Acute Phencyclidine Treatment Induces Extensive and Distinct Protein

Article pubs.acs.org/jpr

Acute Phencyclidine Treatment Induces Extensive and Distinct Protein Phosphorylation in Rat Frontal Cortex Pawel Palmowski,† Adelina Rogowska-Wrzesinska,† James Williamson,† Hans C. Beck,‡,∥ Jens D. Mikkelsen,§,# Henrik H. Hansen,§,⊥ and Ole N. Jensen*,† †

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark ‡ Danish Technological Institute, Kongsvang Allé 29, DK-8000 Aarhus, Denmark § NeuroSearch A/S, Pederstrupvej 93, DK-2750 Ballerup, Denmark S Supporting Information *

ABSTRACT: Phencyclidine (PCP), a noncompetitive N-methyl-D-aspartate receptor antagonist, induces psychotomimetic effects in humans and animals. Administration of PCP to rodents is used as a preclinical model for schizophrenia; however, the molecular mechanisms underlying the symptoms remain largely unknown. Acute PCP treatment rapidly induces behavioral and cognitive deficits; therefore, post-translational regulation of protein activity is expected to play a role at early time points. We performed mass-spectrometry-driven quantitative analysis of rat frontal cortex 15, 30, or 240 min after the administration of PCP (10 mg/kg). We identified and quantified 23 548 peptides, including 4749 phosphopeptides, corresponding to 2604 proteins. A total of 352 proteins exhibited altered phosphorylation levels, indicating that protein phosphorylation is involved in the acute response to PCP. Computational assessment of the regulated proteins biological function revealed that PCP perturbs key processes in the frontal cortex including calcium homeostasis, organization of cytoskeleton, endo/exocytosis, and energy metabolism. This study on acute PCP treatment provides the largest proteomics and phosphoproteomics data sets to date of a preclinical model of schizophrenia. Our findings contribute to the understanding of alterations in glutamatergic neurotransmission in schizophrenia and provide a foundation for discovery of novel targets for pharmacological intervention. KEYWORDS: phencyclidine, schizophrenia, LC−MS, phosphorylation, proteomics, rat, PCP

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INTRODUCTION

alogia, avolition, impaired attention, information processing, learning and memory) and social/occupational dysfunction. Current treatment is symptomatic and targeted predominantly against positive symptoms, while negative and cognitive symptoms tend to be resistant to current antipsychotics.5 One of the most commonly used pharmacological models of the illness is based on the use of N-methyl-D-aspartate (NMDA) receptor antagonists (i.e., phencyclidine or ketamine) in rodents. The effects of phencyclidine (PCP), as observed in human addicts, largely overlap with schizophrenia, covering a wide range of positive, negative, and cognitive symptoms.6

Schizophrenia is a complex and heterogeneous psychiatric disorder. Although the earliest descriptions date back to ancient times, the ailment was defined late in the 19th century, and the name “Schizophrenia” was proposed in 1908.1 Affecting about 1% of the population,2 schizophrenia is among the top ten leading causes of disease-related disability in the world,3 but despite its universality and extensive studies over the past century, disease etiology and pathophysiology remain unclear. Schizophrenia is still being diagnosed based on behavioral symptoms that, according to the DSM-IV,4 include: characteristic symptoms (positive symptoms, i.e., delusions, hallucinations, disorganized speech, grossly disorganized or catatonic behavior; negative/cognitive symptoms, i.e., affective flattening, © 2014 American Chemical Society

Received: October 30, 2013 Published: February 12, 2014 1578

dx.doi.org/10.1021/pr4010794 | J. Proteome Res. 2014, 13, 1578−1592

Journal of Proteome Research

Article

Inhibitor Cocktail Tablets, Roche) and phosphatase inhibitors (PhosSTOP, Phosphatase Inhibitor Cocktail Tablets, Roche). The tissue samples were homogenized with a tight-fitting Teflon homogenizer (20−25 strokes) in homogenization buffer (0.35 M sucrose, 20 mM HEPES, pH 7.4). After 15 min of incubation, NP-40 was added to a final concentration of 0.2%. The sample was vortexed for 10 s and centrifuged (10 min, 3000g, 4 °C). The supernatant was collected and saved. The crude nuclear pellet was overlaid with 20% Iodixanol (OptiPrep Density Gradient Medium, Sigma), transferred to a homogenizer, and gently disrupted (10−15 strokes). This nuclear suspension was centrifuged again (40 min., 10 000g, 4 °C). The pellet was then washed with homogenization buffer, resuspended in a lysis buffer (20 mM HEPES, 2% SDS, pH 7.4), and sonicated. The first supernatant was further processed by centrifugation (120 000g for 1.5 h). Resulting pellet was washed with homogenization buffer and resuspended in lysis buffer (membranous fraction). SDS was added to the supernatant containing soluble fraction to a final concentration of 2%. Enriched fractions were stored in −80 °C.

These observations allowed a hypothesis of hypoglutamatergic function in schizophrenia to be formulated and an animal model to be developed.7 PCP treatment in rodents causes a subset of behavioral deficits mimicking a number of core symptoms observed in schizophrenia patients, that is, hyperlocomotion, sensorimotor gating deficit (prepulse inhibition), and cognitive dysfunction (impairment of working memory, spatial and nonspatial learning, attention, latent learning and recognition).8 Although psychosis is usually the most apparent clinical aspect of schizophrenia, cognitive impairments are considered to be the core features of the illness. Convergent findings suggest that these disturbances are associated with imbalances in inhibitory γ-amino-butyric acid and excitatory glutamate neurotransmission in the prefrontal cortex.9 Even though the PCP model has been extensively investigated during several decades, the molecular processes behind the observed symptoms are still not fully understood. A drug-treated animal, in contrast with a human schizophrenia patient, is lacking the possible underlying genetic condition; therefore, most of the studies concentrate on changes in gene/ protein expression.10,11 However, observation of human drug addicts showed that the typical onset of PCP effects is rapid (1−5 min) when the drug is injected, with a decay of the major symptoms over 4−6 h.12 Similar pharmacodynamics were observed in animals upon acute exposure.13 The rapid pharmacodynamics following acute PCP exposure, including associated behavioral deficits, may point to the involvement of post-transcriptional and post-translational regulatory events. Unlike the gene transcription and protein expression machineries, post-translational modifications, such as phosphorylation, can rapidly (within minutes) modulate the activity of individual proteins and molecular networks and act as a master control for regulation of biological processes.14 In the present study, we used proteomics and phosphoproteomics methods to determine whether protein phosphorylation in rat frontal cortex was affected by acute PCP treatment. We combined subcellular fractionation techniques and phosphopeptide enrichment by TiO2, with stable isotope labeling by iTRAQ and high-performance LC−MS/MS, to identify and quantify proteins and protein phosphorylation events. We report that proteins involved in some of the key cellular processes are indeed differentially phosphorylated upon acute PCP treatment.

