Down-regulation of SlitâRobo Pathway Mediating Neuronal

Article pubs.acs.org/JAFC

Down-regulation of Slit−Robo Pathway Mediating Neuronal Cytoskeletal Remodeling Processes Facilitates the Antidepressive-like Activity of Gastrodia elata Blume Shih-Hang Lin,† Wei-Cheng Chen,† Kuan-Hung Lu,† Pei-Ju Chen,† Shu-Chen Hsieh,† Tzu-Ming Pan,∥ Shui-Tein Chen,‡,§,# and Lee-Yan Sheen*,†,# †

Institute of Food Science and Technology, National Taiwan University, Taipei, Taiwan Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan ‡ Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan § Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan ∥

ABSTRACT: Nowadays, depression is a serious psychological disorder that causes extreme economic loss and social problems. Previously, we discovered that the water extract of Gastrodia elata Blume (WGE) improved depressive-like behavior by influencing neurotransmitters in rats subjected to the forced swimming test. To elucidate possible mechanisms, in the present study, we performed a proteomics and bioinformatics analysis to identify the related pathways. Western blot-validated results indicated that the core protein network modulated by WGE administration was closely associated with down-regulation of the Slit−Robo pathway, which modulates neuronal cytoskeletal remodeling processes. Although Slit−Robo signaling has been well investigated in neuronal development, its relationship with depression is not fully understood. We provide a potential hint on the mechanism responsible for the antidepressive-like activity of WGE. In conclusion, we suggest that the Slit−Robo pathway and neuronal cytoskeleton remodeling are possibly one of the pathways associated with the antidepressive-like effects of WGE. KEYWORDS: Slit−Robo pathway, Gastrodia elata Blume, depression, antidepressive-like, proteomics

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VAN) prevented the brain from incurring focal ischemic injury7 and epilepsy8 and had neuroprotective activities.9 Furthermore, a recent proteomics study indicated the water extract of G. elata Blume (WGE) enhanced neuroplasticity and synaptic activity of rats without any stress administration.10 Previously, the antidepressant-like effect of the ethanol extract of GE had been mentioned.11 We also noticed that administration with WGE reduced the depressive-like behavior of rats undergoing the forced swimming test and altered the monoaminergic neurotransmitters in the frontal cortex, hippocampus, striatum, and amygdala.12,13 Accordingly, GE could emerge as a potential material for preventing depression or assisting conventional medication to heal depression. Since proteomics facilitates a high-throughput approach, the proteomics techniques such as two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) with bioinformatics analyses, are suitable for the nutraceuticals, pharmacological, and pathological studies of depression. Although our previous studies showed the antidepressive-like effect of WGE in the forced swimming test (FST), the potential molecular pathways are unclear. In this study, we aim to investigate the molecular protein−protein interaction in the frontal cortex and hippocampus, which was influenced by WGE administration. Thus, 2-DE and bioinformatics analysis were employed to analyze the

INTRODUCTION Major depressive disorder (MDD) has seriously threatened human life for decades. The World Health Organization (WHO) predicted that MDD will emerge as a huge burden and disability disorder by the year 2030.1 However, the underlying pathology in depression has not been completely understood. Psychophysiological studies observed that neurotransmitter dysregulation and neuroplasticity retardation have appeared in patients with MDD and animals presenting depressive-like behaviors.2,3 Thus, abnormal status of neurotransmission and neuroplasticity were hypothesized as the pathologies of MDD. On the basis of these hypotheses, antidepressants were designed to restore neurotransmission as the major therapy for MDD. Although these medications ameliorate the illness, they predispose the patients to anxiety, gastrointestinal, eating, and sleeping disorders, which occur simultaneously and make the patients opt out of medical programs.4,5 Hence, nutraceuticals and functional food could be an option for assisting conventional therapies and prevention strategy of MDD. Therefore, investigation of the mechanism by which the bioactivities of these food components influence the neuropsychiatric system is of great significance. Gastrodia elata Blume (GE) has revealed the presence of several phenolic compounds, which are composed of a benzene and para-methylol group, or related compounds such as gastrodin (4-(β-D-glucopyranosyloxy)benzyl alcohol), 4-hydroxybenzyl alcohol (HBA), 4-hydroxybenzaldehyde (HB), and vanillin (VAN), which are normally regarded as the major active compounds and indicators of the quality of GE.6 Researchers reported that GE and/or its four compounds (gastrodin, HBA, HB, and © 2014 American Chemical Society

Received: Revised: Accepted: Published: 10493

July 6, 2014 September 2, 2014 September 8, 2014 September 8, 2014 dx.doi.org/10.1021/jf503132c | J. Agric. Food Chem. 2014, 62, 10493−10503

