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Proteomic Analysis of Alterations Induced by Perinatal Hypoxic− Ischemic Brain Injury Katja Rosenkranz,*,† Caroline May,‡ Carola Meier,§ and Katrin Marcus† †

Department of Functional Proteomics, Ruhr-University Bochum, Germany Medizinisches Proteom-Center, Ruhr-University Bochum, Germany § Department of Anatomy and Cell Biology, Saarland University, Homburg/Saar, Germany ‡

S Supporting Information *

ABSTRACT: Perinatal hypoxic−ischemic brain injury is an important cause of neurological deficits still causing mortality and morbidity in the early period of life. As efficient clinical or pharmaceutical strategies to prevent or reduce the outcome of perinatal hypoxic−ischemic brain damage are limited, the development of new therapies is of utmost importance. To evolve innovative therapeutic concepts, elucidation of the mechanisms contributing to the neurological impairments upon hypoxic−ischemic brain injury is necessary. Therefore, we aimed for the identification of proteins that are affected by hypoxic−ischemic brain injury in neonatal rats. To assess changes in protein expression two days after induction of brain damage, a 2D-DIGE based proteome analysis was performed. Among the proteins altered after hypoxic−ischemic brain injury, Calcineurin A, Coronin-1A, as well as GFAP were identified, showing higher expression in lesioned hemispheres. Validation of the changes in Calcineurin A expression by Western Blot analysis demonstrated several truncated forms of this protein generated by limited proteolysis after hypoxia−ischemia. Further analysis revealed activation of calpain, which is involved in the limited proteolysis of Calcineurin. Active forms of Calcineurin are associated with the dephosphorylation of Darpp-32, an effect that was also demonstrated in lesioned hemispheres after perinatal brain injury. KEYWORDS: hypoxia-ischemia, brain injury, 2D-DIGE, proteomics, mass spectrometry

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INTRODUCTION Hypoxic−ischemic brain injury (HI) is an important cause for mortality and morbidity in the neonatal period. Depending on the extent and localization of the insult, children may develop permanent neurological deficits such as spastic paresis, cerebral palsy, epilepsy, and disorders of sensorimotor coordination.1 Clinical or pharmaceutical strategies to prevent or reduce the long-term effects of hypoxic−ischemic brain damage in the neonatal period are still limited (for an overview, see ref 2). Although many agents have shown positive results in animal models, none of them could yet be transferred to clinical settings.3 Consequently, elucidation of the pathophysiology that follows a hypoxic−ischemic insult in the newborn is of utmost importance. Biochemical events such as energy failure, brain edema, membrane depolarization, increase of neurotransmitter release and inhibition of neurotransmitter uptake, rise of intracellular Ca2+, yield of oxygen-free radicals, and a decrease of blood flow are triggered by hypoxia−ischemia. These changes may lead to brain dysfunction and neuronal death after hypoxic−ischemic brain injury,4 with particular vulnerability of the immature brain being more sensitive to such incidents.5 However, the details of the mechanisms underlying permanent brain injury induced by hypoxia−ischemia in the perinatal period are still unknown. © 2012 American Chemical Society

Using a proteomic approach to identify proteins involved in the formation of hypoxic−ischemic brain injury could provide insight into potential mechanisms of neuronal dysfunction and apoptosis associated with the insult. Furthermore, knowledge of the detailed processes after brain injury may allow development of protective or regenerative therapies. Currently, therapeutic approaches are investigated in clinical as well as animal studies of HI, which aim at an extension of the therapeutic window to days rather than hours (for an overview, see refs 6−8). Therefore, we performed our proteomic analysis two days after induction of HI, at postnatal day (P) 9. In order to identify proteins involved in the mechanisms of hypoxic−ischemic brain injury, we chose two-dimensional difference gel electrophoresis (2D-DIGE) as a method that enables a global comparison of proteins of healthy tissue against those from hypoxic−ischemic tissue of rat brains. Although other studies have analyzed differences in protein abundances in selected brain regions, i.e. cortex and hippocampus after HI,9 we focused our analysis on the total area affected by the lesion. Proteins regulated at the global level are Received: June 29, 2012 Published: November 15, 2012 5794

dx.doi.org/10.1021/pr3005869 | J. Proteome Res. 2012, 11, 5794−5803

Journal of Proteome Research

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gradient, 3 h at 300 V/step, 6 h at 1000 V/gradient, 1.5 h at 6000 V/step, all at 20 °C. After focusing, the strips were equilibrated, placed on top of a SDS-polyacrylamide gel, and run on a Desaphor VA 300 system14−16 with 75 mA per gel at 20 °C.

assumed to be key regulators of HI damage and, therefore, potential target proteins for the development of therapies.

