifugation step (1,000 × g for 10 min) to remove nuclei, cell debris, and intact cells. The supernatant containing mitochondrial suspension was transferred to a new tube and centrifuged with high speed (6,000 × g for 15 min). The pellet were collected and suspended in 0.5 mL buffer (8 M urea, 10 mM DTT, 50 mM NH4HCO3). The lysate was incubated on ice for 30 min, centrifuged at 16,500 × g for 30 min at 4oC. Then the supernatant was collected. At last the protein concentrations were measured using the Bradford assay (Bio-Rad, USA). An immunoblot assay was performed to evaluate the yield of mitochondrial elements in the final preparation and identify potential cross-contamination. Western blotting clearly showed a considerable difference in the protein band patterns between crude extracts and the mitochondria-enriched fraction. Antibody against an abundant marker of the mitochondria, cytochrome C (Santa Cruz Biotechnology, Inc.), was used during the fractionation. Proteins were separated using 12% a SDS-PAGE gel and transferred by electroblotting onto a 0.22 µm PVDF membrane (Bio-Rad, USA). After blocking in 5% skim milk for 2 h at room temperature, the membrane was incubated with primary antibody raised against cytochrome C at 4oC overnight. The membrane was then washed three times with TBST buffer followed by the additional of the appropriate secondary antibody labeled with HRP at room temperature for 2 h. The membrane was further washed with TBST buffer three times and detected using ECL reagents (Millipore, USA).


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Protein digestion, labeling and desalting The

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O-labeling technique was performed three times corresponding to

biological triplicates of mitochondria fraction. For each triplicate samples from three rats were pooled (166.7 µg of proteins each), each of the resulting 500 µg of mitochondrial proteins were reduced with 10 mM DTT, alkylated with 100 mM iodoacetamide, and then diluted and digested by trypsin at an enzyme/protein ratio of 1 : 50 for 20 h at 37ºC. After the digestion, all the peptides from control and SM were evaporated to dryness by SpeedVac

(CHRIST RVC 2-18 CDplus), re-dissolved in

H218O or H216O, and catalyzed by trypsin in the presence of 100 mM K2HPO4/KH2PO4 buffer (pH 4.5) at 37ºC for 16 h. The residual trypsin activity was quenched by a boiling water bath for 10 min, with 5.0% formic acid (v/v) added into it. The 18O-labeled peptides (SM) and unlabeled peptides (control) were mixed at a 1 : 1 ratio and desalted using a C18 solid phase extraction (SPE) column (Sep-Pak C18 1-cc cartridges, (50 mg) Waters, USA) .(19)

Strong cation exchange (SCX) fractionation Strong cation exchange (SCX) chromatography separation of the peptide mixtures was carried out by an Agilent 1100 Series HPLC system with a Zorbax SCX-300 column (2.1 mm × 150 mm，Agilent) at a flow rate of 0.2 mL/min. Mobile phase solvents consisted of (A) 10 mM ammonium formate, 25% acetonitrile, pH 3.0 and (B) 500 mM ammonium formate, 25% acetonitrile, pH 6.6. Once loaded, isocratic conditions at 100% A were maintained for 10 min. Peptides were separated with a linear gradient from 0 to 50% B over 40 min, followed



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by a gradient of 50 - 100% B over 10 min. The gradient was run using 100% solvent B for 10 min, and finally at 100% for 10 min. The fractions were collected after every 2 min while being monitored by UV detector set at wavelength of 280 nm.(20) The fractions were finally remixed into 30 fractions by the intensity of UV detection following dryness completely by SpeedVac (CHRIST RVC 2-18 CDplus).

Chip-cube-ESI-Q-TOF-MS/MS analysis The MS analyses of the fractions were performed using previously proteomic approach.(21) Chip-cube-ESI-Q-TOF-MS/MS analysis was performed in triplicate using an Agilent 1260 nanoflow LC system coupled to Agilent 6538 Q-TOF mass spectrometer (Agilent, USA). Tryptic peptides were separated by SCX for the first dimension. Then fractions were transferred to HPLC vials. Separation of peptides was performed with a nanoflow Agilent 1260 series system, equipped with a Zorbax 300 SB-C18 (5 µm) chip consisting of 160 nL enrichment column and a 75 µm × 150 mm (5 µm) analytical column using the HPLC-Chip technology. After injection of sample, the peptides were loaded (160 nL) on the enrichment column for 8 min with 100% mobile phase A (97% water with 0.1% formic acid) at 4 µL/min. Then the samples were eluted with solvent A (95% water with 0.1% formic acid) and solvent B (90% acetonitrile with 0.1% formic acid) under the linear gradient of 0 – 40% solvent B for 60 min followed by 40 – 80% solvent B for 10 min with a constant nano pump flow of 0.35 µL/min. The gradient was then run using 80% solvent B for another 3 min and finally at 100% solvent A for 7 min. A 10-min post-run delivery of solvent A was used to