Journal of Agricultural and Food Chemistry

Article

4 °C by using the PROTEAN II xi Multi-Cell 2-D electrophoresis system (Bio-Rad). The gels were fixed (10% methanol, 7% acetic acid solution for 30 min) and stained with Sypro Ruby dye (Life Technologies, Grand Island, NY, USA). After the destaining process, a Typhoon scanner 9200 (GE Healthcare) was used to scan the gels (100 μm). PDQuest Basic 2-D analysis software version 8.0 (Bio-Rad) was used for protein spot analysis. The spots on the gel were automatched by PDQuest and manually corrected based on their relative location. Only the significantly expressed spots were selected and subjected to in-gel tryptic digestion. In-Gel Tryptic Digestion. The protein spots that differed significantly were picked up from 2-D gels manually using a UV light box. Then, the gels of the spots were washed with 50% acetonitrile (ACN)−25 mM NH4HCO3 at room temperature for 20 min, and the solution was replaced by 100% ACN. After washing, gel pieces were dried by a SpeedVac concentrator (Savant, Holbrook, NY, USA). Then, the dried gel plugs were crushed and incubated with 1% trypsin (Promega, Madison, WI, USA) in 25 mM NH4HCO3 at 37 °C for 16 h. Peptides were extracted subsequently by 50% ACN−5% trifluoroacetic acid (TFA) from gel crushes and then dried with a SpeedVac concentrator. The dried pellets were rehydrated, resuspended with 0.1% TFA, and then concentrated with a C18 ZipTip (Millipore). After washing the C18 ZipTip with 0.1% formic acid (FA), the peptides were eluted with 75% ACN−0.1% FA. Peptide Sequence Analyses. The matched protein spots of the frontal cortex (loading protein: 400 μg) and hippocampus (loading protein: 400 μg) were subjected to concerted matrix-assisted laser desorption ionization (MALDI) with time-of-flight (TOF) to operate peptide mass fingerprinting (PMF) and CID MS/MS mass spectrometry. In order to catch more possible proteins, the proteins of the hippocampus were loaded as 450 μg into the 2-DE system again, and the spots were submitted to a liquid chromatography electronic spray ionization (LC-ESI)-quantitative-TOF Ultima API (Micromass) instrument. The peptide sequencing analyses were performed by the Core Facilities for Protein Structural Analysis located at the Institute of Biological Chemistry, Academia Sinica, Taiwan. The protocols of LC-ESI-Q-TOF and MALDI-TOF were announced on the Web site of the Core Facilities (http://cfpsa.sinica.edu.tw/eng_04_filedownload.php#F3, material and method of mass analysis section). MS and MS/MS data were submitted to the Mascot protein online search program (Matrix Science, Boston, MA, USA) based on Swissprot and the NCBI database to identify proteins by PMF and MS/MS ion search. Bioinformatics Analysis. Metacore (Thomas Reuters, New York, NY, USA), a systemic bioinformatics software, was employed to evaluate the function of proteins, relationship and interactions between proteins, and the potential signaling maps, based on a huge database. In Metacore, the “GeneGo Process Network” shows the cooperative relationships between identified proteins and the “GeneGo Pathway Maps” provides related signaling cascades. All of the candidates in the lists were ranked by −log(p value) table. A higher −log(p value) indicated by Metacore suggested a higher possibility for its occurrence. Western Blot (WB). The protein extracts were solubilized individually in sample buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, and 0.01% bromophenol blue), heated at 95 °C for 10 min, and subjected to one-dimensional SDS polyacrylamide gel electrophoresis (SDS-PAGE). Following SDS-PAGE, proteins were transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Following transfer, 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween-20 (TBS-T) was used to block the membranes. Then, the membranes were incubated with TBS-T-diluted primary antibodies. The primary antibodies were Slit1 and Slit2 (Genetex, Irvine, CA, USA), Robo1 and Robo2 (Genetex), RhoA (Cell Signaling, Danvers, MA, USA), PFN1 (Cell Signaling), CRMP2 (Cell Signaling), and GAPDH (Genetex). The corresponding secondary antibodies (Cell Signaling) were added for 1 h at room temperature. Membranes were soaked in ECL solution (PerkinElmer, Santa Clara, CA, USA), and the images were obtained using a UVP Autochemi system. Finally, ImageJ software (National Institutes of Health, USA) was utilized for quantification.

potential molecular protein networks. To the best of our knowledge, this is the first proteomics study to report the antidepressive mechanism of WGE.

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MATERIALS AND METHODS

Material Extraction and Composition Determination. WGE was provided by Koda Pharmaceutical Company (Taoyuan, Taiwan). To obtain WGE, 700 g of dried rhizome of GE was boiled twice with 6 and 4.8 L of water for 1 h, respectively. Then, the extract was filtered, freeze-dried, and stored until utilization. Fluoxetine was obtained from Eli Lilly Company (Taiwan). An LC-NET II/ADC (JASCO, Easton, MD, USA) high-performance liquid chromatography (HPLC) system, which consisted of a pump (PU-2089-PLUS) and a UV detector (UV-2075-PLUS), with a Phenomenex Luna C18 column (250 × 4.6 mm, 5 μm, Torrance, CA, USA) and Chrompass analyzing program, was used for analyzing the composition of WGE. An acetonitrile−water solution (10:90, v/v) was used as the mobile phase at 1 mL/min flow rate. The mobile phase was filtered using 0.45 μm Millipore filters, the UV detector was set at 220 nm, and the oven was set at 35 °C to maintain the column temperature. The injected sample volume was 20 μL. Animal and Treatments. Male Sprague−Dawley rats (4 weeks old) were purchased from BioLasco Taiwan Company (Taipei, Taiwan) and housed individually at controlled day/night cycle (12 h light/dark), temperature (23 ± 2 °C), and humidity (60 ± 10%). Free access to food and water was provided and recorded every day. Rats were randomly separated into three groups (7 rats/group): control (CTL), WGE, and fluoxetine (FLX). After 2 weeks of habituation, oral administration was initiated and continued for 21 days with ultrapure water, fluoxetine (20 mg/kg body weight), and WGE (500 mg/kg body weight) to CTL, FLX, and WGE groups, respectively. The dosage was based our preliminary studies. Animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee of National Taiwan University, Taiwan (Approval Number: NTU-IACUC-96-56). The protocol complied with guidelines described in the “Animal Protection Law”, amended on June 29, 2011, Hua-Zong-(1)-Yi-Tzi-10000136211, Council of Agriculture, Executive Yuan, Taiwan. Forced Swimming Test. The protocol of the FST was described previously.12 This method followed the procedure developed by Porsolt et al.14 The behavior of rats during the FST was video recorded. Films were analyzed by ForcedSwimScan software (CleverSys, Reson, VA, USA) for calculating the duration of immobility. Sacrifice and Protein Extraction. After the FST, rats were deeply anesthetized by CO2 and decapitated, and the frontal cortex and hippocampus were dissected from the brain immediately. The dissected tissues were frozen at −80 °C until further analysis. The frozen tissues were ground into a powder with liquid nitrogen and dissolved with lysis buffer containing 7 M urea, 2 M thiourea, 65 mM DTT, 4% CHAPS, and a cocktail of proteinase and phosphatase inhibitors (Roche, Rotkreuz, Switzerland). The samples were centrifuged at 12000g for 2 h, and the precipitates were discarded. Protein extracts were quantified by the Bradford protein assay (Bio-Rad, Hercules, CA, USA). Proteomics and Bioinformatics Analysis. Due to the limitations in the sampling of the 2-DE system, three replicates from CTL and WGE, which had the most opposite duration of immobility in the FST, were further analyzed by 2-DE. Protein extracts were loaded onto the Immobiline DryStrip 18 cm pH 3−10 nonlinear gel strips (GE Healthcare, Pittsburgh, PA, USA) with 350 μL of rehydration buffer (lysis buffer plus 0.5% IPG buffer). The voltage−time program of isoelectric focusing was set at 50 V for 12 h and 100, 250, 500, 1000, 3000, and 5000 V for 1 h, respectively, and 8000 V for a total of 56 000 V × h. Prior to the second dimension, strips were equilibrated for 15 min in buffer (6 M urea, 30% glycerol, 2% SDS, 50 mM Tris-HCl, 0.002% bromophenol blue, and 0.02% w/v DTT), then soaked for 15 min in the same buffer (replacing DTT with 0.025% IAA). Then the second dimension separation was performed using 10 mA/gel electrophoresis for 18 h on 10−18% polyacrylamide gels at 10494

