- Email: [email protected]
Proteomic analysis of PSD-93 knockout mice following the induction of ischemic cerebral injury
NeuroToxicology 53 (2016) 1–11
Contents lists available at ScienceDirect
NeuroToxicology
Full length article
Proteomic analysis of PSD-93 knockout mice following the induction of ischemic cerebral injury Rong Ronga,b,c,1, Hui Yangd,1, Liangqun Ronge , Xiue Weie , Qingjie Lif , Xiaomei Liug , Hong Gaoe, Yun Xua,b,c,* , Qingxiu Zhange,** a
Department of Neurology, Drum Tower Hospital of Nanjing Medical University, Nanjing, 210029 Jiangsu, China Department of Neurology, Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, 210029 Jiangsu, China Jiangsu Key Laboratory for Molecular Medicine, Nanjing, China d Department of Neurosurgery, Xuzhou First People’s Hospital, Xuzhou, 221000 Jiangsu, China e Department of Neurology, Second Affiliated Hospital of Xuzhou Medical College, Xuzhou, 221006 Jiangsu, China f Department of Neurology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, 221000 Jiangsu, China g Department of Pathogenic Biology and Immunology, Lab of Infection and Immunity, Xuzhou Medical College, Xuzhou, 221004 Jiangsu, China b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 October 2015 Received in revised form 4 December 2015 Accepted 5 December 2015 Available online 8 December 2015
Postsynaptic density protein-93 (PSD-93) is enriched in the postsynaptic density and is involved in N-methyl-D-aspartate receptor (NMDAR) triggered neurotoxicity through PSD-93/NMDAR/nNOS signaling pathway. In the present study, we found that PSD-93 deficiency reduced infarcted volume and neurological deficits induced by transient middle cerebral artery occlusion (tMCAO) in the mice. To identify novel targets of PSD-93 related neurotoxicity, we applied isobaric tags for relative and absolute quantitative (iTRAQ) labeling and combined this labeling with on-line two-dimensional LC/MS/MS technology to elucidate the changes in protein expression in PSD-93 knockout mice following tMCAO. The proteomic data set consisted of 1892 proteins. Compared to control group, differences in expression levels in ischemic group >1.5-fold and <0.66-fold were considered as differential expression. A total of 104 unique proteins with differential abundance levels were identified, among which 17 proteins were selected for further validation. Gene ontology analysis using UniProt database revealed that these differentially expressed proteins are involved in diverse function such as synaptic transmission, neuronal neurotransmitter and ion transport, modification of organelle membrane components. Moreover, network analysis revealed that the interacting proteins were involved in the transport of synaptic vesicles, the integrity of synaptic membranes and the activation of the ionotropic glutamate receptors NMDAR1 and NMDAR2B. Finally, RT-PCR and Western blot analysis showed that SynGAP, syntaxin-1A, protein kinase C b, and voltage-dependent L-type calcium channels were inhibited by ischemia– reperfusion. Identification of these proteins provides valuable clues to elucidate the mechanisms underlying the actions of PSD-93 in ischemia–reperfusion induced neurotoxicity. ã 2015 Elsevier Inc. All rights reserved.
Keywords: Proteomic analysis PSD-93 Excitotoxicity Ischemic stroke
1. Introduction
* Corresponding author at: Department of Neurology, Drum Tower Hospital of Nanjing Medical University, 321 Zhongshan Road, Nanjing City, Jiangsu Province 210008, China. Fax: +86 25 83317016. ** Corresponding author at: Department of Neurology, Second Affiliated Hospital of Xuzhou Medical College, 32 Coal Road, Xuzhou City, Jiangsu Province 221006, China. Fax: +86 561 85326105. E-mail addresses: [email protected] (Y. Xu), [email protected] (Q. Zhang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neuro.2015.12.005 0161-813X/ ã 2015 Elsevier Inc. All rights reserved.
Cerebral ischemia–reperfusion induced neuronal injury involves a complex signal transduction network, and its mechanisms remain incompletely understood. Such injury is initiated by the release and aggregation of excitatory amino acids during ischemic brain damage (Brassai et al., 2014). Consequently, Nmethyl-D-aspartate receptors (NMDA receptors or NMDARs) are activated by postsynaptic density (PSD) scaffolding proteins, leading to a series of pathological changes (Zhang et al., 2007, 2010; Zhou et al., 2010; Xu et al., 2004). NMDARs are tetrameric complexes consisted of several subunits. The subunit composition of NMDARs is plastic, leading to a large number of NMDAR
2
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
subtypes. Up to now, seven different subunits have been identified, including GluN1 subunit, four distinct GluN2 subunits (GluN2A, GluN2B, GluN2C and GluN2D), and two GluN3 subunits (GluN3A and GluN3B) (Paoletti et al., 2013). Notably, during early postnatal development, NMDARs switch subunit composition from primarily containing GluN2B subunits to predominantly containing GluN2A subunits in central nerve system, coinciding with the processes of synapse maturation and learning (Dumas, 2005). PSD scaffolding proteins play a vital role in NMDAR activationinduced ischemic brain injury. PSD-93 is a PSD scaffolding protein that belongs to the membrane-associated guanylate kinase family (MAGUKs). PSD-93 plays key role in synaptic development, synaptic plasticity and the scaffolds of postsynaptic complexes (Jiang et al., 2003; Zheng et al., 2011). Moreover, several studies suggest that PSD-93 might be involved in chronic pain, morphine tolerance, morphine withdrawal and learning and memory dysfunction via its effects on synaptic plasticity (Liaw et al., 2008; Tao et al., 2010; Zhang et al., 2003). In addition, PSD-93 is implicated in ischemic brain injury. PSD-93 knockout mice might engage the hypoxic-ischemic injury induced NMDAR signaling pathway by upregulating the protein expression of PSD-95 (Jiang et al., 2003). Recently, our study revealed that PSD-93 interacted with nNOS and NR2A to promote nNOS activation via plateletactivating factor (PAF) to mediate ischemic brain injury (Xu et al., 2004). Additionally, PSD-93 knockout significantly increased the proliferative capacity of cells, which reduces the neuronal death that results from ischemic brain injury (Zhang et al., 2010). Furthermore, PSD-93 knockout exhibited neuroprotective effects against ischemic brain injury that are mediated by the inhibition of the Fyn-mediated phosphorylation of NR2B (Zhang et al., 2014). These findings suggest that PSD-93 plays an important role by suppressing ischemia-induced neuronal excitotoxicity during ischemic brain injury, but the exact mechanisms of this function require further exploration. Recent advances in proteomics technology have enabled the application of a sophisticated labeling-based quantitative method based on iTRAQ labeling coupled with on-line two-dimensional LC/ MS/MS technology to provide important insights into the pathogenesis of major psychiatric and neurodegenerative disorders and ischemic brain injury in patient cohorts (Martins-De-Souza et al., 2010; Johnston-Wilson et al., 2000; Pennington et al., 2008; Castegna et al., 2002) and animal models (Ditzen et al., 2006; Mu et al., 2007; Otte et al., 2011; Patel et al., 2007; Robinson et al., 2011; Szego et al., 2010). Although previous studies have shown that PSD-93 is involved in the neuronal excitotoxicity induced by ischemic brain injury, no studies have attempted to detect the proteomic changes associated with PSD-93 disruption and excitotoxicity. In the present study, iTRAQ-based quantitative proteomics analysis was applied to identify potential protein targets of PSD93 disruption in adult mouse transient middle cerebral artery occlusion (tMCAO) model. To further explore the mechanisms responsible for the function of PSD-93 in MCAO induced excitotoxicity,17 target proteins were selected for gene ontology (GO) function and network analyses. We found that the ras GTPase-activating protein SynGAP (SynGAP), isoform 2 of the voltage-dependent Ltype calcium channel subunit beta-4 (VDLC), syntaxin-1A, and PKCb are involved in PSD-93 mediated neurotoxicity. 2. Materials and methods 2.1. Animals PSD-93 knockout (KO) mice (C57BL/6 genetic background) were produced as described previously (Paoletti et al., 2013). Male PSD93 KO mice and wild-type (WT) littermates (10–12 weeks old)
were obtained by interbreeding PSD-93 heterozygous mice. The genotype of each mouse was confirmed by polymerase chain reaction. All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing University. Mice were housed in cages individually with free food and water supply. The environment was at a temperature (23 2 C), humidity (40– 60%) and a 12/12 h dark–light cycle (light on 9:00–21:00). 2.2. MCAO model Transient focal cerebral ischemia was induced as previously described (Zhang et al., 2012). Briefly, PSD-93 KO mice and WT C57BL/6 mice were anesthetized by intraperitoneal injection of ketamine (100 mg/ml) and xylazine (20 mg/kg) mixture (1:1) at a dose of 1 ml/kg. A 6/0 monofilament nylon suture with a heatrounded tip was inserted through the internal carotid artery into the beginning of the right middle cerebral artery (MCA). The mice were subjected to 2 h of occlusion, and blood flow was then restored by the withdrawal of the filament at 4, 24, 48, or 72 h. Sham control animals were subjected to similar operations without the MCA occlusion. During the experiment, the body temperature was maintained at 37 0.5 C with an infrared heating lamp. The mortality of both PSD-93 KO mice and WT C57BL/6 mice in the model group was approximately 10%. During the reperfusion, post-operative mice had adequate water and food and 24 h awakening-sleep cycle. Those paralysis mice accepted special care and artificial feeding at least 3 times a day. 2.3. Infarct size measurement After anesthesia using sodium pentobarbital, three PSD-93 KO mice and three WT C57BL/6 mice in the model group were executed by cervical dislocation and quickly decapitated at the indicated reperfusion time, respectively. The brains were washed with freezing PBS solution, and then cut into five 2-mm-thick slices and stained with 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) at 37 C for 20 min in the dark to detect infarct volume. Images were acquired with a computer-controlled digital camera (Olympus) and analyzed with Image-ProPlus 6.0 (IPP) software (Media Cybernetics) to calculate the infarct sizes. The infarct volume in all slices was expressed as the percentages of the contralateral hemisphere after correcting for edema. 2.4. Behavioral test Neurological Severity Score (NSS) is a composite of motor, sensory, reflex, and balance tests and was evaluated at 4, 24, 48, and 72 h after MCAO (Chen et al., 2001). Neurological function was graded on a scale ranging from 0 to 18 (normal score, 0; maximal deficit score, 18). Higher scores indicated more severe injury (normal score: 2–3; maximal deficit score: 18). 2.5. Quantitative proteomic analysis Quantitative proteomic analysis was performed with iTRAQ labeling (Applied Biosystems) coupled with an on-line twodimensional nanoLC/MS/MS system (2D-nanoLC-MS/MS) (Agilent, Waldbronn, Germany). Briefly, each treatment group (n = 5) was labeled with iTRAQ reagents (114:WT sham, 115:WT R4 h, 116:PSD93-/- sham and 117:PSD-93-/- R4h) mixed with 100 mg of protein. The mixed samples were cleaned, desalted and vacuum-dried and subsequently analyzed with an on-line two-dimensional nano LC/ MS/MS on a nano-HPLC system coupled with a hybrid Q-TOF mass spectrometer (QSTAR XL, Applied Biosystems) equipped with a nano-ESI source (Applied Biosystems) and a nano-ESI needle
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
(Picotip, FS360-50-20; New Objective Inc., Woburn, MA). This system has been described previously with detailed protocols for instrument preparation and protein identification and quantification (Ji et al., 2010).