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TCA/Acetone Precipitation

Protein precipitation was used for sample preparation to remove low-MW contaminants that were affecting the sensitivity of the workflow. Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Scientific). A volume equivalent to 150 μg of protein was diluted with 9 volumes of ice-cold precipitation mixture (10% TCA, 90% acetone, 10 mM DTT), incubated in −20 °C for 2 h, and centrifuged (20 000g, 4 °C, 20 min). The supernatant was discarded, and the remaining pellet was washed three times with washing solution (10 mM DTT in ice-cold acetone, 1 h of incubation, centrifugation 20 000g, 20 minutes, 4 °C). The final precipitate was air-dried and stored frozen for further processing. Spin Filter Aided in Solution Digestion

The protein precipitate was resuspended in 250 μL of digestion buffer (20 mM TEAB, 0.5% SDC), mixed with DTT solution to a final concentration of 100 mM, and shaken continuously in a Thermomixer (Eppendorf) for 30 min at 60 °C. The solution was transferred to a centrifugal spin filter with an MW cutoff of 10 kDa (YM-10, Millipore) and centrifuged for 15 min at 12 000g. The same conditions were used for all of the following centrifugations. Subsequently, the sample was washed two times with 200 μL of digestion buffer. 150 μL of 50 mM iodoacetamide was added to the sample, followed by shaking for 1 min. After 30 min of incubation in the dark at room temperature, the protein mixture was centrifuged and washed twice with digestion buffer to remove the excess of alkylating agent. Tryptic digestion was performed overnight at 37 °C by adding 1 μg of trypsin (w/w, Promega modified, sequencing grade) per 100 μg of protein (volume 2 proteins per function). Experimental Design

The approach presented in Figure 1A, allowed us to monitor protein abundance and protein phosphorylation changes in response to PCP treatment in rat brain as a function of time and across different cellular compartments. Rat brain tissue samples were individually homogenized and fractionated to enrich for nuclear fraction, membrane fraction (containing proteins bound to cellular and organelle membranes), and soluble fraction (cytoplasmic and extracellular proteins). Biological replicates were pooled in equal amounts (total protein = 25 ug) to ensure adequate sample amounts with good representation of all protein species within a fraction and to reduce sample preparation and LC−MS/MS analysis time. Sample pooling was previously discussed,26,27 and this strategy was successfully used in other proteomics and gene expression studies28−31 to improve robustness and facilitating detection of low abundance species, for example, in biomarker studies. Samples prepared in this manner were precipitated by TCA/ acetone and subjected to trypsin digestion. Resulting peptide mixtures were labeled with iTRAQ tags and combined prior to phosphopeptide enrichment and subsequent LC−MS/MS analysis for peptide sequencing and quantification. An aliquot of each tryptic peptide sample was analyzed separately without phosphopeptide enrichment to determine the protein composition and abundances by LC−MS/MS. All peptide and phosphopeptide mixtures were analyzed in triplicate (three technical replicates). MS/MS files were searched against protein sequence databases using Mascot. PSMs were filtered to generate lists of peptides with their associated iTRAQ reporter ions intensities. Next, on the basis of iTRAQ ratios, we determined

Cluster Analysis

Cluster analysis was performed for proteins and peptides exhibiting a relative change in abundance of >10% for at least one of the time points and with a full “peptide/protein profile” (identified and quantified at all time points within a given compartment). The relative change cutoff was introduced to eliminate potential noise in the data and save only the biologically relevant information. Protein/peptide ratios were log2-transformed and standardized within each profile (μ = 0, σ = 1). For the purpose of clustering, data originating from different cellular compartments were combined. (The information about fraction and regulation type was saved and could be retained after the calculations were completed.) The calculations were performed using the Mfuzz toolbox,23 which is based on the open-source statistical language R.24 We used the fuzzy c-means clustering algorithm, which is a part of the toolbox. The clustering parameters were estimated as described.25 The unassigned profiles were consequently used for the next round of clustering. The iterative process was terminated when the algorithm generated empty clusters. Pathway Analysis

The protein groups regulated in a given experimental condition or belonging to specific clusters were analyzed by Ingenuity Pathway Analysis software (Ingenuity Systems). “Opposite” clusters (while one is up regulated, in the opposite down regulation is observed) were combined prior to analysis. Analysis settings were as follows: data sources = all, species = 1581