dx.doi.org/10.1021/jf503132c | J. Agric. Food Chem. 2014, 62, 10493−10503


Article

Figure 1. (A) Chemical structure of four major active compounds in Gastrodia elata Blume. (B) Chromatogram of the water extract of G. elata Blume (WGE). Different concentrations of WGE and standard were analyzed by HPLC: WGE (top, 15 000 ppm), standards (bottom, 50 ppm). Statistical Analysis. The results are represented as mean ± SD. Student’s unpaired t test (2-DE spots comparison and WB) and oneway ANOVA with Duncan’s multiple test (body weight, daily intakes, and FST) were employed. p < 0.05 (body weight, daily intakes, FST, bioinformatics analysis, and WB) and at least p < 0.1 (2-DE spots comparison) were considered statistically different.

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RESULTS

Active Compounds in WGE. We determined gastrodin, HBA, HB, and VAN, which are considered to be the major active compounds in GE, by HPLC using a C18 column. Similar to gastrodin standard, a signal appeared at 4 min after injection. This peak was further confirmed as gastrodin by MS/MS analysis (data not shown). After quantification, WGE composed with 2.48% gastrodin. However, three other compounds could not be detected in WGE by the HPLC system (Figure 1). Animal Behavior and Daily Intakes. Figure 2 shows the significant difference after FST between each group. CTL demonstrated a higher duration (100.4 ± 23.3 s) of immobility behavior than that of WGE (63.2 ± 19.1 s) and FLX (32.8 ± 5.8 s). In addition, FLX and WGE significantly increased the duration of swimming in comparison to CTL (CTL: 97.04 ± 27.4 s, WGE: 134.2 ± 19.7 s, FLX: 128.2 ± 20.4 s). The duration of struggling behavior was elevated after administration with WGE but was not significant compared to CTL. During experiment, FLX influenced food and water intake and hindered body weight gain. In contrast, WGE administration did not interfere with body weight gain and water intake; even food intake was only slightly affected (Table 1).

Figure 2. Effect of water extract of Gastrodia elata Blume on the immobility behavior in rats in the forced swimming test. abc indicate significant differences between one another in each group (p < 0.05) analyzed by ANOVA and Duncan’s multiple range test.

2-DE and Protein Identification. Forty-six spots had significant differences out of the 316 matched spots in the frontal cortex (loading protein 400 μg, fold change ≥2, at least p < 0.1). Among 46 spots, 30 proteins were identified by MALDI-TOF. In the hippocampus, 29 spots were significantly different in similar conditions to that of the frontal cortex, and 17 proteins were identified. However, 17 protein identities may not be adequate for bioinformatics analyses. To address this issue, we further loaded more proteins (450 μg) for the hippocampus into the 2-DE system and switched to a higher sensitivity MS system (LC-ESI). Finally, 25 proteins, which were not included in the list of 17 proteins from the initial matching, were identified from the 39 differentially expressed spots in the second separation (fold change ≥2, at least p < 0.1). Thus, 10495



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Table 1. Body Weight and Daily Intakea parameter

weekb

body weight (g)

W0 W1 W2 W3 W0 W1 W2 W3 W0 W1 W2 W3

food intake (g)

water intake (g)

CTL 227.2 280.8 341.6 384.0 23.8 27.4 29.0 32.3 39.9 39.7 40.7 42.0

± ± ± ± ± ± ± ± ± ± ± ±

WGE

7.4 D 13.8 Ca 16.6 Ba 18.5 Aa 2.8 C 1.0 Ba 1.3 Ba 2.3 Aa 2.0 3.0 a 2.6 a 3.6 a

221.5 270.5 329.8 370.6 21.3 25.4 26.8 31.1 38.6 38.6 41.7 44.1

± ± ± ± ± ± ± ± ± ± ± ±

12.8 d 18.3 Cab 10.6 Ba 11.4 Aa 1.9 C 2.3 Bb 2.8 Bb 2.3 Aa 2.8 5.4 a 5.0 a 5.4 a

FLX 223.1 257.7 299.3 321.9 22.6 21.3 23.0 26.5 39.6 31.9 32.7 32.1

± ± ± ± ± ± ± ± ± ± ± ±

5.5 D 9.3 Cb 14.8 Bb 18.4 Aab 2.4 B 1.6 Bc 0.9 Bc 2.3 Ab 2.8 A 4.7 Bb 2.9 Bb 2.1 Bb

a

The capital letters represent the significant differences in the specific group in different weeks. The lower case letters symbolize the significant differences between different treatment groups in the same week. All of the results were subjected to ANOVA with Duncan’s post hoc analysis (p < 0.05). bW0: the habituation period, W1−3: the first to the third week.

