TY - JOUR
AU - Pastorelli,, Roberta
AB - Abstract Developmental exposure to polychlorinated biphenyls (PCBs) has been associated with cognitive deficits in humans and laboratory animals by mechanisms that remain unknown. Recently, it has been shown that developmental exposure to 2,2′,3,4,4′,5′-hexachlorobiphenyl (PCB138), a food-relevant PCB congener, decreases the learning ability of young rats. The aim of this study was to characterize the effect of perinatal exposure to PCB138 on the brain proteome profile in young rats in order to gain insight into the mechanisms underlying PCB138 neurotoxicity. Comparison of the cerebellum proteome from 3-month-old unexposed and PCB138-exposed male offspring was performed using state-of-the-art label-free semiquantitative mass spectrometry method. Biological pathways associated with Ca2+ homeostasis and androgen receptor signaling pathways were primarily disrupted. These perturbations may contribute toward a premature ageing-like proteome profile of the cerebellum that is triggered by PCB138 exposure in males. Our proteomic data provide insights into the phenomena that may be contributing to the PCB138 neurotoxicity effects observed in laboratory rodents and correlate with PCB exposure and decreased cognitive functions in humans. As such, this study highlights the importance of PCB138 as a risk factor in developmental neurotoxicity in laboratory rodents and humans. non–dioxin-like PCBs, cognitive functions, proteomics, network analysis There is growing concern that the incidence of neurodevelopmental disorders including attention deficits hyperactivity disorders, autism, and learning disabilities in children worldwide are increasing (Boyle and Cordero, 2005; Schettler, 2001). Although genetic factors may play a key role in their etiology, a large proportion of neurodevelopmental disabilities with no apparent genetic bases and unknown etiology are caused or at least triggered by environmental factors. This has led to an active search for environmental chemical exposures that may affect normal neurodevelopment. From such efforts, polychlorinated biphenyls (PCBs) emerged as a credible risk factor for neurodevelopment disorders. The neurotoxicity of PCBs became evident after several well-documented accidents where pregnant women accidentally exposed to PCB-contaminated food (Faroon et al., 2001; Lung et al., 2005) gave birth to children who suffered from neurodevelopmental disorders. Since then, several epidemiological studies have shown that the nervous system, especially during development, is sensitive to environmental PCB exposure. Population studies performed in the United States and several European countries have reported that levels of exposure to PCBs are positively correlated with decreased IQ scores, impaired learning and memory, decreased neuromuscular function, and lower reading comprehension (Grandjean et al., 2001; Jacobson and Jacobson, 1996; Patandin et al., 1999). Exposure studies with animal models support the results observed in humans (Piedrafita et al., 2008; Roegge et al., 2004; Schantz et al., 1989). Although the production and use of PCBs has been banned in most industrialized countries since the 1970s, PCBs persist in the environment, making them a continuing risk to human health. Recently, it was shown that male and female rats exposed to 2,2′,3,4,4′,5′-hexachlorobiphenyl (PCB138) in utero and lactationally displayed impaired ability to learn a Y-maze conditional discrimination task (Boix et al., 2010). This learning appears to be modulated by the glutamate-nitric oxide-guanosine 3′,5′-cyclic Monophosphate pathway(glutamate-NO-cGMP) pathway (N-methyl-D-aspartate [NMDA]-induced increase in extracellular cGMP) in the cerebellum (Llansola et al., 2009; Yamada et al., 1996). Several studies show a parallel reduction in the function of the glutamate-NO-cGMP pathway in cerebellum in vivo and the ability to learn the Y-maze task in conditions including ageing (Piedrafita et al., 2007) and hepatic encephalopathy (Cauli et al., 2009; Monfort et al., 2001). Because cognitive functions involve temporally and spatially coordinated regulation of multiple protein complexes within the activated neural circuit, it cannot be excluded that other mechanisms could contribute and underlie the spatial learning and memory processes that are impaired following exposure to PCBs. The cerebellum, which plays an important role in cognitive processes such as visual spatial attention, working memory, and procedural learning (Gordon, 2007; Oliveri et al., 2007), is an established target for several toxic compounds. The specific mechanisms through which PCBs exert their cerebellar toxicity have not been extensively characterized. Recent advances in proteomic technology provide us with the opportunity to investigate simultaneous multiple components of protein pathways and cascades that can be used to increase our understanding of the complex mechanisms underlying PCB-induced learning and memory processes. In the current study, we performed large-scale analyses of the rat cerebellum proteome, to gain an insight into the mechanisms underlying perturbation of cognitive functions evoked by developmental exposure to PCB138. The PCB congener used in this study belongs to the food-relevant non–dioxin-like polychlorinated biphenyls (NDL-PCBs), the most abundant PCBs detected in human samples. An evaluation conducted by the European Food Safety Authority (EFSA) revealed that PCB138 is one of the most abundant NDL-PCBs congeners in food and human samples (http://www.efsa.europa.eu/en/scdocs/scdoc/284.htm), suggesting the need of a better understanding of NDL-PCBs toxicology. A comprehensive comparison of the cerebellum proteome from unexposed