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We assessed the efficiency of the cellular fractionation method by a comparative analysis of the lists of proteins identified in the nuclear fraction, membrane fraction, and soluble fraction, respectively. Figure 3A shows that 62.5% of all protein identifications were assignable to one distinct fraction only, providing a good indication of successful enrichment of three distinct cellular fractions. Next, we applied gene ontology (GO) analysis to estimate the specificity of the fractionation protocol. We compared the experimentally observed localization with the predicted localization of the identified proteins (Figure 3B−D). The nuclear fraction was indeed enriched for nuclear proteins because 36% of the proteins in this fraction were annotated as nuclear proteins, whereas an additional 40% were annotated as being associated with the nucleus. The membrane fraction was clearly enriched in membrane proteins and membrane-associated proteins, and we also identified respiratory chain proteins in this fraction, as expected. The soluble fraction mainly contained soluble proteins, including components of the glycolytic pathway. The level of “contaminants” from other fractions was estimated to be on the order of 10−20%. The quantification method was based on stable isotope labeling with iTRAQ. The peptide-labeling efficiency exceeded 97%. (See the Materials and Methods section.) Regulatory events were observed as a decrease or increase in iTRAQ fragment ion intensity ratios. Two MS/MS spectra are shown and illustrate the identification of regulated phosphopeptides (Figure 4). The first MS/MS spectrum (panel A) confidently identified an eight amino acid long peptide that was assigned to calcium/calmodulin-dependent kinase II β, phosphorylated on a threonine residue (mass error = 0.63 ppm; mascot ion score = 32, pRS site probability = 100%). This enzyme is involved in a range of biological activities, acting as a Ca2+-dependent modulator of substrate protein phosphorylation. The identified and down-regulated (cytoplasm, 15′ time point, 114/115 reporter ion ratio, inset) phosphorylation site of CamKIIb (pT287) is known to be responsible for enzyme activation. Autophosphorylation takes place upon interaction with active (calcium bound) calmodulin.32 Another peptide MS/MS spectrum is shown in Figure 4B and identified a histone deacetylase (HDAC4) (mass error = 1.95 ppm; Mascot ion score = 35; pRS site probability = 89%). The tryptic peptide is phosphorylated on serine 3 corresponding to pS466 in HDAC4, which is known to be important for regulation of the enzyme activity and its cellular localization.33 The extent of phosphorylation is reduced in cytoplasm at the 15′ time point (inset, iTRAQ reporter ion ratio). HDAC4 shuttles between the nucleus and the cytoplasm. It is involved in epigenetic repression and plays an important role in transcriptional regulation. Interestingly, HDAC4 is a phosphorylation substrate for CamKII-δ, -γ, -β, or -α form. Because CaMKIIα and β are the main isoforms expressed in neuronal tissues, it is interesting to speculate that the observed decrease in CamKIIβ phosphorylation at pT287 leads to its reduced activity and therefore reduced phosphorylation of pS466 of HDAC4, which is a CamKII substrate site (pS467 - analogical sequence in human34). To identify differentially regulated features, we used a methodology originally developed for microarray experiments22 (illustrated in Figure S4 in the Supporting Information), in which generally a greater variation of signal ratios at lower intensities is observed. The same relation was demonstrated in LC−MS/MS experiments using stable isotope labeling with

the regulation status followed by cluster analysis and pathway analysis to identify distinct brain proteins and protein groups/ networks that were affected by the acute PCP treatment, as outlined in Figure 1B.

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RESULTS

Proteome and Phosphoproteome Analysis of Subcellular Fractions of Rat Frontal Cortex

Our proteomics/phosphoproteomics analysis allowed us to identify (FDR < 0.01) and quantify a total of 23 548 peptides that were assigned to 2604 gene products/proteins. In the database searching process, a mass accuracy threshold of ±10 ppm was used. Figure 2A provides a distribution of delta mass

Figure 2. Data set properties. (A) Mass accuracy in the analyzed data set estimated with a delta mass parameter. (B) Peptide ion score distribution in the analyzed data set. (C) Distribution of phosphorylation sites between amino acid residues. (D) Contribution of multiply phosphorylated peptides to the total number of phosphopeptides.

parameter in the analyzed data set (high-confidence PSMs). The average peptide ion score (Mascot) for identified peptides is 44.2 (median 41.4) with minimum of 10 and maximum of 157. Figure 2B shows the distribution of ion scores in the data set. The technical reproducibility was assessed. Replica versus replica comparisons of peptide ratios (treatment/control), obtained in replicate MS/MS measurements (technical replicates), are provided as scatter plots in the Supporting Information (Figure S1). The obtained coefficients of correlation were in the range 0.73 to 0.78. The phosphopeptide enrichment efficiency was estimated to be 95.8%. Out of the total set of 23 548 identified peptides, 4749 were found to be phosphorylated. Phosphoproteins constituted 58.6% (1524 proteins) of all identified protein species. Figure 2C illustrates the distribution of the observed phosphorylation sites between amino acid residues. Phosphorylation events were predominantly observed on not only serine but also threonine and tyrosine. Peptides phosphorylated at multiple sites accounted for 11% of the identified peptide species, as shown in Figure 2D. 1582



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Figure 3. Fractionation efficiency. (A) Comparison of cellular fractions in terms of protein content. (B−D) Composition of the obtained fractions assessed with theoretical localization of identified proteins derived from GO annotations.

Figure 4. LC−MS/MS spectra of two phosphopeptide identified with high confidence in soluble fractions and regulated at 15 min time point. Regulatory events were observed as a decrease/increase in iTRAQ fragment ion intensity ratios (114/115 reporter ion ratio). Identified peptides are characteristic to (A) Calcium/calmodulin-dependent kinase II β (pT287, delta mass: 0.63, ion score: 32, pRS site probability: 100%) and (B) histone deacetylase HDAC4 (pS466, delta mass: 1.95, ion score: 35, pRS site probability: 89%).

iTRAQ.35 In Figure 5A, the distribution of our phosphopeptide data is presented (reporter ion intensity versus the logarithm of treatment/control ratio), and Figure 5B shows a corresponding curve of local standard deviation. It is evident that the standard deviation of the quantitative ratios depends on the ion intensity and the most intense ion signals have a low standard deviation. Thus, it is not advisable to use a single threshold value for fold-

change, whether defined a priori (e.g., 2-fold or 1.5-fold) or calculated for the whole data set. The locally calculated parameters (within a narrow window of the neighboring data points) allow us to define cutoff values more accurately, taking advantage of the specific data properties. Protein abundance data exhibit the same distribution characteristics (Figures S2 and S3 in the Supporting Information). The attached volcano 1583



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Figure 5. Data distribution. (A) Distribution of phosphopeptide Log2(ratios) within the data set. Data points organized by growing reporter ion intensity. The plotting area was restricted on the X axis to a range −3 to 3. There were few points outside of that range in membrane and soluble fraction at the 15 min time point. (B) Plots of locally calculated standard deviation (window width for calculations was set to 10% of the data points).

