TYPE Original Research PUBLISHED 23 March 2023 DOI 10.3389/fphar.2023.1003557 Analysis of networks in the OPEN ACCESS dorsolateral prefrontal cortex in EDITED BY Javier R. Caso, Universidad Complutense chronic schizophrenia: Relevance de Madrid, Spain REVIEWED BY of altered immune response Dibyadeep Datta, Yale University, United States Luiz Felipe Souza E. Silva, América Vera-Montecinos 1, Ricard Rodríguez-Mias 2, Èlia Vila 1, University of São Paulo, Brazil Judit Villén 2 and Belén Ramos 1,3,4,5* *CORRESPONDENCE 1 Psiquiatria Molecular, Parc Sanitari Sant Joan de Déu, Institut de Recerca Sant Joan de Déu, Sant Boi de Belén Ramos, Llobregat, Spain, 2Department of Genome Sciences, School of Medicine, University of Washington, belen.ramos@sjd.es Seattle, WA, United States, 3Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM SPECIALTY SECTION (Biomedical Network Research Center of Mental Health), Ministry of Economy, Industry and This article was submitted to Competitiveness, Institute of Health Carlos III, Madrid, Spain, 4Department de Bioquímica i Biología Inflammation Pharmacology, Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain, 5Facultat de a section of the journal Medicina, Universitat de Vic-Universitat Central de Catalunya, Vic, Spain Frontiers in Pharmacology RECEIVED 26 July 2022 ACCEPTED 09 March 2023 PUBLISHED 23 March 2023 The dorsolateral prefrontal cortex (DLPFC) has a crucial role in cognitive CITATION functioning and negative symptoms in schizophrenia. However, limited Vera-Montecinos A, Rodríguez-Mias R, information of altered protein networks is available in this region in Vila È, Villén J and Ramos B (2023), Analysis of networks in the dorsolateral schizophrenia. We performed a proteomic analysis using single-shot liquid prefrontal cortex in chronic chromatography-tandem mass spectrometry of grey matter of postmortem schizophrenia: Relevance of altered DLPFC in chronic schizophrenia subjects (n = 20) and unaffected subjects (n = immune response. Front. Pharmacol. 14:1003557. 20) followed by bioinformatic analysis to identify altered protein networks in doi: 10.3389/fphar.2023.1003557 schizophrenia (PXD024939 identifier in ProteomeXchange repository). Our results COPYRIGHT displayed a proteome profile in the DLPFC of 1989 proteins. 43 proteins were © 2023 Vera-Montecinos, Rodríguez- found significantly altered in schizophrenia. Analysis of this panel showed an Mias, Vila, Villén and Ramos. This is an open-access article distributed under the enrichment of biological processes implicated in vesicle-mediated transport, terms of the Creative Commons processing and antigen presentation via MHC class II, intracellular transport Attribution License (CC BY). The use, and selenium metabolism. The enriched identified pathways were MHC class II distribution or reproduction in other forums is permitted, provided the original antigen presentation, vesicle-mediated transport, Golgi ER retrograde transport, author(s) and the copyright owner(s) are Nef mediated CD8 downregulation and the immune system. All these enriched credited and that the original publication categories were found to be downregulated. Furthermore, our network analyses in this journal is cited, in accordance with accepted academic practice. No use, showed crosstalk between proteins involved in MHC class II antigen presentation, distribution or reproduction is permitted membrane trafficking, Golgi-to-ER retrograde transport, Nef-mediated which does not comply with these terms. CD8 downregulation and the immune system with only one module built by 13 proteins. RAB7A showed eight interactions with proteins of all these pathways. Our results provide an altered molecular network involved in immune response in the DLPFC in schizophrenia with a central role of RAB7A. These results suggest that RAB7A or other proteins of this network could be potential targets for novel pharmacological strategies in schizophrenia for improving cognitive and negative symptoms. Abbreviations: ACTR1A, Alpha-centractin; AP2M, AP-2 complex subunit mu; CYFIP2, Cytoplasmic FMR1- interacting protein 2; DLPFC, Dorsolateral prefrontal cortex; FDR, False Discovery Rate; FAB, Frontal Assessment battery; LC/MS-MS, Liquid chromatography-tandem mass spectrometry; LFQ, Level free quantification; MHC II, Major histocompatibility complex class II; SZ, Schizophrenia; RAB7A, Ras-related protein Rab-7a. Frontiers in Pharmacology 01 frontiersin.org Vera-Montecinos et al. 10.3389/fphar.2023.1003557 KEYWORDS DLPFC, schizophrenia, postmortem, molecular network, immune system 1 Introduction in SZ subjects such as a decrease of dendritic cells, involved in the antigen presentation (Fernandez-Egea et al., 1556), an increase in The dorsolateral prefrontal cortex (DLPFC) plays a crucial role the ratio of lymphocytes/neutrophils, an important marker for in schizophrenia (SZ) (Yoon et al., 2008). Prefrontal cortex governs inflammatory damage (Karageorgiou et al., 2019; Sandberg et al., executive functions (Kouneiher et al., 2009), which involve all 2021) and increase in monocytes (Jackson and Miller, 2020) which is rational thinking cognitive processes such as attention, behavior, associated with an increase of proinflammatory cytokines in blood cognitive flexibility and impulse inhibition (Logue and Gould, 2014). (Kowalski et al., 2001). Altered signaling during the immune In the context of SZ, studies have shown cognitive, social, and response could lead to the release of proinflammatory molecules. emotional impairments (Russell et al., 2000; Ragland et al., 2007; Studies in postmortem DLPFC have found high mRNA levels of Ursu et al., 2011), behaviors associated with altered functioning of proinflammatory cytokines in this area, including IL1β, IL6, IL8, and the DLPFC. Evolutionarily, the prefrontal cortex is the last brain IL 1 in SZ subjects (Fillman et al., 2013; Cai et al., 2020)]. Recently, region to develop (Teffer and Semendeferi, 2012). Thus, altered studies have suggested the existence of two sub-biotypes for SZ, cognitive functioning in SZ could be due to the dysfunction in the based on the levels of proinflammatory molecules in the peripheral maturation of connectivity between the prefrontal cortex and other blood sample which could reflect the state of the central nervous brain areas. Studies have demonstrated that maturation of system. The SZ subjects with high levels of proinflammatory connectivity occurs in two periods, the first during the prenatal cytokines have difficulty with language and decreased cortical