and PCB138-exposed rats was performed using a label-free semiquantitative mass spectrometry method (spectral counting). In combination with network enrichment analysis tools, we identified potential functional connections of the dysregulated proteins in the cerebellum of rats perinatally exposed to PCB138, which may contribute to the impairment of learning. MATERIALS AND METHODS Chemicals. PCB138 was purified at the Environmental Chemistry Department, Umea University (Sweden) to 99.9999% purity (European project FOOD-CT_2006-022923 ATHON). Animal treatment and tissue collection. All experimental procedures were approved by the Centro de Investigation Principe Felipe, Valencia, Spain, and adhered to the guidelines of the European Union for care and management of experimental animals (86/609/EEC). Female Wistar rats (Charles River) were treated with PCB138 from gestational day 7 to postnatal day 21 as previously reported (Boix et al., 2010). PCB138 was dissolved in corn oil and administered daily mixed in a sweet jelly bit. Each pregnant rat received 1 mg of PCB138/kg body weight (bw)/day, from gestational day 7 to postnatal day 21, a total of 35 days. Control rats were treated with the sweet jelly containing corn oil, following the same schedule of exposed rats. Pups were weaned at 21 days. Learning and motor coordination test were performed when the pups were 3-month old, an age at which young rats have the ability to perform learning and memory tasks used to test some type of learning controlled by cerebellum (Boix et al., 2010). Following behavioral analysis, rat offspring (3-month-old males) were sacrificed and the cerebellum was isolated, snap frozen and stored at −80°C until proteomic analysis. Control rats and PCB138-exposed rats were sacrificed exactly at the same age. Brain tissue was obtained from the same animals used in cognitive test by Boix et al. (2010). The rat cerebellum samples available for proteomics analysis were two pups cerebella from one litter, two pups cerebella from a second litter, and one pup cerebellum from a third litter for each treatment group (PCB-exposed and unexposed rats). PCB138 content in the cerebellum. Cerebellum samples were taken from the same rats used for learning test and proteomic analysis. Freeze-dried tissue samples were extracted with organic solvent and analyzed by gas chromatography with electron capture detection as previously reported (Boix et al., 2011). PCB138 concentrations were calculated using external standards. Protein extraction. For each individual pup, proteins were extracted by disintegrating 40 mg of frozen cerebellum with a Mikro-Dismembrator S (Sartorius, Florence, Italy) at 3000 revolutions per minute for 40 s. The powder obtained was resuspended in 1.8 ml of lysis buffer (5M urea, 2M thiourea, 2% (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 2% Zwittergent detergent; Calbiochem, La Jolla, CA) with protease inhibitors (complete, mini EDTA-free mixture; Roche Applied Science), homogenized for 1 min, and sonicated for 3 min, and 250 units of benzonase endonuclease (Novagen, San Diego, CA) were added to each sample. The homogenates were mixed for 30 min at 1000 rpm, at 25°C in a Thermomixer apparatus (Eppendorf, Milan, Italy), ultracentrifuged at 100,000 × g, for 30 min at 15°C, and the supernatants collected via methanol/chloroform precipitation. Protein concentration was measured with a modified Bradford method (Bio-Rad, Milan, Italy). In order to reduce technical and biological variation that might mask relevant changes in protein abundances, cerebellum protein extract of individual pups derived from three different litters for each treatment group were pooled. Two composite treatment groups (unexposed and PCB exposed) were created as detailed in Supplementary material. One-dimensional gel electrophoresis and in situ digestion of gel slices. Pooled cerebellum protein samples (15 μg) were run on 1-dimensional gel electrophoresis gel electrophoresis (12% polyacrylamide SDS-polyacrylamide gel electrophoresis) in triplicates to cope with the technical variability of label-free semiquantitative approach. Each gel lane was manually cut into 24 bands (Supplementary material). Excised bands were crushed into small fragments, processed, and submitted to in-gel trypsin digestion and peptide extractions as reported in details by Schiarea et al. (2010). The final sample, hereafter referred to as “digest,” thus contained all peptides recovered from the digestion of a single-gel band. Mass spectrometry, data analysis, protein identification, and quantification. The digest (aliquot 2 μl) was directly analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and all digests from triplicates were analyzed (144 digests in total). Analyses were carried out with an LTQ Orbitrap XL (Thermo Scientific, Waltham, MA), interfaced with a 1200 series capillary pump (Agilent, Santa Clara, CA). LC conditions for peptides separation and MS conditions were as reported by Schiarea et al. (2010). Details about MS/MS database searching, acceptance criteria for protein/peptides identification, and label-free estimation of protein amounts by spectral counting are provided in Supplementary material. Immunoblotting. Cerebellum protein extracts from each pooled experimental group (Supplementary material) were separated on SDS-10% polyacrylamide resolving gels and immunoblotted (Carpi et al., 2009) with rabbit polyclonal antibodies for androgen receptor (AR), inositol 1,4,5-triphospate receptor 1, with mouse monoclonal antibody clone3C12 for Ezrin (Sigma-Aldrich), and with rabbit polyclonal antibody C-19: sc-211 for protein kinase C-gamma (KPCG; Santa Cruz Biotechnology, CA). Peroxidase-conjugated anti-rabbit and anti-mouse IgG