Chronic Cocaine Use Causes Changes in the Striatal Proteome

Article pubs.acs.org/crt

Chronic Cocaine Use Causes Changes in the Striatal Proteome Depending on the Endogenous Expression of Pleiotrophin Marta Vicente-Rodríguez,† Gonzalo Herradón,† Marcel Ferrer-Alcón,‡ María Uribarri,‡ and Carmen Pérez-García*,† †

Pharmacology Laboratory, Department of Pharmaceutical and Health Sciences, Facultad de Farmacia, Universidad CEU San Pablo, Madrid, Spain ‡ BRAINco Biopharma, S.L., Bizkaia Technology Park, Vizcaya, Spain S Supporting Information *

ABSTRACT: The neurotrophic factor pleiotrophin (PTN) is upregulated in different brain areas after the administration of different drugs of abuse, including psychostimulants. PTN has been shown to prevent cocaine-induced cytotoxicity in NG108-15 and PC12 cells. We previously demonstrated that specific phosphoproteins related to neurodegeneration processes are differentially regulated in the mouse striatum by a single cocaine (15 mg/kg) administration depending on the endogenous expression of PTN. Since neurodegenerative processes are usually observed in patients exposed to toxicants for longer duration, we have now performed a striatal proteomic study using samples enriched in phosphorylated proteins from PTN knockout (PTN−/−) mice, from mice with transgenic PTN overexpression (PTN-Tg) in the brain, and from wild type (WT) mice after a chronic treatment with cocaine (15 mg/kg/day for 7 days). We have successfully identified 23 proteins significantly affected by chronic cocaine exposure, genotype, or both. Most of these proteins, including peroxiredoxin-6 (PRDX6), triosephosphate isomerase (TPI1), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), and annexins A5 (ANXA5) and A7 (ANXA7), may be of significant importance because they were previously identified in proteomic studies in animals treated with psychostimulants and/or because they are related to neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease. The data support a protective role of PTN against chronic cocaine-induced neural alterations.

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INTRODUCTION The neuroadaptations caused by cocaine and other drugs of abuse underlie drug dependence, drug-induced neurotoxicity, and neurodegenerative processes associated with chronic drug consumption.1 Cocaine inhibits the dopamine transporter (DAT) in neuron terminals, thus increasing the extracellular dopamine level and the potentiation of dopamine transmission in the mesocorticolimbic system. Key areas in this system include the ventral tegmental area (VTA) that projects mainly to the nucleus accumbens (NAc) and prefrontal cortex (PFC). An excessive dopaminergic stimulation caused by psychostimulants in this circuit correlates with damage of dopaminergic neuronal bodies and terminals in substantia nigra and the striatum, respectively.2 Neuroprotective and adaptative responses to these actions of psychostimulants include cerebral upregulation of different neurotrophic factors including pleiotrophin (PTN) and midkine (MK).3 Interestingly, amphetamine induces an increased striatal astrocytosis in MK−/− mice4 and dopaminergic cell loss in the substantia nigra and an enhanced loss of striatal dopaminergic terminals in PTN−/− mice compared to WT mice.5 In addition, PTN prevents cocaine- and amphetamineinduced cytotoxicity in NG108-15 and PC12 cell cultures.6−8 Thus, evidence suggest that PTN and MK efficiently counteract © XXXX American Chemical Society

psychostimulant-induced neurotoxicity which is in agreement with the known actions of these cytokines as survival factors for dopaminergic neurons in other contexts such as neurodegenerative diseases.9−12 Some of the signaling pathways triggered by PTN are important for the neuroprotective roles of these cytokines against psychostimulants-induced neurotoxicity.13 PTN binds receptor protein tyrosine phosphatase (RPTP) β/ζ (also known as PTPRZ1) and inactivates its intrinsic tyrosine phosphatase activity.14 As a result, PTN causes rapid increases in the phosphorylation levels of substrates of RPTPβ/ζ known to be important in neuron survival including Fyn kinase, anaplastic lymphoma kinase (ALK), and β-catenin.3 Recently, we used a proteomic approach to study protein phosphorylation, in which we combined phosphoprotein enrichment, by immobilized metal affinity chromatography (IMAC), with two-dimensional gel electrophoresis and mass spectrometry, in order to identify the phosphoproteins regulated in the striatum of PTN knockout and wild type (WT) mice treated with a single dose of cocaine (15 mg/kg, i.p.). We identified 7 differentially expressed Received: March 27, 2015

A

DOI: 10.1021/acs.chemrestox.5b00130 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology

15% polyacrylamide gels, transferred to nitrocellulose membranes, and probed with anti-PTN (1:500) antibodies (R&D systems, Minneapolis, MN). Membranes were then reprobed with anti-actin antibodies at a 1:5,000 dilution (Chemicon, Temecula, CA). After incubation with appropriate secondary antibodies (1:5,000) conjugated with horseradish peroxidase, the immunoreactive proteins were visualized using the ECL method according to the manufacturer’s instructions (Amersham, San Francisco, CA). PTN levels were quantified by densitometry in each animal sample using Image Lab image acquisition and analysis software (Bio-Rad, Hercules, CA) and normalized with actin protein levels. PTN protein expression analysis was performed by 2-way ANOVA followed by Bonferroni’s posthoc tests, considering brain area and genotype as variables. Cocaine Treatment. WT, PTN−/−, and PTN-Tg mice (n = 11−14 per group) were administered i.p. with 15 mg/kg cocaine HCl (Alcaliber, Madrid, Spain) or saline (control, 10 mL/kg), once a day for 7 days. The dose of cocaine was chosen because a single administration of 15 mg/kg cocaine was shown to induce significant changes in phosphoproteins related to neurodegeneration processes and oxidative stress depending on the endogenous levels of PTN.15 The six experimental groups depending on genotype and treatment were WT saline (WS), WT cocaine (WC), PTN−/− saline (PS), PTN−/− cocaine (PC), PTN-Tg saline (TS), and PTN-Tg cocaine (TC). Twenty-four hours after the last administration of cocaine or saline, mice were sacrificed, their brains removed, and the striatum dissected and preserved at −80 °C until the proteomic study. Protein Identification: Proteomic Analysis. Since we were interested in the identification of differentially expressed phosphorylated proteins, we used a proteomic approach, previously employed in our laboratory,15,30 in which we combined phosphoprotein enrichment, by IMAC, with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This approach has been previously used by others for similar experimental purposes.31,32 Extraction and Enrichment in Phosphorylated Proteins. For the extraction and enrichment in phosphorylated proteins, we used the Pierce Phosphoprotein Enrichment Kit (Thermo Scientific, USA), which is based on IMAC and results in highly efficient purification of phosphoprotein containing phosphotyrosine, phosphoserine, and phosphothreonine residues.33 Tissue samples (n = 11−14) of each experimental group were pooled, homogenized by sonication, centrifuged (10,000 rpm, 20 min, 4 °C), and the supernatants, containing up to 4 mg of total protein, collected. We followed rigorously the previous protocol used in our laboratory for the identification of striatal phosphoproteins regulated by a single administration of amphetamine and cocaine.15,30 Final protein concentration was measured by the Pierce BCA Protein Assay Kit (Thermo Scientific, USA). 2D-PAGE. For protein separation, we employed the 2D gel electrophoresis protocol routinely used in our laboratory.30,34 Briefly, 300 μL of each sample obtained in the previous step were taken for the rehydration and simultaneous loading of the proteins on an IPG strip (17 cm, 3−10 NL, Bio-Rad, USA) at 50 V, 20 °C, for 12 h in a PROTEAN IEF cell (Bio-Rad, USA). Then, the voltage was increased to 10000 V and focused for a total of 60000 Vh. Prior to SDS−PAGE, the strips were equilibrated in a solution containing 0.375 M Tris− HCl (pH 8.8), 6 M urea, 2% SDS, and 20% glycerol (Bio- Rad, USA). The equilibration was carried out in two steps. DTT (2%, Bio-Rad, USA) was added in the first step and iodoacetamide (2.5%, Bio-Rad, USA) in the second one to the equilibration solution. The SDS− PAGE was run in polyacrylamide gels (180 × 200 × 1 mm, 12%) for 6 h at 200 V, 20 °C, in a PROTEAN Plus Dodeca cell (Bio-Rad, USA). The gels were stained with the “‘Silver Stain”’ kit (Bio-Rad, USA), according to manufacturer’s protocol.35 Silver staining was performed in a Dodeca Stainer (Bio-Rad, USA). Five gels from each experimental group were scanned using the densitometer GS-800 (Bio-Rad, USA). Spots were detected, quantified, and matched automatically with the PDQuest v8 software (BioRad, USA), and manually checked. Normalization of the optical density of each spot and statistical analysis were conducted as described before.34 Spots that showed statistical differences in optical

phosphoproteins including endoplasmic reticulum resident protein 60 (ERP60), 5′(3′)-deoxyribonucleotidase, glutamate dehydrogenase 1 (GLUD1), peroxiredoxin-6 (PRDX6), and aconitase which are known to be involved in the development of neurological and neurodegenerative disorders.15 In addition, it has been hypothesized that cocaine abuse could play a role in the development of Parkinson’s disease based on the capacity of cocaine to increase the levels of α-synuclein in dopaminergic neurons.16−18 Also, it has been previously established that the molecular mechanisms of neurodegeneration include the production of reactive oxygen species,19 and acute cocaine is known to increase the production of reactive oxygen species in the striatum.20 Interestingly, in our previous study, we determined that PTN−/− mice are more prone to significant alterations in proteins related to oxidative stress (i.e., aconitase, ERP60, PRDX6, and GLUD1) caused by a single injection of cocaine, suggesting that PTN may play an important role in cocaine-induced neurotoxicity and neurodegeneration in vivo. However, one limitation of that study was that significant changes in the proteins of interest were identified after an acute administration of cocaine (15 mg/kg), whereas neurodegeneration processes are usually observed in patients exposed to toxicants for longer duration.21 To fill this gap, we have now performed a proteomic study in samples enriched in phosphorylated proteins from PTN−/− and WT mice after chronic cocaine exposure. In addition, we have used in this study mice with transgenic PTN overexpression (PTN-Tg) in relevant areas of the mesolimbic system such as PFC.22 Methamphetamine-induced neuroadaptations in the dorsal striatum are related to changes in the glutamatergic innervation by afferents in the PFC,23 whereas the ventral striatum is heavily innervated by afferents arising in the PFC which is relevant for the motivational significance of reward-related cues.24,25 Thus, a mouse model with specific PTN transgenic overexpression in the PFC is useful to better establish the relevance of PTN in the changes of key phosphorilated proteins induced by chronic cocaine in the striatum.

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EXPERIMENTAL PROCEDURES

Animals. PTN−/− mice on a C57BL/6 background were generated as previously described.26 PTN-Tg mice on a C57BL/6 background were generated by pronuclear injection as recently described.22,27 Briefly, the pCMV-sport6 vector including the PTN cDNA sequence was obtained from imaGenes (IRAVp968B05104D). The acceptor vector used was pTSC-a2, which contained the regulatory regions responsible for tissue specific expression of Thy-1 gene, which drives neuron-specific expression of transgenes28,29 and induces the expression of the transgene mainly in neurons from cortex and hippocampus.22 A 2−4-fold PTN mRNA and protein specific overexpression in the cortex and hippocampus was established by quantitative real time-polymerase chain reaction (qRT-PCR), in situ hybridization, and by Western blot.22,27 For this study, we used male PTN−/−, PTN-Tg, and WT animals of 9−10 weeks (20−25 g). Mice were housed under controlled environmental conditions (22 ± 1 °C and a 12-h light/12-h dark cycle) with free access to food and water. All the animals used in this study were maintained in accordance with European Union Laboratory Animal Care Rules (86/609/ECC directive), and the protocols were approved by the Animal Research Committee of USP-CEU. Western Blot. We performed Western blot analyses to confirm the PTN overexpression in a relevant area (PFC) and to confirm readable levels of expression of PTN in the striatum of PTN-Tg mice. Striata and PFC from PTN-Tg and WT mice (n = 3−4/group) were removed and frozen in dry ice and stored to −80 °C until the protein extraction procedure. Protein extraction and quantification were performed as previously described.22 Equilibrated protein samples were loaded onto B