Figure 6. Overview of regulated proteins. (A) Comparison of protein groups regulated by phosphorylation or whole molecule level. (B) Number of regulations exceeding two-fold in each experimental group.

time point to a level that was sustained up to 240 min. The magnitude of changes, observed as the number of features that exhibited more than two-fold regulation (Figure 6B), confirms that the most dramatic regulatory events (both protein abundance and phosphorylation changes) take place in the cytoplasm and in the plasma membrane at the 15 min time point. Nuclear proteins were only affected to a low extent by acute PCP treatment. Next, we investigated whether the observed phosphorylation changes were a result of activation of particular protein kinases. We searched for phosphorylation motifs using the Motif-X tool36 for the complete data set and for the subset of significantly regulated phosphopeptides. A number of sequence motifs were identified by analysis of the complete phosphopeptide data set, whereas only a few were recovered in the subset of regulated phosphopeptides. The latter included calcium/ calmodulin-dependent kinases, mitogen-activated kinases, and cyclin-dependent kinases (RxxS and xxS/TPxx. motifs37,38) that are among the main mediators of NMDA receptor signaling events (Figure S7 in the Supporting Information).

plots illustrate the outcome of the regulation status determination (Figures S5 and S6 in the Supporting Information). Our data analysis workflow revealed that in the course of the experiment a total of 555 proteins were regulated in PCP treated rats. Over 54% of these proteins exhibited differential phosphorylation, as shown in Figure 6A. We assessed whether alterations in protein abundance contributed to the observed changes in phosphopeptides by comparing protein abundance data and phosphorylation data. We found that only 5% of the cases exhibited a coexisting change in protein level and decided to ignore this small contribution during subsequent data analysis. Next, the time-course profile of the reaction to PCP treatment was investigated. As shown in Figure 4A and the attached volcano plots (Figure S5 in the Supporting Information), a high degree of phosphorylation-mediated regulation was observed for phosphorylation determined 15 min after PCP injection (Figure 4B), predominantly in the soluble protein fraction and the membrane protein fraction. For these experimental conditions, a more scattered distribution of data points can be noticed, resulting in a general increase in standard deviation. The response was attenuated at the 30 min 1584


Journal of Proteome Research Regulated proteins found in this study that are known to be directly or indirectly sensitive to changes in the calcium level in the cell. Fold-change values were corrected for protein level changes.

histone deacetylase synapse-localized Ras/Rap GTPase-activating protein

Camk2B (pT287) Camk2G (pT287) HDAC4 (pS466) SynGAP (pS146) calcium/calmodulin-dependent protein kinase II

a

membrane 15−30 min membrane 15−30 min soluble 15 min membrane 15 min −2.3 −1.6 −3.3 2.3

Phosphorylation by CaMKII promotes nuclear export and results in activation of the target genes. Phosphorylated when in complex with CaMKII. Ca2+/calmodulin binding to CaMKII dissociates the complex and drives the dephosphorylation of SynGAP. S146 was not previously reported but may potentially controlled by CaMKII (RxxS motif).

membrane 15 min GRIN2B (pS1303)

myristoylated alanine-rich C-kinase substrate NMDA receptor

MARCKS

−2.9

−1.8

soluble 15 min

calcium dependence compartment/time point phosphorylation

2.3

protein gene name protein description

NRGN

regulation

Table 1. Calcium Sensorsa 1585

neurogranin

PCP-induced effects on specific subsets of identified proteins were investigated. Among the highly PCP-regulated proteins, we found subunits of NMDA receptor (the main target of PCP) and AMPA receptor to be differentially phosphorylated. Fifteen minutes after PCP injection, the phosphorylation of GRIA2/GLUR2 (AMPA receptor) on tyrosine 876 and GRIN2B (NMDA receptor) on serine 1303 was reduced. We identified other glutamate receptors subunits/subtypes, including GRIA1, GRIA2, GRIN1, and GRIN2A; however, they did not exhibit any regulation at the protein or phosphorylation level. The glutamate receptors are known to be regulated by phosphorylation.6 We confirmed several known and identified many predicted phosphorylation sites that were not previously reported in rat but having analogs in other species. We also identified novel phosphorylation sites in, that is, Grin2a(S1384) and Grin2b(S1477). These findings are summarized in Figure S8 in the Supporting Information. The identified phosphorylation sites were located in the cytoplasmic domains of these ion channels, thereby confirming their predicted topology. A number of other ion channels and ion transporters were found to be differentially phosphorylated, including voltage-dependent Ca2+ channels (CACNA1, CACNG2, CACNG4) and sodium/calcium exchanger (SLC8A2), as summarized in Table S1 in the Supporting Information. PCP modulates the intracellular Ca2+ concentration and interferes with calcium-dependent processes by a direct interaction with the NMDA receptor.39 Because we could not monitor in vivo calcium concentration in brain cells, we used certain proteins as indicators of a lowered cellular calcium level. Table 1 summarizes regulated proteins that were observed in this study and previously reported to be regulated in a calciumdependent manner. These include not only calcium/calmodulin-binding proteins, for example, neurogranin,40 but also molecules that are substrates of calcium dependent kinases (i.e., NMDAR41) and elements of calcium/calmodulin-dependent signaling pathways (i.e., HDAC434). In the synapse, glutamate receptors and downstream signal transduction are organized by the protein assembly of the postsynaptic density (PSD). PSD is an electron-dense structure located at the synaptic contacts between neurons. Its considerable complexity includes cytoskeletal and scaffold proteins, receptors, ion channels, and signaling molecules.42−44 We compared our results with a data set integrating multiple studies of the PSD.45 It contains 466 proteins, which were validated by their detection in two or more studies, forming what was designated the “consensus PSD”. We identified 166 proteins and 189 phosphoproteins belonging to this consensus PSD group of proteins. Of these, 71 were regulated at the protein level and 81 by phosphorylation, which is a relatively large part (24%) of all regulated proteins identified in our experiment. We also observed the regulation of molecules participating in other aspects of cellular function. The most prominent and highly regulated groups are involved in the processes of endo/ exocytosis, cytoskeleton dynamics and organization, energy metabolism, and protein expression/degradation. Selected examples of regulated proteins, involved in these processes, are listed in Table 2. Complete lists of regulated protein features are provided in Tables S2 (changes in protein abundance) and S3 (differential phosphorylation) in the Supporting Information.