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MATERIAL AND METHODS

Levine Model of Perinatal Hypoxic−Ischemic Brain Injury

Scanning and Image Analysis

All surgical and experimental protocols on animals were approved by the appropriate institutional review committee (LANUV Recklinghausen, Germany) and met the guidelines of the German animal protection law. All animals were kept in a temperature-controlled environment on a 12 h light/12 h dark cycle with food and water ad libitum. The Levine model10,11 was used to achieve reproducible hypoxic−ischemic injury in neonatal Wistar rats on postnatal day seven (P7) and was performed as described previously.12 Briefly, seven-day-old Wistar rat pups were deeply anesthetized by inhalation of isoflurane. The left common carotid artery was exposed, double ligated, and severed. Afterward, the rats were exposed to a hypoxic gas mixture (8% oxygen/92% nitrogen) for 80 min. The environmental temperature was strictly maintained at 36 °C. Two days after induction of the insult, i.e. at P9, rats were anesthetized and decapitated. Brains were dissected, and macroscopic brain injury was assessed immediately.13 Brains were trimmed (−3 mm frontal), and left and right hemispheres were separated and frozen in liquid nitrogen. Samples were immediately stored at −80 °C until further processing.

Gels were scanned using a Typhoon 9400 scanner (GE Healthcare). Excitation and emission wavelengths were chosen specifically for each of the dyes according to recommendations of the manufacturer. Images were preprocessed using the ImageQuant software (GE Healthcare). Intragel spot detection and intergel matching were performed using the Differential Ingel Analysis (DIA) and Biological Variation Analysis (BVA) mode of DeCyder 6.5 software (GE Healthcare), respectively. The estimated spot number was set to 10.000 and the volume filter to 30.000. Furthermore, setting 100−200 landmarks improved automatic matching. Spot intensities were normalized to the internal standard, and the significance of protein regulation was determined using the Student’s t test. Protein spots differentially expressed (p ≤ 0.05, average ratio ≥1.5) between left and right hemispheres in at least five of seven samples were further analyzed by mass spectrometry (MS). In-Gel Tryptic Digestion and Protein Identification Using LC-ESI Mass Spectrometry

For identification of proteins, all differentially expressed 2D protein spots were manually cut from the gel. Proteins were ingel digested with trypsin (Promega, Mannheim, Germany) and extracted from the gel, and nanoLC-ESI MS/MS analyses were performed as described previously.17 The extracted peptides were first preconcentrated (0.3 mm i.d. × 1 mm, PepMapcolumn, 0.1% TFA at a flow rate of 30 μL/min for 5 min) and, in the following, separated (75 μm i.d. × 150 mm, PepMap column) on the Ultimate HPLC System (all Dionex LC Packings, Idstein, Germany) consisting of Famos, Switchos, and Ultimate online coupled to a Finnigan LCQdeca XP ion trap and HCT ultra (Bruker Daltonic, Bremen, Germany) instrument. The flow rate for separation was set to 300 nL/min using the following solvent system: solvent A (0.1% (v/v) formic acid) and solvent B (0.1% (v/v) formic acid, 84% (v/v) acetonitrile (ACN)). The gradient was set to 5−15% solution B in 5 min, 15−40% solution B in 60 min, and 40−95% B in 5 min. All experimental data were stored in the Proteinscape database (version 1.3, Bruker Daltonics). Automatic spectra interpretation for peptide identification was done using MASCOT V.2.2 (Matrixscience, London, U.K.) with a fragment ion and peptide mass tolerance of ±1.5 Da. Searches were performed allowing one missed cleavage after tryptic digestion. All data were searched against a database created by DecoyDatabaseBuilder18 containing the IPI.rat.V3.87 protein sequence database (39.925 entries; http://www.ebi.ac.uk/IPI/ IPIrat.html) with one additional shuffled decoy for each protein, resulting in a database containing 78.850 entries. FDR analysis was used to identify validate candidates. Using the decoy database, we limited and determined the FDR. Additional parameters were used for protein identification after FDR analysis: proteins were considered to be identified if at least two peptides (with an peptide ion score above 30) were explained by the spectra and if the protein ion score was higher than 90.

Protein Extraction

For the differential proteome study, seven left (lesion) and right (control) hemispheres of P9 rats undergoing HI at P7 were investigated. Frozen tissue sample was transferred to a cooled mortar with liquid nitrogen together with 1.2 volumes of lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris, pH 8.5) and ground to a fine powder. The suspension was treated with ultrasound for 10 min at 4 °C. To remove insoluble components, the homogenate was centrifuged for 30 min at 4 °C at 226.000g (Beckmann Optima, rotor: MLA130, 65.000 rpm). After centrifugation, the supernatant was transferred to a new reaction tube. The pellet was resolved in lysis buffer and poured back into the mortar, and the abovementioned procedure was performed again. Both supernatants were combined and stored at −80 °C. Protein Labeling for 2D-DIGE

The protein concentration of each sample was determined by amino acid analysis. 50 μg aliquots were prepared and stored at −80 °C. For subsequent labeling, tissue homogenates were thawed and labeled with CyDye minimal dyes (GE Healthcare, Freiburg, Germany) according to the manufacturer’s protocol with 400 pmol of dye per 50 μg of protein. Left hemispheres were labeled with Cy3, and right hemispheres with Cy5. The internal standard was prepared from equal amounts of all samples and labeled with Cy2. 2D-DIGE

