Comparative proteomics and correlated signaling network of rat ...

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DOI 10.1002/pmic.200700514

Proteomics 2008, 8, 582–603

RESEARCH ARTICLE

Comparative proteomics and correlated signaling network of rat hippocampus in the pilocarpine model of temporal lobe epilepsy Xin-Yu Liu, Jin-Liang Yang, Li-Juan Chen*, Ying Zhang, Ming-Li Yang, Yong-Yang Wu, Fu-Qiang Li, Ming-Hai Tang, Shu-Fang Liang and Yu-Quan Wei State Key Laboratory of Biotherapy, West China Hospital and College of Life Sciences, Sichuan University, Chengdu, China

In temporal lobe epilepsy (TLE), the seizure origin typically involves the hippocampal formation. The pilocarpine-induced TLE provides a model to investigate the molecular and functional characterization of epileptogenesis by mimicking the human epileptic condition. Here, we employed a 2-D gel-based proteomic technique to profile proteome changes in the rat hippocampus after pilocarpine treatment. Using MALDI MS and MS/MS, 57 differentially expressed proteins were identified, which were found either up-regulated and/or down-regulated at the two time points 12 h (acute period; Ap) and 72 h (silent period; Sp) compared with the control. These proteins can be related to underlying mechanism of pilocarpine-induced TLE, indicating cytoskeleton modification, altered synaptic function, mitochondrial dysfunction, changed ion channel, and chaperone. Five of the identified proteins, synaptosomal-associated protein 25 (SNAP25), synapsin-2 (SYN2), homer protein homolog 2 (HOMER2), a-internexin (INA), and voltage-dependent anion channel 2 (VDAC2) were investigated by semiquantitative RT-PCR, and SNAP25 and INA were further validated by Western blot and immunohistochemistry staining. Furthermore, association of these pilocarpine-induced proteins with biological functions using the Ingenuity Pathway Analysis (IPA) tool showed that nucleic acid metabolism, system development, tissue and cell morphology were significantly altered. IPA of the canonical networks indicated that six membrane proteins (e.g., SNAP25, SYN2, and HOMER2) participated in three biological networks as starting proteins. Our results offer a clue to identify biomarkers for the development of pharmacological therapies targeted at epilepsy.

Received: May 31, 2007 Revised: November 2, 2007 Accepted: November 2, 2007

Keywords: Hippocampus / MALDI-Q-TOF / Pilocarpine / Signaling network / Temporal lobe epilepsy

Correspondence: Dr. Jin-Liang Yang, State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Keyuan Road 4, No. 1, Chengdu, Sichuan 610041, China E-mail: [email protected] Fax: 186-28-85164060 Abbreviations: Ap, acute period; GAPDH, glyceraldehyde-3phosphate dehydrogenase; HOMER2, homer protein homolog 2; IF, intermediate filament; INA, a-internexin; i.p., intraperitoneally; IPA, Ingenuity Pathway Analysis; MALDI-Q-TOF, MALDI quadrupole TOF; SE, status epilepticus; SNAP25, synaptosomalassociated protein 25; Sp, silent period; SRS, spontaneous recurrent seizure; SYN2, synapsin-2; TLE, temporal lobe epilepsy; VDAC2,voltage-dependent anion channel 2

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1

Introduction

Temporal lobe epilepsy (TLE), the most common type of epilepsy in adult humans, is not only characterized clinically by the progressive development of spontaneous recurrent seizures (SRSs) from temporal lobe foci, but also characterized pathologically by unique morphological alterations in the hippocampus [1]. Because a favorable response to unilateral temporal lobe resection is observed in .75% of patients and the typical pathologic abnormality is hippo-

* Additional corresponding author: Dr. Li-Juan Chen, E-mail: [email protected]

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campal neuronal loss and gliosis [2], and moreover hippocampal sclerosis is found in the TLE patients that is recapitulated in animal models of TLE [3], much attention has been paid to the epileptogenic hippocampus. Not only the role of hippocampal neurogenesis in physiological and pathological conditions has been intensely investigated [4], but also several mechanisms that are responsible for hippocampal injury and subsequent epileptogenesis have been proposed, which include events that are largely a consequence of activation of the N-methyl-D-aspartate (NMDA) receptor [5, 6] and the metabotropic glutamate receptors (mGluRs) [7–9]. In the pilocarpine model of epilepsy in rats, epileptogenesis is triggered by status epilepticus (SE) that is induced chemically using a cholinergic substance, pilocarpine [10]. Prolonged SE in rats causes neuronal death, hippocampal mossy fiber sprouting, and SRSs. This model replicates several features of human TLE, including the similarities in pathology, behavioral abnormalities, and the occurrence of both partial and generalized seizures [11]. Hence, the pilocarpine model of epilepsy has been considered one of the best models for studies of the relationships between epilepsy and hippocampus [6]. In this model, the pilocarpine paradigm can be divided into three distinct periods: (i) an acute period (Ap; 6–24 h) after pilocarpine administration, characterized by a limbic SE; (ii) a silent period (Sp) with a progressive normalization of behavior that lasts ,15 days; and (iii) a chronic period with SRSs [12]. Despite of a long time study at the physiological, cellular, and molecular level in epilepsy, its pathogenesis remains poorly understood. Moreover, the molecular mechanism and the pathways underlying the induction of seizures are unclear, and comprehensive protein expression analysis of hippocampus in these three distinct periods has not been performed as yet. Therefore, it is necessary to profile the protein expression patterns in the rat hippocampus in the different period after pilocarpineinduced epilepsy using proteomics technology. Proteomics technology provides a powerful tool for us to elucidate complex biological mechanisms, and to find proteins changed by cell, tissue or organism’s response to internal states, external stimulations or developmental changes and to profile the differential protein expression. The information offered by proteomics provides context-based understanding of protein networks and has been proven to be a valuable approach [13, 14]. Currently, it has been widely employed in the area of disease research, particularly to reveal disease relevant biomarkers. So far, many proteomic studies were involved in hippocampus [15–19], however, only few reports of proteomic studies were available regarding the epilepsy and the epileptic hippocampus [20–26]. In order to get insight into the protein expression profile of rat hippocampus after pilocarpine treatment, and to further explore the molecular mechanisms and specific regulatory networks that are important during the development of epilepsy, we first reported the change patterns of hippocampal proteome in the two periods (Ap and Sp) by proteomics approach and correlated networks by Ingenuity Path© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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way Analysis (IPA) against a curated database. To further validate proteomic data, we performed semiquantitative RTPCR, Western blot, and immunohistochemistry analysis on a set of selected protein genes or proteins targets. In conclusion, the changed expression patterns of the hippocampus proteins may help to generate novel mechanistic insight into the pathogenesis of TLE. The ultimate goal is to develop clinically relevant biomarkers for epilepsy.

2

Materials and methods

2.1 Chemical and drugs Immobiline Dry-Strips (17 cm, 3–10 NL), IPG buffer, DryStrip cover fluid, urea, thiourea, ammonium bicarbonate, and 2-D SDS-PAGE standards were purchased from BioRad (Hercules, CA, USA). DTT, TFA, and iodoacetamide (IAA) were obtained from Merck (Darmstadt, Germany). CHAPS, glycerol, agarose, ammonium persulfate, glycine, acrylamide, Bis, TEMED, SDS, Tris base, and CBB R-250 were obtained from Amresco (Solon, OH, USA). Pilocarpine, trypsin (sequencing grade) and protease inhibitors were obtained from Sigma Chemical (St. Louis, MO, USA). ACN and methanol were from Fisher Chemicals (NJ, USA). MilliQ water (Millipore, Bedford, MA). Other chemicals are domestic products (analytical grade). 2.2 Animals Thirty-six male Sprague–Dawley rats (weight 250–300 g, obtained from the Experimental Animal Center, Sichuan University) were used in this study. After arriving at our department, rats were housed in groups of two per cage under controlled conditions (temperature: 20 6 27C; humidity: 55 6 5%), under a 12 h light–dark cycle with lights on at 6.00 a.m. The rats were given free access to food and water, and they were allowed to adapt to laboratory conditions for at least one week before starting the experiments. In the handling and care of all animals, all possible steps were taken to avoid animals’ suffering at each stage of the experiment and efforts were made to use the minimum number of animals. All procedures were approved by the Institutional Animal Care and Use Committee of Sichuan University and Project of Sichuan Animal Experiment Committee, License 045, China. 2.3 Pilocarpine treatment Pilocarpine was administered according to Cavalheiro [27]. Briefly, the rats were pretreated with methyl-scopolamine bromide (1 mg/kg, intraperitoneal; i.p.) 30 min before administration of pilocarpine. Methyl-scopolamine bromide was administered as a standard procedure to counteract the peripheral cholinergic effects, i.e., to diminish the mortality rate due to respiratory insufficiency (bronchospasm and www.proteomics-journal.com

