The Proteomic Analysis of Primary Cortical Astrocyte Cell Culture after Morphine Administration Piotr Suder,*,† Anna Bodzon-Kulakowska,† Pawel Mak,⊥ Anna Bierczynska-Krzysik,†,# Michal Daszykowski,‡ Beata Walczak,‡ Gert Lubec,§ Jolanta H. Kotlinska,| and Jerzy Silberring† Neurobiochemistry Department, Faculty of Chemistry, Jagiellonian University, Ingardena 3 Street, 30-060 Krakow, Poland, Department of Chemometrics, Institute of Chemistry, Silesian University, 9 Szkolna Street, 40-006 Katowice, Poland, Department of Pharmacology and Pharmacodynamics, Medical University, Staszica 4 Street, 20-081 Lublin, Poland, Department of Pediatrics, Medical University of Vienna, Waehringer Guertel 18, 1090 Vienna, Austria, and Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7 Street, Krakow, Poland Received May 19, 2009
Astrocytes are supportive cells, necessary for ensure optimal environment for neural cells functioning. They are involved in extracelular K+ level regulation and neurotransmitters removal. They are also dependent for myelination and synapses formation. They may make a contribution in signal propagation in the central nervous system, for example, through Ca2+ signaling. With the use of neonatal pure astrocyte cell culture, we investigated changes in astrocyte’s proteomes under the influence of morphine. We found 10 major proteins, which show different expression between physiological cell culture and morphine treatment. With 2D gel electrophoresis and nanoLC-ESI-MS/MS, we identified proteins and characterized their potential role in morphine dependence. Observed differences were also confirmed by Western blotting. Our data suggests a role for astrocytes in the formation of the morphine dependence at the molecular level. This finding may support interpretation of causes of morphine dependence formation based only on behavioral data. Keywords: morphine • addiction • cell culture • proteome • neurons • astrocytes
Introduction The central nervous system is mainly composed of neurons and three types of the glial cells: astrocytes, microglia and oligodendrocytes. For a very long time, astrocytes were thought to be the supportive cells for neurons, necessary to ensure optimal environment for their functioning. The recent findings suggest that those cells seem to be equal partners for neurons in information processing. Taking this into account, it is not surprising that their dysfunction may lead to neurological disorders and may take part in development of addiction.1 Morphine is widely applied in clinic to attenuate severe pain, but it is also illegally used due to its ability to decrease the level of stress, or to produce relaxation and euphoria. Its repetitive administration leads to the development of tolerance and dependence. Severe withdrawal symptoms accompanying * To whom correspondence should be addressed. Piotr Suder, Neurobiochemistry Department, Faculty of Chemistry, Jagiellonian University, Ingardena 3 st., 30-060 Krakow, Poland. Fax: 0048 12 634 05 15. E-mail:
[email protected]. † Neurobiochemistry Department, Faculty of Chemistry, Jagiellonian University. ⊥ Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University. # Current address: Institute of Biotechnology and Antibiotics, Staroscinska 5 st., 02-516, Warsaw, Poland. ‡ Silesian University. § Medical University of Vienna. | Medical University. 10.1021/pr900443r CCC: $40.75
2009 American Chemical Society
the cessation of morphine usage usually force the addicted person to continue taking the drug seeking a relief, which makes addiction very difficult to treat. Even when a person overcomes those symptoms, biochemical changes in the brain caused by the substance may lead to craving and relapse even after a very long period of abstinence. There is a growing number of evidence reporting the role of astrocytes in pain control and in phenomena associated with morphine addiction, such as rewarding effect of drug, tolerance, dependence and withdrawal. Morphine was reported to activate astrocytes2 and enhance production of proinflammatory cytokines, such as interleukin-1 (IL-1), and interleukin-6 (IL-6). Administration of substances that cause inhibition of their action, or substances suppressing activation of glial cells (such as propentofylline), was shown to attenuate the effect of tolerance to the analgetic