3
USA). The primers were synthesized by Shanghai Invitrogen Biotechnology Co., Ltd. Relative gene expression was analyzed with the 2DDCt method, and each sample was tested in triplicate. 2.8. Mitochondria extraction
2.6. Bioinformatics analysis Scatterplot matrices and density plots were created with the R programming language to verify the correlations between the data samples. All differentially expressed proteins were put into the Database for Annotation, Visualization and Integrated Discovery (DAVID: http://david.abcc.ncifcrf.gov/), which utilizes gene ontology (GO) to identify biological processes, molecular functions, and cellular components (Focking et al., 2006). Additionally, we used the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING; http://www.string-db.org/) database to evaluate the protein–protein interaction networks of the differentially expressed proteins. 2.7. Real-time PCR Real-time PCR was performed as described previously (Wang et al., 2009). Total RNA was extracted using Trizol reagent and subsequently reversed transcribed to cDNA using a PrimeScriptTM RT reagent kit. Quantitative PCR was performed using SYBR Premix Ex TaqTM on an ABI 7500 PCR instrument (Applied Biosystems,
For the analyses of VDAC1 and VDAC3, the mitochondrial and cytosolic fractions were extracted according to the manufacturer’s instructions (Beyotime, Nanjing, China). Briefly, fresh cortices from 5 mice in each group were washed twice with cold PBS and homogenized on ice, and the nuclei, unbroken cells, and cell debris were centrifuged at 600 g for 10 min at 4 C. The supernatant was spun again at 13,000 g for 20 min at 4 C. The supernatant was transferred carefully, and the final deposit was resuspended in mitochondrial lysis buffer and used as the mitochondrial fraction. 2.9. Western blot analysis Western blotting was performed as described previously (Zhao et al., 2013). Equal amounts of prepared protein from each sample (n = 5) were separated by sodium dodecyl sulfate-PAGE and blotted onto polyvinylidene fluoride membranes. After blocking for 2 h in 5% non-fat dry milk, the membranes were incubated at 4 C overnight with primary antibodies. Next, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit, anti-goat or anti-mouse secondary antibodies, and the reactions
Fig. 1. PSD -93 KO mice exhibited decreased infarct areas and neurological deficits following MCAO compared to WT mice. The infarcted areas were measured with TTC staining, and the neurological deficits were measured with NSS at different time points following focal ischemia. (A and B) PSD-93 KO mice exhibited decreased infarcted volumes at different time points following transient focal ischemia compared to WT mice. N = 5 per group *p < 0.05 vs. WT group. (C) PSD-93 KO had alleviated neurological deficits. N = 10 per group *p < 0.05 vs. WT group.
4
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
Fig. 2. Scatterplot matrices and density curves of all of the verified proteins with correlation analyses. (A) The protein expression patterns of WT (R4h/Sham) and PSD-93-/(R4h/Sham) mice were highly consistent. A similar consistency was observed between sham (PSD-93-/-/WT) and R4h (PSD-93-/-/WT) mice. Pink indicated stronger correlations, and pale yellow indicated weaker correlations. (B) The protein density distributions of the four samples were similar, and all were normally distributed.
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
were visualized using enhanced chemiluminescence detection reagents (Bioworld, USA). The intensities of the blots were quantified with ImageJ software. 2.10. Statistical analysis The data are expressed as the mean SD and were analyzed with SPSS 16.0 statistical analysis software (SPSS, Chicago, IL, USA). Differences between multiple groups were examined with one-way analyses of variance (ANOVAs) followed by Newman–Keuls multiple range tests. Differences were considered significant at P < 0.05. 3. Results 3.1. PSD-93 disruption decreased infarct areas and neurological deficits in MCAO mice To verify whether PSD-93 deficiency led to neuroprotection following ischemic stroke in vivo, TTC staining was used to measure the infarct size following 24 h of reperfusion after 2 h of MCAO. Compared to WT mice, PSD-93 KO mice exhibited significantly reduced infarct size (24 h: 0.23 0.01 in KO vs. 0.41 0.01 in WT, p < 0.01; Fig. 1A and C). Furthermore, neurological deficits of the mice were measured by NSS. As shown in Fig. 1B, PSD-93 KO mice exhibited improved behavioral performances at 48 h and 72 h compared to control (48 h: 8.33 1.53 in KO vs. 11.67 0.58 in WT, p < 0.05; 72 h: 8.00 1.00 in KO vs. 11.67 0.58 in WT, p < 0.01; Fig. 1B). 3.2. Correlation analyses of the data with scatterplot matrices and density curves To investigate the correlations between the variables, our quantitative data were analyzed with scatterplot matrices. As shown in Fig. 2A, WT (R4h/Sham), PSD-93-/- (R4h/Sham), Sham (PSD-93-/-/WT) and R4h (PSD-93-/-/WT) groups were all highly consistent. To further investigate the density distribution of the protein ratio of each sample, density map analyses were applied. These maps indicated that the distributions of the samples of the four groups were similar and normally distributed (Fig. 2B). These
5
results indicated that the design of experiment was reasonable and the iTRAQ stability was reliable. 3.3. The proteins differentially expressed between sham (PSD-93-/-/ WT) and R4h (PSD-93-/-/WT) groups To explore the effects of PSD-93 deletion on protein expression, we identified 45 and 21 proteins that were differentially expressed based on cutoffs of >1.5-fold and <0.66-fold change between PSD93 KO and WT shams in the R4h group, respectively (Fig. 3). Twenty-three proteins were shared between the sham and R4h groups as shown in the Venn diagram. To further investigate the role of PSD-93 in ischemia-induced neuronal excitotoxicity, we selected 17 proteins to confirm their different expression levels in PSD-93 KO and WT mice. Among these proteins, the expression levels of isoform 2 of the voltage-dependent L-type calcium channel subunit beta-4 (VDLC), ras GTPase-activating protein SynGAP (SynGAP), Synaptotagmin-1, Syntaxin-1A, Complexin-3, Isoform 2 of the sodium-driven chloride bicarbonate exchanger (NCBE), voltage-dependent anion-selective channel protein 3 (VDAC3), isoform Mt-VDAC1 of the voltage-dependent anionselective channel protein 1 (VDAC1), Sn1-specific diacylglycerol lipase alpha (DAG), keratin, type II cytoskeletal 78-like, Sphingomyelin phosphodiesterase 3 (SMPD3), isoform Beta-I of Protein kinase C beta type (PKCb-1), and Brain protein 44 (Brp44) were upregulated; while the expression levels of ATP-binding cassette sub-family F member 1 (ABCB1), Down syndrome cell adhesion molecule homolog (Dscam), Pyridoxine-50 -phosphate oxidase (PNPO), and Fatty acid-binding protein, brain (FABP7) were down-regulated. 3.4. Gene ontology analysis of the proteins differentially expressed in PSD-93 KO and WT mice To reveal the function of these differentially expressed proteins, we used the UniProt database (http://www.uniprot.org/) for GO analysis and found that these proteins are involved in the regulation of synaptic transmission and neurons (ontology: biological process), the transport of neurotransmitters, synaptic vesicles and ions (ontology: biological process), cell–cell signaling (ontology: biological process), the modification of organelle membrane components (ontology: cellular component) and the binding of related components (ontology: molecular function) (Fig. 4). PSD-93 serves as a scaffolding protein that is primarily located in the postsynaptic density and participates in synaptic transmission and plasticity. Therefore, PSD-93 might mediate NMDAR-induced neuronal excitotoxicity by disturbing transmission, transport, the stability of the membrane and the assembly of signaling modules. 3.5. Protein–protein interaction network analysis of the differentially expressed proteins
Fig. 3. Venn diagram of the proteins differentially expressed between sham (PSD93-/-/WT) and R4h (PSD-93-/-/WT) mice. Forty-five and 21 unique proteins were differentially regulated in PSD-93 KO mice compared to WT mice in the sham and R4h groups, respectively. Twenty-three proteins were shared between the sham and R4h groups.
To further understand the function of the 17 differentially expressed proteins in terms of protein–protein interactions, we submitted these proteins to the STRING database (http://string-db. org/) to conduct network analysis. As demonstrated in Fig. 5, the synapse-associated proteins Syt1, Stx1a and Slc4a10 interacted with more proteins than did the other proteins. Their interaction proteins were most strongly related to synaptosomal-associated proteins and were involved in the transport of synaptic vesicles and the integrity of synaptic membranes. Additionally, PKCb exhibited stronger associations with the ionotropic glutamate receptors NMDAR1 and NMDAR2B, which might explain how PSD93 is involved in neuronal excitotoxicity.