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Chemical Research in Toxicology density (P < 0.05) between two groups, with an expression foldchange of 2 or more, and could be clearly visualized in the gels were cut out for mass spectrometry identification using pipet tips. Mass Spectrometry Analysis of Protein Spots. Proteins selected for analysis were in-gel reduced, alkylated, and digested with trypsin (Roche, Germany). One microliters of the supernatant was spotted onto a matrix-assisted laser desorption/ionization (MALDI) sample plate and allowed to air-dry at RT. Then, 0.4 μL of a 3 mg/mL solution of α-cyano-4 hydroxy-cinnamic acid in 50% acetonitrile, and 0.1% TFA (Sigma, Spain) were added to the dried peptide digest spots and allowed again to air-dry at RT. MALDI-time-of-flight (TOF) mass spectrometry analyses were performed at the Proteomics Facility UCM-PCM of Madrid, a member of ProteoRed network. A 4800 Plus Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, MDS Sciex, Canada) was used, operated in positive reflector mode with an accelerating voltage of 20,000 V. Peptides from the autodigestion of trypsin were used to calibrate internally all mass spectra. The analysis by MALDI-TOF/TOF mass spectrometry produces peptide mass fingerprints and the peptides observed with a signal to noise greater than 10 can be collated as a list of monoisotopic molecular weights. Proteins identified by peptide mass fingerprints with some ambiguity were subjected to tandem mass spectrometry sequencing analyses. From the mass spectrometry spectra, a suitable precursor was selected for tandem mass spectrometry analyses with CID on (atmospheric gas was used) 1 KV ion reflector mode and precursor mass Windows ±5 Da. The plate model and default calibration were optimized for the tandem mass spectrometry spectra processing. For protein identification, the nonredundant Uniprot/Swiss-Prot (release 57.15; 535698 sequences; 190107059 residues) database was searched using MASCOT 2.1 (matrixscience.com) through the Global Protein Server v3.6 from Applied Biosystems. Search parameters were oxidized with methionine as the variable modification and carbamidomethyl cystein as the fixed modification; peptide mass tolerance 50 ppm for peptide mass fingerprints were searched and 80−100 ppm for tandem mass spectrometry were searched; 1 missed trypsin cleavage site; and tandem mass spectrometry fragment tolerance of 0.3 Da. We accepted positive protein identification when the probability based Mowse Score was greater than the score fixed by MASCOT as significant with p < 0.05. The similarity between theoretical MW and pI values and the experimental ones was checked.

Figure 1. Pleiotrophin protein levels in prefrontal cortex (PFC) and striatum (Str) of WT and PTN-Tg mice. PTN protein levels were determined by Western blot with anti-PTN antibodies. Actin amounts were determined using anti-actin antibodies. The graph shows the ratio PTN/actin of optical density (OD) measurements corresponding to the total PTN and actin protein levels, respectively. Data show the mean ± SEM (a.u.) of 3−4 individual samples from every experimental group. ** P < 0.01 vs WT; # P < 0.05 vs PFC; ### P < 0.001 vs PFC.

higher than 50 au that could be well visualized, manually excised, and, therefore, analyzed by mass spectrometry. Of all spots that showed significant differences in normalized optical density among the different experimental groups, we selected those with an expression fold-change of 2 or more. Twenty-six spots corresponding to 23 different proteins were successfully identified by either peptide mass fingerprinting or tandem mass spectrometry (Table 1). These spots are indicated in one representative gel showed in Figure 2. The phosphorylation in different residues of all the proteins included in the identified spots has been previously demonstrated according to PhosphositePlus.36 The comparison of the 2-D patterns between different treatments within the same genotype resulted in 5 proteins showing significant differences in optical density in WT mice, 12 proteins in PTN−/− mice, and 3 proteins in PTN-Tg mice (comparisons WC vs WS; PC vs PS; and TC vs TS, respectively, Table 2). In WT mice, 4 of the 5 proteins identified were significantly upregulated after chronic cocaine treatment. Remarkably, the 12 proteins identified in PTN−/− mice were significantly downregulated after cocaine treatment, whereas the opposite pattern occurred in PTN-Tg mice, where the 3 proteins differently expressed were upregulated after cocaine treatment. When comparing genotypic differences within the same treatment (saline or cocaine), we identified significant differences in the optical density of 26 spots corresponding to 23 proteins because spots 5103 and 6109, 6104 and 6220, and 7008 and 8003 corresponded to the same proteins: phosphoglycerate mutase 1 (PGAM1), carbonic anhydrase 2 (CA2); and peptidylprolyl cis−trans-isomerase (PPIA) (Table 2). In the case of PGAM1 and PPIA, both spots exhibited the same pattern in the change of protein levels. However, both spots corresponding to CA2 showed opposite patterns of expression changes. In all cases, both spots were located close to each other in the gel with limited changes in their horizontal position (Figure 2), suggesting differences in their Ip. These changes in the Ip are usually