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exaggerated excretion in the brochial). Then, the experimental rats (n = 24) were injected (i.p.) with a single dose of pilocarpine at 350 mg/kg (diluted in sterile saline), and the control rats (n = 12) were injected with the same volume of saline instead of pilocarpine. Continuous seizure activity for over 30 min (SE) was defined as an effective response to pilocarpine. 2.4 Sample preparation Two groups of 18 rats each (12 pilocarpine-injected rats and 6 control rats) were sacrificed under deep flurothane anesthesia at 12 and 72 h after pilocarpine-induced SE, respectively. The brains were removed from the skull and the hippocampus tissues were rapidly dissected and immediately frozen on dry ice and stored in an 2807C freezer. Hippocampus was homogenized with a glass/Teflon homogenizer in 0.5 mL of 20 mM Tris, 7 M urea, 2 M thiourea, 4% w/v CHAPS, 10 mM DTT, 1 mM EDTA, 1 mM PMSF, and phosphatase inhibitors (0.2 mM Na2VO3 and 1 mM NaF), and centrifuged at maximum speed for 10 min. The obtained supernatants containing proteins were then precipitated with acetone (1:4, overnight, 2207C followed by centrifugation at 12 0006g, 5 min, at 47C). After removing residual acetone by air-drying, the protein pellets were suspended in urea buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 40 mM Tris base, 65 mM DTT, 0.5% Triton X-100) and a cocktail of protease inhibitors (0.7 g/mL pepstain A, 0.5 g/mL leupeptin, 0.3 mg/mL EDTA, and 100 g/mL PMSF), and centrifuged at 40 0006g for 30 min at 47C. The supernatant was recovered and stored at 2707C prior to 2-DE. Protein concentrations of the supernatant were determined by protein Assay Reagent (BioRad, Richmond, CA, USA). 2.5 2-DE For 2-DE, a total of 12 rats was used including 4 control rats, 4 rats from the Ap and 4 rats from the Sp after pilocarpineinduced SE. Samples (1 mg for preparative gels) were diluted in rehydration buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 40 mM Tris base, 65 mM DTT, 0.5%v/v ampholyte pH 3–10 with a trace of bromophenol blue) to give a final volume of 350 mL, and were applied on immobilized 17 cm pH 3–10 nonlinear gradient strips by in-gel passive rehydration for 12 h. IEF was run subsequently for 1 h at 200 V, 1 h at 500 V and 1 h at 1000 V; then a gradient was applied from 1000 to 8000 for 1 h and finally at 8000 V for 8 h to reach a total of 69 kV?h. All IEFs were carried out at 207C. Following IEF separation, the gel strips were incubated with gentle shaking in an equilibration solution (6 M urea, 2%SDS, 375 mM Tris pH 8.8, 20% glycerol, 2% DTT with a trace of bromophenol blue) for 15 min and then the strips were put in the same buffer containing 2.5% IAA instead of DTT for 15 min again. The equilibrated gel strips were placed on the top of a 12.5%T slab gels and sealed with 0.5% agars pre© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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pared in running buffer, SDS-PAGE was performed for 30 min at a constant current of 10 mA and then at 25 mA until the bromophenol blue reached the bottom of the gels. Immediately after the second dimension run, gels were fixed for 18 h in 50% methanol containing 10% acetic acid. The gels were then stained with CBB for 12 h on rocking shaker. Excess dye was washed out from the gel with distilled water. The 2-DE was performed on each hippocampus individually, and the experiments were replicated four times. 2.6 2-D gel image analysis and statistics The 12 gels were scanned with the Images Scanner GS800 (BioRad) at 300 dpi resolution. Spot detection, quantification, and comparison of 2-D protein patterns were done with the PDQuest software (version 7.2, BioRad). The comparison report of quantitative differences between the gel images of the controls and the pilocarpine-induced groups was then generated. From the several options of PDQuest normalization, the intensities of spots were normalized to the total intensity of all matched spots within each gel. The student’s t-test was applied to compare the spot relative volume (% vol, the ratio of the volume of a spot to the volume of the spots from the entire gel) in gels derived from individual control and pilocarpine-induced animal. The relative volume (% vol) of the variably expressed proteins spots were figured out and the mean % vol ratios of these spots and SDs were provided to elucidate the expression change in Tables 1 and 2. Significant spots that showed changed consistently and at least 2.0fold difference in % vol between the groups were selected for protein identification. 2.7 In-gel protein digestion To identify the protein spots of interest, preparative 2-DE gels were manually excised with pipette tips. The gel pieces were destained at room temperature in 50 mM NH4HCO3 buffer, pH 8.8, containing 50% ACN for 1–2 h, and dehydrated with 100% ACN. The shrunken gel pieces were reswelled in 50 mM NH4HCO3 buffer, dehydrated again in 100% ACN, and dried in a vacuum centrifuge (Concentrator 5301, Eppendorf, Hamburg, Germany) for a few minutes. For gel pieces that were heavily stained, the rehydration/dehydration step was repeated once. The gel pieces were rehydrated in 10 mL trypsin solution (50 mM NH4HCO3, pH 8, containing 12.5 mg/mL) for 1 h, followed by addition of 10 mL 50 mM NH4HCO3 buffer to completely immerse the gel pieces. After incubation overnight at 377C, 0.5 mL incubation buffer was mixed with 0.5 mL matrix solution (CHCA, 2 mg/mL in 50% ACN, and 0.5% TFA) and pipetted directly onto the stainless steel sample plate of the mass spectrometer. The samples were analyzed by MALDI-Q-TOF MS and MS/MS analysis. In cases where the MS signals were weak, the peptides were enriched by C18 ZipTip (Millipore). The bound peptides were eluted from the ZipTip using 1 mL CHCA, which was directly deposited onto the metal plate [28]. www.proteomics-journal.com

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Table 1. Identification of the differentially expressed proteins in rat hippocampus at the time point 12 h in the Ap in the pilocarpine model of TLE by MALDI-Q-TOF MS/MS

Spot Protein Gene no. ID name

Protein name

MS/MS Sequence Peptides Theoretical score coveragea) matched pI/Mr (Da) (%)

SDc) Fold changeb)

MS/MS sequence datad)

Down-regulated proteins 1

P31000

VIM

Vimentin

48

5

2

5.06/53601.54 2.03*

60.429

2

P68136

ACTA1

a-Actin-1

39

10

3

5.23/42051.03 5.83**

60.099

3

P04691

TUBB2B

Tubulin beta-2B chain

67

7

3

4.79/49937.06 2.17*

60.104

4

P07323

ENO2

g-Enolase

48

9

3

5.03/47009.34 2.15*

60.613

5

P49432

PDHB

8

2

5.94/38848.08 3.67*

60.815

6

P60711

ACTB

Pyruvate dehydrogenase 75 E1 Actin, cytoplasmic 1 132 (beta-actin) a-Enolase 38 60 kDa heat shock protein 40

7

2

5.29/41736.73 2.01*

60.857

4 6

1 2

6.16/46996.67 3.75** 5.91/60955.49 4.71**

60.285 60.027

8#A P04764 10#A P63039

ENO1 HSPD1

11

P05065

ALDOA

Fructose-bisphosphate aldolase A

46

20

5

8.40/39220.75 2.06*

60.546

12

P60892

PRPS1

Ribose-phosphate pyrophosphokinase I SNAP-25

38

9

2

6.56/34703.04 2.69*

60.941

205

31

5

4.66/23528.08 2.07*

60.031

Lactoylglutathione lyase

53

24

3

5.12/20688.42 4.84**

60.143

59 154

8 35

1 5

5.77/17157.29 2.31* 5.12/23407.41 2.52**

60.501 60.073

13#B P60881

SNAP25

14#B Q6P7Q4 GLO1

15 P13668 STMN1 Stathmin 16#A Q5M9P6 ARHGDIA Rho GDP-dissociation inhibitor 1

17#A Q05982

NME1

Nucleoside diphosphate kinase A

97

31

4

5.96/17192.74 3.11**

60.033

18

P41498

ACP1

17

2

6.10/18020.42 2.77*

60.458

19

Q9EQX9 UBE2N

Low molecular weight 50 phosphotyrosine protein phosphatase Ubiquitin-conjugating 62 enzyme E2

30

3

6.13/17123.79 2.39*

61.07

Hemoglobin subunit alpha-1/2

21

2

7.93/15197.34 2.23*

60.684

20#A P01946

HBA2

112

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

R.TNEKVELQELNDR.F R.ISLPLPNFSSLNLR.E R.AVFPSIVGRPR.H K.SYELPDGQVITIGNER.F K.QEYDEAGPSIVHR.K R.FPGQLNADLR.K K.LAVNMVPFPR.L R.LHFFMPGFAPLTSR.G R.AAVPSGASTGIYEALELR.D R.IEEELGEEAR.F R.FAGHNFRNPSVL R.IMEGPAFNFLDAPAVR.V K.TYYMSAGLQPVPIVFR.G K.SYELPDGQVITIGNER.F K.QEYDESGPSIVHR.K R.AAVPSGASTGIYEALELR.D K.ISSVQSIVPALEIANAHR.K R.KPLVIIAEDVDGEALSTLVLNR.L -.PHPYPALTPEQK.K K.ADDGRPFPQVIK.S K.IGEHTPSSLAIMENANVLAR.Y K.FSNEEIAMATVTALR.R K.CPLLKPWALTFSYGR.A R.NCTIVSPDAGGAK.R R.VYAILTHGIFSGPAISR.I R.TLVMLDEQGEQLER.I K.AWGNNQDGVVASQPAR.V R.VVDEREQMAISGGFIR.R R.EQMAISGGFIR.R R.ENEMDENLEQVSGIIGNLR.H K.DFLLQQTMLR.I K.FSLYFLAYEDKNDIPK.D R.GFGHIGIAVPDVYEACKR.F R.ASGQAFELILSPR.S K.SIQEIQELDKDDESLR.K R.VAVSADPNVPNVIVTR.L K.QSFVLKEGVEYR.I K.IDKTDYMVGSYGPR.A R.AEEYEFLTPMEEAPK.G R.TFIAIKPDGVQR.G K.DRPFFSGLVK.Y R.VMLGETNPADSKPGTIR.G R.GDFCIQVGR.N R.IDSAATSTYEVGNPPDYR.G K.HGIHMQHIAR.Q R.YFHVVIAGPQDSPFEGGTFK.L K.LELFLPEEYPMAAPK.V K.SNEAQAIETAR.A K.IGGHGGEYGEEALQR.M K.TYFSHIDVSPGSAQVK.A