on animal locomotion of treatments, an open field test was performed. During a 5 min observation for the open field test, the travel distance and velocity of rats were not affected by administrations (data not shown). This result indicates that the influence of the behaviors in the FST was not coming from the change of locomotion. In the present study, we detected the composition of WGE in order to realize the active components that could be responsible for the antidepressive effect of WGE. The HPLC fingerprint indicated that among the four candidates only gastrodin could be detected in the WGE (Figure 1). This profile was similar to Zhang’s study.15 In contrast, HBA, HB, and VAN could be detected in the diethyl ether fraction of a methanolic extract of GE.16 Zhang’s research indicated that gastrodin ameliorated depressive-like behavior and up-regulated the expression of brain-derived neurotrophic factor (BDNF).17 In addition, the reduction of glutamate-induced apoptosis in differentiated PC12 cells via regulating CaMKII/ASK-1/p38 and MAPK/p53 signals by gastrodin had been noticed.18 However, the blood brain barrier hinders compounds acting on the central nervous system (CNS), which is the difficulty in drug development for mood disorders. Pharmacokinetic studies showed that gastrodin could be detected in plasma after gavage with WGE15 and quickly appeared in brain microdialysates after gastrodin administration in rats.19,20 Furthermore, gastrodin could be absorbed via glucose transporters 1 (GLUT1) that are distributed in the brain and intestine, on the basis of its glycoside structure.21 These findings suggested that WGE could be absorbed and directly act on the CNS, implying that gastrodin is the major active compound of WGE. Thus, gastrodin was surmised to be the major potential active component for the antidepressive-like effect of WGE. The relationship between neurocytoskeletal remodeling, neuroplasticity, and depression has been discovered.22,23 In addition to the original pharmaceutical designs, many antidepressants not only influenced the level of neurotransmitters but also stimulated neurocytoskeletal rearrangement and elevated neuroplasticity.24−26 In the present study, many cytoskeletonrelated proteins were up-regulated by 2−5-fold after chronic administration with WGE. According to the network analysis, the detection of cytoskeleton rearrangement with the highest score in bioinformatics analysis indicated the potential interaction between identified proteins (Figure 4A). Hence, WGE could enhance neurocytoskeletal rearrangement and be associated with

42 total proteins were identified from the hippocampus. The information on identified proteins such as protein name, peptide matching percentage, and fold change is shown in Tables 2 and 3 for the frontal cortex and hippocampus, respectively. The zoom-in spot images on 2-DE gel and quantitative charts of specific identified proteins are shown in Figure 3. Bioinformatics Analysis. To elucidate the mechanism underlying the antidepressive-like activity of WGE, the identified proteins were uploaded to Metacore. A cytoskeleton-related process emerged with a high ranking in the analysis of functional classifications based on the GeneGo Process Network (Figure 4A). Furthermore, Slit−Robo signaling emerged with the highest score in the predicted pathways analyzed by the GeneGo Pathway Map of Metacore (Figure 4B). The gel images of these identified proteins related to cytoskeleton remodeling and the Slit−Robo pathway are shown in Figure 3. Besides, we found similar results when the two regions were assessed individually (data not shown). Therefore, the Slit−Robo pathway probably mediates the antidepressive-like activity of WGE. The proteins shown on the Slit−Robo signaling map were built using the Metacore database (Figure 5). The tubulin and actin subunits, actin-related protein 3 (ARP3), dihydropyrimidinase-related protein 2 (DPYL2; also named CRMP2), and profilin1 (PFN1), which were identified in 2-DE, are presented with a “ratio bar (WGE compared to CTL)” on the upper right corner of the symbols. Western Blot. WB was employed to validate the expression of the proteins associated with Slit−Robo signaling, which emerged with the highest score for the predicted pathways. In addition to CRMP2 and PFN1 identified in 2-DE, Slit1, Slit2, Robo1, Robo2, and RhoA were also analyzed (Figure 6). The result showed that Slit1 was markedly down-regulated in both regions after WGE administration. However, the expression of Slit2 was not equally down-regulated. WGE did not influence the expression of Robo1 and Robo2, the receptors of Slits. The down-regulation of the negative regulator RhoA was discovered in the WGE group. In contrast, incremental changes in the expression of CRMP2 and PFN1 were observed.

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DISCUSSION Previously, we found that G. elata Blume reduced depressivelike behavior and influenced the monoaminergic neurotransmitters of the frontal cortex, hippocampus, amygdala, and striatum of rats subjected to FST, a common behavior model for evaluating the antidepressive activity of medicine. To exclude the influence 10496



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Table 2. List of Differentially Expressed Proteins in the Frontal Cortexa SSP

accession no.

0206 1203

P62260 Q5XI54

1302 1612e

B2RYG6 P85108 Q3KRE8 P68370 P50398

b

1616 1619 1711e

2203

P85108 Q3KRE8 Q6P9T8 Q4QRB4 P69897 Q8VD52

2205 2312

O35920 P85969

2411

P59215

2509

Q5RKI1

2613e 3507 3619

P60711 P63259 Q9WTT6 P47942

3710

P62815

3810

D4A133

4203

D3ZG43

4309

P42123

4313 4405

D4ACB8 Q4 V8E4

4506 4609

Q4 V7C7 P62815

8108 8209

P62963 Q9Z2L0

8216

P04905

1303 3203 5105

P14668 P85971 Q9Z0 V6

7416

P16617

protein name

theoretical M/W

theoretical pI

identified byd

mascot score (MS, MS/MS)