HRP (Santa Cruz Biotechnology) were used as secondary antibodies. Proteins were visualized on autoradiography film (Hyperfilm ECL; GE Healthcare, Milan, Italy) and scanned as 14-bit images (Image Scanner II; GE Healthcare). The resulting TIFF images were analyzed using Progenesis software (PG240 v2006l; Nonlinear Dynamics, UK). Expression data were normalized relative to alpha-actin (MAB1501, mouse anti-actin monoclonal antibody; Chemicon International) because the abundance of alpha-actin was not changed by PCB138 exposure as shown by our proteomic data (Tranche file, Supplementary material). Statistical analyses. For Western blot analysis, differences from control and treated samples were assessed by t-test using Prism software (v 5.01; GraphPad Software, Inc., San Diego, CA). Data are represented as the mean ± SD and p value of < 0.05 was considered to be statistically different. Proteomic data were analyzed by multivariate techniques with Simca-P 12 software (Umetrics AB, Umea, Sweden). The data were mean-centred and Pareto-scaled prior to multivariate statistical analysis. The data set was examined using partial least-square discriminant analysis (PLS-DA). The loadings plot was examined to identify which protein (variable) was significant in classifying the data. Differential variables (proteins) responsible for the clustering were then selected based on a threshold of variable importance in the projection (VIP) value (VIP > 1). Pathway and network analysis. The list of differentially expressed proteins was analyzed by pathway analysis using the MetaCore analytical suite version 5.3 (GeneGo, MI). MetaCore is an integrated analytical suite of algorithms, based on a manually curated database of human and rodent protein-protein interactions, transcriptional factors, signaling, metabolism, and bioactive molecules linked to functional processes and diseases (http://www.genego.com/). For network analysis, we used MetaCore's default background as background data set. Differently expressed proteins (Uniprot-Swiss Prot ID) were uploaded on MetaCore, and biological networks were built using two different algorithms: (1) the analyze network algorithm generating subnetworks ranked by a p value and interpreted in terms of Gene Ontology processes to deduce top scoring process that are regulated by differentially expressed proteins and (2) the shortest paths algorithm to map the shortest path for interaction. The resulting networks were evaluated to determine which algorithm succeeded in creating modules that have higher than random saturation with the protein of interest. Networks were then visualized graphically as nodes (proteins) and edges (relationship between proteins) alongside the expression pattern. Details of the bioinformatics analysis pipeline are given in Supplementary material. RESULTS We assessed the effects of developmental exposure to PCB138 on the cerebellum proteome of young male rats that showed reduced learning ability at 3 months of age. To ascertain the effective exposure risk during development, the concentration of PCB138 was measured in the cerebellum analyzed in this proteomic study. Measurements have been performed by gas chromatography with electron capture detection as previously reported (Boix, et al., 2011). The average PCB138 concentration in cerebellum of 3-month-old male rat offspring was 242 ± 63 ng PCB138/g dry weight tissue in exposed animals and 8.1 ± 1.4 ng of PCB138/g dry weight tissue in controls. Values are given as mean ± SEM (n = 5). The difference of PCB138 levels between exposed and nonexposed animals was statistically significant (P < 0.01). At 3 months of age, rats exposed to PCB138 showed a small but statistically significant reduction of their bw when compared with their untreated counterpart (bw expressed as mean ± SEM, PCB138-exposed animals [n = 26] 201.79 ± 25 g; controls [n = 28] 236.23 ± 19 g, P < 0.01). No effect of PCB138 on cerebellum size was observed. Proteomic Profiling Reveals a Temporal Pattern of Change in Cerebellum Protein Levels in the Perinatally Exposed Offspring. Proteomic analysis identified 589 proteins in the cerebellum of 3-month-old male rat offspring. The full dataset of all proteins and the associated raw mass spectrometric files are stored at Tranche (http://proteomecommons.org/tranche/), a public repository for sharing scientific data, as detailed in Supplementary material. The quantitative data trend (normalized spectral counts for each protein in each replicate sample for each experimental group) was explored by PLS-DA to evaluate the protein level of expression in comparison to control offspring. The scores (Supplementary Fig. S1) demonstrated a good separation between control and PCB138 samples, using two principal components (R2(X)cum = 40.7%, R2(Y)cum = 98.9%, Q2 = 34.5%;). The loading scatter plots (data not shown) identified proteins responsible for the cerebellum samples class separation. A threshold was set (VIP > 1) to define those proteins that were significantly important in distinguishing PCB138-exposed or unexposed samples. There were 100 proteins with VIP larger than 1. Their identities and differential expression data are reported in Supplementary tables S1 and S2. We focused on the subset of discriminating proteins (VIP > 1) whose abundance was at least twofold increased or reduced in the rat offspring (Table 1). To assess their quantitation reproducibility in the technical replicate analysis, the average coefficient of variance (CV %) for normalized spectral counts was calculated for each experimental group. The average CV ranged from 26% (unexposed samples) to 35% (PCB138-exposed samples). TABLE 1 Discriminating Proteins (VIP > 1) in Cerebellum from Unexposed and PCB138-Exposed