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RESULTS Previously, it was shown that PTN-Tg mice exhibit a significant increase of PTN protein levels in PFC compared to that of WT mice. Since the striatum is the area of interest in our study, and it is known to be heavily innervated by afferents arising in the PFC, we tested the levels of PTN in the striatum and PFC in PTN-Tg and WT mice (Figure 1). Two-way ANOVA revealed a significant effect of the genotype (F(1,10) = 11.59, P < 0.01) and of the brain area (F(1,10) = 39.45, P < 0.001). We confirmed the upregulation of PTN in PFC of PTN-Tg mice compared to that of WT mice.22 PTN expression was found at higher levels in the striatum compared to that of PFC independently of the genotype and a not significant trend of ∼20% increase of PTN levels was observed in the striatum of PTN-Tg mice compared to that in WT mice (Figure 1). For the identification of differentially expressed proteins in WT, PTN−/−, and PTN-Tg mice treated with cocaine, the samples obtained after pooling 11−14 striata of every experimental group (WS, WC, PS, PC, TS, and TC) were first enriched in phosphoproteins by IMAC. The phosphoprotein yield ranged from 8.0 to 12.0% of the total protein content which is in agreement with previous studies.15,30 Proteins were then separated by 2D-PAGE. The gels (n = 5 per group) obtained from the 6 experimental groups were analyzed simultaneously and matched in the same set. For the statistical analysis, we considered only well-resolved spots, with an optical density C


ubiquitin carboxyl-terminal hydrolase isozyme L1

annexin A7

ATP synthase subunit d, mitochondrial peroxiredoxin-6

aminoacylase-1

1104

1414

2005

3306

D

carbonic anhydrase 2

6104

CA2

PSMA2

proteasome subunit α type-2

6009

PGAM1

ALAD

phosphoglycerate mutase 1

5103

MDH1

δ-aminolevulinic acid dehydratase

malate dehydrogenase, cytoplasmic

4104

DPYSL2

ACY1

PRDX6

ATP5H

ANXA7

UCHL1

ANXA5

abbreviation

5202

dihydropyrimidinase-related protein 2

3617

3106

annexin A5

protein name

0110

Spot n.

P00920

P49722

P10518

Q9DBJ1

P14152

O08553

Q99JW2

O08709

Q9DCX2

Q07076

Q9R0P9

P48036

UniProt ID

29129

26024

36456

28928

36659

62638

45980

24969

18795

50178

25165

35787

theoretical MW

6.49

6.92

6.32

6.67

6.16

5.95

5.89

5.71

5.52

5.91

5.14

4.83

theoretical pI

123

115

76

98

56

176

93

88

72

84

133

56

score

64

52

28

50

30

48

38

41

31

31

72

34

coverage (%)

Table 1. Differentially Expressed Striatal Phosphoproteins Identified by Mass Spectrometrya

10

7

7

8

7

18

11

7

4

12

9

8

peptides matched

DPESEHPSVTLFRICTVQPNPDYGGAITFLEERDSEGYIYARSVSIQYLEAVRRRPEFQALRAGFALDEGLANPTDAFTVFYSERLEGGVAYNVVPATMSASFDFRAMNLTLEPEIFPAATDSRAVGIPALGFSPMNRTPVLLHDHNERLHEDIFLR IVNDDQSFYADIYMEDGLIKQIGENLIVPGGVKMVIPGGIDVHTRFQMPDQGMTSADDFFQGTKDIGAIAQVHAENGDIIAEEQQRSITIANQTNCPLYVTKSAAEVIAQARAVGKDNFTLIPEGTNGTEERMSVIWDKAVVTGKMDENQFVAVTSTNAAKVFNLYPRISVGSDADLVIWDPDSVKTHNSALEYNIFEGMECRGSPLVVISQGKIVLEDGTLHVTEGSGRKPFPDFVYKRGLYDGPVCEVSVTPKNLHQSGFSLSGAQIDDNIPR DLDVAVLVGSMPRVIVVGNPANTNCLTASKSAPSIPKENFSCLTRNVIIWGNHSSTQYPDVNHAKGEFITTVQQRFVEGLPINDFSRELTEEKETAFEFLSSA HGESAWNLENRFSGWYDADLSPAGHEEAKRDAGYEFDICFTSVQKRHYGGLTGLNKAETAAKSYDVPPPPMEPDHPFYSNISKYADLTEDQLPSCESLKDTIARALPFWNEEIVPQIKVLIAAHGNSLR CVLIFGVPSRDEQGSAADSEDSPTIEAVRAGCQVVAPSDMMDGRVEAIKFASCFYGPFRDAAQSSPAFGDRRCYQLPPGARTAVLETMTAFRR AERGYSFSLTTFSPSGKSILYDERHIGLVYSGMGPDYRVLVHRKLAQQYYLVYQEPIPTAQLVQRYNEDLELEDAIHTAILTLKESFEGQMTEDNIEVGICNEAGFRRLTPTEVRDYLAAIA HNGPENWHKDFPIANGDRQSPVDIDTATAQHDPALQPLLISYDKAASKSIVNNGHSFNVEFDDSQDNAVLKGGPLSDSYRKYAAELHLVHWNTKAVQQPDGLAVLGIFLKIGPASQGLQKVLEALHSIKEPITVSSEQMSHFRTLNFNEEGDAEEAMVDNWRPAQPLK

GTVTDFPGFDGRGLGTDEDSILNLLTSRLIVAMMKPSRWGTDEEKFITIFGTRYMTISGFQIEETIDRETSGNLEQLLLAVVKSIPAYLAETLYYAMKNFATSLYSMIK MQLKPMEINPEMLNKLGVAGQWRQIEELKGQEVSPKQTIGNSCGTIGLIHAVANNQDKLEFEDGSVLKQFLSETEKLSPEDRCFEKNEAIQAAHDSVAQEGQCRVDDKVNFHFILFNNVDGHLYELDGRMPFPVNHGASSEDSLLQDAAKEFTEREQGEVR GFGTDEQAIVDVVSNRVLIEILCTRCYQLEFGRDLEKSDTSGHFERLLVSMCQGNRDERQSVNHQMAQEDAQRLYQAGEGRLGTDESCFNMILATREFSGYVESGLKTILQCALNRPAFFAERQMFTQMYQKTLSTMIASDTSGDYRK SWNETFHARLASLSEKPPAIDWAYYRSCAEFVSGSQLRKYPYWPHQPIENL MPGGLLLGDEAPNFEANTTIGRDFTPVCTTELGRLPFPIIDDKGRVVFIFGPDKKLSILYPATTGRNFDEILRVVDSLQLTGTKPVATPVDWK

tandem mass spectrometry sequence data

ND:48 (AD)