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Table 1. Continued

SDc) Fold b) change

MS/MS sequence datad)

Spot Protein Gene no. ID name

Protein name

MS/MS Sequence Peptides Theoretical score coveragea) matched pI/Mr (Da) (%)

21

P39069

AK1

Adenylate kinase isoenzyme 1

124

27

4

7.72/21601.72 2.07*

60.17

K.YGYTHLSTGDLLR.A K.GELVPLETVLDMLR.D K.VDSSNGFLIDGYPR.E K.ATEPVISFYDKR.G

22

P37805

TAGLN3

Transgelin-3

89

20

3

6.53/24712.19 2.81*

60.701

23

P47727

CBR1

NADPH-dependent carbonyl reductase 1 GST Mu 1

55

8

2

8.21/30446.93 2.11*

60.374

98

24

4

8.42/25782.76 2.97**

60.336

Hemoglobin subunit beta-1

61

19

3

7.99/15848.19 3.72**

60.37

K.LVDWIILQCAEDIEHPPPGR.T R.GEPSWFHR.K R.RGFSEEQLR.Q R.FHQLDIDNPQSIR.A K.ELLPIIKPQGR.V R.LLLEYTDSSYEEKR.Y K.LGLDFPNLPYLIDGSR.K K.CLDAFPNLKDFLAR.F R.LLVVYPWTQR.Y K.VINAFNDGLK.H K.LHVDPENFR.L

Neurofilament triplet M protein

57

7

4

4.77/95659.98 2.00*

61.19

25#A P04905

GSTM5

26#B P02091

HBB

Up-regulated proteins 27

P12839

NEF3

28

P34058

HSP90AB1 Heat shock protein HSP 90-beta

44

5

3

5.06/83185.13 2.42*

60.213

29 30

O35276 Q07439

NRP2 HSPA1B

Neuropilin-2 precursor Heat shock 70 kDa protein 1A/1B

29 74

1 11

1 6

5.14/103831 2.08* 5.60/70185.31 2.10*

60.297 60.704

31

Q9WTL3 SEM6C

Semaphorin-6C precursor

32

2

2

8.11/102546

2.05*

60.948

32

Q6P9V9 TUBA3

Tubulin alpha-2 chain

75

9

3

4.94/50151.63 2.03*

60.186

33

P68370

TUBA1

Tubulin alpha-1 chain

68

9

3

4.94/50135.63 2.03*

60.225

34

Q63081

PDIA6

Protein disulfide44 isomerase A6 precursor

10

3

5.00/48173.44 2.13*

60.093

HOMER2

45 65

3 34

1 3

5.76/40563.15 2.90** 6.15/13002.08 2.29**

60.37 60.742

35#A O88801 36#A P80254

HOMER2 DDT

D-dopachrome

decarboxylase P11980

PKM2

Pyruvate kinase isozymes M1/M2

92

15

5

6.69/57686.60 2.03*

60.328

38#A Q63537

SYN2

SYN2

67

4

3

8.73/63456.72 9.13**

60.027

37

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

K.QASHAQLGDAYDQEIR.E K.AQVQLDSDHLEEDIHR.L K.VQSLQDEVAFLR.S R.FSTFSGSITGPLYTHR.Q K.VILHLKEDQTEYLEER.R K.HFSVEGQLEFR.A R.GVVDSEDLPLNISR.E R.IANEQISASSTFSDGR.W K.LLQDFFNGR.D R.FELSGIPPAPR.G K.DAGVIAGLNVLR.I R.LVSHFVEEFKR.K R.TTPSYVAFTDTER.L R.IINEPTAAAIAYGLDR.T R.SCLASLDPYCGWHR.F R.RDLSPASASR.S R.NLDIERPTYTNLNR.L R.LISQIVSSITASLR.F R.IHFPLATYAPVISAEK.A R.NLDIERPTYTNLNR.L R.LIGQIVSSITASLR.F R.IHFPLATYAPVISAEK.A R.TGEAIVDAALSALR.Q K.IFQKGESPVDYDGGR.T K.FALLKGSFSEQGINEFLR.E K.ASHASPADTHLK.S -.PFVELETNLPASR.I R.LCAATATILDKPEDR.V K.FLTEELSLDQDR.I R.NTGIICTIGPASR.S R.LNFSHGTHEYHAETIK.N R.AATESFASDPILYRPVAVALDTK.G R.RFDEILEASDGIMVAR.G K.CLAAALIVLTESGR.S R.SFRPDFVLIR.Q

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Table 1. Continued

Spot Protein Gene no. ID name

Protein name

MS/MS Sequence Peptides Theoretical score coveragea) matched pI/Mr (Da) (%)

39

SDc) Fold b) change

MS/MS sequence datad)

P10719

ATP5B

ATP synthase subunit beta, mitochondrial precursor

192

17

7

5.18/56353.55 2.17*

60.694

R.LVLEVAQHLGESTVR.T K.AHGGYSVFAGVGER.T R.VALTGLTVAEYFR.D R.DQEGQDVLLFIDNIFR.F R.DQEGQDVLLFIDNIFR.F R.FTQAGSEVSALLGR.I R.AIAELGIYPAVDPLDSTSR.I

40#A P81155

VDAC2

4

2

7.44/31745.82 2.04*

60.431

K.LTFDTTFSPNTGK.K K.LTFDTTFSPNTGKK.S

41

P16086

SPTAN1

Voltage-dependent 31 anion-selective channel protein 2 (VDAC-2) Spectrin alpha chain 48

2

4

5.20/284637.5 2.17*

60.885

42

Q04940

NRGN

Neurogranin

62

19

2

6.53/7496.31

INA

INA

64

4

2

5.20/56115.38 3.49**

60.217

RNA-binding protein Musashi homolog 1 Cytochrome c oxidase subunit VIb isoform 1

30

3

1

7.71/39133.53 4.08**

60.439

R.DLAALGDKVNSLGETAQR.L K.HQAFEAELHANADR.I R.SSLSSAQADFNQLAELDR.Q R.ELPTAFDYVEFTR.S R.KGPGPGGPGGAGGAR.G K.GPGPGGPGGAGGAR.G R.ALEAELAALR.Q K.KVESLLDELAFVR.Q K.EVMSPTGSARGR.S

39

17

1

8.96/10071.45 7.35**

60.146

K.TAPFDSRFPNQNQTK.N

50#B P23565

51#B Q8K3P4 MSI1 56#B P56391

#

a) b)

c) d)

COX6B1

2.03*

60.35

Indicates overlapping proteins with significant changes in the two periods (Ap and Sp) compared with the control (N); #A indicates the changed fold in Ap is more than that in Sp; #B indicates the changed fold in Sp is more than that in Ap. Sequence coverage indicates the relative amount of protein sequence covered by the peptides with matching masses, of which came from the top ten precursor ions peptides in abundance in the full scan by MALDI-Q-TOF MS. The fold change column correspond to the expression of each protein in Ap to its expression in N. Results are means of four independent experiments. The mean spot volume % ratios between Ap and N. Up-regulated proteins marked in Fig. 1B, down-regulated proteins marked in the Fig. 1A. *, p,0.05; **, p,0.01. The SD of spot volume % ratios between Ap and N. Peptides matched by using MASCOT MS/MS ion search.

2.8 MALDI-Q-TOF MS The sample–matrix mixture (1.0 mL) was spotted onto the MALDI sample stage and air-dried. Positive ion MALDI QTOF mass spectra were acquired with a Q-TOF Premier Mass Spectrometer (Waters Micromass, Milford, MA, USA) equipped with a standard MALDI source. Ionization was achieved using a nitrogen laser (337 nm) and acquisitions were performed in a V mode. Standard calibration peptide (Glu-fibrinopeptide, m/z 1570.6774) was applied to the MALDI plate as external calibration of the instrument and internal calibration using either trypsin autolysis ions or matrix was applied post acquisition for accurate mass determination. MS spectra were accumulated until a satisfactory S/N had been obtained. MS/MS was performed in a data-dependent mode in which the top ten most abundant ions (threshold 15 counts) for each MS scan were selected for MS/ © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

MS analysis. These parent ions in the mass range from 700 to 3000 m/z were selected to produce MS/MS ion spectra by CID. The collision voltage varied between 34 and 161 eV depending on the mass of the precursor ion. The MS and MS/MS data were acquired and processed using MassLynx 4.1 software (Waters). In a MALDI Survey scan, only one MS scan is performed. Consequently all the information used to extract peak information, which is used to create the MS/MS peak list, must be generated from one combined spectrum. 2.9 Protein identification and database searching The MS/MS data, “peak list” (PKL) files acquired by MassLynx 4.1 software, include the mass values, the intensity, and the charge of the precursor ions (parent ions with 11 charge in this study). These PKL files were analyzed with a licensed copy of the MASCOT 2.0 program (MatrixScience, www.proteomics-journal.com

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Table 2. Identification of the differentially expressed proteins in rat hippocampus at the time point 72 h in the Sp in the pilocarpine model of TLE by MALDI-Q-TOF MS/MS