Male Rats Showing At Least Twofold Increase or Reduction in Abundance Protein name Swiss-Prot accession name Function Fold changes in expression (PCB138 exposed vs. unexposed rats) Alpha-1-inhibitor 3 A1I3_RAT Protease inhibitor −2.66 ATP-citrate synthase ACLY_RAT Synthesis of acetyl-CoA −6.25 Annexin A3 ANXA3_RAT Inhibitor of phospholipase A2 −3.02 Sarcoplasmic/ER calcium ATPase 2 AT2A2_RAT Translocates Ca2+ from the cytosol to the endoplasmatic reticulum lumen 2.05 Plasma membrane calcium-transporting ATPase 1 AT2B1_RAT Transports Ca2+ out of the cell 24.75 Myc box-dependent–interacting protein 1 BIN1_RAT Involved in regulation of synaptic vesicle endocytosis −2.11 Complement C3 CO3_RAT Involved in activation of the complement system −2.29 Dihydropyrimidinase-related protein 3 DPYL3_RAT Involved in remodeling of the cytoskeleton −2.52 Dihydropyrimidinase-related protein 5 DPYL5_RAT Involved in neuronal differentiation and/or axon growth −2.23 Dynamin-3 DYN3_RAT Microtubule-associated force-producing protein −2.89 Ezrin EZRI_RAT Cytoskeletal structure protein −6.46 Peptidyl-prolyl cis-trans isomerase FKBP4 FKBP4_RAT Immunophilin protein co-chaperone activities 2.03 Gelsolin GELS_RAT Ca2+-regulated, actin-modulating protein −2.09 Glutathione S-transferase alpha-3 GSTA3_RAT Involved in xenobiotic catabolic process −3.35 Inositol monophosphatase 1 IMPA1_RAT Phosphatidylinositol biosynthetic process −2.90 Inositol 1,4,5-trisphosphate receptor type 1 ITPR1_RAT Intracellular channel mediating Ca2+ release from the ER 5.57 Protein kinase C gamma type KPCG_RAT Ca2+-activated, phospholipid-dependent, and serine- and threonine-specific enzyme 7.37 Myelin-associated glycoprotein MAG_RAT Adhesion molecule in postnatal neural development −2.15 Myristoylated alanine-rich C-kinase substrate MARCS_RAT Filamentous actin cross-linking protein acting as protein kinase C substrate 4.17 Myelin-oligodendrocyte glycoprotein MOG_RAT Minor component of the myelin sheath −4.30 Nucleoside diphosphate kinase B NDKB_RAT Synthesis of nucleoside triphosphates other than ATP −2.13 Pyruvate carboxylase, mitochondrial PYC_RAT Involved in carbohydrate biosynthesis −2.43 Reticulon-1 RTN1_RAT Involved in membrane trafficking 2.24 Protein S100-B S100B_RAT Ca2+-binding protein involved in neurite extension, stimulation of Ca2+ fluxes, astrocytosis, and axonal proliferation −2.10 Adenosyl homocysteinase SAHH_RAT Plays a key role in the control of methylations 2.48 Superoxide dismutase [Cu-Zn] SODC_RAT Antioxidant function −2.96 Synaptic vesicle glycoprotein 2A SV2A_RAT Regulates vesicle fusion −2.84 Protein name Swiss-Prot accession name Function Fold changes in expression (PCB138 exposed vs. unexposed rats) Alpha-1-inhibitor 3 A1I3_RAT Protease inhibitor −2.66 ATP-citrate synthase ACLY_RAT Synthesis of acetyl-CoA −6.25 Annexin A3 ANXA3_RAT Inhibitor of phospholipase A2 −3.02 Sarcoplasmic/ER calcium ATPase 2 AT2A2_RAT Translocates Ca2+ from the cytosol to the endoplasmatic reticulum lumen 2.05 Plasma membrane calcium-transporting ATPase 1 AT2B1_RAT Transports Ca2+ out of the cell 24.75 Myc box-dependent–interacting protein 1 BIN1_RAT Involved in regulation of synaptic vesicle endocytosis −2.11 Complement C3 CO3_RAT Involved in activation of the complement system −2.29 Dihydropyrimidinase-related protein 3 DPYL3_RAT Involved in remodeling of the cytoskeleton −2.52 Dihydropyrimidinase-related protein 5 DPYL5_RAT Involved in neuronal differentiation and/or axon growth −2.23 Dynamin-3 DYN3_RAT Microtubule-associated force-producing protein −2.89 Ezrin EZRI_RAT Cytoskeletal structure protein −6.46 Peptidyl-prolyl cis-trans isomerase FKBP4 FKBP4_RAT Immunophilin protein co-chaperone activities 2.03 Gelsolin GELS_RAT Ca2+-regulated, actin-modulating protein −2.09 Glutathione S-transferase alpha-3 GSTA3_RAT Involved in xenobiotic catabolic process −3.35 Inositol monophosphatase 1 IMPA1_RAT Phosphatidylinositol biosynthetic process −2.90 Inositol 1,4,5-trisphosphate receptor type 1 ITPR1_RAT Intracellular channel mediating Ca2+ release from the ER 5.57 Protein kinase C gamma type KPCG_RAT Ca2+-activated, phospholipid-dependent, and serine- and threonine-specific enzyme 7.37 Myelin-associated glycoprotein MAG_RAT Adhesion molecule in postnatal neural development −2.15 Myristoylated alanine-rich C-kinase substrate MARCS_RAT Filamentous actin cross-linking protein acting as protein kinase C substrate 4.17 Myelin-oligodendrocyte glycoprotein MOG_RAT Minor component of the myelin sheath −4.30 Nucleoside diphosphate kinase B NDKB_RAT Synthesis of nucleoside triphosphates other than ATP −2.13 Pyruvate carboxylase, mitochondrial PYC_RAT Involved in carbohydrate biosynthesis −2.43 Reticulon-1 RTN1_RAT Involved in membrane trafficking 2.24 Protein S100-B S100B_RAT Ca2+-binding protein involved in neurite extension, stimulation of Ca2+ fluxes, astrocytosis, and axonal proliferation −2.10 Adenosyl homocysteinase SAHH_RAT Plays a key role in the control of methylations 2.48 Superoxide dismutase [Cu-Zn] SODC_RAT Antioxidant function −2.96 Synaptic vesicle glycoprotein 2A SV2A_RAT Regulates vesicle fusion −2.84 Open in new tab TABLE 1 Discriminating Proteins (VIP > 1) in Cerebellum from Unexposed and PCB138-Exposed Male Rats Showing At Least Twofold Increase or Reduction in Abundance Protein name Swiss-Prot accession name Function Fold changes in expression (PCB138 exposed vs. unexposed rats) Alpha-1-inhibitor 3 A1I3_RAT Protease inhibitor −2.66 ATP-citrate synthase ACLY_RAT Synthesis of acetyl-CoA −6.25 Annexin A3 ANXA3_RAT Inhibitor of phospholipase A2 −3.02 Sarcoplasmic/ER calcium ATPase 2 AT2A2_RAT Translocates Ca2+ from the cytosol to the endoplasmatic reticulum lumen 2.05 Plasma membrane calcium-transporting ATPase 