6
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
Fig. 4. GO analysis of the proteins differentially expressed between PSD-93 KO and WT mice following ischemia–reperfusion injury. (A) GO analysis of the target proteins according to biological process. (B) GO analysis of the target proteins according to cellular component. (C) GO analysis of the target proteins according to molecular function.
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
7
Fig. 5. Protein–protein interaction network analysis of the differentially expressed proteins. Syt1, Stx1a and Slc4a10 exhibited higher centrality measures than did the other proteins. The red nodes represented up-regulated proteins, and the green nodes represented down-regulated proteins.
3.6. mRNA expression levels of the proteins differentially expressed between PSD-93 KO and WT mice To verify the differences in protein expression, RT-PCR was performed to detect mRNA expression level. The results showed that the mRNA expression of fatty acid-binding protein (FABP7) (R4h:1.34 0.22 in KO vs. 0.60 0.15 in WT, p < 0.01; WT: 0.60 0.15 in R4h vs. 1 in sham, p < 0.05; KO: 1.34 0.22 in R4 h vs. 0.89 0.18 in sham, p < 0.05), Sn1-specific diacylglycerol lipase alpha (DAG) (R4h: 0.94 0.17 in KO vs. 0.66 0.07 in WT p < 0.05; WT: 0.66 0.07 in R4h vs. 1 in sham, p < 0.05), ras GTPaseactivating protein SynGAP (SynGAP) (R4h: 2.11 0.30 in KO vs. 1.06 0.12 in WT, p < 0.01) and isoform 2 of the voltage-dependent L-type calcium channel subunit beta-4 (VDLC) (R4h:1.00 0.10 in KO vs. 0.78 0.15 in WT, p < 0.01) were significantly altered following ischemia/reperfusion in PSD-93 KO mice (Fig. 6). However, the mRNA expression of the other 11 proteins, including
Syntaxin-1A, Synaptotagmin-1, Complexin-3, were not significantly altered in the R4h KO vs. WT mice.
3.7. Protein expression levels of the proteins differentially expressed between PSD-93 KO and WT mice Finally, we verified the changes of protein expression of the proteins that were identified as differentially expressed based on proteomics analysis. Western blot analysis was performed to detect ten proteins due to the availability of the antibodies. The results demonstrated that the protein expression of SynGAP (R4h: 0.82 0.19 in KO vs. 0.42 0.12 in WT, p < 0.01; WT: 0.42 0.12 in R4h vs. 1 in sham, p < 0.05), Syntaxin-1A (R4h:1.10 0.13 in KO vs. 0.49 0.13 in WT, p < 0.01; WT:0.49 0.12 in R4h vs. 1 in sham, p < 0.05), VDLC (R4h: 0.82 0.14 in KO vs. 0.42 0.12 in WT, p < 0.05;WT:0.42 0.12 in R4h vs. 1 in sham,p < 0.01), and PKCb (R4h: 0.97 0.21 in KO vs. 0.51 0.08 in WT, p < 0.01;
8
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
Fig. 6. RT-PCR analysis of the proteins differentially expressed between PSD-93 KO and WT mice following focal ischemia. The mRNA expression of FABP7, DAG, SynGAP and VDLC were significantly altered following ischemia/reperfusion in PSD-93 KO mice. *p < 0.05 vs. WT group. **p < 0.01 N = 6 repeats.
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
9
Fig. 7. Western blot analysis of the proteins differentially expressed between the different groups of WT and PSD-93 KO mice. The protein expression of SynGAP, Syntaxin-1A, VDLC, and PKC-b were significantly altered in R4h PSD-93 KO group compared with WT group.*p < 0.05 vs. WT group. **p < 0.01 N = 5 repeats.
WT:0.51 0.08 in R4h vs. 1 in sham, p < 0.01) were significantly altered. (Fig. 7). 4. Discussion The molecular mechanisms underlying the role of PSD-93 in ischemic injury have not been definitively elucidated. Therefore, we conducted the current study to better understand the mechanisms of PSD-93 mediated ischemic brain injury. In this study, we applied isobaric tags for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/ MS/MS technology to screen differentially expressed proteins due
to PSD-93 deficiency. The results showed that SynGAP, PKCb, synataxin-1A, and VDLC might be involved in PSD-93-mediated excitotoxicity. Postsynaptic density-93 (PSD-93) is one of the discs large (DLG) membrane associated guanylate kinase (MAGUK) family, which consists of PSD-93/Chapsyn-110, PSD-95/synapse-associated protein (SAP) 90, SAP97, and SAP102. The DLG-MAGUK family proteins share a domain structure that includes alternative N-terminal domains, three consecutive PSD-95/discs large/zona occludens-1 (PDZ) domains, and a src-homology 3 (SH3) domain linked to a guanylate kinase (GK)-like domain (Cho et al., 1992; Kim et al., 1996; Muller et al., 1995; Brenman et al., 1996; Chen et al., 2012).
10
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
The different combinations of the N-terminals of the PSD93 isoforms fine-tune signaling scaffolds to regulate synaptic transmission (Kruger et al., 2013). Furthermore, our previous study revealed that PSD-93 interacted with NR2A and nNOS in cultured cortical neurons and contributed to NMDAR-NOS-mediated neuronal damage triggered by PAF receptor activation (Zhang et al., 2010). Additionally, PSD-93 deficiency blocked NMDARtriggered neurotoxicity by disrupting the NMDAR-Ca2+-NO signaling pathway and reducing the expression of synaptic NR2A and NR2B (Paoletti et al., 2013). Recently, we found that PSD-93 KO exerted profound neuroprotective effects against ischemic brain injury by inhibiting Fyn-mediated phosphorylation of NR2B (Zhang et al., 2014). Overall, these results demonstrate that PSD93 is involved in NMDAR-mediated neuronal excitotoxicity in a manner that is likely related to the assembly of signaling modules in the postsynaptic membrane or alterations in synapse-associated proteins. However, the identities of the proteins that are affected by the changes caused by PSD-93 deficiency during the ischemia– reperfusion process remain unknown. In the last two decades, proteomics has been applied to neuroscience research and help the elucidation of fundamental mechanisms and the discovery of candidate biomarkers and therapeutic targets (Bai et al., 2014, 2013). In the present study, we used iTRAQ quantitative proteomics analysis to screen the proteins that are differentially expressed due to PSD-93 deficiency in a mouse tMCAO model. We demonstrated that PSD-93 deletion reduced cerebral infarction volume and significantly improved neurological function following stroke. The reliability and stability of the iTRAQ analysis were verified by using scatterplot matrices and density map analyses. It is well-known that synaptic transmission, transport, the secretion of neurotransmitters and synaptic vesicles, and the maintenance of the integrity of organelle membrane components are important for synaptic and neuronal function (Shin, 2014; Fernandez-Chacon and Sudhof, 1999; Kriebel et al., 2001). Moreover, cell–cell signaling and cell signaling complexes are necessary for synaptic function and the function of signaling cascade pathways (Turrigiano, 2012; Misra et al., 2010). Our results indicated that PSD-93 deletion might protect against ischemic injury in vivo by affecting synapse-associated proteins. It is generally accepted that network analysis is helpful for gaining insight into the function of differentially expressed proteins that are identified by proteomic analyses. Thus, we further validated four proteins, including SynGAP, Syntaxin-1A, VDLC, and PKC-b, and performed network analyses. The results demonstrated that the synapse-associated protein Stx1a is involved in the transport of synaptic vesicles and the integrity of synaptic membranes via the interacting proteins and PKCb, which acts in calcium signaling pathway, is associated with NMDAR1 and NMDAR2B. Currently, increasing amounts of evidence have shown that calcium overloading and the excessive activation of NMDA receptors are likely the major mechanism of ischemic brain injury (Han et al., 2013; Li et al., 2012). Moreover, in the present study, the differentially expressed proteins are closely related to the proteins involved in calcium overload, NMDAR activation and the integrity of synaptic function. We suggest that the role of PSD-93 in the excitotoxicity induced by tMCAO might be mediated through these four specific differentially expressed proteins. In conclusion, we identified four proteins SynGAP, Syntaxin-1A, VDLC, and PKCb differentially expressed between PSD-93 KO and WT mice 4 h after tMCAO. These identified proteins might be involved in PSD-93 mediated neuroexcitotoxicity in ischemic brain injury. Identification of these proteins provides valuable clues to elucidate the mechanisms underlying the actions of PSD-93 in ischemia–reperfusion induced neurotoxicity.