The proteins were separated by 2D-PAGE using IPG (immobilized pH gradient)-strips in the first dimension as described previously.14,15 The IPG-strips (24 cm, pH 3−11, NL) were loaded with 50 μg of two samples labeled with Cy3 and Cy5 each, plus 50 μg of internal standard labeled with Cy2 (150 μg protein in total). The proteins were focused in an IPGphor unit (GE Healthcare) for a total of 80.000 V·h with the following gradient: 6 h at 100 V/step, 1 h at 300 V/ 5795



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Western Blot Analysis

1D-Western Blot analysis was performed on randomly selected lesioned (n = 3) and control (n = 3) hemispheres as described previously.19 In brief, proteins (10 μg/lane; 50 μg/lane for Calpain I antibody) were separated on SDS-polyacrylamide gels and transferred to nitrocellulose or polyvinylidene fluoride (PVDF) membrane. For 2D-Western Blots, 50 μg protein were separated using IPG-strips (7 cm, pH 3−10, NL). The proteins were focused in an IPGphor unit for a total of 3000 V·h with the following gradient: 20 min at 200 V/step, 15 min at 450 V/step, 15 min at 750 V/step, 2 h at 1000 V/step, all at 20 °C. After focusing, the strips were equilibrated and placed on top of a SDSpolyacrylamide gel. The proteins were transferred to a nitrocellulose membrane. Nonspecific binding sites were blocked, and membranes were incubated with primary antibodies diluted in blocking reagent. After washing, the membranes were incubated with horseradish−peroxidase-conjugated secondary antibodies for 1 h. Visualization was performed by enhanced chemiluminescence detection (GE Healthcare). Primary antibodies were used as follows: Coronin-1A (1:300), β-actin (1:15.000; Sigma-Aldrich, Taufkirchen, Germany), Calcineurin (1:250; BD Biosciences, Heidelberg, Gemany), Calpain I (1:100), Darpp-32 (1:400), p-Darpp-32 (1:100), SH-PTP1 (1:200) (all Santa Cruz Biotechnology, Heidelberg, Germany), Fodrin (1:10.000; Epitomics, Burlingame, CA, USA), and GFAP (1:1000; Millipore, Schwalbach, Germany).

Figure 1. Silver staining of two-dimensional gel showing protein spots significantly altered by hypoxic−ischemic brain injury (HI). Cytoplasmic extracts of control (right) and lesioned (left) hemispheres of rats after HI were separated by two-dimensional gel electrophoresis. Differential expression of spots was evaluated with DeCyder software. Protein spots showing significant changes were identified using LC ESI-MS/MS. White arrows label the spots of which proteins were identified as listed in Table 1.

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RESULTS In this study a well-characterized model of perinatal hypoxic− ischemic brain damage11,20−24 was used to identify proteins affected by hypoxia−ischemia. Using a global 2D-DIGE analysis, essential proteins affected by perinatal HI were identified. Among the regulated proteins, Calcineurin A, Coronin-1A, and GFAP are involved in the most detrimental processes following HI, i.e. apoptosis and inflammation, which render them potential key regulators in perinatal HI. The results of the proteomic approach enabled the analysis of further crucial proteins acting up- or downstream of the identified pathways. Taken together, the results of this study contribute to clarify the mechanisms following perinatal HI.

typical “déjà vu” proteins were identified. Proteins such as dihydropyrimidinase-like proteins and keratins are supposed to be differentially expressed in a nonspecific way in 2D-PAGEbased studies of different neurodegenerative disorders.25,26 In the present study, dihydropyrimidinase-like proteins were identified in several isoforms and spots, sometimes higher in lesioned sometimes higher in control hemispheres. Therefore, these proteins are not included in Table 1 and Figure 1. All proteins identified in this study, including nonspecific proteins, are listed in Supporting Information Table 1. We classified the proteins with significant changes in expression levels based on their published biological functions (Table 2). The proteins identified to be altered by HI comprised proteins involved in the formation of the cytoskeleton (such as Tubulin and Coronin-1A), proteins that act as molecular chaperones (e.g., T-complex protein 1, heat shock 70 kDA protein 1A), as well as proteins with metabolic function (glucose-6-phosphate 1-dehydrogenase, Superoxide dismutase 1). Furthermore, proteins associated with cerebral plasticity, namely Calcineurin A (serine/ threonine-protein phosphatase 2B) and GFAP (glial fibrillary acidic protein), were identified (Table 2). For further analysis, we chose three of the regulated proteins, which play a fundamental role in different brain functions. Coronin-1A is an actin-binding protein, which has recently been identified as a marker for macrophages and microglia, the immune cells of the brain.27 GFAP is an intermediate filament protein that plays an important role in astrocyte morphology and plasticity as well as their reaction after brain injury. Calcineurin A, a Ca2+/Calmodulin-dependent phosphatase, is implicated in crucial neuronal functions such as production of

Identification of Differences in Protein Abundance after Perinatal Hypoxic−Ischemic Brain Injury