no. of peptides sequence identified (MS, coverage rate (%) MS/MS) (MS, MS/MS)c

fold change (WGE/CTL)f

14-3-3 protein epsilon (1433E) leucine-rich repeat-containing protein 48 (LRC48) ubiquitin thioesterase (OTUB1) tubulin β-2A chain (TBB2A) tubulin β-2B (TBB2B) tubulin α-1A chain (TBA1A) Rab GDP dissociation inhibitor alpha (GDIA) tubulin β-2A chain (TBB2A) tubulin β-2B chain (TBB2B) tubulin β-4B chain (TBB4B) tubulin β-3 chain (TBB3) tubulin β-5 chain (TBB5) pyridoxal phosphate phosphatase (PLPP) calpain-9 (CAN9) β-soluble NSF attachment protein (SNAB) guanine nucleotide-binding protein G (o) subunit alpha (GNAO) eukaryotic initiation factor 4A-II (IF4A2) actin, cytoplasmic 1 (ACTB) actin, cytoplasmic2 (ACTG) guanine deaminase (GUAD) dihydropyrimidinase-related protein 2 (DPYL2) V-type proton ATPase subunit B, brain isoform (VATB2) V-type proton ATPase catalytic subunit A (VATA) NADH dehydrogenase [ubiquinone] iron−sulfur protein 3, mitochondrial (NADU3) L-lactate dehydrogenase B chain (LDHB) chaperonin subunit 8 Theta (CCT8) coiled-coil domain- containing protein 104 (CC104) actin-related protein 3 (ARP3) V-type proton ATPase subunit B, brain isoform (VATB 2) profilin 1 (PFN1) voltage-dependent anion-selective channel protein 1 (Vdac) glutathione S-transferase Mu 1 (GSTM1)

29 174 60 798

Up-regulation 4.63 MS, MS/MS 4.66 MS

58, 85 62, N/D

10, 2 6, N/D

36%, 11% 20%, N/D

2.01** 2.47**

31 270 49 907 49 953 50 136 50 537

4.85 4.78 4.78 4.94 5.00

MS, MS/MS MS/MS MS/MS MS/MS MS, MS/MS

63, 49 N/D, 74 N/D, 74 81, 148 75, 207

6, 1 N/D, 2 N/D, 2 12, 4 10, 4

38%, 5% N/D, 7% N/D, 7% 38%, 14% 31%, 13%

3.58** 3.16**

49 907 49 953 49 801 50 419 49 671 33 115

4.78 4.78 4.79 4.82 4.78 5.44

MS, MS/MS MS, MS/MS MS/MS MS MS, MS/MS MS, MS/MS

73, 94 73, 94 N/D, 94 60, N/D 63, 94 54, 70

11, 2 11, 2 N/D, 2 9, N/D 10, 2 7, 1

34%, 7% 34%, 7% N/D, 7% 25%, N/D 31%, 7% 32%, 3%

2.14**

78 989 33 470

5.14 5.32

MS/MS MS, MS/MS

N/D, 59 56, 68

N/D, 9 7, 2

N/D, 19% 34%, 12%

4.46*** 2.41**

40 069

5.34

MS/MS

N/D, 102

N/D, 2

N/D, 9%

4.70***

46 125

5.33

MS/MS

N/D, 89

N/D, 2

N/D, 8%

2.72*

41 737 41 793 51 016 62 278

5.26 5.31 5.56 5.95

MS/MS MS/MS MS MS/MS

N/D, 79 N/D, 79 65, N/D 75, 109

N/D, 2 N/D, 2 6, N/D 9, 3

N/D, 9% N/D, 9% 20%, N/D 29%, 9%

2.53*

56 551

5.57

MS/MS

120, 178

16, 4

42%, 11%

3.55***

68 265

5.42

MS/MS

N/D, 194

N/D, 4

N/D, 11%

2.88*

30 226

6.67

MS/MS

N/D, 64

N/D, 2

N/D, 9%

3.58***

36 612

5.70

MS/MS

60, 58

8, 1

33%, 5%

2.11**

59 589 39 590

5.44 4.93

MS MS

57, N/D 48, N/D

8, N/D 6, N/D

19%, N/D 29%, N/D

47 357 56 551

5.61 5.57

MS, MS/MS MS, MS/MS

57, 71 110, 103

7, 2 15, 2

27%, 8% 39%, 6%

2.61*** 2.69***

14 957 30 756

8.08 8.62

MS/MS MS/MS

N/D, 40 N/S, 106

N/D, 1 N/D, 2

N/D, 12% N/D, 14%

4.03* 2.22*

25 914

8.27

MS/MS

N/D, 68

N/D, 2

N/D, 13%

2.73*

annexin A5 (ANXA5) 6-phosphogluconolactonase (6PGL) thioredoxin-dependent peroxide reductase (PRDX3) phosphoglycerate kinase 1 (PGK1)

35 745 27 234 28 295

68, 52 N/D, 64 N/D, 82

7, 1 N/D, 8 N/D, 2

34%, 5% N/D, 41% N/D, 9%

−4.16*** −3.23* −2.17*

73, 52

9, 3

39%, 11%

−2.38**

44 538

Down-regulation 4.93 MS, MS/MS 5.54 MS/MS 7.14 MS/MS 8.02

MS, MS/MS

8.48** 2.92* 2.47*

3.27* 2.49*

>100*** 2.51*

a

These proteins were differentially expressed in the water extract of Gastrodia elata Blume (WGE) and control (CTL) groups and were detected by MALDI-TOF mass spectrometry. bThe SSP is the serial number assigned by the gel image analysis software. cN/D means that the spot was not detected by MS or MS/MS. dThe protein identities were identified from MS and/or MS/MS method in the Mascot protein online search program. e All the protein identities that were matched successfully from the Mascot protein online search program are presented (more than one protein was identified from one spot). fAsterisk represents the significance of the spot comparing between WGE and CTL (***p < 0.01, **p < 0.05, *p < 0.1).