ND:66 (AD)

ND:65

ND:48,63 (AD) DN:64

ND:48,62 (AD)

ND:48,61 (AD) DAB:34

ND:39,40 (PD)41−43 (AD) DAB:34,38 DN:15 ND:60

DN:30,56,58 DAB:.34

ND:59 (PD) DN:30,56

ND:47 (PD and AD)48 (AD)

ND:57 (AD)

association with ND, DN and/or DAB

Chemical Research in Toxicology Article


phosphoglycerate mutase 1

triosephosphate isomerase

actin-related protein 2/3 complex subunit 2

glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic carbonic anhydrase 2

dihydrolipoyl dehydrogenase, mitochondrial

6109

6110

6111

6114

6504

E

α-crystallin B chain

fructose-bisphosphate aldolase C

7012

7205

succinate-semialdehyde dehydrogenase, mitochondrial

CRYAB

peptidyl-prolyl cis−transisomerase

7008

7403

PPIA

nucleoside diphosphate kinase B

7005

ALDH5A1

ALDOC

NME2

GTP-binding nuclear protein Ran

RAN

DLD

CA2

GPD1

ARPC2

TPI1

PGAM1

abbreviation

7003

6220

protein name

Spot n.

Table 1. continued

Q8BWF0

P05063

P23927

P17742

Q01768

P62827

O08749

P00920

P13707

Q9CVB6

P17751

Q9DBJ1

UniProt ID

56503

39769

20056

18131

17466

24579

54751

29129

38176

34450

32684

28928

theoretical MW

8.53

6.67

6.76

7.74

6.97

7.01

7.99

6.49

6.75

6.84

5.56

6.67

theoretical pI

140

282

76

74

96

89

78

140

85

186

60

125

score

37

73

33

54

74

50

30

30

20

54

25

58

coverage (%)

12

17

5

7

9

8

9

4

5

11

5

9

peptides matched

SIVNNGHSFNVEFDDSQDNAVLKAVQQPDGLAVLGIFLKEPITVSSEQMSHFRTLNFNEEGDAEEAMVDNWRPAQPLK TVCIEKNETLGGTCLNVGCIPSKALLNNSHYYHMAHGKDFASRNQVTATKADGSTQVIDTKAEVITCDVLLVCIGRRPFTQNLGLEELGIELDPKIPNIYAIGDVVAGPMLAHKSEEQLKEEGIEFKVCHAHPTLSEAFREANLAAAFGKPINF HLTGEFEKKYVATLGVEVHPLVFHTNRGPIKFNVWDTAGQEKDGYYIQAQCAIIMFDVTSRNVPNWHRVCENIPIVLCGNKVDIKNLQYYDISAKSNYNFEKPFLWLAR TFIAIKPDGVQRGLVGEIIKRASEEHLKQHYIDLKDRPFFPGLVKYMNSGPVVAMVWEGLNVVKVMLGETNPADSKPGTIRGDFCIQVGRNIIHGSDSVESAEK SCAHDWVYE VSFELFADKVPKALSTGEKGFGYKIIPGFMCQGGDFTRSIYGEKFEDENFILKTEWLDGKHVVFGKVKEGMNIVEAMERKITISDCGQL RPFFPFHSPSRAPSWIDTGLSEMRHFSPEELKVKVLGDVIEVHGKHEERQDEHGFISR PHSYPALSAEQKKELSDIALRGILAADESVGSMAKRLSQIGVENTEENRRQVLFSADDRVKCIGGVIFFHETLYQKDDNGVPFVRVDKGVVPLAGTDGETTTQGLDGLLERISDRTPSALAILENANVLARYASICQQNGIVPIVEPEILPDGDHDLKRCQYVTEKYSPEEIAMATVTALRRTVPPAVPGVTFLSGGQSEEEASLNLNAINRALQASALNAWRDNAGAATEEFIKRAEMNGLAAQGRYEGSGDGGAAAQSLYIANHAY SYASGPGGLHADLLRGDSFVGGRWLPAPATFPVYDPASGAKLGTVADCGVPEARAAYDAFNSWKGVSVKRIYGDIIYTSAKEVGEVLCTDPLVSKILLHHAANSVKRNAGQTCVCSNRVGNGFEEGTTQGPLINEK RHQSGGNFFEPTLLSNVTRVAEQLEVGMVGVNEGLISSVECPFGGVKEGSKYGIDEYLEVK

HGESAWNLENRFSGWYDADLSPAGHEEAKRDAGYEFDICFTSVQKRHYGGLTGLNKAETAAKSYDVPPPPMEPDHPFYSNISKYADLTEDQLPSCESLKDTIARALPFWNEEIVPQIKRVLIAAHGNSLRNLKPIKPMQFLGDEETVR DLGATWVVLGHSERRHVFGESDELIGQKVSHALAEGLGVIACIGEKVVLAYEPVWAIGTGKTATPQQAQEVHEKLR MILLEVNNRELQAHGADELLKRDSIVHQAGMLKRRNCFASVFEKYFQFQEEGKEGENRAVIHYRDDETMYVESKVTVVFSTVFKDDDDVVIGKVFMQEFKEGRASHTAPQVLFSHREPPLELKDTDAAVGDNIGYITFVLFPRDNTINLIHTFRDYLHYHIK VTMWVFEEDIGGRKLTEIINTQHENVKLGIPMSVLMGANIASEVAEEKDLMQTPNFRVCYEGQPVGEFIR


DAB:71

ND:48,62 (AD)

ND:70 (AD)

DN:30 ND:48,52 (AD)

ND:53,69 (AD)

ND:69 (AD)

ND:48,62 (AD)

ND:48 (AD)

DN:68

ND:67 (PD)

ND:48,49,51 (AD)

ND:48,63 (AD) DN:64


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due to differences in the number of phosphorylated residues or in the type of residues that are phosphorylated.37 Remarkably, all of the 23 identified proteins have been previously shown to be involved in neurodegenerative disorders and/or drug-induced neurotoxicity and/or addictive behaviors (Table 1). In the section to follow, we discuss the possible involvement of the most interesting proteins identified here in the modulation of chronic cocaine effects depending on PTN expression.