For differential proteome analysis, soluble proteins were extracted from lesioned (left) or control (right) brain hemispheres. Lesioned hemispheres (n = 7) were labeled with Cy3, and control hemispheres (n = 7), with Cy5. The internal standard composed of all samples analyzed was labeled with Cy2 to achieve high accuracy in protein quantification. About 2000 protein spots were detected using DeCyder 6.5 software. A total of 44 differentially protein spots (p < 0.05) with at least a 1.5-fold up- or down-regulation in a minimum of five of seven samples was identified (Supporting Information Figure 1). For 25 differentially expressed protein spots, a higher abundance in lesioned hemispheres (negative fold change) was detected, whereas 19 protein spots showed a higher expression in control hemispheres (positive fold change). All differentially expressed protein spots were analyzed using nanoLC ESI-MS/ MS, and 16 proteins were identified in these protein spots (Figure 1; Table 1). Besides the proteins listed in Table 1, 5796



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Table 1. Proteins Identified To Be Influenced by Hypoxic−Ischemic Brain Injurya spot no. 411 507 537 574 574 595 595 606 638 726 726 726 770 888 1713 1758 1758

protein name

accession no.

exp pI

exp MW

obs pI

obs MW

seq cov (%)

Mascot score

peptide count

fold change

Log2 fold change

heat shock 70 kDa protein 1A/1B T-complex protein 1 subunit α glucose-6-phosphate 1-dehydrogenase T-complex protein 1 subunit ε serine/threonine-protein phosphatase (Calcineurin) tubulin α-1C chain Na+/H+ exchange regulatory cofactor NHE-RF1 Coronin-1A tubulin α-1C chain Rap1 GTPase-GDP dissociation stimulator 1 isoform 1 of glial fibrillary acidic protein isoform 2 of glial fibrillary acidic protein ATP synthase subunit α, mitochondrial actin cytoplasmic 1 eukaryotic translation initiation factor 5A-1 superoxide dismutase (Cu−Zn) Prefoldin 5

IPI00196751 IPI00200847 IPI00231637 IPI00470301 IPI00559849

5.5 5.8 5.9 5.4 5.9

70.1 60.3 59.3 59.5 57.5

5.5 5.8 5.9 5.5 5.5

70 60 60 58 58

3.9 24.1 4.9 3.3 4.3

112.9 658.6 95.8 125.4 94.1

3 14 3 2 2

−1.84 1.54 1.52 −6.82 −6.82

−0.88 0.62 0.60 −2.77 −2.77

IPI00364046 IPI00200898

4.8 5.6

49.9 38.8

5.4 5.4

57 57

9.6 6.5

234.3 99.6

5 2

−1.8 −1.8

−0.85 −0.85

IPI00210071 IPI00364046 IPI00369496

6 4.8 5.5

51 49.9 52.2

6.0 5.5 5.4

58 53 49

18 9.1 6.3

277.4 217.2 153.5

7 5 3

−5.26 −1.6 −1.65

−2.4 −0.68 −0.72

IPI00190943 IPI00210211 IPI00396910 IPI00189819 IPI00211216

5.2 5.6 9.7 5.2 4.9

49.9 49.5 59.7 41.7 16.8

5.4 5.4 6.7 5.3 5.1

49 49 49 43 16

36.5 17.5 7.2 14.1 10.4

1103.2 405.8 263.5 247.6 118

23 8 4 6 3

−1.65 −1.65 1.59 1.54 1.75

−0.72 −0.72 0.67 0.62 0.81

IPI00231643 IPI00870220

5.9 5.9

15.9 17.3

5.7 5.7

15 15

34.4 5.2

268.1 94.1

6 2

−1.87 −1.87

−0.90 −0.90

a

Proteins were extracted from left and right hemispheres of HI-treated rats at postnatal day 9, separated by 2D-DIGE, and identified by LC-ESI-MS. To every significantly regulated spot (average ratio ≥ 1.5; p-value 30 were accepted. Protein identifications with an ion score >90 and a minimum of two peptides identified were approved.

Validation of Proteomic Data by Western Blot Analysis

Table 2. Functional Classification of Proteins Significantly Altered after Hypoxic−Ischemic Brain Injury protein name

Coronin-1A, GFAP, and Calcineurin A were found to be changed after induction of hypoxic−ischemic brain injury as identified by 2D-DIGE analyses. The enlarged representative illustrations in Figures 2A, 3A and 4A showed a clear increase in Coronin-1A, GFAP and Calcineurin A expression in lesioned hemispheres as identified by 2D-DIGE analysis. In Figures 2B, 3B, and 4B the standardized log abundances are presented, showing the expression in left and right hemispheres for every 2D-DIGE gel as analyzed with the DeCyder software. These changes in protein expression were further validated on three randomly selected animals by Western Blot analysis using protein specific antibodies. For Coronin-1A, the results obtained with Western Blot analysis were in agreement with the data by 2D-DIGE. In lesioned hemispheres, expression of Coronin-1A was increased compared to control hemispheres (Figure 2C), suggesting more activated microglial cells in the lesioned hemisphere. 1D-Western Blot analysis of GFAP also reflects the results obtained by the proteomic approach. In lesioned hemispheres, a higher expression of GFAP was detected compared to control hemispheres (Figure 3C). Furthermore, Western Blot analysis revealed additional differences between left and right hemispheres (Figure 3C). In lesioned hemispheres, GFAP was detected as multiple bands, which was much less pronounced in control hemispheres (Figure 3C, *). The additional immunosignals in lesioned hemispheres point to protein degradation or to post-translational modifications of GFAP. To analyze this in more detail, we performed 2D-Western Blots using GFAP antibodies (Figure 3D). The differences detected with 1DWestern Blots could also be shown in 2D-Western Blots. The