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remove sample carryover. Q-TOF analysis was performed in positive ionization mode with a capillary voltage of 1700 V, ion fragmentary voltage of 200 V and drying gas temperature of 325ºC, with a flow rate of 5.0 L/min. Mass spectra were collected in the centroid mode over the mass range between 290 and 1250 m/z with a scan rate of 1 spectrum/sec. MS/MS spectra were collected in centroid mode over the mass range between 100 and 3,000 m/z. Fragmentation of protonated molecular ions was conducted in auto MS/MS mode with the 5 highest precursors of each spectrum selected for the tandem MS with an active exclusion of 0.5 min. The collision energy voltage slope was 3.7 V plus an offset of 2.5 V for +2 ions, whereas a slope of 3.6 V plus an offset of -4.8 V was used for +3 and > +3 ions.(16)

Data analysis and bioinformatics The mass spectrometry data were queried against the RATTUS NORVEGICUS species sequences of the Swiss-Prot database (sequences downloaded in October 2013; 3563 entries) using the Spectrum Mill search engine (Rev.B.04.00.127), and the DAVID 6.7 Bioinformatics tool was used to perform gene functional categorization and

pathway

analysis.(22)

(http://www.pantherdb.org/)

Protein

classification

annotation

with

system

(version

the 9.0)

PANTHER statistical

overrepresentation test tool was used to search for significant differences between biological processes and molecular functions.(23, 24) A spectrum mill peptide score greater than 9 and a 70% scored peak intensity (SPI) are commonly considered reliable for proteins. The following parameters were used in this search: two missed cleavage sites, trypsin digestion, monoisotopic mass values, Rattus norvegicus, carbamidomethylation of cysteine as fixed modification, oxidation of methionine and



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O-labeling as variable modification, peptide mass tolerance of 20 ppm, fragment

mass tolerance of 50 ppm, and peptide charge of +1, +2 and +3. Spectrum Mill cutoff scores were determined using a reverse database strategy producing a false positive rate of less than 5.0% for double- and triple-charged peptides. The peak area of the first isotopic peak of the double

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O-labeled peptide and

16

O-labeled peptide in MS

scans was used to quantify the differential peptides. Relative quantification was performed by comparison of the peak area of the doubly 16O-labeled and 18O-labeled peptide. Protein level ratios were obtained based on the mean of the peptide ratios. Analysis of the area ratios was performed using Student’s two-sampled t test. Differences were statistically significant at p values less than 0.05.

Determination of cellular oxidative stress levels Oxidative stress was determined by analyzing hippocampus homogenates of all model rats. Superoxide dismutase (SOD) activity in the hippocampus homogenates was measured using the xanthine oxidase test.(25) The thiobarbituric acid test was used to detect malondialdehyde (MDA) levels, and hydrogen peroxide (H2O2) levels were assessed using molybdate reactions. Hippocampus homogenates from control and SM rats were de-proteinized and centrifuged to remove the protein after 21 days of lab experimentation. The supernatant was measured using a spectrophotometer. Values are presented as the mean ± SD of six rats, and #p < 0.05 indicates significant differences compared with the control.

Western blot analysis


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Hippocampus mitochondria from control and SM rats were dissolved in RIPA lysis buffer. The supernatants were collected after centrifugation at 16,500 × g for 20 min at 4ºC. Protein concentrations were measured using the BCA protein assay kit (Thermo Scientific Pierce, USA). Proteins were separated using 12% a SDS-PAGE gel and transferred by electroblotting onto a 0.22 µm PVDF membrane (Bio-Rad, USA). After blocking in 5% skim milk for 2 h at room temperature, the membrane was incubated with primary antibody raised against DJ-1 (Abcam, USA), COX 5A (Abcam, USA) or GAPDH (ZSGB-BIO, China) at 4ºC overnight. The membrane was then washed three times with TBST buffer followed by the additional of the appropriate secondary antibodies labeled with HRP at room temperature for 2 h. GAPDH was detected using anti-GAPDH antibody as an internal control. The membrane was further washed with TBST buffer three times and detected using ECL reagents (Millipore, USA).

RESULTS Comparative assessments of control and SM rats indicated that the oxidative stress markers MDA and H2O2 were significantly increased in the hippocampus of rats exposed to SM (Figure 1). The oxidative stress protective marker SOD was significantly decreased in the hippocampus of SM rats (Figure 1). This study was designed to investigate the effects of long-term exposure (21 days) to SM environments on the rat brain, especially mitochondrial metabolic functions. Mitochondria were prepared according to the method of Brian Cox (2006).(26) Western blots of a mitochondrial marker prepared from hippocampus are shown in



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Supplementary Figure 1. Supernatant and mitochondria fraction exhibited the expected protein band, whereas the other fractions did not. A comparative

18

O-labelled quantitative proteomic strategy was applied to

observe the differential expression of metabolic proteins. A total of 679 proteins were identified as having a spectrum score greater 15 and an FDR of 1% at the protein level. A fold change cutoff of >2 or 2 or 2 or

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