effect of morphine and also reversed withdrawal-induced abnormal pain (hyperalgesia and allodynia).3,4 Single injection of a mixture of astrocytes with astrocyte-conditioned medium into the spinal cord caused acceleration of the development of tolerance to morphine induced analgesia,5 which may confirm the role of astrocytes in those phenomena. Nartia et al. proved that administration of astrocyte-conditioned medium into the nucleus accumbens enhanced the rewarding effect of morphine.6 Moreover, this study showed that place preference produced by morphine was significantly Journal of Proteome Research 2009, 8, 4633–4640 4633 Published on Web 07/31/2009
research articles suppressed by treatment of the animals with propentofylline, which indicates the role of astrocyte activation in the rewarding effect of this substance. Moreover, withdrawal singns triggered by naloxone in the morphine dependent rats were absent when morphine was coadministrated with AV411, a glial activation inhibitor, which can pass a blood brain barrier.7 Despite our growing knowledge, molecular mechanism that are involved in phenomena connected with drug addiction are still very elusive and need further investigations. Proteomics is a very promising strategy in such molecular studies, as it allows the identification of the proteins whose expression is influenced by administration of certain substance. Those proteins may then serve as a biomarkers, or may be the potential targets for new therapy. Up to date, there were several studies investigating changes in proteome after morphine administration. They comprise studies on the whole brain,8 its structures involved in the rewarding system (prefrontal cortex, striatum, nucleus accumbens) and combined with learning (hippocampus)9-11 and in cellular fractions (synaptosomes and postsynaptic density).12,13 Recently, we investigated the changes in proteome of striatal neuronal cell culture.14 In this study, we focused on changes in proteins expression as a result of morphine administration in the primary cortical astrocyte cell culture. As it was mentioned above, studies on astrocytes reveal their pivotal role in the brain which make them a very interesting object of study. Cortical astrocytes express µ, δ and γ opioid receptors by which morphine exerts its influence on the cell, and these receptors remain active in the cell culture.15 Moreover, the cell culture model may be very beneficial in proteomics study due to the simplification of the sample. The proteome of only one type of the cells can be identified in detail as compared to the mixture of various cell types, such as homogenate from the whole tissue. It is known that 2D gel electrophoresis is able to separate around 2000 proteins and its dynamic range is about 103, whereas in a single eukaryotic cell, we can find about 104 proteins at dynamic range of around 105-106. Simplification of the system may improve the analysis and holds the promise of finding the changes among the lowabundance proteins.16 Moreover, cell culture eliminates the problem of high amounts of lipids and abundant proteins which make the proteomic analysis of the whole nervous tissue more difficult. This model also ensures strict control of the environment of the cell in culture (for example, allows for administration of an exact concentration of particular substance).17 On the other hand, there is always a possibility that such in vitro model will not correspond to much more complex in vivo conditions; therefore, the obtained results need to be carefully verified, but the advantages mentioned above outweigh possible drawbacks. In our experiment, morphine in final concentration of 10 µM was applied to the pure, confluent astrocyte cell culture, for 5 days. To separate proteins from the cell culture with morphine application and from the control experiment, we use 2D gel electrophoresis stained with CBB. Obtained gels were analyzed, and proteins from significantly different spots were identified using a nano-LC MS/MS system. Differences in concentrations of two proteins, randomly chosen from analyzed group, were additionally confirmed by Western blotting. Finally, we found 10 proteins which may play an important role in response of astrocytes to morphine administration. 4634
Journal of Proteome Research • Vol. 8, No. 10, 2009
Suder et al.