and perinatal phases and the second during the global maturation of volume (Fillman et al., 2016; Bowen et al., 2019; Childers et al., the circuit to establish functional networks, which lasts until early 2022). These molecules would alter cerebral barriers and lead to adulthood (Teffer and Semendeferi, 2012). In this regard, molecules entering the cerebral parenchyma, thus altering the dysfunction in dopaminergic (Meyer-Lindenberg et al., 2002; homeostatic balance in the brain and contributing to the global Slifstein et al., 2015), GABAergic (Lewis et al., 1999; Tanaka, dysregulation of neuronal networks in SZ. All this evidence supports 2008) and glutamatergic (Li et al., 2019) neurotransmission that the activation of the immune response is performed by a systems has been reported in the prefrontal cortex in SZ. Thus, complex network of molecular interactions and the dysfunction altered neuronal networks underlie SZ and several hypotheses could of this complex network could be responsible for the inflammatory be unified to understand this dysfunction. processes in DLPFC in SZ. Altered expression of genes associated to immune system (Arion The proteomic approaches allow to investigate protein et al., 2007) and inflammatory response (Fillman et al., 2013) has signatures and molecular mechanisms involved in specific brain been described in DLPFC in SZ. Genetic predisposition together areas in SZ. The aim of our study was to identify altered protein with insults during the perinatal such as maternal infection could networks in the DLPFC of SZ patients using single-shot liquid lead to the accumulation of errors during neurodevelopment. chromatography-tandem mass spectrometry analysis. In this Dysfunction of the immune system is one of the hypotheses that study, we compared the proteomic profile in postmortem DLPFC underlie SZ, which proposes that maternal infection during the (Brodmann area 9) of individuals with chronic SZ (n = 20) and gestational period could increase the risk for SZ in offspring exposed unaffected subjects (n = 20). Our results showed a downregulated to viral or bacterial infections in intrauterine life (Brown et al., 2004). network involved in the immune response in DLPFC in which In this regard, high levels of IL-8 during pregnancy have been RAB7A protein has multiple interactions in this network. implicated in a decrease in brain volume and an increase volume of ventricles in schizophrenia subjects (Ellman et al., 2010). Cytokines such as Interferon-Ƴ can modulate and inhibit the differentiation of 2 Materials and methods oligodendrocytes and alter myelinization processes (Chew et al., 2005). Thus, inflammatory processes could alter myelinization and 2.1 Postmortem prefrontal cortex brain lead to neuronal connectivity dysfunction. The involvement of tissue immune dysfunction and inflammation in SZ is supported by emerging evidence suggesting aberrant immune mechanisms Samples from dorsolateral prefrontal cortex of subjects with (O’Donnell et al., 1995; Kroken et al., 2019; Comer et al., 2020; chronic SZ (n = 20, left hemisphere), and unaffected subjects (n = 20, Afia et al., 2021). A genome-wide association study reported a major 10 left hemispheres and 10 right hemispheres). The samples of histocompatibility complex (MHC) locus and Toll-like receptors chronic SZ were obtained from the collection of neurologic tissue of (TLRs), both involved in innate immunity, as genetic risks for SZ Parc Sanitari Sant Joan de Déu Brain Bank (Roca et al., 2008). For (Bramon et al., 1038). Indeed, postmortem studies of SZ subjects unaffected subjects the samples of prefrontal cortex were obtained showed altered expression of elements of TLR4 signaling (García- from the Institute of Neuropathology of Hospital Universitari de Bueno et al., 2016; MacDowell et al., 2017). Moreover, MHC has Bellvitge, Navarra Biomed Biobank and Hospital Clinic IDIBAPS several functions in the central nervous system during development Biobank. The brain banks of Parc Sanitari Sant Joan de Déu, including neurite outgrowth and neuronal plasticity (Shatz, 2009; Institute of Neuropathology of Hospital Universitari de Bellvitge, Elmer and McAllister, 2012). The MHC II has a role in antigen Hospital Clinic IDIBAPS Biobank follow the same protocol based on presentation during adaptative response (Shatz, 2009). In this the guidelines from the Network of European Brain and Tissue context, studies have demonstrated altered immune components Banks for Clinical and Basic Neuroscience (BrainNET Europe). Frontiers in Pharmacology 02 frontiersin.org Vera-Montecinos et al. 10.3389/fphar.2023.1003557 TABLE 1 Demographic, clinical and tissue-related features of cases. Schizophrenia (n = 20) Control (n = 20) Statistic p-value Gender (Male) 100% (n = 20) 100% (n = 20) N/A N/A Age (years) 74 ± 10 74 ± 9 0.06; 38a 0.94 PMD (hours) 5.15 ± 2.59 6.07 ± 2.81 1.07; 38a 0.29 a pH PFC 6.76 ± 0.75 6.67 ± 0.48 0.42; 38 0.67 SZ diagnosis N/A Chronic residual 70% (n = 14) Chronic paranoid 15% (n = 3) Chronic disorganized 5% (n = 1) Chronic catatonic 5% (n = 1) Simple-type 5% (n = 1) Age of onset of SZ (years) 23.9 ± 10 N/A Duration of illeness 50 ± 11 N/A Toxicology N/A Daily AP dose (mg/day)b 683.9 ± 925.9 First generation AP 20% (n = 4) Second generation AP 40% (n = 8) First and Second generation AP 25% (n = 5) AP free 15% (n = 3) Mean ± standard deviation; PMD, postmortem delay; SZ, schizophrenia; AP, antipsychotics; N/A, not applicable. a Mann-Whitney U for non-parametric variables. b Last daily chlorpromazine equivalent dose was calculated based on the electronic records of drugs prescriptions of the patients as described (Gardner et al., 2010). According to their standardized protocols, fresh brain hemisphere patients was calculated based on the electronic records of last drug was cut in coronal slabs of 1 cm of thickness, frozen in dry ice prescriptions administered up to death as described previously (−20°C) and stored at −80°C. In Navarra Biomed Biobank, fresh (Gardner et al., 2010). brain tissue was immersed into isopentane over nitrogen liquid at −180°C for rapidly freeze and were stored at −80°C. Human Brodmann area nine from DLPFC was dissected from coronal slabs 2.2 Label free quantification proteomic stored at −80°C extending from the pial surface to white matter only analysis mass spectrometry including grey matter. pH was measured using pH indicator strips (Merk, range pH 5–10) in all the brain samples immediately before 2.2.1 Protein reduction, alkylation, LysC digestion, performing the