soluble 15 min

Biological Effects of Acute PCP Treatment

Binds apo-calmodulin inhibiting its ability to activate targets. Ng displays a nuclear localization, reduced by binding to cytoplasmic calmodulin. Phosphorylation by PKC or binding to calcium−calmodulin inhibits its association with actin and with the plasma membrane, leading to its presence in the cytoplasm This phosphorylation site modulates the channels activity, phosphorylated by calcium dependent kinases (CaMKII, DAPK1, PKCA). Autophosphorylation on T287 in effect of an interaction with Ca2+/calmodulin causing enzyme activation.

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protein kinases/ phosphatases

gene expression

exo/endocytosis

energy metabolism

cytoskeleton organization

PFKL PFKP PGK1 SDHA

6-phosphofructokinase

phosphoglycerate kinase

succinate dehydrogenase

EEA1 SNAP25

early endosome antigen 1 synaptosomal-associated protein 25

DYRK1A

TOP2B

DNA topoisomerase 2-beta

dual specificity tyrosine-phosphorylation-regulated kinase 1A

NEUROD2

neurogenic differentiation factor 2

CSNK2B DNAJC6

HNRNPUL2

heterogeneous nuclear ribonucleoprotein U-like protein 2

casein kinase II subunit beta putative tyrosine-protein phosphatase auxilin

HNRNPC

heterogeneous nuclear ribonucleoprotein C

SNAP91

CADPS

calcium-dependent secretion activator 1

clathrin coat assembly protein AP180

AMPH

amphiphysin

Aak1

MDH1

malate dehydrogenase

AP2-associated protein kinase 1

ATP5D

MAPT

microtubule-associated protein tau

ATP synthase

DPYSL2

dihydropyrimidinase-related protein 2

symbol ADD1

protein description

alpha-adducin

Table 2. Regulated Proteinsa

pY321

pS209 pT570

pS1453

pS159 pS183 pS7

pS296 pS300 pS253

abundance abundance

pS496 pS514 pS219 pS375 pS486

pS679

abundance

pS775 PS386 pS203

pS241

abundance

pS457 pS586 pT358 pS518 pS522 pT521 pT492

regulation

S 15 min S 240 min S 15 min S 240 min M 15 min M 30 min S 15 min S 15 min

−2.15 −1.36 −2.14 2.37 3.24 3.74 6.18 2.22

N 15 min N 30 min N 240 min N 15 min S 15 min S 15 min S 15 min

−2.36

N 15 min N 30 min N 15 min

−2.14 −2.40 −1.88 −1.24 −1.54 −1.53 1.75 1.23 −1.65 −1.73 −5.67 2.03

S 15 min

S 15 min

M 15 min

−1.43 10.97

S 15 min

S 15 min

S 15 min S 30 min S 15 min M 15 min S 15 min

S 15 min M 15 min S 15 min S 15 min

fraction/time

−2.15 −2.56 −7.31

2.26 1.67 −2.64 3.12 2.47 17.93 3.33 1.27 4.81 −1.41 −3.34

fold change function

Ubiquitously expressed serine/threonine protein kinase Recruits HSPA8/HSC70 to clathrin-coated vesicles and promotes uncoating of clathrin-coated vesicles It may play a significant role in a signaling pathway regulating cell proliferation and may be involved in brain development.

Control of topological states of DNA by transient breakage and subsequent rejoining of DNA strands.

Transcriptional regulator. Mediates calcium-dependent transcription activation by binding to E box-containing promoter.

Acts as a basic transcriptional regulator. Plays a role in mRNA processing and transport.

May play a role in the early steps of spliceosome assembly and pre-mRNA splicing.

Participates in endosomal trafficking. Part of the SNARE core complex. Associates with proteins involved in vesicle docking and membrane fusion. Component of the adapter complex that links clathrin to receptors in coated vesicles.

Required for the Ca2+-regulated exocytosis of secretory vesicles.

Glycolytic enzyme that catalyzes the conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate. Involved in complex II of the mitochondrial electron transport chain and is responsible for transferring electrons from succinate to ubiquinone (coenzyme Q). Plays a regulatory role in clathrin-mediated endocytosis. Regulates phosphorylation of AP-2 subunits. Associated with the cytoplasmic surface of synaptic vesicles.

Catalyzes the reversible oxidation of malate to oxaloacetate, utilizing the NAD/NADH cofactor system in the citric acid cycle. Catalyzes the irreversible conversion of fructose-6-phosphate to fructose-1,6-bisphosphate and is a key regulatory enzyme in glycolysis.

Catalyzes ATP synthesis during oxidative phosphorylation.

Promotes microtubule assembly and stability. Phosphorylation at pT492 elicits its binding to 14-3-3.

Promotes microtubule assembly.

Cytoskeletal protein that promotes the assembly of the spectrin-actin network. Binds Ca2+/calmodulin and is a substrate for protein kinases A and C.

Journal of Proteome Research Article


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Selected molecules representing cellular functions affected by PCP treatment and found to be regulated in this study.