Conflict of interest The authors declare no conflicts of interest. Acknowledgments We thank Dr. Yuanxiang Tao for providing the PSD-93 KO mice and SuZhou BioNovoGene Co., Ltd. (http://www.bionovogene.com) for help with the data analyses. This study was supported by the National Natural Science Foundation of China (Nos. 81301120, and 81200897), the Natural Science Foundation of the Colleges and Universities in Jiangsu Province (No. 13KJB320027) and the Xuzhou Medical Young Talents Project. References Bai, B., Hales, C.M., Chen, P.C., Gozal, Y., Dammer, E.B., et al., 2013. U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A. 110, 16562–16567. Bai, B., Chen, P.C., Hales, C.M., Wu, Z., Pagala, V., et al., 2014. Integrated approaches for analyzing U1-70K cleavage in Alzheimer’s disease. J. Proteome Res. 13, 4526–4534. Brassai, A., Suvanjeiev, R.G., Ban, E.G., Lakatos, M., 2014. Role of synaptic and nonsynaptic glutamate receptors in ischaemia induced neurotoxicity. Brain Res. Bull. 112C, 1–6. Brenman, J.E., Christopherson, K.S., Craven, S.E., McGee, A.W., Bredt, D.S., 1996. Cloning and characterization of postsynaptic density 93, a nitric oxide synthase interacting protein. J. Neurosci. 16, 7407–7415. Castegna, A., Aksenov, M., Aksenova, M., Thongboonkerd, V., Klein, J.B., et al., 2002. Proteomic identification of oxidatively modified proteins in Alzheimer’s disease brain. Part I: creatine kinase BB, glutamine synthase, and ubiquitin carboxyterminal hydrolase L-1. Free Radic. Biol. Med. 33, 562–571. Chen, J., Li, Y., Wang, L., Zhang, Z., Lu, D., et al., 2001. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke 32, 1005–1011. Chen, B.S., Gray, J.A., Sanz-Clemente, A., Wei, Z., Thomas, E.V., et al., 2012. SAP10 mediates synaptic clearance of NMDA receptors. Cell Rep. 2, 1120–1128. Cho, K.O., Hunt, C.A., Kennedy, M.B., 1992. The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein. Neuron 9, 929–942. Ditzen, C., Jastorff, A.M., Kessler, M.S., Bunck, M., Teplytska, L., et al., 2006. Protein biomarkers in a mouse model of extremes in trait anxiety. Mol. Cell. Proteomics 5, 1914–1920. Dumas, T.C., 2005. Developmental regulation of cognitive abilities: modified composition of a molecular switch turns on associative learning. Prog. Neurobiol. 76, 189–211. Fernandez-Chacon, R., Sudhof, T.C., 1999. Genetics of synaptic vesicle function: toward the complete functional anatomy of an organelle. Annu. Rev. Physiol. 61, 753–776. Focking, M., Besselmann, M., Trapp, T., 2006. Proteomics of experimental stroke in mice. Acta Neurobiol. Exp. (Wars) 66, 273–278. Han, Z., Yang, J.L., Jiang, S.X., Hou, S.T., Zheng, R.Y., 2013. Fast, non-competitive and reversible inhibition of NMDA-activated currents by 2-BFI confers neuroprotection. PLoS One 8, e64894. Ji, Y.H., Ji, J.L., Sun, F.Y., Zeng, Y.Y., He, X.H., et al., 2010. Quantitative proteomics analysis of chondrogenic differentiation of C3H10T1/2 mesenchymal stem cells by iTRAQ labeling coupled with on-line two-dimensional LC/MS/MS. Mol. Cell. Proteomics 9, 550–564. Jiang, X., Mu, D., Sheldon, R.A., Glidden, D.V., Ferriero, D.M., 2003. Neonatal hypoxiaischemia differentially upregulates MAGUKs and associated proteins in PSD-93deficient mouse brain. Stroke 34, 2958–2963. Johnston-Wilson, N.L., Sims, C.D., Hofmann, J.P., Anderson, L., Shore, A.D., et al., 2000. Disease-specific alterations in frontal cortex brain proteins in schizophrenia, bipolar disorder, and major depressive disorder. The Stanley Neuropathology Consortium. Mol. Psychiatry 5, 142–149. Kim, E., Cho, K.O., Rothschild, A., Sheng, M., 1996. Heteromultimerization and NMDA receptor-clustering activity of Chapsyn-110, a member of the PSD-95 family of proteins. Neuron 17, 103–113. Kriebel, M.E., Keller, B., Silver, R.B., Fox, G.Q., Pappas, G.D., 2001. Porocytosis: a new approach to synaptic function. Brain Res. Brain Res. Rev. 38, 20–32. Kruger, J.M., Favaro, P.D., Liu, M., Kitlinska, A., Huang, X., et al., 2013. Differential roles of postsynaptic density-93 isoforms in regulating synaptic transmission. J. Neurosci. 33, 15504–15517. Li, L., Yang, R., Sun, K., Bai, Y., Zhang, Z., et al., 2012. Cerebroside-A provides potent neuroprotection after cerebral ischaemia through reducing glutamate release and Ca(2)(+) influx of NMDA receptors. Int. J. Neuropsychopharmacol. 15, 497– 507. Liaw, W.J., Zhu, X.G., Yaster, M., Johns, R.A., Gauda, E.B., et al., 2008. Distinct expression of synaptic NR2A and NR2B in the central nervous system and impaired morphine tolerance and physical dependence in mice deficient in postsynaptic density-93 protein. Mol. Pain 4, 45.
R. Rong et al. / NeuroToxicology 53 (2016) 1–11 Martins-De-Souza, D., Dias-Neto, E., Schmitt, A., Falkai, P., Gormanns, P., et al., 2010. Proteome analysis of schizophrenia brain tissue. World J. Biol. Psychiatry 11, 110–120. Misra, C., Restituito, S., Ferreira, J., Rameau, G.A., Fu, J., et al., 2010. Regulation of synaptic structure and function by palmitoylated AMPA receptor binding protein. Mol. Cell Neurosci. 43, 341–352. Mu, J., Xie, P., Yang, Z.S., Yang, D.L., Lv, F.J., et al., 2007. Neurogenesis and major depression: implications from proteomic analyses of hippocampal proteins in a rat depression model. Neurosci. Lett. 416, 252–256. Muller, B.M., Kistner, U., Veh, R.W., Cases-Langhoff, C., Becker, B., et al., 1995. Molecular characterization and spatial distribution of SAP97, a novel presynaptic protein homologous to SAP90 and the Drosophila discs-large tumor suppressor protein. J. Neurosci. 15, 2354–2366. Otte, D.M., Sommersberg, B., Kudin, A., Guerrero, C., Albayram, O., et al., 2011. Nacetyl cysteine treatment rescues cognitive deficits induced by mitochondrial dysfunction in G72/G30 transgenic mice. Neuropsychopharmacology 36, 2233– 2243. Paoletti, P.1, Bellone, C., Zhou, Q., 2013. NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. Nat. Rev. Neurosci. 14, 383– 400. Patel, S., Sinha, A., Singh, M.P., 2007. Identification of differentially expressed proteins in striatum of maneb-and paraquat-induced Parkinson’s disease phenotype in mouse. Neurotoxicol. Teratol. 29, 578–585. Pennington, K., Beasley, C.L., Dicker, P., Fagan, A., English, J., et al., 2008. Prominent synaptic and metabolic abnormalities revealed by proteomic analysis of the dorsolateral prefrontal cortex in schizophrenia and bipolar disorder. Mol. Psychiatry 13, 1102–1117. Robinson, R.A., Lange, M.B., Sultana, R., Galvan, V., Fombonne, J., et al., 2011. Differential expression and redox proteomics analyses of an Alzheimer disease transgenic mouse model: effects of the amyloid-beta peptide of amyloid precursor protein. Neuroscience 177, 207–222. Shin, O.H., 2014. Exocytosis and synaptic vesicle function. Compr. Physiol. 4, 149– 175. Szego, E.M., Janaky, T., Szabo, Z., Csorba, A., Kompagne, H., et al., 2010. A mouse model of anxiety molecularly characterized by altered protein networks in the brain proteome. Eur. Neuropsychopharmacol. 20, 96–111.
11
Tao, F., Skinner, J., Yang, Y., Johns, R.A., 2010. Effect of PSD-95/SAP90 and/or PSD-93/ Chapsyn-110 deficiency on the minimum alveolar anesthetic concentration of halothane in mice. Anesthesiology 112, 1444–1451. Turrigiano, G., 2012. Homeostatic synaptic plasticity: local and global mechanisms for stabilizing neuronal function. Cold Spring Harb. Perspect. Biol. 4, a005736. Wang, L., Zhang, L., Chen, Z.B., Wu, J.Y., Zhang, X., et al., 2009. Icariin enhances neuronal survival after oxygen and glucose deprivation by increasing SIRT1. Eur. J. Pharmacol. 609, 40–44. Xu, Y., Zhang, B., Hua, Z., Johns, R.A., Bredt, D.S., et al., 2004. Targeted disruption of PSD-93 gene reduces platelet-activating factor-induced neurotoxicity in cultured cortical neurons. Exp. Neurol. 189, 16–24. Zhang, B., Tao, F., Liaw, W.J., Bredt, D.S., Johns, R.A., et al., 2003. Effect of knock down of spinal cord PSD-93/chapsin-110 on persistent pain induced by complete Freund’s adjuvant and peripheral nerve injury. Pain 106, 187–196. Zhang, Q.X., Pei, D.S., Guan, Q.H., Sun, Y.F., Liu, X.M., et al., 2007. Crosstalk between PSD-95 and JIP1-mediated signaling modules: the mechanism of MLK3 activation in cerebral ischemia. Biochemistry 46, 4006–4016. Zhang, M., Xu, J.T., Zhu, X., Wang, Z., Zhao, X., et al., 2010. Postsynaptic density93 deficiency protects cultured cortical neurons from N-methyl-D-aspartate receptor-triggered neurotoxicity. Neuroscience 166, 1083–1090. Zhang, F., Wang, S., Zhang, M., Weng, Z., Li, P., et al., 2012. Pharmacological induction of heme oxygenase-1 by a triterpenoid protects neurons against ischemic injury. Stroke 43, 1390–1397. Zhang, M., Li, Q., Chen, L., Li, J., Zhang, X., et al., 2014. PSD-93 deletion inhibits Fynmediated phosphorylation of NR2B and protects against focal cerebral ischemia. Neurobiol. Dis. 68, 104–111. Zhao, J., Zhang, M., Li, W., Su, X., Zhu, L., et al., 2013. Suppression of JAK2/ STAT3 signaling reduces end-to-end arterial anastomosis induced cell proliferation in common carotid arteries of rats. PLoS One 8, e58730. Zheng, C.Y., Seabold, G.K., Horak, M., Petralia, R.S., 2011. MAGUKs, synaptic development, and synaptic plasticity. Neuroscientist 17, 493–512. Zhou, L., Li, F., Xu, H.B., Luo, C.X., Wu, H.Y., et al., 2010. Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95. Nat. Med. 16, 1439–1443.