dissection of brain tissue for protein extraction. All and desalting SZ subjects were institutionalized donors with a long duration of the Protein extracts were prepared from tissue samples using buffer illness who had no history of neurological episodes. We matched SZ lysis containing ammonium bicarbonate 50 mM, and control groups by gender (only male patients were included), B-glycerophosphate 1M, sodium orthovanadate 100 mM, sodium age, postmortem delay (PMD) and pH (Table 1). Experienced pyrophosphate 200 mM, sodium fluoride 200 mM, tris 100 mM and clinical examiners interviewed each donor antemortem to Rapi Gest surfactant (Waters). Protein concentration was confirm SZ diagnosis according to the Diagnostic and Statistical determined by Bradford assay (Biorad, Hercules, CA, Manual of Mental Disorders (DSM-IV) and International United States). A total amount of 200 µg of protein per each Classification of Diseases 10 (ICD-10). Besides, donor subjects brain protein extract from unaffected subjects and SZ subjects were evaluated antemortem with Positive and Negative Syndrome was used for single-shot liquid chromatography-tandem mass Scale (PANSS) and for neurocognitive measures was used the spectrometry (LC-MS/MS) analysis. The lysate was reduced with Frontal Assessment battery (FAB) with a death to clinical 3 mM dithiothreitol at 55°C for 30 min and alkylated with 20 mM assessment interval of 61 months. All deaths were due to natural iodoacetamide at room temperature for 30 min and quenched with causes. The study was approved by the Institutional Ethics additional 10 mM dithiothreitol for 30 min at room temperature. Committee of Parc Sanitari Sant Joan de Déu. A written Peptide samples for mass spectrometer analysis were prepared from informed consent was obtained from each subject. The last daily reduced and alkylated lysate by adapting a single-pot solid-phase chlorpromazine equivalent dose for the antipsychotic treatment of enhanced sample preparation (SP3) similar to Hughes et al. (2014) Frontiers in Pharmacology 03 frontiersin.org Vera-Montecinos et al. 10.3389/fphar.2023.1003557 ™ and using a KingFisher Flex (Thermo Fisher) automated robotic to 0.1. The experimental strategy used in this proteomic study is magnetic bead handler (Leutert et al., 2019). shown in Figure 1. The quantified proteins were imported into Specifically, 50 µg of lysate protein was diluted to 0.5 mg/mL Perseus software platform (version 1.6.1.3) to check data quality concentration in 80% EtOH and loaded onto 500 µg of conditioned obtained by LC/MS/MS and to visualize the data distribution magnetic carboxylated sp3 beads. Beads were subsequently washed (Tyanova et al., 2016). We performed four analyses for these 3 times with 100 µL of 80% EtOH prior to on bead digestion/elution altered proteins in SZ: i) Correlation matrix: the normalized using 75 µL of 50 mM AMBIC buffer and LysC at 10 ng/µL. LFQ intensity data were used to estimate a correlation Digestion was carried out for 4 h at 37°C, beads were further coefficient matrix between controls and SZ patients. The washed with 75 µl of water and wash was combined with eluate. Pearson correlation coefficients were used to defined Eluate was acidified with formic acid and freeze dried prior to unsupervised hierarchical cluster analysis and were plotted as a resuspending in MS sample buffer. Peptides were resuspended in 3% heat map; ii) Unsupervised hierarchical clustering analysis formic acid and 4% acetonitrile in water at a 1 µg/µL concentration. generated from quantified proteins in 20 SZ and 20 healthy control samples of postmortem DLPFC. This was carried out 2.2.2 Liquid chromatography coupled to tandem on Z-score transformed normalized LFQ intensity data for each mass spectrometry protein using Euclidean distance; iii) Enrichment analysis of The LC/MS-MS analysis was performed in Q-Exactive biological processes and pathways; and iv) Generation of instrument (Thermofisher Scientific, CA, United States) using networks from significantly enriched pathways by protein- data-dependent Top 20 acquisition method. Peptides were loaded protein interaction. onto a 100 μm ID × 3 cm precolumn packed with Reprosil C18 3 μm beads (Dr. Maisch GmbH), and separated by reverse-phase chromatography on a 100 μm ID × 30 cm analytical column 2.3 Bioinformatic analysis packed with Reprosil C18 1.9 μm beads (Dr. Maisch GmbH). A gradient of 3%–30% acetonitrile in 0.125% formic acid was used To analyze the proteome profile of DLPFC we performed delivered at 225 nL/min over 130 min, with a total 180-min Disease term, Gene Ontology (GO), and Pathways analysis using acquisition time. Peptides were analyzed online on the orbitrap Webgestalt (WEB-based GEne SeT AnaLysis Toolkit). The mass analyser using a top 20 data-dependent acquisition with all MS enrichment analysis, we used the method of Over-Representation spectra being acquired and stored in centroid mode. Full MS scans Analysis (ORA) supported by Fisher’s exact test (Wang et al., 2013). were acquired from 300 to 1,500 m/z at 70,000 FWHM resolution To perform the enriched analysis, we used the whole genome as a with a fill target of 3E6 ions and maximum injection time of 100 ms. reference set. Diseases terms were obtained from PharmacoGenetics The 20 most abundant ions on the full MS scan were selected for Knowledge Base (PharmGKB) and GLAD4U Gene List fragmentation using 2 m/z precursor isolation window and beam- Automatically Derived For You (GLAD4U) databases (Hewett type collisional-activation dissociation (HCD) with 26% normalized et al., 2002; Jourquin et al., 2012). For GO analysis, we collision energy. MS/MS spectra were collected at 17,500 FWHM performed non-redundant enriched categories analysis, for resolution with a fill target of 5E4 ions and maximum injection time pathways analysis we used the Reactome database. Multi-testing of 50 ms. Fragmented precursors were dynamically excluded from correction was performed using Benjamini–Hochberg test (FDR = selection for 35 s. 0.1). For network generation, we used String version 11.0. For Raw files were processed and analyzed by MaxQuant (version screening protein localization in different cell types in cerebral 1.6.2.10). MS/MS spectra were searched with Andromeda against cortex tissue (Endothelial cells, glia Cells and Neuronal Cells) we the uniprot_UP000005640_20180526. fasta with common used the Human Protein Atlas database (Uhlén et al., 1979). The contaminants added. The precursor mass tolerance was set to enrichment analyses were set to FDR = 0.1. For the representation of 7 ppm, and the fragment ion tolerance was set to 20 ppm. Search biological processes and pathways enriched we used the Log2 of the parameters included fully LysC enzyme specificity with up to three fold change of normalized LFQ Intensity between SZ and controls missed cleavages permitted. The minimum required peptide length samples and FDR adjusted -Log10 (q-value). was seven residues. The target-decoy database search strategy was used to guide filtering and estimate False Discovery Rate (FDR). Peptides matches were filtered to ≤0.01 FDR. Proteins with at least 3 Results one peptide were considered identified. Label free quantification (LFQ) was selected for individual protein comparisons between 3.1 Quantitative proteomic analyses in the control and SZ groups. A quality cut off for protein determination dorsolateral prefrontal cortex in chronic was the presence of the protein in the 20 samples per group, thus, schizophrenia proteins no quantified in all the 20 samples per group were excluded from the analysis. The normalized LFQ intensity was To identify altered proteins related to SZ in the DLPFC, we used referred to media of the controls. A significance value for each individual protein extracts from a cohort of SZ subjects (n = 20) and quantified protein was calculated from Student’s t-test and unaffected subjects (n = 20). No differences were observed between correction of significance values of the quantified protein data the SZ and control groups for any demographic or tissue-related set was performed following the Benjamini and Hochberg methods variables (Table 1). Our proteomic analysis in the DLPFC revealed a (Benjamini and Hochberg, 1995). A False Discovery Rate (FDR) total of 4,407 quantified proteins. We excluded from the analysis was computed for all significant values and FDR threshold was set 2,418 proteins that were not quantized in all the 20 samples per Frontiers in Pharmacology 04 frontiersin.org Vera-Montecinos et al. 10.3389/fphar.2023.1003557 FIGURE 1 Experimental design of the proteomic analysis to identify altered pathways in schizophrenia. Protein lysates from the postmortem dorsolateral prefrontal cortex of control subjects (C) (n = 20) and chronic schizophrenia (SZ) patients (n = 20) were processed as depicted. The peptides were separated and analyzed by Liquid Chromatography (LC) coupled to a Q-Exactive Orbitrap tandem mass spectrometer. The relative fold change of peptides was integrated into protein changes. The individual protein signatures for each case and control were used to generate hierarchical clusters. The prioritization of altered proteins (P1-Pn represents generic proteins) in SZ was obtained by comparing protein fold changes between C and SZ groups (significant proteins adjusted to an FDR<0.1). group. 1989 common proteins were quantified (45%) in all the human cerebral cortex. 28 proteins have been previously reported to 20 samples per group and used for subsequent bioinformatics have medium-high level expression in cortical neuronal cells, while analyses (Supplementary Data S1). The proteomic analysis only 10 proteins in glia cells and 11 proteins in endothelial cells revealed that 99.9% of the proteins were identified with two or (Supplementary Data S2). We further classified the altered proteins more peptides and 91.3% with five or more peptides. We found only according to their abundance in excitatory and inhibitory neurons. 12 proteins significantly altered using FDR <0.05 (Data not shown). Only SYNJ1, TXNRD1 and NEFM were found to have different In addition, our analysis using FDR<0.1, showed 43 significantly expression levels between both neuronal types. To analyze the altered proteins in the DLPFC (Supplementary Data S2) of which possible influence of tissue-related features on the altered 9 were upregulated and 34 were downregulated. Altered proteins proteins, we performed correlation analysis in all samples. None were classified according to their cell type protein expression in of the regulated proteins showed significant correlation with PMD Frontiers in Pharmacology 05 frontiersin.org Vera-Montecinos et al. 10.3389/fphar.2023.1003557 had been previously reported in gene expression analyses in SZ from the Schizophrenia Database and 12 of the 43 altered proteins had been previously reported in proteomic studies (Supplementary Data S2). For the individual proteome signature analysis, we first examined the similarity of the individual proteome in the DLPFC through a correlation matrix (Figure 2A). The results showed correlations above a Pearson correlation coefficient of 0.7 with an average coefficient of 0.994 between the SZ and control samples indicating a high quality of the proteomic data. Although two main hierarchical clusters were identified in this analysis, we did not find any clinical or tissue-related feature that explain the segregation of these two clusters. We then analyzed any possible laterality effect on these results. However, we did not find any significant differences between the right (n = 10) and left (n = 10) hemispheres in the control group in our study and no segregation of clustering analysis by hemisphere was observed (Supplementary Figure S1 and Supplementary Data S5). In addition, the brain biobank source of each sample in the unaffected group of subjects was analyzed in this clustering analysis. No segregation by center was observed (Supplementary Figure S1). The unsupervised hierarchical clustering analysis revealed that the proteomic profile detected for each subject did not allow segregation between controls and SZ group (Figure 2B). 3.2 Gene ontology enrichment analysis for biological processes and pathways The gene ontology analysis with the 43 proteins that were altered in the DLPFC showed an enrichment of several biological processes (FDR = 0.1) (Figure 3A; Table 2) involved in vesicle- mediated transport, processing and antigen presentation via MHC class II, intracellular transport and selenium metabolism. The enriched pathways (Figure 3B) were directly related to the enriched biological processes observed. These pathways were related to MHC class II antigen presentation, vesicle-mediated transport, Golgi transport and immune system. All these enriched pathways were found for the downregulated proteins. 