function

Protein Folding heat shock, 70 kDa protein 1A/1B molecular chaperone, cytoprotective T-complex protein 1 subunit α molecular chaperone T-complex protein 1 subunit ε molecular chaperone Prefoldin 5 molecular chaperone, cytoskeleton, actin folding Translation Factor eukaryotic translation initiation protein translation factor 5A-1 Metabolism Enzymes glucose-6-phosphate 1carbohydrate metabolism, glucose dehydrogenase metabolism superoxide dismutase (Cu−Zn) cellular oxygen metabolism, ROS defense Cytoskeleton Formation tubulin α-1C chain cytoskeleton, major constituent of microtubules Coronin-1A cytoskeleton, actin binding protein, marker of inflammation actin cytoplasmic 1 cytoskeleton, cell motility Prefoldin 5 molecular chaperone, cytoskeleton, actin folding Cerebral Plasticity glial fibrillary acidic protein serine/threonine-protein calcium-dependent, calmodulinphosphatase (Calcineurin) stimulated protein phosphatase

free radicals, regulation of the neuronal cytoskeleton, and recycling of synaptic vesicles. All of these proteins were upregulated in lesioned hemispheres after HI and selected for further validation by Western Blot analysis. 5797



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Figure 3. Increased levels of GFAP in lesioned hemispheres after hypoxic−ischemic brain injury. (A) Magnification of GFAP protein spots in left and right hemispheres after 2D-DIGE. (B) Graphic presentation of GFAP expression in left and right hemispheres of all 2D-DIGE gels as analyzed using the DeCyder software. The thick black line represents the median of all gels. (C) Validation of the changes in protein expression using Western Blot analysis. Samples from each group were probed with monoclonal antibodies against GFAP with β-Actin used as loading control. * Labels additional immunosignals of GFAP. (D) 2D-Western Blots of left and right hemispheres using GFAP antibodies. L, lesioned hemispheres; R, control hemispheres.

Figure 2. Postproteomic validation of alterations in Coronin-1A levels after perinatal HI. (A) Enlarged images of representative 2D-gels demonstrating differences in the expression levels of Coronin-1A in left and right hemispheres of P9 rats undergoing perinatal HI. (B) Graphic presentation of the protein abundance in left and right hemispheres in every analyzed 2D-DIGE gel. The thick black line represents the median of all gels. (C) Immunoblot analysis of Coronin-1A expression was performed using the same cytoplasmic extracts used for the 2D-DIGE analyses. β-Actin was used as loading control. L, lesioned hemispheres; R, control hemispheres.

spot pattern recognized by the GFAP antibody in 2D-Western Blots could not be detected in the 2D-DIGE gels. For Calcineurin A, 1D-Western Blot analysis revealed differences in protein expression between left and right hemispheres (Figure 4C). In control hemispheres, a single band was identified at the appropriate molecular weight of 60 kDa. In contrast, the antibody recognized additional bands at lower molecular weight in lesioned hemispheres, in detail at 54, 48, and 46 kDa (Figure 4C). The appearance of additional protein bands is in agreement with the literature: Shioda et al.28 also described limited proteolysis of Calcineurin A in a mouse middle cerebral artery occlusion (MCAO) model referred to as the conversion of Calcineurin A to constitutively active forms. Dependent on the presence of Ca2+ and Calmodulin, different truncated forms of Calcineurin A are generated in vitro.29 In addition, we performed 2D-Western Blots using Calcineurin A antibodies. As shown in Figure 4D, the antibody detected several spots of Calcineurin A. Some of the spots are more pronounced in the right hemispheres, whereas some spots with a lower molecular weight could only be detected in left hemispheres. Most likely, the Calcineurin A form identified by mass spectrometry belongs to one of the truncated forms, which are more prominent in left hemispheres. Concerning the approximate molecular weight, the spots identified in 2DWestern Blots of left hemispheres (Figure 4D) correspond to the additional bands identified by 1D-Western Blot analysis (Figure 4C).

Figure 4. Changes in Calcineurin A protein expression after perinatal HI. (A) Enlarged images of 2D-DIGE protein profile showing changes in Calcineurin A expression in lesioned and control hemispheres after perinatal hypoxic−ischemic injury (HI). (B) Standardized log abundance for Calcineurin A in left and right hemispheres of all 2DDIGE gels. The thick black line shows the median of all analyzed gels. (C) Representative Western Blot analysis of Calcineurin A expression in cytoplasmic extracts of P9 rat brain hemispheres. β-Actin was used as loading control. (D) 2D-Western Blot analysis of left and right hemispheres using Calcineurin A antibodies. L, lesioned hemispheres; R, control hemispheres.