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DAB, drug-induced addictive behaviors; DN, drug-induced neurotoxicity; ND, neurodegenerative diseases (AD, Alzheimer’s disease; PD, Parkinson’s disease).

DISCUSSION In the present work, we have compared the striatal proteome, enriched in phosphorylated proteins of WT, PTN−/−, and PTN-Tg mice that had been exposed for 7 days to cocaine (15 mg/kg/day, i.p.). We have successfully identified 23 proteins significantly affected by cocaine treatment, genotype, or both. We previously used a similar proteomic approach to study the striatum of WT and PTN−/− mice treated acutely with cocaine (15 mg/kg/day, i.p.), and we identified 7 differentially expressed proteins. Only 1 of the 7 proteins identified in the previous study coincides with those identified in the present study, PRDX6.15 Nevertheless, all the proteins identified in the present study are of particular interest because they were previously identified in phosphoproteomic studies in animals treated with psychostimulants because they were related to neurodegenerative disorders such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) or because they are known to be involved in drug-induced neurotoxicity and addictive behaviors. In this discussion, we have focused on proteins that were previously identified in other proteomic studies performed by our group using the striatum of PTN −/− mice treated acutely with psychostimulants (PRDX6,15 nucleoside diphosphate kinase B -NME2-, annexin A7 -ANXA7- and ATP synthase subunit d -ATP5H-).30 Besides and as representative of proteins involved in neurodegenerative diseases, we have also discussed the results obtained for ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), triosephosphate isomerase (TPI1), peptidil-prolil cis/trans-isomerase A (PPIA) and annexin A5 (ANXA5). One of the proteins identified here, PRDX6, was previously found downregulated in PTN−/− mice after a single administration of 15 mg/kg cocaine.15 However, in the present study we have detected lower levels of PRDX6 in cocaine-treated PTN−/− compared to that in WT mice, but we did not detect significant differences caused by cocaine. In contrast, we now found a significant downregulation of PRDX6 in PTN-Tg compared to that in WT mice independently of the treatment received. It is interesting to note that previous reports have linked changes in the levels of expression of PRDX6 and other peroxiredoxin isoforms with the rewarding effects of cocaine in rodents.34,38 PRDX6 belongs to the peroxiredoxin family of sulfhydryl-dependent peroxidases whose levels of expression are altered in the brain of PD and AD patients39,41 in which they seem to exert neuroprotective effects.42 In contrast, it has been suggested that the overexpression of PRDX6 could accelerate the development of AD.43 Taking together the ability of PTN to protect against the neurotoxic effects of cocaine6,8 and that, independently of the treatment, PTN-Tg mice show reduced levels of PRDX6 compared to WT mice, the data suggest that phosphorylation of PRDX6 may be suppressed in a step downstream of the signaling pathways triggered by PTN for this cytokine to exert its neurotrophic effects. All in all, in this case it is crucial to know the influence of tyrosine phosphorylation on the activity of the protein bacause PRDX6 is a

a

PSMA4 proteasome subunit α type-4 8103

Q9R1P0

29737

7.59

97

56

10

VNPTVFFDITADDEPLGRVSFELFADKVPKGSSFHND:48,52 (AD) RIIPGFMCQGGDFTRSIYGEKFEDENFILKVKEGMNIVEAMERKITISDCGQL DAB:72 TTIFSPEGRLLDEVFFSEKIYKLNEDMACSVAGITSDANVLTNELRYLLQYQEPIPCEQLVTALCDIKQAYTQFGGKRPFGVSLLYIGWDKHYGFQLYQSDPSGNYGGWKATCIGNNSAAAVSMLKLSAEKVEIATLTRQKEVEQLIK 6 53 77 7.74 18131 P17742 PPIA peptidyl-prolyl cis−trans-isomerase A 8003

protein name Spot n.

Table 1. continued

abbreviation

UniProt ID

theoretical MW

theoretical pI

score

coverage (%)

peptides matched




F


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Figure 2. Representative silver stained 2D gel of the phosphoproteome from the mouse striatum. Spots labeled with numbers showed significant differences in normalized optical density after comparing the 6 groups of animals and were identified by mass spectrometry. In some cases, the same protein was identified in two different spots: phosphoglycerate mutase 1 (spots 5103 and 6109), carbonic anhydrase 2 (spots 6104 and 6220), and peptidyl-prolyl cis−trans-isomerase (spots 7008 and 8003). The complete list of the identified proteins is shown in Table 1

in the PTN-Tg mice. Basal UCHL1 levels in saline-treated PTN−/− mice were increased compared with those in WT mice, whereas the lowest levels of expression of this protein in cocaine-treated mice were found in PTN-Tg mice (see Table 1 of Supporting Information). The levels of expression of UCHL1 are known to be upregulated in the brain of AD patients48 suggesting the possibility that downregulation of UCHL1 striatal levels in PTN-Tg mice may protect them against the potential neurotoxicity induced by chronic cocaine exposure. Another protein, known to be involved in neurodegeneration, TPI1, was significantly reduced in cocaine-treated

bifunctional protein with both peroxidase and phospholipase A2 activities. Whereas peroxidase activity of PRDX6 is unaffected by phosphorylation,44 the phosphorylation of threonine in position 177 results in an activation of the phospholipase A2 activity of PRDX6,45 and it has been described that phospholipases A2 increase during neurodegeneration.46 UCHL1 is a neuronal desubiquitinase whose genetic mutations have been related to early onset PD.47 When comparing animals of the same genotype, cocaine significantly increased the expression levels of UCHL1 in WT mice, whereas they were decreased in PTN −/− mice and not significantly affected G