1 AT2B1_RAT Transports Ca2+ out of the cell 24.75 Myc box-dependent–interacting protein 1 BIN1_RAT Involved in regulation of synaptic vesicle endocytosis −2.11 Complement C3 CO3_RAT Involved in activation of the complement system −2.29 Dihydropyrimidinase-related protein 3 DPYL3_RAT Involved in remodeling of the cytoskeleton −2.52 Dihydropyrimidinase-related protein 5 DPYL5_RAT Involved in neuronal differentiation and/or axon growth −2.23 Dynamin-3 DYN3_RAT Microtubule-associated force-producing protein −2.89 Ezrin EZRI_RAT Cytoskeletal structure protein −6.46 Peptidyl-prolyl cis-trans isomerase FKBP4 FKBP4_RAT Immunophilin protein co-chaperone activities 2.03 Gelsolin GELS_RAT Ca2+-regulated, actin-modulating protein −2.09 Glutathione S-transferase alpha-3 GSTA3_RAT Involved in xenobiotic catabolic process −3.35 Inositol monophosphatase 1 IMPA1_RAT Phosphatidylinositol biosynthetic process −2.90 Inositol 1,4,5-trisphosphate receptor type 1 ITPR1_RAT Intracellular channel mediating Ca2+ release from the ER 5.57 Protein kinase C gamma type KPCG_RAT Ca2+-activated, phospholipid-dependent, and serine- and threonine-specific enzyme 7.37 Myelin-associated glycoprotein MAG_RAT Adhesion molecule in postnatal neural development −2.15 Myristoylated alanine-rich C-kinase substrate MARCS_RAT Filamentous actin cross-linking protein acting as protein kinase C substrate 4.17 Myelin-oligodendrocyte glycoprotein MOG_RAT Minor component of the myelin sheath −4.30 Nucleoside diphosphate kinase B NDKB_RAT Synthesis of nucleoside triphosphates other than ATP −2.13 Pyruvate carboxylase, mitochondrial PYC_RAT Involved in carbohydrate biosynthesis −2.43 Reticulon-1 RTN1_RAT Involved in membrane trafficking 2.24 Protein S100-B S100B_RAT Ca2+-binding protein involved in neurite extension, stimulation of Ca2+ fluxes, astrocytosis, and axonal proliferation −2.10 Adenosyl homocysteinase SAHH_RAT Plays a key role in the control of methylations 2.48 Superoxide dismutase [Cu-Zn] SODC_RAT Antioxidant function −2.96 Synaptic vesicle glycoprotein 2A SV2A_RAT Regulates vesicle fusion −2.84 Protein name Swiss-Prot accession name Function Fold changes in expression (PCB138 exposed vs. unexposed rats) Alpha-1-inhibitor 3 A1I3_RAT Protease inhibitor −2.66 ATP-citrate synthase ACLY_RAT Synthesis of acetyl-CoA −6.25 Annexin A3 ANXA3_RAT Inhibitor of phospholipase A2 −3.02 Sarcoplasmic/ER calcium ATPase 2 AT2A2_RAT Translocates Ca2+ from the cytosol to the endoplasmatic reticulum lumen 2.05 Plasma membrane calcium-transporting ATPase 1 AT2B1_RAT Transports Ca2+ out of the cell 24.75 Myc box-dependent–interacting protein 1 BIN1_RAT Involved in regulation of synaptic vesicle endocytosis −2.11 Complement C3 CO3_RAT Involved in activation of the complement system −2.29 Dihydropyrimidinase-related protein 3 DPYL3_RAT Involved in remodeling of the cytoskeleton −2.52 Dihydropyrimidinase-related protein 5 DPYL5_RAT Involved in neuronal differentiation and/or axon growth −2.23 Dynamin-3 DYN3_RAT Microtubule-associated force-producing protein −2.89 Ezrin EZRI_RAT Cytoskeletal structure protein −6.46 Peptidyl-prolyl cis-trans isomerase FKBP4 FKBP4_RAT Immunophilin protein co-chaperone activities 2.03 Gelsolin GELS_RAT Ca2+-regulated, actin-modulating protein −2.09 Glutathione S-transferase alpha-3 GSTA3_RAT Involved in xenobiotic catabolic process −3.35 Inositol monophosphatase 1 IMPA1_RAT Phosphatidylinositol biosynthetic process −2.90 Inositol 1,4,5-trisphosphate receptor type 1 ITPR1_RAT Intracellular channel mediating Ca2+ release from the ER 5.57 Protein kinase C gamma type KPCG_RAT Ca2+-activated, phospholipid-dependent, and serine- and threonine-specific enzyme 7.37 Myelin-associated glycoprotein MAG_RAT Adhesion molecule in postnatal neural development −2.15 Myristoylated alanine-rich C-kinase substrate MARCS_RAT Filamentous actin cross-linking protein acting as protein kinase C substrate 4.17 Myelin-oligodendrocyte glycoprotein MOG_RAT Minor component of the myelin sheath −4.30 Nucleoside diphosphate kinase B NDKB_RAT Synthesis of nucleoside triphosphates other than ATP −2.13 Pyruvate carboxylase, mitochondrial PYC_RAT Involved in carbohydrate biosynthesis −2.43 Reticulon-1 RTN1_RAT Involved in membrane trafficking 2.24 Protein S100-B S100B_RAT Ca2+-binding protein involved in neurite extension, stimulation of Ca2+ fluxes, astrocytosis, and axonal proliferation −2.10 Adenosyl homocysteinase SAHH_RAT Plays a key role in the control of methylations 2.48 Superoxide dismutase [Cu-Zn] SODC_RAT Antioxidant function −2.96 Synaptic vesicle glycoprotein 2A SV2A_RAT Regulates vesicle fusion −2.84 Open in new tab Biological Pathways and Protein Networks Perturbed following Perinatal Exposure to PCB138. To understand the biological relevance of the differentially expressed proteins involved in the PCB138-induced neurotoxicity, we concentrated on the 27 proteins, whose abundance was at least twofold increased or reduced in the rat offspring in comparison to controls (listed in Table 1). With this subset of proteins, biological networks were built using the Analyze Network Algorithm, which assigns a biological process to each network. The top scoring Gene Ontology process regulated by these differently expressed proteins was “calcium transport ion” and had 11 proteins significantly altered by PCB 138 (Fig. 1,Supplementary Fig. S2). Indeed the main hub of this network was cytosolic Ca2+ having the maximum number of edges (connections). Moreover, the p53 and intracellular myo-inositol constituted further interesting hubs in the network. We found a decreased expression of IMPA1 that dephosphorylates inositol monophosphate to inositol, suggesting the involvement of the phosphoinositol signaling pathway. FIG. 1. Open in new