Spot Protein no. ID

Gene name

Protein name

Down-regulated proteins 7 P08461 DLAT Dihydrolipoamide acetyltransferase

8#A P04764 9 P10860

ENO1 GLUD1

a-Enolase Glutamate dehydrogenase 1

10#A P63039

HSPD1

60 kDa heat shock protein

13#B P60881

SNAP25

SNAP-25

14#B Q6P7Q4 GLO1

Lactoylglutathione lyase

16#A Q5M9P6 ARHGDIA Rho GDP-dissociation inhibitor 1

17#A Q05982

NME1

Nucleoside diphosphate kinase A

20#A P01946

HBA2

Hemoglobin subunit alpha-1/2 Proteasome subunit Z

24

Q9JHW0 PSMB7

MS/MS Sequence Peptides Theoretical score coveragea) matched pI/Mr (Da) (%)

SDc) Fold changeb)

115

14

5

5.70/58764.14 2.01*

60.621

38 115

4 11

1 4

6.16/46996.67 3.17** 8.05/61415.93 2.35*

60.049 60.578

40

6

2

5.91/60955.49 3.94**

60.128

205

31

5

4.66/23528.08 2.14*

60.019

53

24

3

5.12/20688.42 5.51**

60.243

154

35

5

5.12/23407.41 2.37**

60.153

97

31

4

5.96/17192.74 2.84**

60.074

112

21

2

7.93/15197.34 2.17*

60.571

41

7

2

8.14/29927.46 1.83*

60.124

25#B P04905

GSTM5

GST Mu 1

98

24

4

8.42/25782.76 3.44**

60.536

26#A P02091

HBB

Hemoglobin subunit beta-1

61

19

3

7.99/15848.19 3.29**

60.498

Up-regulated proteins 35#A O88801 HOMER2 HOMER2 36#A P80254 DDT D-dopachrome decarboxylase

45 65

3 34

1 3

5.76/40563.15 2.45** 6.15/13002.08 2.08**

60.838 60.065

38#A Q63537 40#A P81155

SYN2 VDAC2

67 31

4 4

3 2

8.73/63456.72 8.62** 7.44/31745.82 2.01*

60.695 60.029

43

HSPAIL

39

7

4

5.91/70549.18 2.35*

60.479

P55063

SYN2 Voltage-dependent anion-selective channel protein 2 (VDAC-2) Heat shock 70 kDa protein 1L

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

MS/MS sequence datad)

K.AAPAAAAAAPPGPR.V R.DVPLGTPLCIIVEK.Q K.VPLPSLSPTMQAGTIAR.W R.VAPTPAGVFIDIPISNIR.R K.GFDVASVMSVTLSCDHR.V R.AAVPSGASTGIYEALELR.D K.IIAEGANGPTTPEADKIFLER.N K.NLNHVSYGR.L R.DSNYHLLMSVQESLER.K K.HGGTIPVVPTAEFQDR.I K.ISSVQSIVPALEIANAHR.K R.KPLVIIAEDVDGEALSTLVLNR.L R.TLVMLDEQGEQLER.I K.AWGNNQDGVVASQPAR.V R.VVDEREQMAISGGFIR.R R.EQMAISGGFIR.R R.ENEMDENLEQVSGIIGNLR.H K.DFLLQQTMLR.I K.FSLYFLAYEDKNDIPK.D R.GFGHIGIAVPDVYEACKR.F K.SIQEIQELDKDDESLR.K R.VAVSADPNVPNVIVTR.L K.QSFVLKEGVEYR.I K.IDKTDYMVGSYGPR.A R.AEEYEFLTPMEEAPK.G R.TFIAIKPDGVQR.G K.DRPFFSGLVK.Y R.VMLGETNPADSKPGTIR.G R.GDFCIQVGR.N K.IGGHGGEYGEEALQR.M K.TYFSHIDVSPGSAQVK.A K.DGIVLGADTR.A K.LDFLRPYSVPNK.K R.LLLEYTDSSYEEKR.Y K.LGLDFPNLPYLIDGSR.K K.CLDAFPNLKDFLAR.F R.LLVVYPWTQR.Y K.VINAFNDGLK.H K.LHVDPENFR.L

K.ASHASPADTHLK.S -.PFVELETNLPASR.I R.LCAATATILDKPEDR.V K.FLTEELSLDQDR.I R.SFRPDFVLIR.Q K.LTFDTTFSPNTGK.K K.LTFDTTFSPNTGKK.S R.FDLTGIPPAPR.G K.DAGVIAGLNVLR.I R.LVSHFVEEFKR.K R.TTPSYVAFTDTER.L

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Table 2. Continued

Gene name

Protein name

44

P39052

DNM2

Dynamin-2

36

5

3

7.02/98230.38 2.71*

61.03

45

P21575

DNM1

Dynamin-1

117

6

5

6.32/95927.48 3.20*

60.874

46

P02770

ALB

Serum albumin precursor

140

15

8

6.09/68718.74 3.03*

60.002

47

P15999

ATP5A1

79

2

2

9.22/59753.63 2.04*

60.448

48

P26284

PDHA1

ATP synthase subunit alpha Pyruvate dehydrogenase E1 component alpha subunit Cytokeratin-8 INA

45

9

3

8.49/43212.55 2.31*

60.391

32 64

2 4

1 2

5.82/53887.56 2.10* 5.20/56115.38 3.99**

60.837 60.149

RNA-binding protein Musashi homolog 1 3-hydroxyacyl-CoA dehydrogenase type-2

30

3

1

7.71/39133.53 4.93**

60.672

86

20

4

8.91/27114.38 5.71*

60.041

49 Q10758 50#B P23565

KRT8 INA

51#B Q8K3P4 MSI1

MS/MS Sequence Peptides Theoretical score coveragea) matched pI/Mr (Da) (%)

SDc) Fold b) change

Spot Protein no. ID

MS/MS sequence datad)

K.SSVLENFVGRDFLPR.G K.GISPVPINLR.V K.TLNQQLTNHIR.E K.SSVLENFVGRDFLPR.G K.GISPVPINLR.V K.VPVGDQPPDIEFQIR.D K.VLNQQLTNHIR.D K.VLNQQLTNHIRDTLPGLR.N K.DVFLGTFLYEYSR.R R.RHPDYSVSLLLR.L K.APQVSTPTLVEAAR.N R.TGAIVDVPVGDELLGR.V R.LEEGPPVTTVLTR.E K.LPCIFICENNR.Y R.YHGHSMSDPGVSYR.T K.LALDIEIATYR.K R.ALEAELAALR.Q K.KVESLLDELAFVR.Q K.EVMSPTGSARGR.S

52

O70351

HADH2

53 54

P05942 P84817

S100A4 FIS1

Protein S100-A4 Mitochondrial fission 1 protein

51 60

8 23

1 3

5.04/11997 2.50* 8.55/16994.62 3.55**

60.713 60.227

55 P29419 56#B P56391

ATP5I COX6B1

57 39

21 17

1 1

9.35/8123.46 2.14* 8.96/10071.45 9.47**

60.614 60.038

57

CYCS

ATP synthase e chain Cytochrome c oxidase subunit VIb isoform 1 Cytochrome c

K.NQVHTLEDFQR.V R.VINVNLIGTFNVIR.L R.LVAGVMGQNEPDQGGQR.G R.NFLASQVPFPSR.L R.RTDEAAFQK.L K.STQFEYAWCLVR.S R.GIVLLEELLPK.G R.DYVFYLAVGNYR.L R.IERELAEAEDVSIFK K.TAPFDSRFPNQNQTK.N

46

10

1

9.61/11474.24 7.27**

60.168

K.TGPNLHGLFGR.K

P62898

Notations for #, #A, #B, a), and d) in the Table 2 footnote are the same as in the Table1 footnote. b) The fold change column correspond to the expression of each protein in Sp to its expression in N. Results are means of four independent experiments. The mean spot volume % ratios between Sp and N. Up-regulated proteins marked in Fig. 1C, Down-regulated proteins marked in the Fig. 1A. *, p,0.05; **, p,0.01 c) The SD of spot volume % ratios between Sp and N.

London) against Swiss-Prot protein database. Searching parameters were set as follows: enzyme, trypsin; allowance of up to one missed cleavage peptide; the peptide mass tolerance, 1.0 Da and the fragment ion mass tolerance, 0.3 Da; fixed modification parameter, carbamoylmethylation (C); variable modification parameters, oxidation (at Met); auto hits allowed (only significant hits were report); results format as peptide summary report. Proteins were identified on the basis of two or more peptides whose ions scores both exceeded the threshold, p,0.05, which indicates identification at the 95% confidence level for these matched peptides [29]. If proteins were identified by a single peptide, then the spectrum was manually inspected. For a protein to be confirmed: © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

(i) the assignment must be based on four or more y- or b-series ions; (ii) the protein molecular mass must be consistent with gel migration data; and (iii) the result of MS/MS ion search is consistent with that of PMF search. MS spectra as well as MS/MS spectra were searched against Swiss-Prot database by MASCOT software using a peptide tolerance of 0.5 Da (Supporting Information part 1 and 2). 2.10 Semiquantitative RT-PCR For semiquantitative RT-PCR, a total of six rats was used including two control rats, two rats from the Ap and two rats from the Sp after pilocarpine-induced SE. Total RNA was www.proteomics-journal.com