DISCUSSION Posttranslational modifications greatly influence protein function in most eukaryotic organisms. The ability of reversible post-translational modifications to rapidly modulate enzyme activity and protein−protein interactions impacts many cellular functions. Signaling networks, including growth factor and hormone-mediated signaling systems, are regulated by reversible protein phosphorylation.46 Despite its important biological role, there has been until now no large-scale phosphorylation analysis published in the context of schizophrenia or animal models of this disease. In this study, we investigated the acute response to NMDA receptor antagonist (PCP) treatment, addressing both protein abundance changes and phosphorylation-mediated regulation of biological processes by using quantitative mass-spectrometrybased proteomics. Analysis of quantitative data allowed us to identify a series of cellular processes and functions that are directly and acutely affected by PCP treatment. We identified 1080 proteins and 1524 phosphoproteins and found that 555 proteins were regulated at the protein level or the posttranslational level, that is, by phosphorylation. A total of 134 of these molecules were previously reported to be associated with PCP-based treatment in animals, and 70 proteins were previously associated with schizophrenia by proteomics and genomics studies (references in Supporting Tables S2 and S3 in the Supporting Information). As expected, we observed the regulation of many proteins of the post-synaptic density or proteins associated with it, confirming that NMDA receptor is the primary target for PCP and its intracellular effects are due to inhibition of glutamatergic signaling. Taken together, these observations demonstrate the validity of the experimental procedures and provide a proof-of-concept of our analytical and bioinformatics approach. More than 300 proteins were regulated by phosphorylation, including glutamate receptor subunits that are critical mediators of the cognitive deficits in schizophrenia. Cognitive functions depend on the activity of subsets of PFC pyramidal neurons synchronized by fast-spiking GABA neurons.9 Glutamate terminals arising from other brain areas impinge upon pyramidal glutamatergic neurons and GABA interneurons in

a

M 15 min M 30 min −1.68 −1.67 YWHAH

cell signaling

14-3-3 protein eta

abundance

M 15 min S 15 min S 15 min M 15 min 1.66 −4.26 −2.23 2.26 RPL14 UBE2O RAPGEF2 SYNGAP1 60S ribosomal protein L14 ubiquitin-conjugating enzyme E2 O Rap guanine nucleotide exchange factor 2 Ras/Rap GTPase-activating protein

pS139 pS536 pS758 pS146

S 15 min −2.50 abundance PSMD2 26S proteasome non-ATPase regulatory subunit 2

function

The proteins and phosphopeptides observed in the three subcellular fractions were used for cluster analysis, with the aim to identify discrete changes in protein abundance and protein phosphorylation levels, which would otherwise be missed. Molecules within a cluster show a similar regulation profile and are possibly involved in related molecular processes. Cluster analysis identified 26 clusters representing distinct protein groups (Table S4 in the Supporting Information). Finally, differentially regulated proteins were subjected to pathway analysis to identify molecular processes and biological functions affected by PCP treatment. This analysis revealed enrichment of proteins involved in the organization of cytoskeleton, energy metabolism, ion transport/homeostasis, and endo- and exocytosis. Alterations in these fundamental and important cellular processes point toward higher order perturbations at the levels of neurotransmission, synaptic plasticity, and ultimately in learning, coordination, or behavior. A complete list of cellular functions that were identified by this analysis is summarized in Tables S4 and S5 in the Supporting Information. This global analysis confirmed that PCP induces dramatic effects in particular membrane protein complexes and Ca2+-dependent signaling modules and processes.

Calcium-dependent, calmodulin-stimulated protein phosphatase. 20S core alpha subunit of proteasome, involved in the ATP-dependent degradation of ubiquitinated proteins. A regulatory subunit of the 26 proteasome, involved in the ATP-dependent degradation of ubiquitinated proteins Ribosomal protein, component of the 60S subunit. Catalyzes the covalent attachment of ubiquitin to other proteins. Guanine nucleotide exchange factor (GEF) for Rap1A, Rap1B, and Rap2B GTPases. Member of the NMDAR signaling complex plays a role in NMDAR-dependent control of AMPAR potentiation, AMPAR membrane trafficking, and synaptic plasticity. Adapter protein implicated in the regulation of a large spectrum signaling pathways. PPP3CB PSMA3 protein synthesis/ degradation

calcineurin proteasome subunit alpha type-3

pS479 pS250

M 15 min M 30 min S 15 min M 15 min −1.30 −1.52 −3.48 2.39 MINK1 misshapen-like kinase 1

pS654

S 15 min

fraction/time

2.22 GSK3A

pS21

Article

glycogen synthase kinase-3 alpha

fold change regulation symbol protein description

Table 2. continued

Implicated in the control of several regulatory proteins including glycogen synthase, and transcription factors, such as JUN. It also plays a role in the WNT and PI3K signaling pathways as well as regulates the production of beta-amyloid peptides. Negative regulator of Ras-related Rap2-mediated signal transduction to control neuronal structure and AMPA receptor trafficking. Activates the JNK and MAPK14/p38 pathways.


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Figure 7. Calcium signaling. (A) Summary of calcium-dependent signaling pathways and their targets. Some of the observed regulations were included. (B) Process of AMPA receptor trafficking is dependent on an interplay between Ras and Rap signaling pathways that is regulated by interaction between CaMKII and SynGAP (synapse-localized Ras/Rap GTPase-activating protein). Direct SynGAP phosphorylation by CaMKII regulates its activity.75 Ras-dependent pathway increases, while a Rap-dependent pathway decreases, the number of active AMPARs in postsynaptic membranes.76 Upon decreased calcium concentrations (situation on the right) SynGAP is phosphorylated. Phosphorylation deactivates SynGAP, which in turn activates Rap. Activated Rap drives p38 MAPK, and as a result, AMPARs are being actively removed from the synapse.77 At the same time (situation on the left) phosphorylation of SynGAP, by CaMKII, increases its Ras GTPase-activating activity by 70−95%.75 GDP-bound Ras would then stop activating its downstream effectors p42−44 MAPK and lead to decreased synaptic delivery of AMPARs.78 Alternatively, influx/efflux of Ca2+ ions can control RapGEF (i.e., calcium sensitive RAPGEF279 dephosphorylated at regulatory pS960).