3.3 Network generation from enriched pathways in the dorsolateral prefrontal cortex FIGURE 2 Quantitative proteomic analysis in the prefrontal cortex in schizophrenia. (A) The correlation matrix shows the correlation coefficiency Network analysis of significantly enriched pathways from the between the 1989 quantified proteins in each compared pair of samples. (B) 43 proteins altered in the dorsolateral prefrontal cortex showed an Unsupervised hierarchical clustering analysis from prefrontal cortex proteins. This analysis was performed with matrix processing according to overlap among the several altered pathways (Figure 4). A mixed the Euclidean distance and z-score aggregation method. We used module was observed in which the main overlap was generated by 1989 quantified proteins. Green color clusters represent downregulated the immune system pathway. This pathway showed about 43% proteins. Red color clusters represent upregulated proteins. The intensity scale represents the z-score, positive scores show significantly upregulated overlap with membrane trafficking proteins, 37% with MHC class proteins labeled in red, while negative scores significantly downregulated II antigen presentation, 12% with Nef-mediated proteins are labeled in green. SZ, schizophrenia; C, control. CD8 downregulation and 31% with Golgi ER retrograde transport. All 13 proteins of the network were proteins with known expression in cortical neurons being eight of them with or pH (FDR<0.1) (Supplementary Datas S3, S4). Further, no medium to high expression levels (Supplementary Data S2). significant correlation was observed between altered proteins and Furthermore, RAB7A showed eight interactions with proteins antipsychotic dose (Data not shown). 42 of the 43 altered proteins involved in all enriched pathways. Frontiers in Pharmacology 06 frontiersin.org Vera-Montecinos et al. 10.3389/fphar.2023.1003557 FIGURE 3 Enrichment analyses from the dorsolateral prefrontal cortex proteome in chronic schizophrenia. The bubble chart shows enriched categories from 43 altered proteins in schizophrenia. (A) Non-redundant enriched biological processes categories were: antigen processing and presentation of exogenous peptide antigen via MHC class II (GO:0019886), vesicle-mediated transport (GO:0016192), intracellular transport (GO:0046907) and selenium compound metabolic process (GO:0001887). (B) The enriched pathway categories in schizophrenia were: MHC class II antigen presentation (R-HSA-2132295), vesicle-mediated transport (R-HSA-5653656), Golgi-to-ER retrograde transport (R-HSA-8856688), Nef-mediated CD8 downregulation (R-HSA-182218) and immune system (R-HSA-168256). The X-axes show the mean of normalized LFQ intensity in schizophrenia relative to the control group for all the proteins that belonged to each category. The Y-axes show the -log10 enrichment p-value. The bubble size is directly proportional to the number of proteins represented in each enriched category, biological process or pathway. The green color represents downregulated proteins. SZ, schizophrenia; C, control; LFQ, label free quantification. 3.4 Correlation analysis between proteins transport, Nef-mediated CD8 downregulation and the immune altered in the dorsolateral prefrontal cortex system. All altered proteins of the network were proteins with and executive function and negative known expression in cortical neurons being more than half of symptoms them with medium to high levels in this cell type. RAB7A showed eight interactions with proteins of all these pathways. We performed a correlation analysis between the protein levels Thus, our results provide an altered molecular networks involved and the Frontal Assessment battery (FAB) and negative symptom in immune response in the DLPFC in schizophrenia with a central scores. We did not find any significant correlation between proteins role of RAB7A. altered in the DLPFC and FAB scale, which measures only executive function. However, we found a significant correlation between negative symptom scale and Cytoplasmic FMR1-interacting 4.1 Network generation from pathways protein 2 (CYFIP2) protein (r = −0.738, FDR< 0.1). enriched in the dorsolateral prefrontal CYFIP2 showed a significant association with some specific cortex domains of negative symptom scale: blunted affect (r = −0.625, FDR< 0.1), passive/apathetic social withdrawal (r = −0.666, FDR< The immune system is essential for the correct maintenance of 0.1), and lack of spontaneity and flow of conversation (r = −0.781, brain homeostasis. Our results from the proteome of the DLPFC FDR< 0.1). showed enrichment for biological processes and pathways related mainly to the immune response. It is well known that intrauterine or perinatal infections can increase vulnerability to SZ (Brown and 4 Discussion Derkits, 2010). An imbalance in the immune system has been proposed as one possible hypothesis that underlies this disorder Our study has allowed us to identify and characterize the altered (Kinney et al., 2010). Thus, our findings support an imbalance in the proteome profile in the DLPFC in chronic SZ. We identified a panel immune system in the prefrontal cortex in schizophrenia. of 43 altered proteins in this region. 65% of the proteins belong to proteins with medium-high expression levels in neurons indicating a 4.1.1 Major histocompatibility complex class II in higher impact on neuronal functioning. The enriched altered antigen presentation pathways were mainly related to the immune system. The The immune response involves major histocompatibility network generated from the enriched pathway showed a mixed complex class II antigen presentation (MHC-complex class II). module with interactions between MHC class II antigen This process is essential in the adaptative immune response presentation, membrane trafficking, Golgi-to-ER retrograde (Bondy, 2020). The MHC class II molecule is assembled in the Frontiers in Pharmacology 07 frontiersin.org Vera-Montecinos et al. 10.3389/fphar.2023.1003557 TABLE 2 Non-redundant gene ontology analysis of the 43 proteins altered in the dorsolateral prefrontal cortex. Classification Category Proteins overlap in category Total Observed E p-value FDR number number BIOLOGICAL Antigen processing and presentation ACTR1A; AP2M1; AP2S1; DCTN5; 97 5 0.24 4.32 × 0.02 PROCESSES of exogenous peptide antigen via RAB7A 10−6 MHC class II Vesicle-mediated transport RAB6B; SYNJ1; ACTR1A; PLCG1; 1942 15 4.89 4.28 × 0.06 AP2M1; ACLY; AP2S1; ATG7; ARF3; 10−5 DCTN5; GPI; RAB5C; CYFIP2; LIN7C; RAB7A Intracellular transport RAB6B; RAN; SYNJ1; ACTR1A; AP2M1; 1803 14 4.54 8.06 × 0.08 AP2S1; ARF3; PPP1CC; NEFL; DCTN5; 10−5 RAB5C; TIMM10; SRSF9; RAB7A Selenium compound metabolic SCLY; TXNRD1 6 2 0.02 9.24 × 0.08 process 10−5 PATHWAYS MHC class II antigen