tabDownload slide The top-scored biological network, generated by using the analyze network algorithm, associated with the GO process “Calcium transport ion” illustrating the connections among the differently expressed proteins (at least 2-fold induction/reduction) in male cerebellum in response to PCB138 exposure. Nodes represent proteins and the different shapes of the nodes represent the functional class of the proteins. Circles represent single protein or group of proteins identified in our analysis. Lines connecting the nodes indicate interactions of activation or induction or modification or direct binding; the arrowheads indicate the direction of the interaction. Broken line circles highlight network hubs as indicated by network statistics. The protein nodes in the figure refer to the following proteomics abbreviations: Ca-ATPase2=AT2A2, GSTA1= GSTA3, IP3R1= ITPR1, NDPKB= NDKB, PKC-gamma= KPCG, PMCA1= AT2B1, VIL2= EZR. FIG. 1. Open in new tabDownload slide The top-scored biological network, generated by using the analyze network algorithm, associated with the GO process “Calcium transport ion” illustrating the connections among the differently expressed proteins (at least 2-fold induction/reduction) in male cerebellum in response to PCB138 exposure. Nodes represent proteins and the different shapes of the nodes represent the functional class of the proteins. Circles represent single protein or group of proteins identified in our analysis. Lines connecting the nodes indicate interactions of activation or induction or modification or direct binding; the arrowheads indicate the direction of the interaction. Broken line circles highlight network hubs as indicated by network statistics. The protein nodes in the figure refer to the following proteomics abbreviations: Ca-ATPase2=AT2A2, GSTA1= GSTA3, IP3R1= ITPR1, NDPKB= NDKB, PKC-gamma= KPCG, PMCA1= AT2B1, VIL2= EZR. To evaluate protein-protein interaction network among the proteins having at least twofold difference in expression against their respective counterparts (listed in Table 1), MetaCore software was used to map the shortest paths of interactions among these differentially expressed proteins; 24 out of 27 proteins were brought together in the network (Fig. 2) and revealed that 3 downregulated proteins, ACLY, EZR1 and GELS, were directly connected to the AR network interaction. Moreover, many of the remaining proteins in the network were indirectly linked to the AR through other transcriptional factors. Indeed AR together with c-Myc and p53 constituted the most relevant hubs of this network. Seven target proteins (SOD1, FKBP4, PYC, BIN1, CRMP4, AT2A2, and NDPKB) were directly linked to c-Myc and five proteins (NDPKB, AT2A2, S100B, GSTA1, and GELS) to p53. FIG. 2. Open in new tabDownload slide Protein network generated by the shortest path algorithm using the list of differently expressed proteins (more than 2-fold induction/reduction) in male cerebellum in response to PCB138 exposure. Nodes represent proteins and the different shapes of the nodes represent the functional class of the proteins. Circles represent single protein or group of proteins identified in our analysis. Lines connecting the nodes indicate interactions of activation or induction or modification or direct binding; the arrowheads indicate the direction of the interaction. Thick line refers to canonical pathways present in the network automatically traced by MetaCore. Broken line circles highlight network hubs as indicated by network statistics. The protein nodes in the figure refer to the following proteomics abbreviations: Ca-ATPase2= AT2A2, CRMP4= DPYL3, GSTA1= GSTA3, NDPKB= NDKB, SOD1= SODC, VIL2= EZR1. FIG. 2. Open in new tabDownload slide Protein network generated by the shortest path algorithm using the list of differently expressed proteins (more than 2-fold induction/reduction) in male cerebellum in response to PCB138 exposure. Nodes represent proteins and the different shapes of the nodes represent the functional class of the proteins. Circles represent single protein or group of proteins identified in our analysis. Lines connecting the nodes indicate interactions of activation or induction or modification or direct binding; the arrowheads indicate the direction of the interaction. Thick line refers to canonical pathways present in the network automatically traced by MetaCore. Broken line circles highlight network hubs as indicated by network statistics. The protein nodes in the figure refer to the following proteomics abbreviations: Ca-ATPase2= AT2A2, CRMP4= DPYL3, GSTA1= GSTA3, NDPKB= NDKB, SOD1= SODC, VIL2= EZR1. Validation of cerebellum protein targets for PCB138. To verify the protein targets of PCB138 as identified by LC-MS/MS, three proteins, showing strong differences in abundance, were selected from the male cerebellum protein expression lists and evaluated by Western blot. Changes in protein expression for ITPR1 and KPCG (increased protein expression induced by PCB138) and EZR1 (reduced protein expression induced by PCB138) were consistent with proteomic analysis in male rats (Fig. 3, panel A). Based on our network analysis, we hypothesized that AR may be a key regulator for some of the downstream proteins whose levels were changed by the PCB138 exposure in male rats. Western blot analysis confirmed the reduction of the AR protein level in male PCB138-exposed cerebellum (Fig. 3, panel B). FIG. 3. Open in new tabDownload slide Western blot analyses. (Panel A) Expression changes of ezrin (EZR1), inositol 1,4,5-trisphosphate receptor type 1 (ITPR1), and KPCG in cerebellum from unexposed (Ctr) and PCB138-exposed male rats. (Panel B) Expression changes of AR in cerebellum from unexposed (Ctr) and PCB138-exposed male rats. Protein