Actin remodeling is facilitated by dendritic branching and synaptogenesis during neuronal growth. We discovered that actin, actin-related protein 3 (APR3), cyclase-associated protein 2 (CAP2), and profilin1 (PFN1), which were related to actin

neuroplasticity and an antidepressive-like effect. On the basis of the bioinformatics analysis, the identified proteins that were associated with actin filament, microtubule, and neurofilament processes will be discussed in the following section. 10497



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Table 3. List of Differentially Expressed Proteins in the Hippocampusa b

SSP

accession no.

3517d 3808d

P07335 D4A133

5216d 6718d

P31399 Q5RKI0

8146d

P10111

8148d 8233d

P62963 P04905

9316d

A7VJC2

1107e,h

P11240 P55051

1109

e

Q6PEC4

1113e

P62870

1307e

Q5M7T6

1615e,h, 1616e,h,

Q6P9 V9 P10719 Q6P9T8 P85108 P69897 Q3KRE8 B4F7C2 Q4QRB4 Q5XIF6 P18418 P19527

1703e 1705e 2410e 2413e 2519e 2610e,h

P60711 O35179

2520e

Q6P9 V9 P68370 Q5XIF6 Q68FR8 Q4QRB4 P69897 Q68FY0

2715e

P50398

2720e

Q68FY0

2723e

P63018

3632e

P47942

6525e 6617e

P04764 P52481

6620e

Q62950

7432e 7617e 7621e 7622e

P51635 P10860 P62630

protein name creatine kinase B-type (KCRB) V-type proton ATPase catalytic subunit A (VATA) ATP synthase subunit d (ATP5H) WD repeat-containing protein 1 (WDR1) peptidyl-prolyl cis−trans isomerase A (PPIA) profilin 1 (PFN1) glutathione S-transferase Mu 1 (GSTM1) heterogeneous nuclear ribonucleoproteins A2/B1 (ROA2) cytochourome c oxidase subunit 5A (COX5A) fatty acid-binding protein, brain (FABP7) S-phase kinase-associated protein 1 (SKP1) transcription elongation factor B polypeptide 2 (ELOB) ATPase, H+ transporting, lysosomal 38 kDa, V0 subunit d1 (protein Atp6v0d1) tubulin α-1B chain (TBA1B) ATP synthase subunit β (ATPB) tubulin β-2C chain (TBB2C) tubulin β-2A chain (TBB2A) tubulin β-5 chain (TBB5) tubulin β-2B chain (TBB2B) tubulin β-4 chain (TBB4B) tubulin β-3 chain (TBB3) tubulin α-4A chain (TBA4A) calreticulin (CALR) neurofilament light polypeptide (NFL) actin, cytoplasmic 1 (ACTB) endophilin-A1 (SH3G2)

theoretical M/W 42 725 68 265

theoretical pI

identified by

f

Up-regulation 5.39 MS, MS/MS 5.42 MS


no. of peptide identification (MS, MS/MS)

sequence coverage fold change (WGE/ rate (%) (MS, CTL)g MS/MS)c

130, 121 105, N/D

14, 3 14, N/D

48%, 12% 32%, N/D

2.04** 6.42*

18 763 66 181

6.17 6.15

MS, MS/MS MS

82, 40 62, N/D

6, 3 10, N/D

49%, 34% 30%, N/D

2.44** 2.44**

17 863

8.34

MS/MS

N/D, 98

N/D, 2

N/D, 20%

2.34*

14 957 25 914

8.48 8.27

MS, MS/MS MS/MS

67, 29 N/D, 72

5, 2 N/D, 2

53%, 21% N/D, 13%

3.01* 5.87*

37 478

8.97

MS, MS/MS

80, 158

9, 3

37%, 15%

2.07*

16 130

6.08

MS/MS

170

6

26%

3.01*

14 864

5.46

MS/MS

104

2

19%

18 672

4.44

MS/MS

197

7

32%

4.05***

13 170

4.87

MS/MS

50

2

12%

2.11*

40 301

4.89

MS/MS

436

13

33%

2.25*

50 152 56 318 49 799 49 875 49 671 49 921 49 586 50 386 49 924 47 995 61 471

4.94 5.19 4.79 4.78 4.78 4.78 4.78 4.82 4.95 4.33 4.62

MS/MS MS/MS MS/MS MS/MS MS/MS MS/MS MS/MS MS/MS MS/MS MS/MS MS/MS

253 2076 4574 4564 4472 4468 4176 2962 277 687 1826

6 29 75 81 76 80 68 59 6 18 36

14% 53% 71% 75% 71% 74% 75% 64% 16% 37% 49%

3.04*** 2.75***

41 737 39 874

5.29 5.26

MS/MS MS/MS

1820 466

30 10

60% 39%

tubulin α-1B chain (TBA1B) tubulin α-1A chain (TBA1A) tubulin α-4A chain (TBA4A) tubulin α-3 chain (TBA3) tubulin β-3 chain (TBB3) tubulin β-5 chain (TBB5) cytochourome b-c1 complex subunit 1, mitochondrial (QCR1) Rab GDP dissociation inhibitor α (GDIA) cytochourome b-c1 complex subunit 1, mitochondrial (QCR1) heat shock protein 71 kDa protein (HSP7C) dihydropyrimidinase- related protein 2 (DPYL2) α-enolase (ENOA) adenylyl cyclase-associated protein 2 (CAP2) dihydropyrimidinase- related protein 1 (DPYL1) alcohol dehydrogenase (AK1A1) glutamate dehydrogenase 1, mitochondrial (DHE3)

50 120 50 136 49 924 49 960 50 386 49 671 52 849

4.94 4.94 4.95 4.97 4.82 4.78 5.57

MS/MS MS/MS MS/MS MS/MS MS/MS MS/MS MS/MS

3348 3184 3075 2619 1778 1543 2940

61 58 54 47 47 30 40

71% 71% 63% 58% 55% 55% 55%

50 537

5.00

MS/MS

62

2

2%

2.54*

52 849

5.57

MS/MS

72

1

2%

2.35**

70 871

5.37

MS/MS

140

2

4%

2.94***

62 278

5.95

MS/MS

94

2

5%

3.65**

47 128 52 879

6.16 6.69

MS/MS MS/MS

121 63

1 2

4% 4%

2.09* 2.26**

62 196

6.64

MS/MS

88

2

2%

2.01*

36 506 61 416

6.84 8.05

elongation factor 1-α1 (EF1A1)