presentation ACTR1A; AP2M1; AP2S1; TUBB3; 121 6 0.42 3.55 × 0.01 DCTN5; RAB7A 10−6 Vesicle-mediated transport ACTR1A; SYNJ1; RAB6B; AP2M1; 667 10 2.33 7,39 × 0.03 AP2S1; TUBB3; ARF3; DCTN5; RAB5C; 10−5 RAB7A Golgi-to-ER retrograde transport ACTR1A; RAB6B; TUBB3; ARF3; 131 5 0.46 8.72 × 0.04 DCTN5 10−5 Nef Mediated CD8 Downregulation AP2M1; AP2S1 7 2 0.02 2.47 × 0.08 10−4 Immune System ACTR1A; PLCG1; PRKACA; AP2M1; 1997 16 6.98 6.63 × 0.09 ACLY; ATP6V1E1; AP2S1; TUBB3; 10−4 ATG7; TRIM2; GPI; CYFIP2; LMNB1; DCTN5; RAB5C; RAB7A For the gene ontology analysis, we used the Gene Ontology Consorcium database: pathways were identified according to the Reactome database and proteins assigned to each pathway are listed. Total number: number of reference proteins in the category/pathway. Observed number: proteins in the data set and also in the category/pathway. E: expected in the category. Adjusted p-value is corrected for multiple test, FDR<0.1. These analyses were carried out using Webgestalt. the reference set was the whole genome. endoplasmic reticulum, transported to the Golgi apparatus and RAB7A is a Rab GTPase that is involved in the regulation of subsequently carried by the endocytic pathway to late endosomes degradative signaling of autophagosomes (Gutierrez et al., 2004). and lysosomes where the MHC class II complexes are loaded with During infection, the microorganism is internalized into the peptides from antigens and sent to the cell surface (Hiltbold and autophagosome, which fuses with lysosomes for the degradation Roche, 2002). MHC II has been proposed as a risk gene for SZ of its contents (Harrison et al., 2003). Altered expression of RAB7A (Consortium, 2009). In this context, our study identified an altered in prefrontal cortex could lead to the dysfunction of the degradation MHC-complex class II antigen presentation pathway in the process, thereby contributing to dysregulation in the innate and prefrontal cortex in SZ. Our analysis identified proteins that were adaptive response in SZ. altered in this pathway such as AP2M, which is involved in MHC II Our analysis also found altered ACTR1A protein in the molecule trafficking (McCormick et al., 2005), a process is crucial in prefrontal cortex in schizophrenia. This protein is an actin- antigen presentation. In line with our study, a previous proteomic related protein that participates in the dynactin complex (Eckley study has also reported altered levels of AP2M in the anterior et al., 1999). A study associated ACTR1A with the regulation of Toll- cingulate cortex in SZ (Föcking et al., 2015). Thus, our study like receptor 2 (TLR2) signaling (Re and Strominger, 2001). This provides additional evidence of a disruption in antigen signaling has an active function in the innate immune response and presentation mediated by MHCII in SZ. the recognition of microbial products in dendritic cells in the brain. However, there is no evidence of the role of ACTR1A in the 4.1.2 Golgi membrane trafficking in immune adaptative immune response. Moreover, ACTR1A could interact response: A central role of RAB7A with DCTN5, another altered protein in our study. Both proteins are Our study identified an alteration of RAB7A in the involved in the dynactin complex (Urnavicius et al., 1979; Eckley prefrontal cortex with multiple interactions with other et al., 1999), which is involved in the activity of dynein during several altered proteins from the network with a role on dynactin intracellular motility processes (Jaarsma and Hoogenraad, 2015). complex formation for motility of Golgi-associated vesicles. Dynactin is recruited to the Golgi membrane by RAB6B and RAB7A Thus, altered RAB7A could be impacting on different GTPases, which contribute to the motility of Golgi-associated processes of immune response related to Golgi-mediated vesicles (Short et al., 2002). Here these two proteins, RAB6B and trafficking in this brain area in SZ. RAB7A, were found altered in the prefrontal cortex in schizophrenia Frontiers in Pharmacology 08 frontiersin.org Vera-Montecinos et al. 10.3389/fphar.2023.1003557 FIGURE 4 Network formed by altered pathways in the dorsolateral prefrontal cortex in schizophrenia. The protein-protein interaction network illustrates a mixed module with interactions and overlaps among proteins formed by downregulated pathways. The interaction overview shows how proteins overlap in the different pathways. Each node represents a protein. The color denotes membership of a module. The colored edge (connections between nodes) represents the type of interaction between nodes. The network was generated using String V.11. with an interaction with both ACTR1A and DCTN5. In this context, study has shown that palmitoylation in the Golgi apparatus is an we found altered pathways related to vesicle-mediate transport and essential step for activation of interferon genes in response to genetic Golgi-to-ER retrograde transport. Altered vesicle-mediated pathogen material (Mukai et al., 2016). In this regard, studies in the transport could be a reflection of the disruption in RAB6B and postmortem prefrontal cortex in SZ subjects also identified an RAB7A, proteins that are altered in this pathway that affect the altered glycosylation process in the Golgi apparatus (Bauer et al., recruitment of dynactin to the Golgi membrane. These pathways 2010; Mueller et al., 2014). Thus, our study showed altered Golgi could be directly related to the immune system, which could apparatus functioning related to the dysfunction in the immune generate the dysfunction in the several steps involved during the response in SZ. immune response in SZ subjects. The Golgi apparatus has recently been proposed as a signaling station that facilitates processes related 4.1.3 The dysregulation of Golgi-to-ER retrograde to the innate immune response (Chen and Chen, 2018) and the transport in synaptic function interface between the Golgi, endoplasmic reticulum and The synapse function depends on correct intracellular mitochondria has been proposed to form an activity core in membrane trafficking (Liu and Storrie, 2012). Our study shows inflammasome activation (Chen and Chen, 2018). Furthermore, a the dysregulation of Golgi-to-ER retrograde transport in DLPFC. Frontiers in Pharmacology 09 frontiersin.org Vera-Montecinos et al. 10.3389/fphar.2023.1003557 Thus, the dysfunction of Golgi-to-ER retrograde transport could mediated by Nef could be responsible for the ability of microbes to contribute to altered membrane trafficking. For normal membrane induce injuries in