bands in the autoradiograms were quantified by scanning densitometry. Expression data were normalized relative to α-actin. Each bar represents the average normalized volume density ± SD derived from three replicated immunoblots. The vertical axis shows arbitrary density unit. Asterisks mark significant expression changes (t-test, *p < 0.01). FIG. 3. Open in new tabDownload slide Western blot analyses. (Panel A) Expression changes of ezrin (EZR1), inositol 1,4,5-trisphosphate receptor type 1 (ITPR1), and KPCG in cerebellum from unexposed (Ctr) and PCB138-exposed male rats. (Panel B) Expression changes of AR in cerebellum from unexposed (Ctr) and PCB138-exposed male rats. Protein bands in the autoradiograms were quantified by scanning densitometry. Expression data were normalized relative to α-actin. Each bar represents the average normalized volume density ± SD derived from three replicated immunoblots. The vertical axis shows arbitrary density unit. Asterisks mark significant expression changes (t-test, *p < 0.01). DISCUSSION The mechanisms by which developmental exposure to individual PCB congeners affect cerebral and cognitive functions are poorly characterized and how molecular and protein changes relate to functional deficits have been difficult to establish. We have applied a global proteomic approach, including pathway and network analysis, to search for candidate effectors and pathways that could be associated to learning ability deficiency in PCB-exposed offspring. So far, very few studies have investigated the link between proteins from several pathways to memory and learning performances (Sunyer et al., 2008a, 2008b; Zheng et al., 2009). This study is significant as it is the first study to incorporate neuroproteomic approaches to investigate neurotoxicity effects of PCBs, such as PCB138. Perinatal exposure to PCB138 results in impaired cognitive functions for rat offspring. PCB138 impaired learning ability tested as decreased ability to learn the Y-maze test but not motor coordination in 3-month-old male rats. Moreover, developmental exposure to PCB138 reduced the amount of NMDA receptors in cerebellum pups, which would contribute to reduced function of the glutamate-NO-cGMP pathway, which in turn would be a main contributor to the impairment of the ability to learn the Y-maze task (Boix et al., 2010). Consistent with this observation, in the present study, we demonstrated that perturbations in the rat proteome profile persist well beyond exposure to PCB138, indicating that permanent protein changes in the cerebellum of 3-month-old male rat offspring results as a consequence of developmental exposure. This disruption during development can therefore induce alterations to learning and other neural functions. Network analysis identified Ca2+signaling transport as the top scoring process altered in PCB138-exposed male cerebellum. Calcium signaling is crucial for normal functioning of neurons. It is well known that PCBs increase intracellular Ca2+, such an increase has been attributed to release from both extracellular and intracellular Ca2+stores, which can be detrimental for neurons (Fonnum and Mariussen, 2009; Kodavanti, 2005). We observed a striking overexpression of ITPR1, a protein that is essential for transferring Ca2+ from endoplasmic reticulum (ER) to cytosol, together with an enhanced level of AT2A2, a protein that mediates the ER Ca2+ uptake. The rise in cytoplasmatic Ca2+ level calls for the action of a system extruding Ca2+ outside the cell, such as AT2B1, whose enhanced expression may prevent Ca2+ cell overload. These findings are consistent with the Ca2+ disturbances following PCBs exposure associated with alterations in ITPR-mediated signals (Inglefield et al., 2001). Moreover, S100B, a calcium-binding astrocytic protein implicated in brain development and neurophysiology, had lower protein level in PCB138-exposed cerebellum. Because specific decrease in Ca2+-handling capacity has been observed in S100B null mice (Xiong et al., 2000), the downregulation of S100B in male cerebellum support the evidence that the maintenance of Ca2+-homeostasis in male offspring is particularly perturbed by PCB138. A second key observation inferred from our network analysis was the importance of the phosphoinositol signaling pathway in PCB138-exposed cerebellum, where the decreased expression of IMPA1 protein would reduce the supply of inositol, which in turn may limit phosphatidylinositol (PI) synthesis. The PI signaling cascade starts with surface receptor–mediated activation of phospholipase C, which catalyses the hydrolysis of the membrane phosphatidylinositol 4,5-biphosphate to inositol 1,4,5-triphosphate (IP3) and to diacylglycerol (DAG). IP3 releases free calcium ions from intracellular stores and DAG activates KPCG, whose expression was indeed enhanced in PCB138-exposed cerebellum as further validated by Western blot analysis. This might be viewed as a mechanism secondary to inositol depletion by accumulated DAG. Interestingly, KPCG signaling pathway has been shown to be affected by dioxin (Kim et al., 2007) and by PCB mixture exposure (Yang et al., 2003). Moreover, KPCG has been implicated in the modulation of learning and memory and spatial memory impairment among aged rats (Colombo and Gallagher, 2002; Rossi et al., 2005). In this regard, it should be stated that KPCG is specifically expressed in the nervous system; consequently, higher levels of KPCG may represent an attempt to compensate for a functional age-related alteration/deregulation affecting the KPCG signaling (Pascale et al., 2007). Taken together, the increased cerebellar expression of KPCG in addition to the perturbations in components of the calcium pathways suggest that perinatal