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Table 2. Striatal Proteins of Mice Showing Significant Differences in Normalized Optical Density When Comparing the Different Experimental Groupsa ratio of normalized optical density Spot n.

protein name

0110 1104 1414 2005 3106 3306 3617 4104 5103 5202 6009 6104 6109 6110 6111 6114

annexin A5 ubiquitin carboxyl-terminal hydrolase isozyme L1 annexin A7 ATP synthase subunit d. mitochondrial peroxiredoxin-6 aminoacylase-1 dihydropyrimidinase-related protein 2 malate dehydrogenase. cytoplasmic phosphoglycerate mutase 1 δ-aminolevulinic acid dehydratase proteasome subunit alpha type-2 carbonic anhydrase 2 phosphoglycerate mutase 1 triosephosphate isomerase actin-related protein 2/3 complex subunit 2 glycerol-3-phosphate dehydrogenase [NAD(+)] cytoplasmic carbonic anhydrase 2 dihydrolipoyl dehydrogenase. mitochondrial GTP-binding nuclear protein Ran nucleoside diphosphate kinase B peptidyl-prolyl cis−trans-isomerase A α-crystallin B chain fructose-bisphosphate aldolase C succinate-semialdehyde dehydrogenase. mitochondrial peptidyl-prolyl cis−trans-isomerase A proteasome subunit α type-4

6220 6504 7003 7005 7008 7012 7205 7403 8003 8103

WC/ WS

PC/ PS

TC/TS

PS/ WS

TS/ WS

TS/ PS

PC/ WC

TC/ WC

TC/ PC

ANXA5 UCHL1 ANXA7 ATP5H PRDX6 ACY1 DPYSL2 MDH1 PGAM1 ALAD PSMA2 CA2 PGAM1 TPI1 ARPC2 GPD1

ns 1.4 ns ns ns ns ns ns 2.3 1.7 ns ns ns ns ns ns

ns 0.6 ns ns ns 0.6 ns ns ns ns 0.5 ns 0.4 0.4 ns 0.3

ns ns ↑ (absent in TS) ns ns ns ns ns ns ns ns ns ns ns ns ns

1.6 1.4 ns 4.4 ns ns ns 2.0 4.3 ns 1.4 ns 2.7 2.0 ns 2.7

ns ns 0 ns 0.4 0.2 0.4 3.2 4.2 ns ns 0.2 2.0 ns 0.5 ns

0.5 0.7 0 0.1 ns 0.2 ns 1.5 ns ns 0.6 0.3 0.7 0.7 0.5 0.4

ns 0.6 ns 6.7 0.5 0.6 ns ns ns 0.3 ns ns ns ns ns ns

ns 0.5 ns 0 0.4 0.2 0.3 2.9 ns 0.2 ns 0.2 1.4 ns ns ns

ns ns ns 0 ns 0.3 ns 3.1 ns ns ns 0.3 1.6 ns ns ns

CA2 DLD RAN NME2 PPIA CRYAB ALDOC ALDH5A1

ns 0.4 ns ns 1.6 ns ns ns

ns 0.5 ns 0.5 0.5 ns 0.4 0.6

ns ns ns ns 1.8 ns ns ns

ns ns ns ns 1.9 ns ns 1.6

22.3 0.4 ns ns ns 11.3 ns ns

16.3 0.5 ns ns 0.6 10.8 ns 0.4

ns ns 0.5 0.5 0.6 0.3 ns ns

20.5 ns ns ns 1.3 15.0 ns ns

20.5 ns ns 1.9 2.1 50.2 2.0 ns

PPIA PSMA4

ns ns

0.6 0.5

2.0 ns

ns 1.6

ns ns

ns 0.5

0.6 0.6

1.4 ns

2.4 ns

abbreviation

a

Numeric values correspond exclusively to the ratio of the normalized optical density of comparison showing significant differences. WS, wild type animals treated with saline; WC, wild type animals treated with cocaine; PS, PTN−/− mice treated with saline; PC, PTN−/− mice treated with cocaine; TS, PTN-Tg mice treated with saline; TC, PTN-Tg mice treated with cocaine; ns, comparison showing not significant differences (n = 5).

NME2 may be involved in the neurotrophic actions of PTN against chronic cocaine exposure. ANXA7 was absent in the striatum of saline-treated PTN-Tg mice but was significantly upregulated after cocaine treatment. Interestingly, ANXA7 was present in saline-treated WT and PTN−/− mice and tended to decrease after cocaine treatment (see Table 1 of Supporting Information). ANXA7 is a member of the annexin family of calcium-dependent phospholipid binding proteins that codes for a Ca2+-dependent GTPase. While several protein kinases have been shown to phosphorylate ANXA7 with high efficiency, only PKC-dependent phosphorylation has a significant positive effect on ANXA7 GTPase.54 Activation of PKC by PTN55 has been suggested to play a role in the neurotrophic actions of PTN against druginduced neurotoxicity.13 Our data suggest that the counteractive mechanism against chronic cocaine treatment, possibly involving activation of ANXA7 and its tissue remodeling function,56 is compromised in the presence of normal levels of PTN (WT mice) or in the absence of endogenous PTN (PTN−/− mice) but is readably induced by cocaine in PTNTg mice compared to that in saline-treated mice. Interestingly, amphetamine downregulates ANXA7 levels in the striatum of the most vulnerable genotype to the neurotoxic effects of amphetamine, the PTN−/− mouse.30 A different protein from the same family, ANXA5, is an apoptotic factor and biomarker