the DLPFC contributing to an altered functioning trafficking, several proteins are finely regulated. In this context, the of this region in SZ. Rab proteins, SNAREs, and COP proteins are essential to regulate the trafficking of proteins and lipids involved in the neuronal structure and synaptic function (Liu and Storrie, 2012; Ridvan 4.2 Altered expression of CYFIP2 in SZ Kiral et al., 2018). In this line, our study found downregulation of RAB6B. This protein has a key role in the Golgi-to-ER retrograde Here we found that CYFIP2 is downregulated in prefrontal transport (Zhang et al., 1493; Lu et al., 2021). Furthermore, the cortex in SZ. We found a significant correlation between altered Golgi-to-ER retrograde transport could be due to Golgi CYFIP2 and negative symptoms in our SZ cohort. Negative fragmentation leading to altered membrane trafficking, symptoms are the most resistant to currently available sphingomyelin biosynthesis (Rasika et al., 2019), transport to pharmacological interventions in SZ (Correll and Schooler, 2020; axons and dendrites (Pottorf et al., 2018), and dendrite growth Malashenkova et al., 2021). Negative symptoms in PANSS include and branching (Wang et al., 2020). The Golgi complex is also seven specific domains, including blunted affect, emotional essential for glycosylation. In this context, a recent study in SZ withdrawal, poor rapport, passive/apathetic social withdrawal, has shown abnormal glycosylation of AMPA receptors which are difficulty in abstract thinking, lack of spontaneity and flow of involved in the postsynaptic response in excitatory signaling conversation, and stereotyped thinking (Shankar and Nate, 2007). (Mueller and Meador-Woodruff, 2020). The abnormal Our results show that CYFIP2 correlates with three domains related glycosylation could be due to the dysfunction of the Golgi to negative symptoms, blunted affect, passive/apathetic social complex. Altered membrane trafficking could lead to the withdrawal, and lack of spontaneity and flow of conversation, existence of synaptic abnormalities which have been previously suggesting that altered CYFIP2 expression in DLPFC could documented in SZ (Kao and Porton, 2009; Hall et al., 2020). participate in the abnormal behavior related to negative Several studies including genetic and proteomic studies have symptoms in SZ. reported a disruption in the expression of synaptic vesicle CYFIP2 belongs to the CYFIP family which has been related to proteins in this disorder (Pennington et al., 2008; Fromer et al., autism and intellectual disability (Abekhoukh and Bardoni, 2014). 2014; Egbujo et al., 2016; Onwordi et al., 2020). These deficits have However, the function of CYFIP2 in psychiatric disorders is not been also observed in the prefrontal cortex (Yan and Rein, 2021). clear. CYFIP2 is highly expressed in the cortex brain (Zhang et al., This region is very sensitive to neurochemical changes (Arnsten, 2019) CYFIP2 participates in the regulation of actin dynamic which is 2009), and dysfunction in synaptic transmission in this area could essential for the biological processes related to neurodevelopment such as impact prefrontal cortex function. Thus, altered Golgi-to-ER axonal migration, branching and synapse morphology (Spence and retrograde transport in SZ could alter the synaptic function Soderling, 2015) Here we found that CYFIP2 was known to be through the disruption of vesicle-mediated neurotransmitter highly expressed in both excitatory and inhibitory neurons based on release in the presynaptic terminal and the post-translational the information available for single cell analysis in the Human Protein modification of AMPA receptors in the postsynaptic membrane, Brain Atlas (Uhlén et al., 1979). The expression of the CYFIP2 is detected which could lead to altered of neuronal connectivity in this disorder. in layers two and three of the prefrontal cortex (Zhang et al., 2020). In the context of SZ, decreased number of neurons has been identified in the 4.1.4 Nef mediated CD8 downregulation layer three of DLPFC (van Berlekom et al., 2020). Besides, a study in the Here we also found enriched Nef-mediated BA46 in DLPFC found morphological alterations in the pyramidal cells, CD8 downregulation pathway in prefrontal cortex in however, the authors did not find an alteration in the spatial organization schizophrenia. Nef (Negative regulatory factor) is a protein that of pyramidal cells, suggesting a normal neuronal migration in SZ enhances viral particle infectivity (Sacha et al., 2007) CD8+ is a subjects (Larsen et al., 2022)In agreement with our study, molecule present in T cells that is related to the immune response CYFIP2 has also been found downregulated in a proteomic study in against infected cells mediated by MHC I (Smolders et al., 2018). the cingulate anterior (Föcking et al., 2015). However, the relation Thus, the Nef-mediated CD8 downregulation mechanism is between CYFIP2 negative symptoms has not been studied in SZ. interfering with the ability of CD8+ T cells to inhibit viral Thus, our study provides evidence that CYFIP2 is altered in replication causing a depressed immune response to the viral prefrontal cortex with a possible impact on morphological alterations infections (Palesch et al., 2020). Aberrant immune response of pyramidal neurons in layers two and three in schizophrenia and the during viral infection could induce the infection of glial cells and presence of negative symptoms. Further studies are needed to explore the activation of astrocytes with subsequent release of CYFIP2 as a potential target for the development of therapeutic proinflammatory molecules and reactive oxygen species (Borrajo interventions to compensate negative symptoms in schizophrenia et al., 2021) and contributing to neuronal damage in the central related to morphological changes on pyramidal neurons. nervous system. Furthermore, Nef is transported in extracellular vesicles (EVs) during viral infection. These EVs could be transported from astrocytes into neurons which could become more vulnerable 4.3 Proteomic signature in the dorsolateral to viral infection (Sami Saribas et al., 2017). Nef could also inhibit prefrontal cortex the EVs formation from CD4+ T cells which could alter the recognition of circulating microbes (Borrajo et al., 2021). Thus, The unsupervised hierarchical clustering analysis revealed that dysfunction of the immune response to viral and bacterial infections the proteomic profile detected for each subject did not allow the Frontiers in Pharmacology 10 frontiersin.org