Materials and Methods Cell Culture. If not otherwise stated, all materials and chemicals mentioned in Materials and Methods were purchased from Sigma-Aldrich Company (Germany). Primary cortical astrocyte cell culture was prepared as described.18 It was shown that this procedure results in a pure astrocytes cell culture. Briefly, eight 1-day-old Male Wistar Rat pups were decapitated and their brains were removed from the skull and placed in the Hanks Balanced Salt Solution (HBSS; Sigma). After careful separation of cortex, the tissue was cut into small pieces. Porcine trypsin was added to a final concentration of 0.25%. The dish was placed at 37 °C for about 20 min to ensure the cleavage of connections between single cells. To stop the process, soybean trypsin inhibitor (STI) was added in the final concentration of 10 mg/mL. Next, 1.0 mg/mL of Deoxyribonuclease I was used to cleave DNA leaking from damaged cells. After centrifugation, the obtained pellet was resuspended using a fire-polished Pasteur pipet in the final medium containing DMEM with addition of 10% of FBS and antibiotics mix (penicillin 100 IU/mL, streptomycin 100 µg/mL, amphotericin 0.25 µg/mL). The suspension was left for 5 min to allow the decantation of the unseparated cell clusters. The supernatant was centrifuged (3 min, 100g, at room temperature). The pellet was resuspended once again in the new volume of the medium. Cells were counted and plated in the poly-L-lysine coated Layton bottles (Nalge-NUNC) at density of about 4 × 105 cells/ cm2. After 6 days of culturing, when the cells formed confluent monolayer; cytosine-beta-D-arabinofuranoside (AraC, Sigma) was added to inhibit the divisions of oligodendrocytes. Layton bottles were shaken on a rotary shaker placed in the incubator for at least 18 h to separate micro- and oligodendroglia from astrocytes. Culture medium with detached micro- and oligodendroglia was removed and the remaining, attached astrocytes were washed 4 times with warm PBS. After that, astrocytes were released by vigorous shaking with 0.25% trypsin solution in PBS for about 20 min. After trypsinization, solutions containing astrocytes were collected and the same volume of growth medium (containing 10% FBS) was added to stop trypsin activity. Finally, cells were centrifuged. The pellet was then dispersed by gentle syringing in a new portion of growth medium and transferred to the new dishes. Split ratio used was 1:3. When the cultures were close to confluence, half of them were treated with morphine at concentration of 10 µM, for 5 days. It was shown that this period of time and the concentration of morphine influence the astrocytes cells in culture and is considered as a chronic morphine administration.19 At the end, we obtained 10 Layton bottles with the control cell cultures and 10 with the cell cultures exposed to morphine. Then, cells were washed with PBS and harvested with cell scraper. Remaining PBS was removed by centrifugation and the pellets were frozen prior to analysis. Cell culture pellet from a single Layton bottle contained about 0.4 mg of proteins. For a single 2D gel electrophoresis, 0.7 mg of proteins was necessary; therefore, proteins obtained from two Layton bottles were separated on a single 2D gel. Two-Dimensional Gel Electrophoresis. Samples were prepared for 2D gel electrophoresis as described previously.10 Briefly, cortical astrocytes were suspended in 1 mL of sample buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 10 mM DTT, 1 mM EDTA, 1 mM PMSF and a mixture of protease inhibitors (Roche Diagnostics, Mannheim, Germany), sonicated on ice (4 cycles for 0.03 s each) and left for 1 h at room
research articles
Proteomic Analysis of Primary Cortical Astrocyte Cell Culture temperature in darkness to allow each constituent of the sample to solubilize. Then, the suspension was centrifuged at 14 000g for 60 min at 12 °C to remove nucleic acids and insoluble material. Desalting of the sample was performed using an Ultrafree-4 centrifugal filter unit (Millipore, Bedford, MA). Protein content in the samples was measured by the Bradford method.20 2D gel electrophoresis was performed as reported by Yang et al.21 First dimension was done on immobilized pH 3-10 nonlinear gradient strips, 18 cm long (Amersham Bioscience, Uppsala, Sweden). Exactly 0.7 mg of proteins was applied on each of them. Focusing started at 200 V and the voltage was gradually increased to 8000 at 4 V/min. Then, the voltage was kept constant for further 3 h (approximately 150 000 Vh in total). Strips were then equilibrated for 15 min in the buffer containing 6 M urea, 20% glycerol, 2% SDS, 2% DTT and