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prepared from the frozen rat hippocampus using TRIzol Reagent kit (Invitrogen, Carlsbad, CA, USA). After chloroform and isopropanol extractions, the RNA pellet was washed with 1 mL of 75% ethanol, centrifuged at 75006g for 5 min at room temperature, and air dried in sterile conditions for 5– 10 min. The RNA pellets were dissolved in 20 mL of diethylpyrocarbonate treated ddH2O and stored at 2807C until further analysis. The quantity of total RNA isolated was determined by absorbance at 260 nm with a spectrophotomer (Beckman Coulter, Fullerton, CA). The first-strand cDNA was synthesized by Superscript III kit (Invitrogen) using 5 mg of total RNA and random oligo-d(T) as primer in a final volume of 20 mL according to the manufacturer’s protocol. This cDNA solution was then stored at 2207C and used as the template for analysis of gene expression by PCR. The rat homer protein homolog 2 (HOMER2), a-internexin (INA), VDAC-2, SNAP25, and SYN-2 primers were designed using primer 5 software (Table 2 in Supporting Information part 3). To avoid the amplification of contaminant genomic DNA, primers from different exons, separated by long introns were used. PCR conditions were as follows: denaturation at 957C for 10 min, followed by 35 cycles of denaturation at 947C for 1 min, annealing at 567C for 1 min and elongation at 727C for 1 min. A final step of elongation at 727C for 10 min was performed and stopped at 47C. The PCR products were visualized following electrophoresis on a 1.5% agarose gel and ethidium bromide staining. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene and as an internal control. The intensity of the bands was determined using the FluorChem software (version 5.0, Alpha Innotech) and normalized to the band intensity for GAPDH. The experiments were repeated three times. 2.11 Western blot The protein quantitation of INA and SNAP-25 were selected to be validated by Western-blot analysis because the expression changes of them were more obvious than that of the other proteins and the obtaining of their antibodies was convenient. A total of nine rats were used including three control rats, three rats from the Ap and three rats from the Sp after pilocarpine-induced SE. Hippocampal tissue samples were firstly precipitated with acetone as described before (Section 2.4). Then protein pellets were suspended in chilled lysis solution containing 50 mM Tris-HCl (pH 8.0), 1% SDS, 1% DTT, and a cocktail of protease inhibitors as listed above, and centrifuged at 40 0006g for 30 min at 47C. At last, the supernatant was recovered, and the protein concentration was determined according to the method above. After protein samples in lysis buffer were mixed with the same volume of Laemmli sample buffer (BioRad) and incubated at 957C for 7 min, 10 mg of each protein sample was load in each well and separated using 12.5% SDS-PAGE in a Mini PROTEAN cell (BioRad), and then proteins were electroblotted onto PVDF membranes (Millipore) by wet blotting (300 mA for 45 min). After incubation in blocking buffer © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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(16TBS, 0.1% Tween-20, and 5% w/v dried skimmed milk) for 1 h at room temperature, membranes were incubated in a 1:500 dilution of primary antibody (mouse monoclonal antibody INA Ab-1 and SNAP-25 Ab-1, Neomarkers, Fremont, CA, USA) overnight at 47C, respectively. Membranes were then washed with TBST and incubated in a 1:2000 dilution of a HRP-conjugated antimouse IgG1 (Southern Biotechonology Associates, Birmingham, AL, USA) for 45 min at room temperature. Bands containing rat hippocampus proteins were visualized using an ECL detection system (Western Lighting™, PerkinElmer Life Science, Boston, MA) on X-ray film. The film signals were digitally scanned and then quantified using FluorChem software. The experiments were performed in triplicate and proteins of a rat hippocampus were used in a Western blot analysis. 2.12 Immunohistochemistry A total of nine rats, including three control rats, three rats from the Ap and three rats from the Sp after pilocarpineinduced SE, were used for immunohistochemistry study. Rats were sacrificed under deep flurothane anesthesia at the time points of 12 and 72 h after pilocarpine-induced SE. Brains were removed and immersed in 4% paraformaldehyde fixative for at least 10 h, and then brains were sliced in 1–2 mm slices, embedded in paraffin blocks and cut into 8 mm sections. For immunohistochemistry processing, sections were deparaffinized, blocked in 5% normal horse serum (NHS, for monoclonal antibody) in 0.3% Triton X-100 and 0.1% NaN3 in PBS for 1 h. The following primary antibodies were used: mouse monoclonal antibody INA Ab-1 and SNAP-25 Ab-1 (Neomarkers) diluted 1:2000 in 1% NHS, 0.3% Triton X-100 and 0.1% NaN3 in PBS. After incubated in primary antibodies, sections were incubated in biotinylated horse antimouse IgG, diluted 1:200 in 1% NHS and 0.1% NaN3 in PBS. Finally, sections were reacted in the avidinbiotin peroxidase complex (ACB, Elite kit, Vector Laboratories, Burlingame, CA), for color development, 3,30 -diaminobenzidine kit (Vector Laboratories) were used as a chromagen, and sections were counterstained with hematoxylin. Sections were then mounted with glass cover slips using PBS/glycerol (1:9). The immunostained tissue sections were viewed under an Olympus Ax-70 DMC Ie CCD camera to a PC monitor. The image of the area containing the hippocampus was captured with DMC Ie Low light software at a 406magnification. The intensity of the immunostain on slides was individually scored by three blinded observers on a scale of 0–4. The scores obtained were not statistically analyzed and were used only to evaluate trends of staining intensity. 2.13 Statistics All experimental data were presented as means 6 SD and analyzed by Student’s t-test using SPSS software (version 13.0, SPSS). Statistical significance was defined as p,0.05. www.proteomics-journal.com

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2.14 Bioinformatic pathways analysis

3.2 Comparative proteomic analysis

Differentially expressed proteins were evaluated by IPA (Ingenuity Systems, Mountain View, CA). IPA constructs hypothetical protein interaction clusters on the basis of a regularly updated “Ingenuity Pathways Knowledge Base (IPKB)”. IPKB is a very large curated database that consists of millions of individual relationships between proteins, culled from the biologic literature [30, 31]. This database also integrates a broad range of systems biology including protein function, cellular localization, and small molecule and disease inter-relationships. The networks are displayed graphically as nodes (individual proteins) and edges (the biologic relationships between the nodes). In practice, a data set that contains the Swiss-Prot number of differentially expressed proteins is uploaded into IPA. IPA then builds hypothetical networks from these proteins, and other proteins from the database that are needed fill out a protein cluster. Proteins in the input proteins mapped to the networks are called “focus proteins”. Network generation ranked by score is optimized for inclusion of as many proteins from the inputted expression profile as possible and aims for highly connected networks. IPA computes a p-score for each possible network according to the fit of that network to the inputted proteins. The p-scores are derived from p-values. Say there are n proteins in the network and f of them are focus proteins. The pvalue is the probability of finding f or more focus proteins in a set of n proteins randomly selected from the Global Molecular Network. It is calculated using Fisher’s exact test. Since interesting p-values are typically quite low (e.g., 1028), it is visually easier to concentrate on the exponent. Therefore, the p-score is defined as: p-score = 2log10 (p-value) and scores of 2 or higher have at least a 99% confidence of not being generated by random chance alone. A p-value cut-off of 0.01 was considered for the analysis in this study.

To gain a better understanding of the molecular mechanisms in the pilocarpine model of TLE, we used a 2-DEbased proteomics approach to determine protein expression profiles in the hippocampus of rats receiving pilocarpine (pilocarpine-treated group) and saline (control group) through i.p. injection. In total, 12 2-DE gels corresponding to 12 animals (4 control rats, both 4 rats of each at 12 and 72 h after pilocarpine treatment) were analyzed simultaneously and matched in the same set. In general, we detected approximately 800–1200 spots in each gel. After a gel had been chosen as a master image that had the largest number of well-resolved and good-quality spots, the spots in the remaining gels were automatically matched using PDQuest software. The image analysis with statistical evaluation revealed that the average number of spots and SD detected on 2-DE gels were 1076 6 151 in the control, 1063 6 179 in the Ap and 1153 6 164 in the Sp, respectively. More than 90% of overlapped rates of 2-DE spots were achieved in these parallel gels from each hippocampus sample. These data indicated that parallel 2-DE images were reproducible and acceptable for differential analysis of 2-DE spots. When 2-DE patterns of the experimental groups were compared with 2-D patterns of the control group by automatically and manual matching, 78 spots were consistently differentially expressed between the normal versus Ap including 37 up-regulated and 41 down-regulated, and 45 spots were consistently differentially expressed between the normal versus Sp including 28 up-regulated and 17 down-regulated (
2-fold change, t-test, p,0.05). As shown in Tables 1 and 2, a total of 57 distinct proteins were finally identified with MALDI-Q-TOF MS and MS/MS analysis corresponding to 41 unique protein and 16 overlapping proteins compared with the control. Among the 41 unique proteins, 26 proteins (14 down-regulated, 12 up-regulated) were significantly altered in the Ap and 15 proteins (3 down-regulated, 12 up-regulated) were significantly altered in the Sp. In the 16 overlapping proteins, 9 down-regulated proteins and 7 up-regulated proteins were significantly changed in the two periods (Ap and Sp), respectively. In total, 42 and 31 proteins were found either up-regulated or down-regulated in the Ap and the Sp, respectively. Figure 1 shows representative reference 2-D gel images for the rat hippocampus. Figure 2 describes overlapping proteins with significant difference comparing Ap and Sp to the normal control such as synaptosomal-associated protein 25 (SNAP25), synapsin-2 (SYN2), HOMER2, INA, and voltagedependent anion channel 2 (VDAC2). Compared with the control, SNAP25 protein was significantly down-regulated (p,0.05) and INA protein was significantly up-regulated (p,0.01) both in the two periods (Ap and Sp) after pilocarpine treatment. However, though SYN2, HOMER2, and VDAC2 proteins were up-regulated, the changed level at 12 h is more than that at 72 h (p,0.05 for VDAC2, p,0.01 for SYN2 and HOMER2).