Further Proteins Associated with Those Proteins Identified Directly by Proteomic Analysis

The identification of potential key regulators of perinatal HI using 2D-DIGE led our analysis to additional proteins involved in the identified pathways. 5798



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Figure 5. Analysis of additional associated proteins affected by perinatal HI. Expression levels of additional proteins influenced by perinatal hypoxic− ischemic brain damage were analyzed in samples of lesioned and control hemispheres with antibodies against (A) Calpain I, (B) α-Fodrin to analyze calpain activity, (C) Darpp-32 phosphorylated at Thr-34 (p-Darpp-32), (D) total Darpp-32, and (E) SH-PTP1. β-Actin was used as loading control. L, lesioned hemispheres; R, control hemispheres.

Increased Expression and Activation of Calpain I after Hypoxic−Ischemic Brain Injury

control hemispheres (Figure 5C). Examination of the expression of total Darpp-32 demonstrated a decrease of this protein in lesioned hemispheres compared to control hemispheres (Figure 5D). The decrease in phosphorylated Darpp-32 detected in lesioned hemispheres emphasized the crucial function supposed for Calcineurin A.

As the degradation of Calcineurin A observed in lesioned hemispheres in our study was suggested to be generated by Calpain mediated limited proteolysis,28 we analyzed the expression of Calpain I in our model of perinatal HI. Western Blot analysis using antibodies against the catalytic and regulatory subunit of Calpain I resulted in a higher abundance of both Calpain I subunits in lesioned hemispheres compared to control hemispheres (Figure 5A). Calpains, calcium-activated cysteine proteases, have been implicated in acute neuronal injury along with limited specific proteolysis of target proteins.30,31 In addition to the Ca2+/ Calmodulin dependent activation of Calcineurin A, it has been reported that Calcineurin A is further proteolyzed by activated Calpain.28,29 The α-subunit of Fodrin, a protein of 240 kDa, a major component of the cortical cytoskeleton, has been shown to be cleaved to 150 and 120 kDa Fodrin breakdown products (FBPs) by activated Calpains during apoptosis.32 To determine the activity of Calpains, we analyzed the levels of the FBPs. Western Blot analysis yielded higher amounts of the 150 and 120 kDa FBPs in lesioned hemispheres, whereas this degradation was not as pronounced in control hemispheres (Figure 5B). These results point to an activation of Calpain I together with a higher abundance of this protein in lesioned hemispheres after perinatal HI.

Increased Expression of the Protein Tyrosine Phophatase SH-PTP1 in Lesioned Hemispheres

Previous results suggest that SH-PTP1 (also known as PTP1C and SHP), a protein tyrosin phophatase, plays an important role in the regulation of glial activation and proliferation in the ischemic CNS.36 Therefore, we analyzed the expression of this protein phosphatase in our model of perinatal HI. Western Blot analysis revealed elevated levels of SH-PTP1 in lesioned hemispheres compared to control hemispheres (Figure 5E), which correlated with the higher expression of GFAP in lesioned hemispheres after perinatal HI (Figure 3C).

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DISCUSSION Despite substantial progress in obstetric and neonatal care that had led to a reduction in neonatal morbidity and mortality, the risk of perinatal brain injury has not been reduced. This is due to the better survival of extremely premature infants and severely hypoxic neonates, which are more susceptible to brain injury.37 Cerebral palsy, seizure disorders, sensory impairment, and cognitive limitation are still some of the consequences of brain injury acquired in this early period of life.38−40 Therapeutic strategies to prevent or reduce the long-term effects of perinatal hypoxic−ischemic brain injury (HI) are limited in this particular phase of life. Several pharmaceutical agents which showed ameliorations in animal models of HI failed in clinical trials (for an overview, see ref 3). To date, promising clinical data are available for the use of moderate hypothermia in term infants. When started within 6 h after birth and continued for at least 72 h, hypothermia can provide neuroprotection and improve survival.41−44 Because hypothermia does not exhibit overall protection from neurological consequences, no widely effective therapeutic approach for human neonates exists. Therefore, it is of major importance to

Decreased Expression of Darpp-32 and p-Darpp-32 after Hypoxic−Ischemic Brain Injury

Calcineurin A is associated with the dephosphorylation of several proteins, one of them being Darpp-32 (dopamine- and cAMP-regulated phosphoprotein).33,34 The phosphorylation of Darpp-32 at a single threonine residue (Thr-34) plays an important role in mediating the actions of dopamine and inhibiting protein phosphatase-1, which controls the state of phosphorylation and activity of a number of physiologically important substrates.35 Therefore, we evaluated the phosphorylation levels of Thr-34 of Darpp-32 in our model of perinatal HI. Western Blot analysis resulted in lower levels of phosphorylated Darpp-32 (p-Darpp-32) in lesioned than in 5799