the strength of the synaptic response.53 AMPA receptor trafficking is presumably one of the key mechanisms of synaptic plasticity. A number of studies indicate that NMDA receptor opening and rise in postsynaptic calcium concentration during repetitive synaptic activity lead to regulated trafficking of postsynaptic AMPA receptors into (for LTP) and out of (for LTD) excitatory synapses.54 Multiple signaling cascades are thought to be activated by alterations in postsynaptic calcium that may trigger both the regulated receptor addition and removal, including Ras/Rap, MAPK, PKA, and PKC.53 It is likely that complicated interactions between different signaling pathways determine whether the final output results in a net increase or decrease in synaptic AMPARs. Affected synaptic and cellular plasticity would influence long-range connections in the brain and may induce abnormalities in other neurotransmitter systems.55

the PFC, regulating their activity and modifying the output signal, that is, to limbic areas, which seem to be of special relevance in modulating motor, emotional, and mnemonic functions.47 By blocking the NMDA receptor, PCP disrupts GABAergic and glutamatergic neurotransmissions causing dysfunctional information processing. On a molecular level, by blocking the NMDAR-mediated ion transport, PCP affects calcium levels in neurons.39 Calcium homeostasis is known to play a central role in schizophrenia.48,49 This hypothesis is supported by this study and by previous proteomics studies identifying alterations in the expression of Ca2+-related proteins in a brain tissue of patients.50,51 Calcium (Ca2+) is a ubiquitous signal propagated by downstream proteins, like Ras protein family, protein kinases, and their substrates (Figure 7A), to regulate almost every cellular function, including gene expression and proteins synthesis, cell death, energy metabolism, cell movement, and proliferation (reviewed in ref 52). Apart from the NMDA receptor, the abnormal calcium level may be due to altered activity of various other plasma membrane calcium transporters and ion channels. These alterations may also be a downstream effect of the inhibited NMDAR signaling. A good example of such a mechanism is the calcium-dependent process of AMPA receptor trafficking. Recent hypotheses regarding this process are summarized in Figure 7B. We identified PCP-induced alterations in proteins involved in AMPAR trafficking, that is, SynGAP, CaMKII, and RapGEF2. AMPA receptors (AMPARs) mediate most excitatory currents, and hence they have a major influence on

Endo/Exocytosis

Our study demonstrated regulation (predominantly by phosphorylation) of proteins involved in endo- and exocytosis upon PCP stimulation of rat brain. We here report several novel observations that may be relevant in the context of schizophrenia and associated animal models: synaptotagmin (SYT1, −1.78), syntaxin (STX1B, −1.53x, pS14), syntaxin binding protein 5 (STXBP5, −2.1x, pS1059), Synapsin (SYN1, 3.8x, pS425), and SNAP91. Endo- and exocytosis were both previously reported to be involved in schizophrenia56 as well as in PCP-based rat model.57 Molecules known to be associated with the disease were also found to be regulated (references in Supporting Tables 2 and 3 in the Supporting Information), that 1588



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phosphofructokinase, and elements of electron transport chain, that is, succinate dehydrogenase, and ATP synthase. The affected and desynchronized metabolic enzymes may result in reactive oxygen species production, changing the redox state of SZ tissues and leading to oxidative stress,67 commonly considered as a possible disease-causing factor. We observed alterations in the protein level of enzyme-involved ROS defense system, that is, superoxide dismutase 2 (SOD2, −2.17x). Impairments in energy metabolism seem to be a common trait of psychiatric disorders and were previously identified by functional assays, gene and protein expression studies, as well as linkage analysis in schizophrenia patients (reviewed in ref 67), although it is still not concluded whether it represents a cause or a consequence of the disease. In PCP-based animal model, it was shown that administration of PCP to rats results in reduced rates of oxygen uptake (decreased activity) in mitochondria isolated from their brains.68 Ca2+ plays an important role in signal transduction from cytosol to mitochondria.69 Activation or deactivation of metabotropic receptors in cell membrane, that is, serotonergic, glutamatergic, and muscarinic receptors, may influence the calcium levels in mitochondria70 that regulate intramitochondrial Ca2+-sensitive targets and may promote significant and long-lasting changes in energy production.71 Ca2+ has the ability to stimulate oxidative phosphorylation via Ca2+-sensitive intramitochondrial dehydrogenases (FAD-glycerol phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase, and oxoglutarate dehydrogenase) and F0/F1ATPase.72,73 It is hypothesized that the down-regulation of TCA cycle, may lead to an increase in cytosolic H + concentrations, modifying the activity of 6-phosphofructokinase and inhibiting glycolysis.74

is, AP2-associated protein kinase, which regulates clathrinmediated endocytosis by cycles of phosphorylation/dephosphorylation (Aak1), amphiphysin (AMPH), synaptosomalassociated protein (SNAP25), calcium-dependent secretion activator (CADPS), and calcineurin (PPP3CB). Endo- and exocytosis are the key processes involved in the signaling mechanisms of the synapse, crucial for both presynaptic and postsynaptic functions: in presynaptic axon terminals, required for neurotransmitter release and for the recycling of vesicles back to the reserve pool; postsynaptically, for trafficking and recycling of various neurotransmitter receptors, that is, already mentioned AMPAR. It is hypothesized that it is in fact a disorder of altered trafficking and that disrupted clathrinmediated endocytosis is responsible for the pathophysiology of the disease (reviewed in ref 56). Exo/endocytotic machinery in neuronal tissues is also sensitive to changes in intracellular Ca2+ concentration. Exocytosis in the nerve terminal is stimulated by calcium entry through voltage-dependent calcium channels and involves synaptotagmin as a calcium sensor. It takes part in synaptic vesicle docking to the presynaptic membrane via interaction with β-neurexin58 or SNAP-2559 and can displace complexin from the SNARE complex in the presence of calcium as one of the last steps in exocytosis.60 Activation of calcineurin Ca2+/ calmodulin-dependent protein phosphatase 2B, is an initiating signal for synaptic vesicle endocytosis,61 leading to dephosphorylation of proteins implicated in endocytosis, so-called “dephosphins”. Among the dephosphins are dynamin 1, amphiphysin, AP180, synaptojanin, epsin, and eps15.62 Organization of Cytoskeleton