3

Results

3.1 Animal model For each pilocarpine-treated animal, clinical signs of seizure activity were observed. All rats exhibited a well-defined pattern of behavior after pilocarpine treatment. About 5 min, the animals developed piloerection, diarrhea, and other signs of cholinergic stimulation. In the following 15–20 min, akinesia, ataxic lurching, tremor, head bobbing, masticatory automatisms with myoclonus of facial muscles and wet dog shakes were noted. The rats then progressed to status with episodes of head and bilateral forelimb clonus, rearing, and falling around 25–35 min after injection of pilocarpine. These behavioral changes progressed to motor TLE seizures, and these signs evolved to SE in 90–100% of animals and lasted up to 12 h without remission. Our results from the pilocarpine model of TLE are consistent with the previous study [27]. © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 1. Representative 2-DE gel images of hippocampal protein extracts from the control and the pilocarpine-induced epileptic rats. One milligram of total proteins of each sample was separated by 2-DE on a pH 3–10 nonlinear IPG strip in the first dimension and on a 12.5% SDS-PAGE gel in the second dimension. Gels were stained with CBB. Numbered protein spots were identified using MALDI-Q-TOF MS and MS/MS and correspond to the respective spot number found in Tables 1 and 2. (A) The normal control. (B) At the time point 12 h (Ap). (C) At the time point 72 h (Sp).

3.3 Validation studies It is essential to validate some differentially expressed proteins after pilocarpine treatment using other methods such as semiquantitative RT-PCR or quantitative real-time RTPCR, western blot and/or immunohistochemistry (IHC) analysis. IHC approach is especially powerful, not only to validate the differential expression, but also to demonstrate protein cellular localization and rule out potential contamination. 3.3.1 Semiquantitative RT-PCR for differentially expressed protein identities The differential protein expression pattern in the different period was further studied. We performed semiquantitative RT-PCR analysis of five selected genes to verify whether the differential expression level of corresponding proteins observed in 2-DE gels were paralleled at their transcript level. Figure 3 shows the mRNA expression patterns of the genes for HOMER2, INA, VDAC-2, SNAP25, and SYN2. Comparing Ap and Sp to the normal control, HOMER2 mRNA expression was significantly increased 13- and 10-fold (both p,0.01), respectively; INA mRNA expression was significantly increased 1.7-fold (p,0.05) and 1.9-fold (p,0.01), respectively; SYN2 mRNA expression is significantly increased 2.2-fold (p,0.01) and 1.6-fold (p,0.05), respectively, and VDAC2 mRNA expression is significantly © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

increased approximately 1.7-fold (both p,0.05), respectively. However, SNAP25 mRNA was significantly decreased 1.5fold (p,0.05) in the Sp, and no significantly changes were observed in the Ap (Fig. 3B). The possible reasons are that the expression of SNAP25 was regulated at late stage by mRNA level, or the decrease in protein abundance was not regulated by the transcription of this gene after pilocarpine treatment, though the level of mRNA was down-regulated. 3.3.2 Validation by western blot The differentially expressed proteins identified by 2-DE combined with MALDI MS and MS/MS were further confirmed using Western blot analysis for SNAP25 and INA. Their expression in rat hippocampus at 0 h (the control), 12 and 72 h after pilocarpine treatment was examined. Comparing the treatment group to the control, Fig. 4 demonstrates clearly that SNAP25 was remarkably down-regulated at the two time points 12 h (p,0.05) and 72 h (p,0.01), and the protein expression of INA was increased significantly (both p,0.01). The above results were consistent with 2-DE results. Hence, the results of western blot analysis confirmed the reliability of the proteomic analysis. 3.3.3 Immunohistochemical analysis To obtain more information about cellular location, tissue distribution, and expression level of the identified proteins www.proteomics-journal.com

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and the Sp, with the staining intensity decreasing slightly from the normal to Ap and significantly altering from Ap to Sp. As shown in the magnified images (Fig. 5D), SNAP25 was detected to be located in the cell membrane and distribution in the whole tissue. Figures 5E–G show that INA was detected to be expressed in the normal group, the Ap and the Sp, with the signal significantly increasing from the normal to Ap while not obviously altering between in Ap and in Sp. Figure 5H shows that INA is located in the cytoplasma, and also distributed in the whole hippocampus tissue. Additionally, comparing the treatment groups to the control, Figs. 3B and 4B show also that the trend of expression of these selected genes is consistent between mRNA level and protein level, as well as that of the staining intensity of IHC (Fig. 5). These results agree well with those obtained from 2-DE (Fig. 2B) and mass spectrometric analyses (Tables 1 and 2), and therefore confirming the proteomic data.

Figure 2. A comparative analysis of changes in abundance of hippocampal proteins from the control and the pilocarpineinduced epileptic rats. The images of each changed protein spot were compared with the control. (A) 2-DE gel images of five selected protein indicated by arrows in the panels. Each panel shows an enlarged view of the gel spots from Fig.1 and ranked from the left to the right: the normal control (N), Ap, and Sp. (B) Volume density analysis graphs: the data were expressed as mean 6 SD of four repeats. Significant differences are indicated: *p,0.05, **p,0.01, comparing the Ap to the control; #p,0.05, ##p,0.01, comparing the Sp to the control.

in hippocampus, SNAP25 and INA were selected for immunohistochemical analysis. As shown in Figs. 5A–C, SNAP25 was detected to be expressed in the normal control, the Ap

Figure 3. (A) Identification of the expression patterns of five selected protein genes SNAP25, INA, HOMER2, VDAC-2, and SYN2 in the control and the epileptic rat hippocampus at the two time points 12 h (Ap) and 72 h (Sp) at mRNA levels by semiquantitative RT-PCR. Total RNA was isolated from hippocampus, reverse transcribed, and amplified with specific primers indicated in Table S2. GAPDH was used as an internal control. Densitometry analysis was performed using FluorChem software. (B) The intensity of bands was quantified and normalized by that of GAPDH. The data were expressed as mean 6 SD of three independent experiments. Significant differences are indicated: *p,0.05, **p,0.01, comparing the Ap to the control; #p,0.05, ##p,0.01, comparing the Sp to the control.

Figure 4. Western blot analysis to validate the differential displays for SNAP25 and INA in the control and the epileptic rat hippocampus at the two time points 12 h (Ap) and 72 h (Sp). Total proteins (10 mg/lane) extracted from rat hippocampus were separated by SDS-PAGE for each sample and probed with the primary antibody of these two proteins. Densitometry analysis was performed using FluorChem software. (A) A representative western blot visualizing SNAP25 and INA expression levels. (B) Band density was digitized, and means 6 SD of the triplicate experiments were shown. Significant differences are indicated: *p,0.05, **p,0.01, comparing the Ap to the control; #p,0.05, ##p,0.01, comparing the Sp to the control.

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Figure 5. Representative photographs of immunohistochemical analysis of INA and SNAP-25 protein expressions and subcellular location in the control and the epileptic rat hippocampus at the two time points12 h (Ap) and 72 h (Sp). Formalin-fixed, paraffinembeded sections were stained with mouse mAb INA Ab-1 and SNAP-25 Ab-1. All tissues were counterstained with hematoxylin and viewed by light microscopy. (A–C) INA expression in the normal control hippocampus (A), hippocampus in the Ap (B), and hippocampus in the Sp (C); (E–H) SNAP25 expression in the normal control hippocampus (E) hippocampus in the Ap (F) and hippocampus in the Sp (G). (D) Black arrows in the magnified image indicate that INA is located in the cytoplasm. (H) Black arrows in the magnified image indicate that SNAP25 is located in the cellular membrane.

3.4 Signaling network analysis As for the 57 differentially expressed proteins associated with epilepsy, we conducted them as a group for the biologically related network, signaling and metabolic pathway analysis. We uploaded the set of these proteins with their Swiss-Prot numbers into IPA. The IPA output includes biological network, signaling and metabolic pathways, and a statistical assessment of their significance. In this study, out of 57 differentially expressed proteins, 46 were eligible for network analysis based on the Ingenuity Pathway Knowledge Base criteria. The remaining 11 proteins © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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had no any information available in the database and did not fall into larger networks. A merge network (Fig. 6A), which included 46 focus proteins and 84 association proteins predicted by IPA, and seven biological networks with highly significant scores of 23, 21, 17, 8, 2, 2, and 2, respectively, were created (Table 3). Located within these seven networks were 14, 13, 11, 5, 1, 1, and 1 focus proteins, respectively (Table 3). The highest scoring network 1 (with a score of 23) incorporated 14 focus proteins (Fig. 6B). The most significant functions associated with this network were DNA replication, recombination, and repair, nucleic acid metabolism, small molecule biochemistry. Not surprisingly, the network also predicted involvement of the transcription factor TP53 which has been previously associated with the response to DNA damage and has also been shown to be induced in hippocampus by brain irradiation [32]. As shown in Fig. 6A, ALB is known to be extracellular protein; NME1 is located in the nuclear; PDHA1 is unknown for its location, and six (SNAP25, HOMER2, SYN2, DNM2, NRP2, and SPTAN1) are plasma membrane proteins. These six membrane proteins were used as the starting point to generate three biological networks (Fig. 6B, and Fig. S1A–B of the Supporting Information). In these network figures, the gray icons indicate that a protein is a focus protein, which comes from the list of differentially expressed proteins. In Table S3 (Table 3 in Supporting Information part 3), we have indicated both the gene symbol produced by ingenuity as well as the specific network to which each protein belongs. In addition, other epilepsy-associated canonical signaling or metabolic pathways defined by IPA as having statistically significant representation are listed in Table S4 (Table 4 in Supporting Information part 3).