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protein. Site-specific phosphorylation of intermediate filament proteins including GFAP has been shown to regulate the dynamic properties that play a fundamental role in astrocytic plasticity.57,58 Therefore, the potential dephosphorylation of GFAP following HI likely leads to fatal consequences and has to be analyzed in more detail using a phosphospecific antibody. As SH-PTP1, a protein tyrosine phosphatase, was shown to be up-regulated after cerebral ischemia in reactive astrocytes,36,59 we decided to analyze the expression of SH-PTP1 in our model of perinatal HI. SH-PTP1 was up-regulated in lesioned hemispheres similar to the observed expression of GFAP.59 Tyrosine phosphorylation is known to play a fundamental role in the activation of astroglial and microglial cells in the injured CNS.60 Given that GFAP has tyrosine phophorylation sites, it is possible that SH-PTP1 is involved in the assembly or disassembly of GFAP filaments. The potential interplay of GFAP and SH-PTP1 has to be analyzed in further detail, likely to reveal important details about the inflammatory reaction, particularly in view of the astroglial activation. In our proteome analysis, we also identified Calcineurin A with differential abundance in lesioned and control hemispheres. Calcineurin, a Ca2+/Calmodulin-dependent phosphatase, comprises more than 1% of total protein content in brain tissue, pointing to an important role in CNS functions, including neurite extension, synaptic plasticity, and learning and memory.61−63 Brain ischemia causes accumulation of intracellular Ca2+, and increased intracellular Ca2+ levels lead to activation of Ca2+-dependent enzymes, such as protein kinases, proteases, and phophatases, such as Calcineurin. All of these proteins are linked to morphological, biochemical, and physiological changes, resulting in cell death.64−67 Validation of Calcineurin A changes by 1D-Western Blot revealed additional bands of the protein with lower molecular weight in lesioned hemispheres, whereas only one immunosignal was detectable in control hemispheres. Appearance of the 48 and 46 kDa bands of Calcineurin A has also been reported in a MCAO model and was referred to as the conversion of this protein to constitutively active forms generated by Calpain mediated limited proteolysis in the presence of Ca2+.28,68,69 In the presence of Ca2+ and Calmodulin, the 54 kDa form of Calcineurin is generated.29 Excessive stimulation of Calcineurin A leads to the release of this protein into the cytosol, where it may cause neuronal apoptosis via dephosphorylation of key cytosolic proteins, such as Bcl-2 associated death protein (BAD) and cAMP response element binding protein (CREB).70 Furthermore, Calcineurin A plays a role in the dephosphorylation of GFAP.57 Summing up these data, it becomes apparent that the protein phosphatase Calcineurin A is a key factor involved in the detrimental events following perinatal HI and that the activation of Calcineurin A could account for the outcome of perinatal brain injury. Identifying optimal therapeutic strategies resulting in the inhibition of Calcineurin A would potentially prevent harmful processes following perinatal brain damage. Cyclosporine A (CsA) and FK506 (Tacrolimus), both routinely used as immunosuppressive drugs after organ transplantation, were shown to possess neurotrophic and neuroprotective characteristics to specific neurons in vitro and in vivo.71 Data from various studies indicate that it is the common inhibition of Calcineurin activity that mediated the neuroprotective effects observed on neuronal cells.72,73 The clinical potential of these Calcineurin inhibitors to promote neuronal survival and

develop suitable therapies for the treatment of hypoxic− ischemic brain injury in the newborn, particularly those which can be administered at delayed time points after HI.6−8 The pathologic mechanisms of perinatal HI are similar to that described for stroke, and they occur in two major phases: In the first phase, excitotoxicity and necrotic cell death take place. The second phase is characterized by inflammation, glial activation, and apoptosis.45 However, perinatal HI displays some unique aspects: (1) The immature brain is more sensitive to the events occurring in the acute phase of injury.5 (2) The inflammatory reaction starts much earlier in the immature brain, and the process of inflammation is much more severe than in the adult brain.46,47 The details of the underlying mechanisms are unknown, but development of therapies requires extensive knowledge of the proteins and pathways involved in perinatal HI. In the rat model of perinatal hypoxic− ischemic brain injury employed in this study, the resulting brain damage as well as the underlying cascade of pathological events is highly similar to the human situation.48 Using a proteomic approach, we were able to identify proteins that are globally affected by perinatal HI. Proteins significantly regulated after perinatal HI included molecular chaperones, proteins involved in metabolic processes or in the formation of the cytoskeleton. In addition, we identified proteins associated with essential functions in the CNS, such as cerebral plasticity, and involved in the detrimental processes of perinatal HI, namely apoptosis and inflammation. Our proteomic and Western Blot results demonstrated that Coronin-1A, an actin-binding protein, is up-regulated in lesioned hemispheres 48 h after perinatal HI. Members of the Coronin family appear to function in association with the membrane cytoskeleton through interactions with filamentous actin and the Arp2/3 protein complex.49 In addition, Coronin1A has recently been identified as a novel and effective imunohistochemical marker for microglial cells in the CNS.27 Microglia are the immune cells of the CNS, and the upregulation of Coronin-1A in lesioned hemispheres as identified with our proteomic approach reflects the inflammatory response observed after HI.21 As inflammation seems to contribute to the fatal effects of brain ischemia,50−52 especially in the newborn, the identification of novel factors involved in the inflammatory response provides a deeper insight into the mechanisms following perinatal HI. However, the definite understanding of the mechanisms of action of Coronin-1A remains unknown. Another protein identified in our proteome analysis was GFAP, which was up-regulated in lesioned hemispheres. GFAP is an intermediate filament protein mainly expressed in astrocytes and involved in cerebral plasticity.53 The activation of astroglial cells is seen in close relation to the process of inflammation and connected to the activation of microglia, with both being associated with HI.54 The increase in GFAP expression, as demonstrated in lesioned hemispheres in this study, is defined as a hallmark of reactive gliosis after brain injury and a key step in astrocyte activation.55 Besides other critical functions of astrocytes, which are hampered due to their activation, the formation of the glial scar is one of the most detrimental factors preventing recovery from hypoxia-ischemia.56 Interestingly, using Western Blot analysis, GFAP was detected as multiple bands in lesioned hemispheres, indicative of potential protein degradation or maybe due to posttranslational modifications. It could be possible that these additional bands of GFAP are desphosphorylated forms of the 5800