The largest group of proteins that we found to be altered in our experiments is involved in cytoskeleton organization and dynamics. This group is part of a large family of cytoskeletal proteins and associated factors, many of which are rather abundant in the cell. The cytoskeleton plays a key role in maintaining the highly asymmetrical shape and structural polarity of neurons that are essential for neuronal physiology. Cumulative evidence suggests that neurodegenerative and psychiatric illnesses are associated with cytoskeletal alterations in neurons, which, in turn, lose synaptic connectivity and the ability to transmit incoming axonal information.63 There are reports suggesting the similarity of pathogenesis in schizophrenia and Alzheimer disease,64 supported by studies in a ketamine-induced animal model of schizophrenia.65 We detected increased phosphorylation of MAPT (protein modulating stability of axonal microtubules) upon PCP treatment. Moreover, stress-induced-phosphoprotein (STIP1, −1.5x, pS481) and ubiquitin-conjugating enzyme (UBE2O, −4.35x, pS481), proteins participating in the ubiquitination of hyperphosphorylated TAU, leading to filamentous polymers,66 were affected. The pathway analysis software also identified MAPT as a top upstream regulator of the disturbed molecular pathways. Tauopathies, that is, Alzheimer’s disease and dementia, were among the top diseases associated with regulated proteins.

Other Observations

Except for those already discussed, many other biological functions and proteins were observed to be regulated in this study. A number of molecules involved in signaling networks were affected, that is, 14-3-3 proteins (YWHAH: −1.7x, YWHAB: −1.38x, YWHAQ: −1.28x) and calcium-independent protein kinases (PRKCE, 2.1x, pS329; PRKCD, pS504, pS643, pS662). Protein expression and degradation processes were affected. Changes were observed in ribosome phosphorylation (RPL14) as well as in proteasome subunit PSMA3. In summary, our proteomics and phosphoproteomics study recovered known features and revealed many novel molecular aspects of PCP treatment or schizophrenia, which may be directly related to disturbance in cellular calcium level, thereby demonstrating the ability of the experimental strategy to recover relevant molecular details of the PCP-treated rat brain.

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CONCLUSIONS

This is, to our knowledge, the largest proteomics study of an acute PCP rat model of schizophrenia. This is also the first study to report molecular changes both at the protein level and posttranslational modification level. We used proteomics to provide molecular evidence that calcium homeostasis, cytoskeleton organization, energy metabolism, and endo/exocytosis are key components of the cellular response to acute NMDA receptor antagonist treatment, which contributes to validation of the PCP-based animal model and the glutamatergic hypothesis of schizophrenia. We also identified a number of proteins, potentially involved in PCP mode of action and schizophrenia pathology that were not previously reported.

Energy Metabolism

We noted a differential regulation of proteins that participate in energy metabolism as an effect of PCP treatment. Their altered levels can potentially lead to an overall disturbance and contribute to the pathological state. We detected regulation, after 15 min of drug exposure, of proteins involved in the TCA cycle, that is, malate dehydrogenase, glycolytic enzymes, that is, 1589



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Bioanalytical Sciences). We thank our colleagues in the Protein Research Group for many useful discussions.

Understanding of alterations in glutamate neurotransmission in schizophrenia may lead to the discovery of novel targets for pharmacological intervention and provide an alternative to currently used antipsychotics, which allow us to manage positive symptoms, but their effect on patients cognitive functions is relatively small or absent. Protein phosphorylation is extensively involved in the response to acute PCP treatment, as demonstrated by direct detection of differential phosphorylation of more than 300 proteins. This finding is relevant for future use of acute pharmacological animal models of psychiatric disorders, which are still predominantly investigated in the context of protein expression changes. In these models, the time between exposure to a pathogenic factor and the measurement may be too short for some changes in protein abundance to occur, although properties of affected molecules are altered by post-translational modification and contribute to the development of symptoms. Proteomics and modificationspecific proteomics will be important tools to investigate the molecular details and etiology of schizophrenia and other neurological diseases.

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ABBREVIATIONS DTT, dithiothreitol; LC, liquid chromatography; MW, molecular weight; MS, mass spectrometry; PFC, prefrontal cortex; PSD, postsynaptic density; PSM, peptide spectrum match; SDC, sodium deoxycholate; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; TEAB, triethylammonium bicarbonate; TFA, trifluoroacetic acid

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ASSOCIATED CONTENT

S Supporting Information *

Reproducibility test. Distribution of phosphopeptide Log2(ratios) within the dataset and plots of standard deviation. Data distribution in phosphoenriched nuclear fraction. Volcano plots for phosphopeptide data. Motif extracted from the localized and regulated phosphorylation sites. Maps of known phosphorylation sites in protein sequence of glutamate receptors. Ion transporters found to be differentially phosphorylated in membrane fraction. Gene products regulated on a protein level and by phosphorylation under the experimental conditions. Biological and molecular functions associated by IPA with co-clustering gene products. Molecular and biological functions associated by IPA with regulated proteins. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Tel: +45 6550 2368. E-mail [email protected]. Present Addresses ∥

H.C.B.: Centre for Clinical Proteomics, Department for Clinical Biochemistry and Pharmacology, Odense University Hospital, Sdr. Boulevard 29, DK-5000 Odense, Denmark. ⊥ H.B.H.: Gubra Aps, Agern Allé 1, DK-2970 Hoersholm, Denmark # J.D.M.: Neurobiology Research Unit, University Hospital Rigshospitalet, Copenhagen, Denmark. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Danish Ministry of Science, Technology and Innovation (grant no. 08-034107). The ONJ laboratory is also supported by grants from the Danish Research Councils and the Villum Foundation (Center for 1590



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Acute Phencyclidine Treatment Induces Extensive and Distinct Protein

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