for next 15 min in the same buffer containing 2.5% iodoacetamide instead of DTT. Separation in the second dimension was performed on 9-16% gradient SDS-PAGE gels. The gels (180 × 200 × 1.5 mm) were run at 40 mA per gel. After protein fixation in 50% methanol and 10% acetic acid for 12 h, the gels were stained with colloidal Coomassie blue (Novex, San Diego, CA) for 8 h. Excess of the dye was washed out with distilled water. At the end, gels were scanned with the Image Scanner (Amersham Bioscience). In total, five gels from the different cell cultures (biological replications) treated with morphine and five from the control ones were analyzed. Statistical Gel Image Analysis and Protein Quantification. The analysis of the studied set of images of the 2D gel electropherograms was performed using the strategy described in details in our previous paper.22 The main steps of this strategy are as follows: (1) denoising of images in the wavelet domain, (2) background elimination using penalized asymmetric least-squares, (3) warping of images using fuzzy warping method, (4) spots detection and mask construction for the average image, (5) unfolding of images and reduction to the mask, (6) identification of significant spots based on univariate correlation with variable y, describing the belongingness of objects to the studied classes, (7) multivariate analysis of the data. Analysis of data started with preprocessing of individual images, that is, with their denoising and background elimination. Denoising was performed in the wavelet domain using Symmlet 3 and Translation Invariant decomposition of images. To eliminate features associated with noise, a universal threshold and soft thresholding policy were applied.23-27 Background estimation and elimination was performed using the method proposed by Eliers28,29 with the following parameters: 10 basis functions in directions x and y; second degree derivatives and the penalty term equal to 50 in both directions. After preprocessing, the images were aligned using the fuzzy warping method30-32 with the second degree polynomial transformation. As a target, one image from the control group was selected, and all the remaining images were warped to it. On the basis of the identified corresponding spots, the piecewise alignment was performed The average image (constructed for the scaled individual images) was used for identification of spots and for the binary mask construction. Threshold value for mask construction was calculated using the Otsu’s method.33
Table 1. The Correlation Coefficient Values, the Significant Levels Correlations and the Ratios of the Mean Intensities of Morphine to Control Group spot
correlation
significance (p-value)
ratio of intensities (morphine/control)
1 2 4 5 6 7 8 9 10 11 12 14 16 18
0.811 0.732 0.700 0.740 0.815 0.653 0.806 0.661 0.679 0.836 0.686 0.750 0.785 0.779
0.004 0.016 0.034 0.014 0.004 0.040 0.005 0.037 0.031 0.003 0.028 0.012 0.007 0.008
1.3594 1.5391 1.5979 1.5859 1.5150 1.2394 1.5621 1.5057 1.9791 1.8737 1.2822 1.3746 1.3466 1.4682
Then, the pixels within the white region of the binary mask (21 731 altogether) were extracted from all images, unfolded to the vector form and processed for identification of the pixels significant for data discrimination. For each individual pixel, the correlation between the observed intensities and the dependent variable, y, describing belongingness of samples to the studied two classes (morphine and control), were calculated. To evaluate the significance of correlation between individual ‘spots’ (instead of pixels) and variable y, the following approach was applied: each spot, containing pixels significantly correlated with y (the significance level lower than 0.01) was characterized by the sum of intensities for all its pixels, and then the correlation of intensities of this spot for the 10 studied images with variable y was calculated. The correlation coefficient values, the significant levels of those correlations and the ratios of the mean intensities of the two studied classes are summarized in Table 1. As each individual sample is described by the intensities of 21 731 pixels (extracted from the corresponding image according to the constructed binary mask), we can estimate the joint predictive power of pixels based on multivariate approach, such as discriminant Partial Least Squares (D-PLS).34 The D-PLS model, constructed for all 21 731 pixels has, however, a very bad predictive power, due to the big portion of variance in X not related to y. With the number of independent variables (pixels) reduced to 570 (only pixels correlated with y at the significance level