exposure to PCB138 may led to premature ageing cerebellum proteome profiles. This, would be consistent with the impaired ability to learn the Y-maze task observed in rats, where 7-month-old unexposed rats showed a similar inability to learn the task as 3-months old exposed rats (Piedrafita et al., 2007). In support of our premature ageing hypothesis, it has been shown that NMDA receptor subunit NR1 is decreased in the cerebellum of these PCB138-exposed 3-month-old rat offspring (Boix et al., 2010). Consistent with this finding, studies have reported that protein levels of NR1 decline in the hippocampus and cerebral cortex of ageing mice compromising synaptic communication (Magnusson et al., 2010; Zhao et al., 2009). The alteration in NMDA receptor is therefore consistent with the notion that multiple Ca2+-mediated processes are disrupted by developmental PCB138 exposure that might mimic and/or accelerate ageing processes. The changes in the abilities to handle Ca2+ would ultimately influence neuronal firing and the ability to propagate action potentials, which in turn would affect the ability of the cerebellum to alter its structure or function (i.e., its plastic nature) and the network dynamics of neural ensembles that support cognition. Interestingly, data over the adult life span in humans and nonhuman primates suggest that the cerebellum shows earlier senescence than the hippocampus, with cerebellum-essential tasks having age-related deficits at earlier ages than hippocampus-essential tasks (Woodruff-Pak et al., 2010). PCB138-induced protein expression alterations may also be mediated through changes in the activity of transcription factors. Protein interaction network analysis highlighted several regulatory proteins, but AR drew our attention because of its involvement in brain functions and ageing. Due to its exceptional synaptogenic potential (Hajszan et al., 2008; Hatanaka et al., 2009), the AR and androgen signaling pathways play a key role in learning, memory, and other brain functions that deteriorate with increasing age (Pike et al., 2008). Reduction of AR protein has been reported in the brain cortex of old mice (15 months old) as compared with adult mice (6 months old) (Kumar and Thakur, 2004). Moreover, PCB138 has been shown to exert an anti-androgenic activity being able to compete with the binding of the natural ligand to AR (Bonefeld-Jorgensen et al., 2001). In our study, PCB138 decreased the level of cytoskeleton proteins EZR1 and GELS together with the metabolic enzyme, ACLY. These proteins are directly regulated by AR (Chuan et al., 2006; Massie et al., 2007; Nishimura et al., 2003). Our proteomic and network analysis thus suggest that the AR level and/or activity may be decreased. Based on the stringent criteria used in our proteomic analysis to identify differentially expressed proteins, the AR protein (together with proteins involved in the glutamate-NO-cGMP pathway) were not included in our list of high-confidence identified differentially expressed proteins. Due to the stochastic nature of data-dependent MS acquisition, failure to identify peptides to a protein does not necessarily indicate that the protein is not present in the sample. Using less stringent protein identification parameters (i.e., identification with only one search engine such as Mascot, analysis not performed against a decoy database) both the AR and proteins involved in the glutamate-NO-cGMP pathway were identified (data not shown). Consistent with this comparison, Western blot analysis revealed that the level of AR protein was dramatically decreased in PCB138 male cerebellum compared with the untreated controls, suggesting that steroid receptors may contribute to mediating neurotoxicity induced by PCB138. In conclusion, our findings reinforce the concept that development exposure to PCB138 has long-lasting effects on the nervous system, with consequences on cerebellum proteome profile and cognitive functions of the offspring. We propose that the observed alterations in Ca2+ signaling (with consequent changes in second messengers cascades) and in network of regulatory proteins (with consequent changes in gene/protein expression) may point toward a common theme: the generation of a premature ageing-like proteome profile of cerebellum triggered by the developmental PCB138 exposure. Future research is required to evaluate if senescence is gender or PCB congener specific. Our proteomic data provide insights into the phenomena that may be contributing to the PCB138 neurotoxicity effects observed in laboratory rodents and support the concern that EFSA has raised about the NDL-PCB toxicity. FUNDING European Commission (FOOD-CT-2006-022923 ATHON). We thank Dr Patrick Andersson and Mia Stenberg (Environmental Chemistry Department, Umea University, Sweden) for purifying PCB138. We thank Dr Robin Wait, Kennedy Institute of Rheumatology Division, Imperial College of London, for critical review of the manuscript. References Boix J , Cauli O , Felipo V . Developmental exposure to polychlorinated biphenyls 52, 138 or 180 affects differentially learning or motor coordination in adult rats. Mechanisms involved , Neuroscience , 2010 , vol. 167 (pg. 994 - 1003 ) Google Scholar Crossref Search ADS PubMed WorldCat Boix J , Cauli O , Leslie H , Felipo V . 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TI - Cerebellum Proteomics Addressing the Cognitive Deficit of Rats Perinatally Exposed to the Food-Relevant Polychlorinated Biphenyl 138
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfr156
DA - 2011-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/cerebellum-proteomics-addressing-the-cognitive-deficit-of-rats-gVoUTpoW0y
SP - 170
EP - 179
VL - 123
IS - 1
DP - DeepDyve
ER -