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contribute to functional recovery after brain injury, especially in the newborn, has to be proven. On the basis of the fact that Calcineurin A is involved in many processes in the CNS, we analyzed the expression of further up- and downstream acting proteins such as calpain and Darpp-32 in our model of perinatal HI. Because constitutively active forms of Calcineurin A are supposed to be generated by limited proteolysis mediated by Calpain, we decided to analyze the expression and activity of Calpain I by Western Blot. Calpains are calcium-dependent cysteine proteases that become activated during ischemia, resulting in the proteolysis of a variety of proteins.30,74 The Calpain family of proteases is causally linked to postischemic neurodegeneration.31 Activated Calpain catalyzes the cleavage of the α-subunit of Fodrin, a 240 kDa protein, into 150 and 120 kDa breakdown products.75 This effect could be observed in lesioned hemispheres in our study in correlation with a higher expression of Calpain I after perinatal HI. A recent study has demonstrated that reducing the expression of an individual calpain isoform can decrease postischemic neuronal cell death and preserve hippocampal function in a model of global brain ischemia,76 emphasizing the significant role of calpains in brain injury. Thus, calpains are potential targets for the development of a therapeutic approach. Due to the fact that increased Calcineurin A activity has previously been associated with the dephosphorylation of dopamine- and cAMP-regulated phosphoprotein (Darpp)32,34,77 we analyzed the expression of total and phosphorylated Darpp-32 after perinatal HI. Beside the decrease of Darpp-32 phosphorylated at Threonin 34, we found a reduction of total Darpp-32 in lesioned hemispheres. The decrease in total Darpp-32 together with its reduced phopshorylated form may contribute to the impaired behavioral effects observed after perinatal HI or to the higher risk of schizophrenia observed after perinatal complications.78−80 Darpp-32 has crucial functions in the dopamine-signaling pathway, and abnormalities in this pathway were reported in several neurological and psychiatric diseases such as Parkinson's and schizophrenia.35 The actions of Darpp-32 in perinatal HI have to be analyzed in more detail. However, the fact that many components and principles of the dopamine signaling pathway are known gives hope to identify mediators to block the potentially negative effects of Darpp-32 after perinatal HI. Using a global 2D-DIGE-based approach, we were able to identify proteins altered after perinatal HI. Identification of these proteins contributes to a better understanding of the mechanisms underlying perinatal HI and enables analysis of further proteins acting up- or downstream in these pathways and, subsequently, identification of interfering strategies. Thus, the 2D-DIGE-based proteome analysis was essential to elucidate the complex mechanisms of perinatal brain injury and, thereby, to identify new molecular targets of pharmacological interventions for potential delayed therapy.

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AUTHOR INFORMATION

Corresponding Author

*Address: Department of Functional Proteomics, RuhrUniversity Bochum, Universitaetsstrasse 150, D-44801 Bochum, Germany. Phone: +49 234 3229281. Fax: +49 234 32144496. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Frederic Brosseron, Kathy Pfeiffer, and Cornelia Schumbrutzki for their excellent technical assistance. This study was supported by a grant of the Stem Cell Network North Rhine Westphalia, Germany (to C. Meier) and by the Medical Faculty of Ruhr-University Bochum, Germany (FoRUM). C. May was supported by the European Regional Development Fond (ERDF) of the European Union and the “Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen” (ParkChip, FZ 280381102) and was funded from P.U.R.E. (Protein Unit for Research in Europe), a project of North Rhine Westphalia, a federal state of Germany.

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ABBREVIATIONS Darpp-32, dopamine- and cAMP-regulated phosphoprotein 32 MW; GFAP, glial fibrillary acidic protein; HI, hypoxic− ischemic brain injury; L, left/lesioned hemispheres; R, right/ control hemispheres

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Full list of the identified proteins; and 2D-DIGE gel showing all significantly altered spots (arrows) as identified with the DeCyder software. This material is available free of charge via the Internet at http://pubs.acs.org. 5801



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