50 114

9.10

MS/MS MS/MS MS/MS MS/MS

54 83 59 65

2 2 2 2

8% 2% 2% 2%

2.23* 2.18** 7.1** 2.49**

10498

2.43** 4.34** 2.19** 2.60*** 3.42** 4.05***

2.14**



Article

Table 3. continued b

SSP

accession no.

protein name

theoretical M/W

8608e

P15999

ATP synthase subunit α, mitochondrial (ATPA)

59 754

2817d

Q66HF1

79 412

2820d

P46462

3210d 3809d 4608d

P85971 P48721 P11598

5224d

Q9Z0 V6

6826d 6827d 7717d

P21575

NADH-ubiquinone oxido- reductase 75 kDa subunit, mitochondrial (NDUS1) transitional endoplasmic reticulum ATPase (TERA) 6-phosphogluconolactonase (6PGL) stress-70 protein (GRP75) protein disulfide-isomerase A3 (PDIA3) thioredoxin-dependent peroxide reductase (PRDX3) dynamin 1 (DYN1) pyruvate kinase isozymes M1/M2 (KPYM)

P11980

theoretical pI

identified by

f

Up-regulation 9.22 MS/MS Down-regulation 6.65 MS, MS/MS


no. of peptide identification (MS, MS/MS)

sequence coverage fold change (WGE/ rate (%) (MS, CTL)g MS/MS)c

121

1

2%

2.93**

93, 68

14, 3

30%, 6%

−2.17***

89 349

5.14

MS

75, N/D

11, N/D

23%, N/D

−2.08**

27 234 73 858 56 588

5.54 5.81 5.88

MS MS, MS/MS MS/MS

64, N/D 54, 181 N/D, 84

8, N/D 10, 4 N/D, 3

41%, N/D 21%, 7% N/D, 7%

−4.17** −2.56** −2.32***

28 295

7.14

MS/MS

N/D, 55

N/D, 1

N/D, 5%

−2.08***

97 295

6.44

57 818

6.63

MS, MS/MS MS, MS/MS MS, MS/MS

72, N/D 125, 47 96, 127

13, N/D 17, 1 13, 4

23%, N/D 29%, 1% 34%, 11%

−3.33** −2.08** −2.12**

a These proteins were differentially expressed between the water extract of Gastrodia elata Blume (WGE) and control (CTL) groups. bThe SSP is the serial number assigned by the gel image analysis software. cN/D means that the spot was not detected by MS or MS/MS. dTotal loading protein was 400 μg, and the spots were analyzed by MALDI-TOF mass spectrometry. eTotal loading protein was 450 μg, and the spots were analyzed by LC-ESI mass spectrometry. fThe protein identities were identified from MS and/or MS/MS method in the Mascot protein online search program. gAsterisk represents the significance from spot comparing between WGE and CTL (***p < 0.01, **p < 0.05, *p < 0.1). hAll the protein identities that were matched successfully from the Mascot protein online search program are presented (more than one protein was identified from one spot; more than one spot was identified as the same protein).

molecular effects have also been reported in pharmacological studies of antidepressants such as nortriptyline in rats.24 Actin dynamic programs include the CAP family. According to Peche et al., CAP1 abundantly exists in many tissues, but CAP2 is specifically highly expressed in brain, heart, and skeletal muscle.27 The cooperative model between CAP, actin, and cofilin in the promotion of actin dynamics and synaptogenesis was established.28 In the same study, Moriyama et al. also noted that the functions of CAP1 and CAP2 are analogous. However, research indicated that a defect in CAP is correlated with mood disorders.29 Similar to CAP and ARP, the expression of PFN1 was also increased in the WGE group. PFN1 regulates the mechanisms related to fixing actins on the phosphoinositide of the plasma membrane and assists in the formation of actin filaments during neuronal growth. Thus, PFN1 was shown to be an essential factor for neuronal differentiation, synaptic plasticity, and stabilizing the dendritic spine morphology.30 In contrast, actin-related genes were down-regulated in the mice that are sensitive to depression and the post-mortem cerebrum of mood disorder victims.29 In our study, we showed that WGE stimulates the expression of proteins associated with actin processes (Tables 2 and 3), which in turn facilitates its antidepressive-like effect. Like the roles of actin during neuronal growth, microtubules are polymerized to elongate the axon and dendritic trunk. In the present study, we found that the essential factor, CRMP2, was up-regulated in both the frontal cortex and hippocampus. However, dysfunction of CRMP2 has a strong correlation with psychiatric disorders. Recent proteomics data indicated that CRMP2 is down-regulated severely in rats with depressive-like behavior induced by UCMS.31 Post-mortem research also noted a reduction in the expression of CRMP2 in the frontal cortex of patients with MDD and bipolar disorder.32 Hence, CRMP2 is considered to be a potential biomarker for MDD. Another CRMP protein, CRMP1, was also up-regulated in the

Figure 3. Magnified gel images and quantification of differentially expressed proteins related to cytoskeleton remodeling in the frontal cortex and hippocampus. The matched spots are located in the center of each zoom-in gel image with yellow squares. The charts beside the gel images show the quantification results. The red and green bars represent respectively the expressed density of the matched spots for each of three replications of the control and WGE. The protein name in blue signifies that protein is involved in the Slit−Robo pathway. CRMP2, dihydropyrimidinase-related protein-2; ARP3, actin-related protein 3; PFN1, profilin-1; NFL, neurofilament light chain; CAP2, adenylyl cyclase-associated protein 2; CRMP1, dihydropyrimidinaserelated protein-1.

processes, were up-regulated in the WGE group. ARP2 and ARP3 form an ARP 2/3 complex and control the dependent branch function of actin filaments in rapidly developed neuronal regions such as lamellipodia and filopodia. These 10499



Article

Figure 4. Metacore-analyzed results presented as −log(p value) chart of matched protein identities for potential molecular networks and pathways. A higher value in the chart represents a higher correlation and possibility. (A) GeneGo Process Network; (B) GeneGo Pathway Maps (orange chart: frontal cortex; blue chart: hippocampus).