Contents lists available at ScienceDirect
NeuroToxicology
Full length article
Proteomic analysis of PSD-93 knockout mice following the induction of ischemic cerebral injury Rong Ronga,b,c,1, Hui Yangd,1, Liangqun Ronge , Xiue Weie , Qingjie Lif , Xiaomei Liug , Hong Gaoe, Yun Xua,b,c,* , Qingxiu Zhange,** a
Department of Neurology, Drum Tower Hospital of Nanjing Medical University, Nanjing, 210029 Jiangsu, China Department of Neurology, Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, 210029 Jiangsu, China Jiangsu Key Laboratory for Molecular Medicine, Nanjing, China d Department of Neurosurgery, Xuzhou First People’s Hospital, Xuzhou, 221000 Jiangsu, China e Department of Neurology, Second Affiliated Hospital of Xuzhou Medical College, Xuzhou, 221006 Jiangsu, China f Department of Neurology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, 221000 Jiangsu, China g Department of Pathogenic Biology and Immunology, Lab of Infection and Immunity, Xuzhou Medical College, Xuzhou, 221004 Jiangsu, China b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 October 2015 Received in revised form 4 December 2015 Accepted 5 December 2015 Available online 8 December 2015
Postsynaptic density protein-93 (PSD-93) is enriched in the postsynaptic density and is involved in N-methyl-D-aspartate receptor (NMDAR) triggered neurotoxicity through PSD-93/NMDAR/nNOS signaling pathway. In the present study, we found that PSD-93 deficiency reduced infarcted volume and neurological deficits induced by transient middle cerebral artery occlusion (tMCAO) in the mice. To identify novel targets of PSD-93 related neurotoxicity, we applied isobaric tags for relative and absolute quantitative (iTRAQ) labeling and combined this labeling with on-line two-dimensional LC/MS/MS technology to elucidate the changes in protein expression in PSD-93 knockout mice following tMCAO. The proteomic data set consisted of 1892 proteins. Compared to control group, differences in expression levels in ischemic group >1.5-fold and <0.66-fold were considered as differential expression. A total of 104 unique proteins with differential abundance levels were identified, among which 17 proteins were selected for further validation. Gene ontology analysis using UniProt database revealed that these differentially expressed proteins are involved in diverse function such as synaptic transmission, neuronal neurotransmitter and ion transport, modification of organelle membrane components. Moreover, network analysis revealed that the interacting proteins were involved in the transport of synaptic vesicles, the integrity of synaptic membranes and the activation of the ionotropic glutamate receptors NMDAR1 and NMDAR2B. Finally, RT-PCR and Western blot analysis showed that SynGAP, syntaxin-1A, protein kinase C b, and voltage-dependent L-type calcium channels were inhibited by ischemia– reperfusion. Identification of these proteins provides valuable clues to elucidate the mechanisms underlying the actions of PSD-93 in ischemia–reperfusion induced neurotoxicity. ã 2015 Elsevier Inc. All rights reserved.
Keywords: Proteomic analysis PSD-93 Excitotoxicity Ischemic stroke
1. Introduction
* Corresponding author at: Department of Neurology, Drum Tower Hospital of Nanjing Medical University, 321 Zhongshan Road, Nanjing City, Jiangsu Province 210008, China. Fax: +86 25 83317016. ** Corresponding author at: Department of Neurology, Second Affiliated Hospital of Xuzhou Medical College, 32 Coal Road, Xuzhou City, Jiangsu Province 221006, China. Fax: +86 561 85326105. E-mail addresses: [email protected] (Y. Xu), [email protected] (Q. Zhang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neuro.2015.12.005 0161-813X/ ã 2015 Elsevier Inc. All rights reserved.
Cerebral ischemia–reperfusion induced neuronal injury involves a complex signal transduction network, and its mechanisms remain incompletely understood. Such injury is initiated by the release and aggregation of excitatory amino acids during ischemic brain damage (Brassai et al., 2014). Consequently, Nmethyl-D-aspartate receptors (NMDA receptors or NMDARs) are activated by postsynaptic density (PSD) scaffolding proteins, leading to a series of pathological changes (Zhang et al., 2007, 2010; Zhou et al., 2010; Xu et al., 2004). NMDARs are tetrameric complexes consisted of several subunits. The subunit composition of NMDARs is plastic, leading to a large number of NMDAR
2
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
subtypes. Up to now, seven different subunits have been identified, including GluN1 subunit, four distinct GluN2 subunits (GluN2A, GluN2B, GluN2C and GluN2D), and two GluN3 subunits (GluN3A and GluN3B) (Paoletti et al., 2013). Notably, during early postnatal development, NMDARs switch subunit composition from primarily containing GluN2B subunits to predominantly containing GluN2A subunits in central nerve system, coinciding with the processes of synapse maturation and learning (Dumas, 2005). PSD scaffolding proteins play a vital role in NMDAR activationinduced ischemic brain injury. PSD-93 is a PSD scaffolding protein that belongs to the membrane-associated guanylate kinase family (MAGUKs). PSD-93 plays key role in synaptic development, synaptic plasticity and the scaffolds of postsynaptic complexes (Jiang et al., 2003; Zheng et al., 2011). Moreover, several studies suggest that PSD-93 might be involved in chronic pain, morphine tolerance, morphine withdrawal and learning and memory dysfunction via its effects on synaptic plasticity (Liaw et al., 2008; Tao et al., 2010; Zhang et al., 2003). In addition, PSD-93 is implicated in ischemic brain injury. PSD-93 knockout mice might engage the hypoxic-ischemic injury induced NMDAR signaling pathway by upregulating the protein expression of PSD-95 (Jiang et al., 2003). Recently, our study revealed that PSD-93 interacted with nNOS and NR2A to promote nNOS activation via plateletactivating factor (PAF) to mediate ischemic brain injury (Xu et al., 2004). Additionally, PSD-93 knockout significantly increased the proliferative capacity of cells, which reduces the neuronal death that results from ischemic brain injury (Zhang et al., 2010). Furthermore, PSD-93 knockout exhibited neuroprotective effects against ischemic brain injury that are mediated by the inhibition of the Fyn-mediated phosphorylation of NR2B (Zhang et al., 2014). These findings suggest that PSD-93 plays an important role by suppressing ischemia-induced neuronal excitotoxicity during ischemic brain injury, but the exact mechanisms of this function require further exploration. Recent advances in proteomics technology have enabled the application of a sophisticated labeling-based quantitative method based on iTRAQ labeling coupled with on-line two-dimensional LC/ MS/MS technology to provide important insights into the pathogenesis of major psychiatric and neurodegenerative disorders and ischemic brain injury in patient cohorts (Martins-De-Souza et al., 2010; Johnston-Wilson et al., 2000; Pennington et al., 2008; Castegna et al., 2002) and animal models (Ditzen et al., 2006; Mu et al., 2007; Otte et al., 2011; Patel et al., 2007; Robinson et al., 2011; Szego et al., 2010). Although previous studies have shown that PSD-93 is involved in the neuronal excitotoxicity induced by ischemic brain injury, no studies have attempted to detect the proteomic changes associated with PSD-93 disruption and excitotoxicity. In the present study, iTRAQ-based quantitative proteomics analysis was applied to identify potential protein targets of PSD93 disruption in adult mouse transient middle cerebral artery occlusion (tMCAO) model. To further explore the mechanisms responsible for the function of PSD-93 in MCAO induced excitotoxicity,17 target proteins were selected for gene ontology (GO) function and network analyses. We found that the ras GTPase-activating protein SynGAP (SynGAP), isoform 2 of the voltage-dependent Ltype calcium channel subunit beta-4 (VDLC), syntaxin-1A, and PKCb are involved in PSD-93 mediated neurotoxicity. 2. Materials and methods 2.1. Animals PSD-93 knockout (KO) mice (C57BL/6 genetic background) were produced as described previously (Paoletti et al., 2013). Male PSD93 KO mice and wild-type (WT) littermates (10–12 weeks old)
were obtained by interbreeding PSD-93 heterozygous mice. The genotype of each mouse was confirmed by polymerase chain reaction. All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing University. Mice were housed in cages individually with free food and water supply. The environment was at a temperature (23 2 C), humidity (40– 60%) and a 12/12 h dark–light cycle (light on 9:00–21:00). 2.2. MCAO model Transient focal cerebral ischemia was induced as previously described (Zhang et al., 2012). Briefly, PSD-93 KO mice and WT C57BL/6 mice were anesthetized by intraperitoneal injection of ketamine (100 mg/ml) and xylazine (20 mg/kg) mixture (1:1) at a dose of 1 ml/kg. A 6/0 monofilament nylon suture with a heatrounded tip was inserted through the internal carotid artery into the beginning of the right middle cerebral artery (MCA). The mice were subjected to 2 h of occlusion, and blood flow was then restored by the withdrawal of the filament at 4, 24, 48, or 72 h. Sham control animals were subjected to similar operations without the MCA occlusion. During the experiment, the body temperature was maintained at 37 0.5 C with an infrared heating lamp. The mortality of both PSD-93 KO mice and WT C57BL/6 mice in the model group was approximately 10%. During the reperfusion, post-operative mice had adequate water and food and 24 h awakening-sleep cycle. Those paralysis mice accepted special care and artificial feeding at least 3 times a day. 2.3. Infarct size measurement After anesthesia using sodium pentobarbital, three PSD-93 KO mice and three WT C57BL/6 mice in the model group were executed by cervical dislocation and quickly decapitated at the indicated reperfusion time, respectively. The brains were washed with freezing PBS solution, and then cut into five 2-mm-thick slices and stained with 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) at 37 C for 20 min in the dark to detect infarct volume. Images were acquired with a computer-controlled digital camera (Olympus) and analyzed with Image-ProPlus 6.0 (IPP) software (Media Cybernetics) to calculate the infarct sizes. The infarct volume in all slices was expressed as the percentages of the contralateral hemisphere after correcting for edema. 2.4. Behavioral test Neurological Severity Score (NSS) is a composite of motor, sensory, reflex, and balance tests and was evaluated at 4, 24, 48, and 72 h after MCAO (Chen et al., 2001). Neurological function was graded on a scale ranging from 0 to 18 (normal score, 0; maximal deficit score, 18). Higher scores indicated more severe injury (normal score: 2–3; maximal deficit score: 18). 2.5. Quantitative proteomic analysis Quantitative proteomic analysis was performed with iTRAQ labeling (Applied Biosystems) coupled with an on-line twodimensional nanoLC/MS/MS system (2D-nanoLC-MS/MS) (Agilent, Waldbronn, Germany). Briefly, each treatment group (n = 5) was labeled with iTRAQ reagents (114:WT sham, 115:WT R4 h, 116:PSD93-/- sham and 117:PSD-93-/- R4h) mixed with 100 mg of protein. The mixed samples were cleaned, desalted and vacuum-dried and subsequently analyzed with an on-line two-dimensional nano LC/ MS/MS on a nano-HPLC system coupled with a hybrid Q-TOF mass spectrometer (QSTAR XL, Applied Biosystems) equipped with a nano-ESI source (Applied Biosystems) and a nano-ESI needle
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
(Picotip, FS360-50-20; New Objective Inc., Woburn, MA). This system has been described previously with detailed protocols for instrument preparation and protein identification and quantification (Ji et al., 2010).