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Discussion

To date, TLE animal models can be separated into two major groups: induction of SE by application of an excitotoxic compound (kainic acid, pilocarpine) followed by development of a spontaneous epilepsy condition and electrically stimulated chronic recurrent seizures referred to as kindling induced TLE [33]. An important feature of experimental TLE is the possibility to examine the dynamic development of recurrent seizures. Although the complexity of alterations in TLE hippocampus suggests numerous genes and signaling cascades to be involved in the pathogenesis, early stages of epilepsy development are not available for functional genome analysis in humans. However, animal models of TLE appear particularly helpful to study molecular mechanisms of the highly dynamic processes such as the development of pharmacoresistance. Since the discovery that a specific neuropathologic syndrome of TLE is associated with hippocampus sclerosis, there has been a controversy whether the sclerosis is the cause or consequence of repeated complex partial seizures [34]. To characterize TLE from a molecular point of view and to unravel the molecular events, we perwww.proteomics-journal.com

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Figure 6. Signaling networks/function analysis of rat hippocampal proteins regulated by pilocarpine treatment. The identified differentially expressed proteins were analyzed by the IPA tools as described in Section 2. Protein–protein associations are indicated by edges containing single lines, whereas proteins that act upon another protein (controlling their expression) are indicated by arrows. The shaded node in bold are those proteins identified with high confidence. Nodes are represented by shapes (see next page). Relationships between nodes are represented as edges. Proteins with a gray background were detected while other interacting proteins with a clear background were not detected in this study. The focus proteins were indicated by their gene names. (A) A merge biological network, containing the largest number (46) of focus proteins. (B) The biological network associated with DNA replication, recombination, and repair, nucleic acid metabolism, small molecule biochemistry. (C) Node shapes.

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mer’s disease and may reflect or lead to neuropathological changes as, e.g., neuronal death and synaptosomal loss [35, 36]. The cytoskeleton is a highly dynamic structure not only forming the scaffold, the basis of cell morphology and plasticity but plays a major role in transport and signaling [37]. The cytoskeleton is comprised of three major types of cytoplasmic structural proteins: microtubules, actin, and intermediate filaments (IFs). Here, we identified expression changes in b-actin, a-actin-1, tubulin beta chain, tubulin alpha-2 chain, tubulin alpha-1 chain, all of which are microtubule or actin proteins. Microtubules, mainly composed of heterodimers of a- and b-tubulin, perform essential and diverse functions in eukaryotic cells including chromosome segregation and determine cellular morphology and size, axonal transport, neuronal polarity [38], mortality, and organelle distribution [39]. Other research suggested also that the oxidation of tubulin leading to loss of protein function could result in loss of neuronal connections and communication, as well as compromised cellular structure which would play important roles in neurodegeneration [40]. The actin cytoskeleton is a complex structure serving the control of cellular shape, distribution of membrane proteins, intracellular trafficking mechanisms [41] as well as generation and motility of growth cones, spines, and dendrites [37, 42].

formed a proteomic analysis on the epileptic rat hippocampus, and obtained 57 differentially expressed proteins, which can be categorized into five main groups by biological functions. 4.1 Changed proteins with structural molecule activity Derangement of the brain’s cytoskeleton including several structures has been reported in different forms of neurodegenerative disease including Down Syndrome and Alzhei-

Table 3. The top seven biological networks in rat hippocampus in the pilocarpine-induced epilepsy model

Network Genes in networka) IDs

Scoreb)

Focus gene

Top functions

1

ACTA1,ACTB,ACTC,ACTG1,ACTG2,AK1,CAV1,COL4A1, EEF2,GSTM5,HAS2,HSPA1B,HSPD1,INA,JMJD1C,KLF11, MAPK1,MYCN,NEF3,NEFH,NME1,NRGN,PDE4B,PSMB7, RPL11,RPL28,RPLP0,RPS4X,RXRA,S100A4,SNAP25, STAU1,STMN1,TP53,TTF1

23

14

DNA replication, recombination, and repair, nucleic acid metabolism, small molecule biochemistry

2

ACP1,ATP5A1,ATP5A2,ATP5B,ATP5C1,ATP5C2,ATP5D, ATP5E,ATP5J,CBR1,CCL20,DBNL,DLAT,DLD,DNM1,DNM2, ELF3,ERRFI1,FNBP1,HNRPH2,KRT8,MICAL1,PDHA1, PDK1,PDK2,PKD1,PKM2,SHANK1,SLC9A2,SP3,SPTAN1, SRC,SYN2,VIM,YWHAZ

21

13

Cellular movement, cellular assembly and organization

3

AKT1,ALDOA,ARHGDIA,ATP5I,COX17,COX5B,COX6B1, CYCS,ENO1,HIF1A,HIF3A,HOMER2,HSP90AB1,IL1RAP, JRK,MYC,NDRG1,NRP2,PGF,PGK1,PRDX2,RAC1,RORC, RPL7,RPL13,RPL26,RPLP1,RPS7,RPS12,RPS19,SLC25A5, SUCLA2,TUBA3,VDAC2,VEGF

17

11

Cardiovascular system development, organ development, organism development

4

ALB,APP,CEBPB,GATA1,HADH2,HBA1,HBA2,HBB,HBB-AR, HBD,HBE1,HBG1,HBG2,HBQ1, HBZ,MSI1,NUMB

8

5

Hematological disease, hematological system development, tissue morphology

5

E2F1,ENO2

2

1

Cancer, cell cycle, cellular assembly and organization

6

PDIA6,XBP1

2

1

Cell morphology, cellular assembly and organization, cellular development

7

TRAF2,UBE2N,UBE2V1,UBE2V2

2

1

PTM, protein degradation, protein synthesis

a) The focus proteins are indicated with gene names and shown in bold italic. b) A score of .2 was considered significant (p,0.01).

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Among these differential proteins, INA and neurofilament NF-M belong to the IF proteins. During development of the mammalian nervous system, INA mRNA and protein are expressed earlier and more abundantly than the neurofilament M proteins. Because of its early expression, it has been suggested that INA may stabilize neurons and their processes and provide a scaffold for the coassembly of other IF proteins during development [43]. In the mature nervous system, INA shows a distribution pattern restricted to neurons, partially overlapping but distinct from that of the NFM protein. Although many larger neurons express INA along with the NF-M protein, in some mature neurons, INA is the only IF protein expressed, suggesting a role for this protein in neuron maturation and neuronal regeneration after injury [43]. These IFs abnormalities lead to deleterious effects and even to cell death. However, their functional role remain elusive, possibly it is related to neurite elongation [44]. In the present study, the complexity of the function of these structure proteins and the possible link between them and other identified proteins (e.g., synapsin-associated proteins and dynamin) suggest that these proteins may have an extensive and important role in the hippocampus function, possibly participating in information storage and memory, and produces long-term plasticity pathological changes to improve the development of epileptogenesis. The aberrant expression of cytoskeleton proteins means also that structural and functional changes in neural circuits can accompany the epilepsy. 4.2 Changed proteins in synaptic function HOMER2, a Homer/Vesl family protein up-regulated during seizure, is regulated during long term potentiation in the hippocampus, and one form of synaptic plasticity thought to underlie memory formation [45, 46]. As scaffolding proteins, the homer proteins have been characterized as adaptor proteins in signaling complexes associated with metabotropic glutamate receptors, G protein-coupled receptors [47], inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs) [48]. Because homer proteins trigger the localization of metabotropic glutamate receptor subtype 5 (mGlu5 receptor) to the postsynaptic plasma membrane [49], Homer2 may mediate clustering of mGluRs at synaptic junctions and signaling during SE [50]. SYN2, one of a family of neuron-specific phosphoproteins, has been demonstrated to regulate the supply of synaptic vesicles (SV) available for exocytosis by binding to both SV and actin cytoskeleton in a phosphorylation-dependent manner [51]. Kosik and coworkers [52] showed that synapsin II was involved in the formation and maintenance of synapses in hippocampal neurons. Furthermore, other research also indicated that synapsin II is necessary for full expression of modulatory effects on synaptic transmission originating from the readily releasable vesicles in various types of excitatory vertebrate neu© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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ron [53]. In an animal model of TLE, GABAergic inhibitory synaptic transmission is known to be suppressed because of the reduced synaptic input to the inhibitory cells [54]. Therefore, up regulation of SYN2 expression support indirectly that synaptic vesicle turnover at the inhibitory synaptic terminals can be altered by synapsin II differential expression. SNAP25, a key component of synaptic vesicle-docking/ fusion machinery, plays a critical role in exocytosis and neurotransmitter release, and is crucial for the regulation of intracellular calcium dynamics and, possibly, of network excitability [55]. Wilson [56] examined the distribution of SNAP25 mRNA and protein in the hippocampal formation of the adult rat following kainic acid lesions. Their results showed that SNAP25 could be regards as a novel useful marker of major hippocampal pathways and of axonal plasticity in neurological disorders such as Alzheimer’s disease and TLE. In other study, superimposed cholinergic lesions did not affect the return to normal SNAP-25 levels after a long-term entorhinal cortex lesion, which may indicate that changes in SNAP-25 may represent early markers of synaptic loss following afferent lesions to the hippocampus during the epileptogenesis [57]. Further studies in epilepsy and other neurodegenerative disorders could confirm the importance of SNAP-25 in the normal functioning of synaptic hippocampal connectivity. In summary, the altered expression of these three proteins possibly reflects synaptic impairment and some changes in presynaptic efficacy in TLE. Such changes might be closely associated with the pathogenesis of epilepsy. Moreover, networks 1–3 (Fig. 6B, and Fig. S1A–B of the Supporting Information) also show their important roles in TLE. 4.3 Proteins associated with mitochondrial function Mitochondrial dysfunction usually leads to inadequate supply of energy, alterations of cellular ion homeostasis, and induces apoptotic and nonapoptotic cell death [58]. The related research [59] suggests that the mitochondrial dysfunction in the hippocampus is associated with prolonged seizure during experimental TLE and mitochondria are more vulnerable to epilepsy. The accumulating evidences show also that mitochondrial dysfunction in the CNS is associated with epileptic seizures [60–63]. Mitochondrial dysfunction is likely to cause varied courses toward neuronal degeneration and death. Proteins released from mitochondria into the cytosol are important inducers of apoptosis by the outer membrane VDAC2 [64]. Furthermore, the mitochondrial intermembrane space proteins are also released and promote apoptosis [65]. Therefore, both apoptotic and excitotoxic neuronal injury results in cleavage of chromosomal DNA into fragments. However, the biological network 1 (Fig. 6B) was related to DNA replication, recombination, and repair, may participated in this pathological process. www.proteomics-journal.com