PTN−/− mice compared to that in saline-treated mice and increased in saline-treated PTN−/− compared to that in WT and in PTN-Tg mice. TPI1 is essential in the glycolytic pathway, and its levels of expression are significantly altered in AD patients.48−50 Oxidative stress and, as a result, nitrotyrosination of TPI1 have been robustly linked to amyloid β-peptide-induced toxicity and τ pathology.50,51 Our data support a role of TPI1 in cocaine-induced neurotoxic and/or neurodegenerative processes depending on endogenous PTN. In PTN−/− mice, cocaine induced a significant downregulation of PPIA, whereas, conversely, this protein was upregulated in WT and PTN-Tg mice after cocaine treatment, although in WT mice, the upregulation was only significant in one of the spots where this protein was detected. Alterations in PPIA levels have been linked to aging related disorders including AD.52 PPIA is an enzyme involved in protein folding that is downregulated in the brain of AD patients.48,52 Our data suggest the possibility that cocaine-induced downregulation of PPIA may be involved in neurodegenerative processes induced by chronic cocaine when endogenous PTN is lacking. Another protein that is significantly downregulated by chronic cocaine treatment in PTN−/− mice is NME2. It is interesting to note that NME2 is involved in the proliferation of neuronal precursors and neurite formation and is found to be downregulated in the brain of AD patients.53 The data suggest H


Article

Chemical Research in Toxicology for AD and Lewy body dementia57 and, overall, is found at lower levels in PTN-Tg mice independently of the treatment, this decrease being significant when comparing PTN-Tg and PTN−/− saline treated mice. This reduction in the levels of ANXA5 correlates with the known neurotrophic actions of PTN in neurodegenerative processes and drugs-induced neurotoxicity.13 The ATP synthase, subunit d (ATP5H), was found significantly upregulated in PTN−/− mice independently of the treatment received compared with that in WT mice, whereas PTN-Tg mice showed the lowest levels of expression of this protein, being undetectable after cocaine treatment. These differences were already detected in a similar study with amphetamine.30 In that study, amphetamine tended to decrease the levels of expression of ATP5H, and the highest levels of expression of this protein were found in the striatum of PTN−/− independent of the treatment received. Alterations in ATP synthase are related to impaired cellular energy metabolism resulting in increases in the production of reactive oxygen species, which in turn causes apoptosis and necrosis in nerve cells.58 Thus, the data suggest that constitutive elevation of ATP5H levels in the striatum of PTN−/− mice compared to those in WT and PTN-Tg mice may partially underlie the increased vulnerability of PTN−/− mice to psychostimulantinduced neurotoxicity in this genotype.13 Taken together, the data obtained in PTN−/− and PTN-Tg mice support further studies to test the possibility that PTN is an important modulator of ATP5H. In summary, the present study identifies 23 proteins differentially regulated in the mouse striatum by chronic cocaine exposure depending on the endogenous expression of PTN. All of these proteins are known to be related with neurodegeneration processes, drug-induced neurotoxicity and addictive behaviors. As compared with previous studies, some of these proteins are differentially regulated by acute and chronic treatments with cocaine. Some of the proteins identified here are directly related to oxidative stress, and their variations especially affect PTN−/− mice, suggesting a protective role of endogenous PTN against cocaine-induced neural alterations which correlate with the known neuroprotective effects of PTN against cocaine-induced neurotoxicity in cultures. Further studies, including the influence of phosphorylation on specific residues, are needed to validate these proteins as possible targets against neural alterations induced by cocaine.

■

Innovación of Spain and by grant USP-BS-APP03/2014 from Universidad CEU San Pablo and Banco de Santander. M.V.-R. is supported by fellowship from Fundación Universitaria San Pablo CEU. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; ACY1, aminoacylase-1; ALAD, δ-aminolevulinic acid dehydratase; ALDH5A1, succinate-semialdehyde dehydrogenase. mitochondrial; ALDOC, fructose-bisphosphate aldolase C; ANXA5, annexin A5; ANXA7, annexin A7; ARPC2, actinrelated protein 2/3 complex subunit 2; ATP5H, ATP synthase subunit d. mitochondrial; CA2, carbonic anhydrase 2; CRYAB, α-crystallin B chain; DLD, dihydrolipoyl dehydrogenase. mitochondrial; DPYSL2, dihydropyrimidinase-related protein 2; GPD1, glycerol-3-phosphate dehydrogenase [NAD(+)] cytoplasmic; IMAC, immobilized metal affinity chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MDH1, malate dehydrogenase. cytoplasmic; MK−/−, mice knock out; MK, midkine; NME2, nucleoside diphosphate kinase B; PC, PTN−/− mice treated with cocaine; PFC, prefrontal cortex; PGAM1, phosphoglycerate mutase 1; PPIA, peptidyl-prolyl cis−trans-isomerase A; PRDX6, peroxiredoxin-6; PS, PTN−/− mice treated with saline; PSMA2, proteasome subunit α type-2; PSMA4, proteasome subunit α type-4; PTN−/−, PTN knockout; PTN, pleiotrophin; PTN-Tg, transgenic PTN overexpression; RAN, GTP-binding nuclear protein Ran; RPTP β/ζ, receptor protein tyrosine phosphatase β/ζ:; TC, PTN-Tg mice treated with cocaine; TPI1, triosephosphate isomerase; TS, PTN-Tg mice treated with saline; UCHL1, ubiquitin carboxyl-terminal hydrolase isozyme L1; WC, wild type animals treated with cocaine; WT, wild type

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ASSOCIATED CONTENT

S Supporting Information *

Values of normalized optical density of striatal proteins showing significant differences when comparing the different groups of animals. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.chemrestox.5b00130.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Laboratory Pharmacology, Faculty of Pharmacy, Universidad ́ CEU San Pablo, Urb. Monteprincipe, 28668 Boadilla del Monte, Madrid, Spain. Tel: 34-91-3724700 ext. 4812. Fax: 34-91-3510475. E-mail: [email protected]. Funding

This work has been supported by grants SAF2009-08136 and CENIT CEN-20101023 from Ministerio de Ciencia e I


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