3
USA). The primers were synthesized by Shanghai Invitrogen Biotechnology Co., Ltd. Relative gene expression was analyzed with the 2DDCt method, and each sample was tested in triplicate. 2.8. Mitochondria extraction
2.6. Bioinformatics analysis Scatterplot matrices and density plots were created with the R programming language to verify the correlations between the data samples. All differentially expressed proteins were put into the Database for Annotation, Visualization and Integrated Discovery (DAVID: http://david.abcc.ncifcrf.gov/), which utilizes gene ontology (GO) to identify biological processes, molecular functions, and cellular components (Focking et al., 2006). Additionally, we used the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING; http://www.string-db.org/) database to evaluate the protein–protein interaction networks of the differentially expressed proteins. 2.7. Real-time PCR Real-time PCR was performed as described previously (Wang et al., 2009). Total RNA was extracted using Trizol reagent and subsequently reversed transcribed to cDNA using a PrimeScriptTM RT reagent kit. Quantitative PCR was performed using SYBR Premix Ex TaqTM on an ABI 7500 PCR instrument (Applied Biosystems,
For the analyses of VDAC1 and VDAC3, the mitochondrial and cytosolic fractions were extracted according to the manufacturer’s instructions (Beyotime, Nanjing, China). Briefly, fresh cortices from 5 mice in each group were washed twice with cold PBS and homogenized on ice, and the nuclei, unbroken cells, and cell debris were centrifuged at 600 g for 10 min at 4 C. The supernatant was spun again at 13,000 g for 20 min at 4 C. The supernatant was transferred carefully, and the final deposit was resuspended in mitochondrial lysis buffer and used as the mitochondrial fraction. 2.9. Western blot analysis Western blotting was performed as described previously (Zhao et al., 2013). Equal amounts of prepared protein from each sample (n = 5) were separated by sodium dodecyl sulfate-PAGE and blotted onto polyvinylidene fluoride membranes. After blocking for 2 h in 5% non-fat dry milk, the membranes were incubated at 4 C overnight with primary antibodies. Next, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit, anti-goat or anti-mouse secondary antibodies, and the reactions
Fig. 1. PSD -93 KO mice exhibited decreased infarct areas and neurological deficits following MCAO compared to WT mice. The infarcted areas were measured with TTC staining, and the neurological deficits were measured with NSS at different time points following focal ischemia. (A and B) PSD-93 KO mice exhibited decreased infarcted volumes at different time points following transient focal ischemia compared to WT mice. N = 5 per group *p < 0.05 vs. WT group. (C) PSD-93 KO had alleviated neurological deficits. N = 10 per group *p < 0.05 vs. WT group.
4
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
Fig. 2. Scatterplot matrices and density curves of all of the verified proteins with correlation analyses. (A) The protein expression patterns of WT (R4h/Sham) and PSD-93-/(R4h/Sham) mice were highly consistent. A similar consistency was observed between sham (PSD-93-/-/WT) and R4h (PSD-93-/-/WT) mice. Pink indicated stronger correlations, and pale yellow indicated weaker correlations. (B) The protein density distributions of the four samples were similar, and all were normally distributed.
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
were visualized using enhanced chemiluminescence detection reagents (Bioworld, USA). The intensities of the blots were quantified with ImageJ software. 2.10. Statistical analysis The data are expressed as the mean SD and were analyzed with SPSS 16.0 statistical analysis software (SPSS, Chicago, IL, USA). Differences between multiple groups were examined with one-way analyses of variance (ANOVAs) followed by Newman–Keuls multiple range tests. Differences were considered significant at P < 0.05. 3. Results 3.1. PSD-93 disruption decreased infarct areas and neurological deficits in MCAO mice To verify whether PSD-93 deficiency led to neuroprotection following ischemic stroke in vivo, TTC staining was used to measure the infarct size following 24 h of reperfusion after 2 h of MCAO. Compared to WT mice, PSD-93 KO mice exhibited significantly reduced infarct size (24 h: 0.23 0.01 in KO vs. 0.41 0.01 in WT, p < 0.01; Fig. 1A and C). Furthermore, neurological deficits of the mice were measured by NSS. As shown in Fig. 1B, PSD-93 KO mice exhibited improved behavioral performances at 48 h and 72 h compared to control (48 h: 8.33 1.53 in KO vs. 11.67 0.58 in WT, p < 0.05; 72 h: 8.00 1.00 in KO vs. 11.67 0.58 in WT, p < 0.01; Fig. 1B). 3.2. Correlation analyses of the data with scatterplot matrices and density curves To investigate the correlations between the variables, our quantitative data were analyzed with scatterplot matrices. As shown in Fig. 2A, WT (R4h/Sham), PSD-93-/- (R4h/Sham), Sham (PSD-93-/-/WT) and R4h (PSD-93-/-/WT) groups were all highly consistent. To further investigate the density distribution of the protein ratio of each sample, density map analyses were applied. These maps indicated that the distributions of the samples of the four groups were similar and normally distributed (Fig. 2B). These
5
results indicated that the design of experiment was reasonable and the iTRAQ stability was reliable. 3.3. The proteins differentially expressed between sham (PSD-93-/-/ WT) and R4h (PSD-93-/-/WT) groups To explore the effects of PSD-93 deletion on protein expression, we identified 45 and 21 proteins that were differentially expressed based on cutoffs of >1.5-fold and <0.66-fold change between PSD93 KO and WT shams in the R4h group, respectively (Fig. 3). Twenty-three proteins were shared between the sham and R4h groups as shown in the Venn diagram. To further investigate the role of PSD-93 in ischemia-induced neuronal excitotoxicity, we selected 17 proteins to confirm their different expression levels in PSD-93 KO and WT mice. Among these proteins, the expression levels of isoform 2 of the voltage-dependent L-type calcium channel subunit beta-4 (VDLC), ras GTPase-activating protein SynGAP (SynGAP), Synaptotagmin-1, Syntaxin-1A, Complexin-3, Isoform 2 of the sodium-driven chloride bicarbonate exchanger (NCBE), voltage-dependent anion-selective channel protein 3 (VDAC3), isoform Mt-VDAC1 of the voltage-dependent anionselective channel protein 1 (VDAC1), Sn1-specific diacylglycerol lipase alpha (DAG), keratin, type II cytoskeletal 78-like, Sphingomyelin phosphodiesterase 3 (SMPD3), isoform Beta-I of Protein kinase C beta type (PKCb-1), and Brain protein 44 (Brp44) were upregulated; while the expression levels of ATP-binding cassette sub-family F member 1 (ABCB1), Down syndrome cell adhesion molecule homolog (Dscam), Pyridoxine-50 -phosphate oxidase (PNPO), and Fatty acid-binding protein, brain (FABP7) were down-regulated. 3.4. Gene ontology analysis of the proteins differentially expressed in PSD-93 KO and WT mice To reveal the function of these differentially expressed proteins, we used the UniProt database (http://www.uniprot.org/) for GO analysis and found that these proteins are involved in the regulation of synaptic transmission and neurons (ontology: biological process), the transport of neurotransmitters, synaptic vesicles and ions (ontology: biological process), cell–cell signaling (ontology: biological process), the modification of organelle membrane components (ontology: cellular component) and the binding of related components (ontology: molecular function) (Fig. 4). PSD-93 serves as a scaffolding protein that is primarily located in the postsynaptic density and participates in synaptic transmission and plasticity. Therefore, PSD-93 might mediate NMDAR-induced neuronal excitotoxicity by disturbing transmission, transport, the stability of the membrane and the assembly of signaling modules. 3.5. Protein–protein interaction network analysis of the differentially expressed proteins
Fig. 3. Venn diagram of the proteins differentially expressed between sham (PSD93-/-/WT) and R4h (PSD-93-/-/WT) mice. Forty-five and 21 unique proteins were differentially regulated in PSD-93 KO mice compared to WT mice in the sham and R4h groups, respectively. Twenty-three proteins were shared between the sham and R4h groups.
To further understand the function of the 17 differentially expressed proteins in terms of protein–protein interactions, we submitted these proteins to the STRING database (http://string-db. org/) to conduct network analysis. As demonstrated in Fig. 5, the synapse-associated proteins Syt1, Stx1a and Slc4a10 interacted with more proteins than did the other proteins. Their interaction proteins were most strongly related to synaptosomal-associated proteins and were involved in the transport of synaptic vesicles and the integrity of synaptic membranes. Additionally, PKCb exhibited stronger associations with the ionotropic glutamate receptors NMDAR1 and NMDAR2B, which might explain how PSD93 is involved in neuronal excitotoxicity.