Figure 5. Pathway map for Slit−Robo signaling and the downstream factors. Proteins labeled with “1” and “2” represent the proteins identified from 2-DE of the frontal cortex and hippocampus, respectively. The ratio bars on the upper right corner of the symbols represent the expression ratio between the water extract of Gastrodia elata Blume (WGE) and the control (CTL) (WGE/CTL). c-Abl, c-Abl nonreceptor tyrosine kinase; CDK5, cyclin-dependent kinase 5; ROCK, Rho-associated protein kinase; CRMP2, dihydropyrimidinase-related protein-2; Arp 2/3, actin-related protein 2/3 complex; RhoA, Ras homologous member A; c-Src, Proto-oncogene tyrosine-protein kinase Src; CDC42, cell division cycle 42; N-WASP, neural Wiskott−Aldrich syndrome protein; FAK1, focal adhesion kinase 1.

processes.34 Hence, up-regulation of CRMP1 and -2 reflect the activation of microtubule-dependent activity in the brain after WGE administration. Neurofilament light chain (NFL), which forms intermediate filaments and composes the axon structure with medium

hippocampus. The function of CRMP1 is similar to CRMP2, which stimulates neuronal pathfinding and axon elongation. Research has proved that a deficiency of CRMP1 also leads to a defect in neuronal development.33 In contrast, antidepressants up-regulate the expression of CRMP proteins and tubulin-based 10500



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Figure 6. Western blot analysis of the regulators in the Slit−Robo pathway in the (A) frontal cortex and (B) hippocampus. The value on the bottom of each group and the chart images represent the ratio of expressed levels, which were normalized with the expression of GAPDH and relative to the control group. * indicates significant difference between control and WGE (p < 0.05) analyzed by Student’s unpaired t test.

neural plate to construct the networks. Thus, the Slit−Robo signal is generally regarded as an essential process for embryonic neuronal development. Interestingly, slit and robo are not only expressed in the embryonic developmental phase but maintain a high level in postnatal stages and adults.38 Marillat et al. also demonstrated that the expression of slit1 and robo2 changes in the hippocampus from postnatal day 10, the stage when synapses are rapidly generated. It seems that Slit−Robo signaling has some other activities in addition to mediating embryonic neuronal development. In fact, some research revealed that Slit−Robo may suppress neuroplasticity. Lin et al. discovered that dopaminergic neuronal extension and neurite growth are restricted by Slits and stimulated by Netrin.39,40 Furthermore, this phenomenon was also validated in a stem cell-derived neuron mode.40 In addition, a negative regulator, RhoA, is also involved in the Slit−Robo pathway. Down-regulation of RhoA correlates to neuronal growth. Cheng et al. observed that BDNF down-regulated the expression of RhoA, which stimulated neuronal growth.41 Besides, L-glutamate (L-Glu), an excitatory transmitter, promotes and stabilizes dendritic growth and spine formation, results in neuroplasticity, and is correlated with Rac1/Cdc42 activation

(NFM) and heavy chains (NFH), was highly up-regulated after WGE administration (Table 3). Studies have showed that, similar to other cytoskeletal units, negative regulation of neurofilaments is possibly related to neurodegenerative diseases.35 Foot-shock-induced depressive-like behavior in rats presented with lower expression of NFL but not NFM and NFH in the hippocampus.36 Moreover, antidepressants enhanced the expression of NFL and synaptic remodeling.37 Thus, incremental increase in the expression of NFL may be related to cytoskeleton remodeling and a neuroplasticity-stimulating effect of WGE. The Slit−Robo pathway emerged with the highest score in the bioinformatics analysis of the predicted mechanisms for the antidepressive-like effects of WGE (Figure 4B). During neuronal growth, the axon is elongated and guided to another target neuron by the attractive and repulsive activities of Netrin and Slits, respectively. These actions involve excited cytoskeletal remodeling. The repulsive property of Slits includes the deactivation or down-regulation of positive regulators such as CRMP2 and PFN1 and activation of negative factors such as Ras homologous member A (RhoA). This program operates especially in the embryo, so that axons grow and allow the 10501



Article

and reduction of RhoA activity.42 According to these studies, down-regulation of the Slit−Robo pathway plays a potential role in neuroplasticity. The WB results validated the expression of mediators involved in Slit−Robo signaling, which was matched by Metacore (Figure 6). To the best of our knowledge, there was no study indicating the potential relationship between the Slit−Robo pathway, pathology, and pharmacology for depression. In this study, based on the bioinformatics analyses and validation processes, stimulating neurocytosekeletal remodeling and enhancing neuroplasticity due to down-regulation of the Slit−Robo pathway might be one of the mechanisms to achieve the antidepressive-like activity of WGE.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 886-2-3366-4129. Fax: 886-2-2362-0849. E-mail: [email protected]. Author Contributions #

Shui-Tein Chen and Lee-Yan Sheen have contributed equally.

Funding

This research work was partially funded by the National Science Council (NSC97-2313-B-002-014-MY3) and National Taiwan University (aim for top university program, number 102R-7620), Taiwan. Proteomic mass spectrometry analyses were performed by Core Facilities for Protein Structural Analysis, Institute of Biological Chemistry, Academia Sinica, Taiwan, supported by a grant from National Science Council (NSC100-2325-B-001-029). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED FST, forced swimming test; WGE, water extract of Gastrodia elata Blume; CTL, control; FLX, fluoxetine; 2-DE, twodimensional electrophoresis

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REFERENCES

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