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In this study, we found that heat shock protein HSP 90beta (HSPAB1), pyruvate kinase isozymes M1/M2 (PKM2), ATP synthase subunit beta (ATP5B), VDAC2, heat shock 70 kDa protein 1L (HSPAIL), ATP synthase subunit alpha (ATP5A1), pyruvate dehydrogenase E1 component alpha subunit (PDHA1), 3-hydroxyacyl-CoA dehydrogenase type-2 (HADH2), mitochondrial fission 1 protein (FIS1), ATP synthase e chain (ATP5I), and cytochrome c (CYCS) up-regulated, and pyruvate dehydrogenase E1 (PDHB), dihydrolipoamide acetyltransferase (DLAT), glutamate dehydrogenase 1 (GLUDI1), 60 kDa heat shock protein (HSPD1), hemoglobin subunit alpha-1/2 (HBA2), and adenylate kinase isoenzyme 1 (AK1) down-regulated, all of which are associated with mitochondria. However, functional role(s) of many proteins in these mitochondria-associated proteins remain elusive in epilepsy as yet, and needed to be further studied. In short, our results showed that aberrant expression of mitochondria-associated proteins leads to mitochondrial impairment and dysfunction, which is related to neuron cell loss and hippocampus lesion. 4.4 Proteins associated with ion channel Voltage-dependent anion channel 2 (VDAC2) protein is small, abundant pore-forming protein found in the outer membrane of mitochondria as well as in plasma membrane [66]. In addition to its function as a channel protein for solutes or ions, mammalian VDAC2 has been reported to show multiple functions [67]. The possible involvement of VDAC2 in the regulation of apoptosis has also been considered [68], because it has been reported to interact with Bcl2 family proteins, critical regulators of apoptosis, or to function as one of the components of the permeability transition pore, whose opening is presumed to induce the release of cytochrome c from mitochondria to trigger apoptosis. Some of the key proteins comprising this function were dynamin2, a-enolase, cytokeratin-8, Hsp60, and protein disulfide isomerase in these differentially expressed proteins. Here, we show up regulation of VDAC2 in the epileptic rat hippocampus. Opening of voltage-gated ion channels located in the neuronal membrane is mandatory when an excitatory synapse becomes activated. In view of this, regulations of ionic conductance are discussed as one of the mechanisms modulating the propagation of neuronal information in the long-term [69]. Especially, according to the recent study [70], it is likely that pilocarpine interacts with the choline receptor to activate certain signaling pathways (e.g., the network 3 in Fig. S1B of the Supporting Information) to increase the expression of VDAC2, and then binds directly or indirectly to VADC2 to induce mitochondrial dysfunction, release of oxidative species and, ultimately, nonapoptotic, oxidative cell death. This kind of cell death is consistent with the neuron loss in the hippocampus. Hence, VDAC2 play an important role in hippocampus impairment and mitochondrial dysfunction, which is worth to be further studied as a biomarker for epilepsy. © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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4.5 Changed heat shock proteins (HSPs) In the nervous system, heat shock proteins are induced in a variety of pathological states, such as cerebral ischemia, neurodegenerative diseases, epilepsy, and trauma. Hsp60 is a mitochondrial chaperone protein that is involved in mediating the proper folding and assembly of mitochondrial proteins, especially in response to oxidative stress [71]. Additionally, Hsp60 has been proposed to play a role as an antiapoptotic protein [72]. Expression of Hsp60 is significantly decreased in Alzheimer’s disease [73]. Here, we found Hsp60 to be significantly decreased in the epileptic rat hippocampus, because the loss of function of Hsp60 could lead to increased protein misfolding and aggregation, as well as an increased vulnerability to oxidative stress. This is particularly important due to the lack of mechanisms to protect mitochondrial from oxidative stress and the vicinity of mitochondrial proteins to ROS generated during normal oxidative phosphorylation and more so in concert with mitochondrial dysfunction. Induction of heat shock/stress proteins is a key feature of a universal mechanism of cellular defense to injury known as the “stress response”. For example, Hsp70 was increased following induction of SE in the rat by treatment with fluorothy1 [74] or by systemic injection of kainic acid [75]. Two studies demonstrated that Hsp70 is rapidly and transiently expressed in dying neurons [76], and detected in the pyramidal cell layers of CA1 and CA3 regions of hippocampus [77] in the kainic acid model of epilepsy. More recently, investigators reported that induction of Hsp70 protein expression coincided with the development of epileptic tolerance [78], and the overexpression of rat Hsp70 reduced neuronal injury after ischemia and seizures [79]. Furthermore, the accumulation of the Hsp70 was also found to be an excellent marker for prolonged seizure related to metabolic activity of neurons, in the model of kainic acid induced SE [80]. These studies provided an inhibition of a function associated with neuronal cell death. However, the involvement of Hsp70 in epileptic related neuronal death is still poorly understood. Thus the higher Hsp70 levels in epileptic rat hippocampus may be linked to their reduced sensitivity to pilocarpine promotion in this study. Similarly, in the kainic acid model of epilepsy, Hsp90 was also induced in adult Sprague–Dawley rats and has an active role as a molecular chaperone in the signal transduction pathway for steroid receptors [75]. Therefore, expression changes of heat shock proteins reflect the risk of hippocampal neurons to undergo seizure induced neuronal degeneration and neuroprotective effect. 4.6 Protein networks As in CNS disorders, the dysfunctional or hippocampus impairment may result from the changes in multiple members of the deranged protein signal transduction pathways. Therefore, an understanding of the pathways www.proteomics-journal.com

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and networks that involve extracellular, plasma membrane, cytoplasm, and nuclear proteins would facilitate the development of a disease biomarker panel for clinical applications. Our proteome data produced seven networks containing 46 focus proteins in total. However, eleven proteins were not contained in the merge network. The reasons that we do not detect all the proteins in this merge network could be that they are not expressed in high enough amounts to be detected on a 2-D gel, or perhaps any differential expression is simply not large enough to be determined by this technology. These seven biological networks are assigned functions based on the known functions of their members and the statistical likelihood that a specific function would be represented with that frequency by chance. With scores of 23, 21, 17, and 8 (Fig. 6B, and Fig. S1A–C of the Supporting Information), the probability of creating these networks by chance was very low. The most significant global functions associated with the highest number of changing proteins in networks 2–4 (Fig. S1A–C of the Supporting Information) were the cell and tissue morphology, cellular assembly and organ development, which demonstrated that those cytoskeleton proteins, chaperonin, mitochondria proteins, ion channel proteins and synapse-associated proteins play an important role in TLE. Therefore, the pathway analysis reveals that our dataset indeed provides extensive coverage for important signaling pathways (e.g., calcium signaling pathway, Table 4 in Supporting Information part 3) and protein networks. Taken together, the present study showed aberrant expression of structural proteins, heat shock proteins, channel proteins, mitochondrial and synapse-associated proteins, etc. in the epileptic rat hippocampus. These findings demonstrated the significance of abnormal expression of structural protein in hippocampus lesion, mitochondrial proteins in apoptosis, as well as synaptic transmission, memory formation, and plasticity, when elucidating these physiological and pathophysiological properties associated with epilepsy. Our data support directly the idea that hippocampus injury, as well as mitochondrial dysfunction, play an important role at the protein level in the development of epilepsy. It also point out specific networks that may be worth further investigation to gain a deeper insight into the origins of the dysfunction and the structural abnormality in rat hippocampus region, and to explore biomarkers for epilepsy.

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Conclusion

A number of aspects of the epilepsy are poorly understood, which includes its mechanisms of signaling and its complex protein networks. However, technical developments in the field of proteomics are poised to generate advances in our understanding of protein expression, function, and organization in signaling processes and regulatory net© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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works, thus, providing deeper insight into how cellular protein networks are regulated in the epileptic hippocampus under the pathological conditions. Here, we applied 2-DE to analyze the difference of the protein profiles, and used MALDI-Q-TOF MS to identify differentially expressed proteins, and combined with IPA to explore rapid molecular biological network, signaling and metabolic pathway, potentially associated with the development of TLE. Considering the similarity between the model of TLE in rat and TLE in human, some of these identified proteins could be involved in the pathological mechanisms underlying TLE. The data presented demonstrate that systems biology is a powerful means of discovering important genes, proteins and pathways involved in complex biological processes such as epileptogenesis. We believe that this global approach to interrogating the epileptogenesis process may yield novel insight into the complex and multiple pathways involved in this process, and potentially lead to exciting new strategies and targets for clinical intervention.

We thank Dr. Zu-Jun Yang (School of Life Science and Technology, University of Electronics Science and Technology of China) for critically reviewing the manuscript. This work was supported by National Key Basic Research Program of China, 2004CB518800 and 2004CB517806, Project of National Natural Sciences Foundation of China, National 863 Projects. The authors have declared no conflict of interest.

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