6
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
Fig. 4. GO analysis of the proteins differentially expressed between PSD-93 KO and WT mice following ischemia–reperfusion injury. (A) GO analysis of the target proteins according to biological process. (B) GO analysis of the target proteins according to cellular component. (C) GO analysis of the target proteins according to molecular function.
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
7
Fig. 5. Protein–protein interaction network analysis of the differentially expressed proteins. Syt1, Stx1a and Slc4a10 exhibited higher centrality measures than did the other proteins. The red nodes represented up-regulated proteins, and the green nodes represented down-regulated proteins.
3.6. mRNA expression levels of the proteins differentially expressed between PSD-93 KO and WT mice To verify the differences in protein expression, RT-PCR was performed to detect mRNA expression level. The results showed that the mRNA expression of fatty acid-binding protein (FABP7) (R4h:1.34 0.22 in KO vs. 0.60 0.15 in WT, p < 0.01; WT: 0.60 0.15 in R4h vs. 1 in sham, p < 0.05; KO: 1.34 0.22 in R4 h vs. 0.89 0.18 in sham, p < 0.05), Sn1-specific diacylglycerol lipase alpha (DAG) (R4h: 0.94 0.17 in KO vs. 0.66 0.07 in WT p < 0.05; WT: 0.66 0.07 in R4h vs. 1 in sham, p < 0.05), ras GTPaseactivating protein SynGAP (SynGAP) (R4h: 2.11 0.30 in KO vs. 1.06 0.12 in WT, p < 0.01) and isoform 2 of the voltage-dependent L-type calcium channel subunit beta-4 (VDLC) (R4h:1.00 0.10 in KO vs. 0.78 0.15 in WT, p < 0.01) were significantly altered following ischemia/reperfusion in PSD-93 KO mice (Fig. 6). However, the mRNA expression of the other 11 proteins, including
Syntaxin-1A, Synaptotagmin-1, Complexin-3, were not significantly altered in the R4h KO vs. WT mice.
3.7. Protein expression levels of the proteins differentially expressed between PSD-93 KO and WT mice Finally, we verified the changes of protein expression of the proteins that were identified as differentially expressed based on proteomics analysis. Western blot analysis was performed to detect ten proteins due to the availability of the antibodies. The results demonstrated that the protein expression of SynGAP (R4h: 0.82 0.19 in KO vs. 0.42 0.12 in WT, p < 0.01; WT: 0.42 0.12 in R4h vs. 1 in sham, p < 0.05), Syntaxin-1A (R4h:1.10 0.13 in KO vs. 0.49 0.13 in WT, p < 0.01; WT:0.49 0.12 in R4h vs. 1 in sham, p < 0.05), VDLC (R4h: 0.82 0.14 in KO vs. 0.42 0.12 in WT, p < 0.05;WT:0.42 0.12 in R4h vs. 1 in sham,p < 0.01), and PKCb (R4h: 0.97 0.21 in KO vs. 0.51 0.08 in WT, p < 0.01;
8
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
Fig. 6. RT-PCR analysis of the proteins differentially expressed between PSD-93 KO and WT mice following focal ischemia. The mRNA expression of FABP7, DAG, SynGAP and VDLC were significantly altered following ischemia/reperfusion in PSD-93 KO mice. *p < 0.05 vs. WT group. **p < 0.01 N = 6 repeats.
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
9
Fig. 7. Western blot analysis of the proteins differentially expressed between the different groups of WT and PSD-93 KO mice. The protein expression of SynGAP, Syntaxin-1A, VDLC, and PKC-b were significantly altered in R4h PSD-93 KO group compared with WT group.*p < 0.05 vs. WT group. **p < 0.01 N = 5 repeats.
WT:0.51 0.08 in R4h vs. 1 in sham, p < 0.01) were significantly altered. (Fig. 7). 4. Discussion The molecular mechanisms underlying the role of PSD-93 in ischemic injury have not been definitively elucidated. Therefore, we conducted the current study to better understand the mechanisms of PSD-93 mediated ischemic brain injury. In this study, we applied isobaric tags for relative and absolute quantitation (iTRAQ) labeling coupled with on-line two-dimensional LC/ MS/MS technology to screen differentially expressed proteins due
to PSD-93 deficiency. The results showed that SynGAP, PKCb, synataxin-1A, and VDLC might be involved in PSD-93-mediated excitotoxicity. Postsynaptic density-93 (PSD-93) is one of the discs large (DLG) membrane associated guanylate kinase (MAGUK) family, which consists of PSD-93/Chapsyn-110, PSD-95/synapse-associated protein (SAP) 90, SAP97, and SAP102. The DLG-MAGUK family proteins share a domain structure that includes alternative N-terminal domains, three consecutive PSD-95/discs large/zona occludens-1 (PDZ) domains, and a src-homology 3 (SH3) domain linked to a guanylate kinase (GK)-like domain (Cho et al., 1992; Kim et al., 1996; Muller et al., 1995; Brenman et al., 1996; Chen et al., 2012).
10
R. Rong et al. / NeuroToxicology 53 (2016) 1–11
The different combinations of the N-terminals of the PSD93 isoforms fine-tune signaling scaffolds to regulate synaptic transmission (Kruger et al., 2013). Furthermore, our previous study revealed that PSD-93 interacted with NR2A and nNOS in cultured cortical neurons and contributed to NMDAR-NOS-mediated neuronal damage triggered by PAF receptor activation (Zhang et al., 2010). Additionally, PSD-93 deficiency blocked NMDARtriggered neurotoxicity by disrupting the NMDAR-Ca2+-NO signaling pathway and reducing the expression of synaptic NR2A and NR2B (Paoletti et al., 2013). Recently, we found that PSD-93 KO exerted profound neuroprotective effects against ischemic brain injury by inhibiting Fyn-mediated phosphorylation of NR2B (Zhang et al., 2014). Overall, these results demonstrate that PSD93 is involved in NMDAR-mediated neuronal excitotoxicity in a manner that is likely related to the assembly of signaling modules in the postsynaptic membrane or alterations in synapse-associated proteins. However, the identities of the proteins that are affected by the changes caused by PSD-93 deficiency during the ischemia– reperfusion process remain unknown. In the last two decades, proteomics has been applied to neuroscience research and help the elucidation of fundamental mechanisms and the discovery of candidate biomarkers and therapeutic targets (Bai et al., 2014, 2013). In the present study, we used iTRAQ quantitative proteomics analysis to screen the proteins that are differentially expressed due to PSD-93 deficiency in a mouse tMCAO model. We demonstrated that PSD-93 deletion reduced cerebral infarction volume and significantly improved neurological function following stroke. The reliability and stability of the iTRAQ analysis were verified by using scatterplot matrices and density map analyses. It is well-known that synaptic transmission, transport, the secretion of neurotransmitters and synaptic vesicles, and the maintenance of the integrity of organelle membrane components are important for synaptic and neuronal function (Shin, 2014; Fernandez-Chacon and Sudhof, 1999; Kriebel et al., 2001). Moreover, cell–cell signaling and cell signaling complexes are necessary for synaptic function and the function of signaling cascade pathways (Turrigiano, 2012; Misra et al., 2010). Our results indicated that PSD-93 deletion might protect against ischemic injury in vivo by affecting synapse-associated proteins. It is generally accepted that network analysis is helpful for gaining insight into the function of differentially expressed proteins that are identified by proteomic analyses. Thus, we further validated four proteins, including SynGAP, Syntaxin-1A, VDLC, and PKC-b, and performed network analyses. The results demonstrated that the synapse-associated protein Stx1a is involved in the transport of synaptic vesicles and the integrity of synaptic membranes via the interacting proteins and PKCb, which acts in calcium signaling pathway, is associated with NMDAR1 and NMDAR2B. Currently, increasing amounts of evidence have shown that calcium overloading and the excessive activation of NMDA receptors are likely the major mechanism of ischemic brain injury (Han et al., 2013; Li et al., 2012). Moreover, in the present study, the differentially expressed proteins are closely related to the proteins involved in calcium overload, NMDAR activation and the integrity of synaptic function. We suggest that the role of PSD-93 in the excitotoxicity induced by tMCAO might be mediated through these four specific differentially expressed proteins. In conclusion, we identified four proteins SynGAP, Syntaxin-1A, VDLC, and PKCb differentially expressed between PSD-93 KO and WT mice 4 h after tMCAO. These identified proteins might be involved in PSD-93 mediated neuroexcitotoxicity in ischemic brain injury. Identification of these proteins provides valuable clues to elucidate the mechanisms underlying the actions of PSD-93 in ischemia–reperfusion induced neurotoxicity.
Conflict of interest The authors declare no conflicts of interest. Acknowledgments We thank Dr. Yuanxiang Tao for providing the PSD-93 KO mice and SuZhou BioNovoGene Co., Ltd. (http://www.bionovogene.com) for help with the data analyses. This study was supported by the National Natural Science Foundation of China (Nos. 81301120, and 81200897), the Natural Science Foundation of the Colleges and Universities in Jiangsu Province (No. 13KJB320027) and the Xuzhou Medical Young Talents Project. References Bai, B., Hales, C.M., Chen, P.C., Gozal, Y., Dammer, E.B., et al., 2013. U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A. 110, 16562–16567. Bai, B., Chen, P.C., Hales, C.M., Wu, Z., Pagala, V., et al., 2014. Integrated approaches for analyzing U1-70K cleavage in Alzheimer’s disease. J. Proteome Res. 13, 4526–4534. Brassai, A., Suvanjeiev, R.G., Ban, E.G., Lakatos, M., 2014. Role of synaptic and nonsynaptic glutamate receptors in ischaemia induced neurotoxicity. Brain Res. Bull. 112C, 1–6. Brenman, J.E., Christopherson, K.S., Craven, S.E., McGee, A.W., Bredt, D.S., 1996. Cloning and characterization of postsynaptic density 93, a nitric oxide synthase interacting protein. J. Neurosci. 16, 7407–7415. Castegna, A., Aksenov, M., Aksenova, M., Thongboonkerd, V., Klein, J.B., et al., 2002. Proteomic identification of oxidatively modified proteins in Alzheimer’s disease brain. Part I: creatine kinase BB, glutamine synthase, and ubiquitin carboxyterminal hydrolase L-1. Free Radic. Biol. Med. 33, 562–571. Chen, J., Li, Y., Wang, L., Zhang, Z., Lu, D., et al., 2001. Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke 32, 1005–1011. Chen, B.S., Gray, J.A., Sanz-Clemente, A., Wei, Z., Thomas, E.V., et al., 2012. SAP10 mediates synaptic clearance of NMDA receptors. Cell Rep. 2, 1120–1128. Cho, K.O., Hunt, C.A., Kennedy, M.B., 1992. The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein. Neuron 9, 929–942. Ditzen, C., Jastorff, A.M., Kessler, M.S., Bunck, M., Teplytska, L., et al., 2006. Protein biomarkers in a mouse model of extremes in trait anxiety. Mol. Cell. Proteomics 5, 1914–1920. Dumas, T.C., 2005. Developmental regulation of cognitive abilities: modified composition of a molecular switch turns on associative learning. Prog. Neurobiol. 76, 189–211. Fernandez-Chacon, R., Sudhof, T.C., 1999. Genetics of synaptic vesicle function: toward the complete functional anatomy of an organelle. Annu. Rev. Physiol. 61, 753–776. Focking, M., Besselmann, M., Trapp, T., 2006. Proteomics of experimental stroke in mice. Acta Neurobiol. Exp. (Wars) 66, 273–278. Han, Z., Yang, J.L., Jiang, S.X., Hou, S.T., Zheng, R.Y., 2013. Fast, non-competitive and reversible inhibition of NMDA-activated currents by 2-BFI confers neuroprotection. PLoS One 8, e64894. Ji, Y.H., Ji, J.L., Sun, F.Y., Zeng, Y.Y., He, X.H., et al., 2010. Quantitative proteomics analysis of chondrogenic differentiation of C3H10T1/2 mesenchymal stem cells by iTRAQ labeling coupled with on-line two-dimensional LC/MS/MS. Mol. Cell. Proteomics 9, 550–564. Jiang, X., Mu, D., Sheldon, R.A., Glidden, D.V., Ferriero, D.M., 2003. Neonatal hypoxiaischemia differentially upregulates MAGUKs and associated proteins in PSD-93deficient mouse brain. Stroke 34, 2958–2963. Johnston-Wilson, N.L., Sims, C.D., Hofmann, J.P., Anderson, L., Shore, A.D., et al., 2000. Disease-specific alterations in frontal cortex brain proteins in schizophrenia, bipolar disorder, and major depressive disorder. The Stanley Neuropathology Consortium. Mol. Psychiatry 5, 142–149. Kim, E., Cho, K.O., Rothschild, A., Sheng, M., 1996. Heteromultimerization and NMDA receptor-clustering activity of Chapsyn-110, a member of the PSD-95 family of proteins. Neuron 17, 103–113. Kriebel, M.E., Keller, B., Silver, R.B., Fox, G.Q., Pappas, G.D., 2001. Porocytosis: a new approach to synaptic function. Brain Res. Brain Res. Rev. 38, 20–32. Kruger, J.M., Favaro, P.D., Liu, M., Kitlinska, A., Huang, X., et al., 2013. Differential roles of postsynaptic density-93 isoforms in regulating synaptic transmission. J. Neurosci. 33, 15504–15517. Li, L., Yang, R., Sun, K., Bai, Y., Zhang, Z., et al., 2012. Cerebroside-A provides potent neuroprotection after cerebral ischaemia through reducing glutamate release and Ca(2)(+) influx of NMDA receptors. Int. J. Neuropsychopharmacol. 15, 497– 507. Liaw, W.J., Zhu, X.G., Yaster, M., Johns, R.A., Gauda, E.B., et al., 2008. Distinct expression of synaptic NR2A and NR2B in the central nervous system and impaired morphine tolerance and physical dependence in mice deficient in postsynaptic density-93 protein. Mol. Pain 4, 45.
R. Rong et al. / NeuroToxicology 53 (2016) 1–11 Martins-De-Souza, D., Dias-Neto, E., Schmitt, A., Falkai, P., Gormanns, P., et al., 2010. Proteome analysis of schizophrenia brain tissue. World J. Biol. Psychiatry 11, 110–120. Misra, C., Restituito, S., Ferreira, J., Rameau, G.A., Fu, J., et al., 2010. Regulation of synaptic structure and function by palmitoylated AMPA receptor binding protein. Mol. Cell Neurosci. 43, 341–352. Mu, J., Xie, P., Yang, Z.S., Yang, D.L., Lv, F.J., et al., 2007. Neurogenesis and major depression: implications from proteomic analyses of hippocampal proteins in a rat depression model. Neurosci. Lett. 416, 252–256. Muller, B.M., Kistner, U., Veh, R.W., Cases-Langhoff, C., Becker, B., et al., 1995. Molecular characterization and spatial distribution of SAP97, a novel presynaptic protein homologous to SAP90 and the Drosophila discs-large tumor suppressor protein. J. Neurosci. 15, 2354–2366. Otte, D.M., Sommersberg, B., Kudin, A., Guerrero, C., Albayram, O., et al., 2011. Nacetyl cysteine treatment rescues cognitive deficits induced by mitochondrial dysfunction in G72/G30 transgenic mice. Neuropsychopharmacology 36, 2233– 2243. Paoletti, P.1, Bellone, C., Zhou, Q., 2013. NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. Nat. Rev. Neurosci. 14, 383– 400. Patel, S., Sinha, A., Singh, M.P., 2007. Identification of differentially expressed proteins in striatum of maneb-and paraquat-induced Parkinson’s disease phenotype in mouse. Neurotoxicol. Teratol. 29, 578–585. Pennington, K., Beasley, C.L., Dicker, P., Fagan, A., English, J., et al., 2008. Prominent synaptic and metabolic abnormalities revealed by proteomic analysis of the dorsolateral prefrontal cortex in schizophrenia and bipolar disorder. Mol. Psychiatry 13, 1102–1117. Robinson, R.A., Lange, M.B., Sultana, R., Galvan, V., Fombonne, J., et al., 2011. Differential expression and redox proteomics analyses of an Alzheimer disease transgenic mouse model: effects of the amyloid-beta peptide of amyloid precursor protein. Neuroscience 177, 207–222. Shin, O.H., 2014. Exocytosis and synaptic vesicle function. Compr. Physiol. 4, 149– 175. Szego, E.M., Janaky, T., Szabo, Z., Csorba, A., Kompagne, H., et al., 2010. A mouse model of anxiety molecularly characterized by altered protein networks in the brain proteome. Eur. Neuropsychopharmacol. 20, 96–111.
11
Tao, F., Skinner, J., Yang, Y., Johns, R.A., 2010. Effect of PSD-95/SAP90 and/or PSD-93/ Chapsyn-110 deficiency on the minimum alveolar anesthetic concentration of halothane in mice. Anesthesiology 112, 1444–1451. Turrigiano, G., 2012. Homeostatic synaptic plasticity: local and global mechanisms for stabilizing neuronal function. Cold Spring Harb. Perspect. Biol. 4, a005736. Wang, L., Zhang, L., Chen, Z.B., Wu, J.Y., Zhang, X., et al., 2009. Icariin enhances neuronal survival after oxygen and glucose deprivation by increasing SIRT1. Eur. J. Pharmacol. 609, 40–44. Xu, Y., Zhang, B., Hua, Z., Johns, R.A., Bredt, D.S., et al., 2004. Targeted disruption of PSD-93 gene reduces platelet-activating factor-induced neurotoxicity in cultured cortical neurons. Exp. Neurol. 189, 16–24. Zhang, B., Tao, F., Liaw, W.J., Bredt, D.S., Johns, R.A., et al., 2003. Effect of knock down of spinal cord PSD-93/chapsin-110 on persistent pain induced by complete Freund’s adjuvant and peripheral nerve injury. Pain 106, 187–196. Zhang, Q.X., Pei, D.S., Guan, Q.H., Sun, Y.F., Liu, X.M., et al., 2007. Crosstalk between PSD-95 and JIP1-mediated signaling modules: the mechanism of MLK3 activation in cerebral ischemia. Biochemistry 46, 4006–4016. Zhang, M., Xu, J.T., Zhu, X., Wang, Z., Zhao, X., et al., 2010. Postsynaptic density93 deficiency protects cultured cortical neurons from N-methyl-D-aspartate receptor-triggered neurotoxicity. Neuroscience 166, 1083–1090. Zhang, F., Wang, S., Zhang, M., Weng, Z., Li, P., et al., 2012. Pharmacological induction of heme oxygenase-1 by a triterpenoid protects neurons against ischemic injury. Stroke 43, 1390–1397. Zhang, M., Li, Q., Chen, L., Li, J., Zhang, X., et al., 2014. PSD-93 deletion inhibits Fynmediated phosphorylation of NR2B and protects against focal cerebral ischemia. Neurobiol. Dis. 68, 104–111. Zhao, J., Zhang, M., Li, W., Su, X., Zhu, L., et al., 2013. Suppression of JAK2/ STAT3 signaling reduces end-to-end arterial anastomosis induced cell proliferation in common carotid arteries of rats. PLoS One 8, e58730. Zheng, C.Y., Seabold, G.K., Horak, M., Petralia, R.S., 2011. MAGUKs, synaptic development, and synaptic plasticity. Neuroscientist 17, 493–512. Zhou, L., Li, F., Xu, H.B., Luo, C.X., Wu, H.Y., et al., 2010. Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95. Nat. Med. 16, 1439–1443.