Vera-Montecinos et al. 10.3389/fphar.2023.1003557 segregation between controls and SZ samples. Similar segregation institutionalized or not remain poorly understood. Therefore, pattern between SZ subjects and controls has been observed in other further studies with both groups institutionalized would be of “omics” gene expression studies of postmortem prefrontal cortex interest. Finally, our study included elderly individuals due to the (Santarelli et al., 2020) suggesting that changes in different neuron type of sample available. subpopulations in prefrontal cortex could be responsible of this mixed pattern. In this context, a recent study suggests that SZ could be categorized into two sub-biotypes at a molecular level depending 5 Conclusion on the DLPFC transcriptome, subset 1: similar DLPFC transcriptome to the unaffected subjects and subset 2: different Our results in dorsolateral prefrontal cortex showed enriched DLPFC transcriptome to the unaffected subjects (Bowen et al., pathways that involved proteins related to MHC class II antigen 2019). In our study, although no segregation between SZ and presentation, immune system and transport. Furthermore, the unaffected subject groups was found, a subgroup of SZ samples enriched pathways were integrated into a unique immune classified within the control group while another subgroup of response module in the network analyses. Also, our results 11 samples segregated from hierarchical clusters of unaffected suggest that RAB7A could have a central role in the immune subjects with the exception of two controls (#C4 and C#10) response due to its interaction with proteins of all the enriched (Figure 2B) suggesting that maybe the proteomic signature could pathways. Thus, our proteomic study provides a possible also allow to discriminate between two subtypes of SZ subjects. In dysfunction in the immune response in dorsolateral prefrontal this line, a second meta-analysis study from postmortem DLPFC cortex in schizophrenia, suggesting that this region could be an found that a subset of SZ subjects showed differential expression of area susceptible to damage in schizophrenia subjects due to a failure inflammatory molecules in comparison to another subset of SZ in immune protection in this area. These results could provide a set subjects and to unaffected subjects (Childers et al., 2022). Our results of proteins related to dysfunction in the immune response or showed a limited set of proteins involved in the immune response involved in negative symptoms in schizophrenia. that could be related to a sub-set of SZ subjects that are susceptible to the proinflammatory cytokines due to the aberrant molecular mechanism of the immune response. Alternately, the mixture Data availability statement presence of two SZ subtypes could explain why there were a reduced number of altered proteins detected. The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material. 4.4 Limitations The use of human postmortem brain constitutes a useful tool to Ethics statement dissect the molecular pathways disrupted in psychiatric disorders. However, it has limitations. First, confounding factors including age, The studies involving human participants were reviewed and postmortem delay, and pH must be carefully explored. In our study, approved by Institutional Ethics Committee of Parc Sanitari Sant we did not find any association between these variables and the Joan de Déu. Written informed consent for participation was not significantly altered proteins in the DLPFC. Second, the possible required for this study in accordance with the national legislation effect of laterality in our samples cannot be ruled out since all of SZ and the institutional requirements. samples were from left hemisphere. However, we did not find any difference in the proteomic profile when comparing 10 right and 10 left hemisphere samples of control healthy individuals. Third, Author contributions patients with chronic schizophrenia had been treated with long- term, heterogeneous antipsychotics medications. However, we did Author AV-M: acquired and analyzed data, interpreted the results not find any significant association with the chlorpromazine and co-wrote the first draft of the manuscript. Author RR-M: equivalent doses. Fourth, our study only included men, who do designed, conducted and analyzed the proteomic experiments and not represent a real population of this disorder. There was no brain contributed to the discussion of the results. EV: contribute to sample sample availability of females for SZ group in this biobank. Future processing, clinical data analysis and discussion of the results. Author studies including both genders are needed to elucidate the proteomic JV: supervised the proteomic analyses and contributed to the changes in females compared to males. Fifth, our study includes discussion of the results. Author BR: designed the study, samples from different brain biobanks, however, no substantial contributed to the interpretation and discussion of the results, and differences in clustering analysis were observed between centers. co-wrote the first draft of the manuscript. All authors have read and Sixth, our study includes institutionalized SZ subjects and non- agreed to the published version of the manuscript. institutionalized unaffected subjects. The lowest social contact of SZ subjects institutionalized could contribute to the cognitive alteration (Maia, 2010). A study has shown that differences in subcortical Funding alterations between institutionalized and non-institutionalized (Zhao et al., 2022) However, the differences in the cortical This research was funded by a Miguel Servet grants (MS16/ neuronal circuits and behavior between SZ subjects 00153-CP16/00153- CPII21/00008) and PI18/00213 to BR Frontiers in Pharmacology 11 frontiersin.org Vera-Montecinos et al. 10.3389/fphar.2023.1003557 financed and integrated into the National R + D + I and funded by Conflict of interest the Instituto de Salud Carlos III (Spanish Ministry of Health)— General Branch Evaluation and Promotion of Health The authors declare that the research was conducted in the Research–and the European Regional Development Fund absence of any commercial or financial relationships that could be (ERDF). This work was also supported by CONICYT- construed as a potential conflict of interest. Doctorado Becas Chile 2015 (72160426 grant number) to AV- The handling editor JRC declared a shared consortium M, Predoctoral Fellowship Program from ISCIII (PFIS) FI19/ [National Network of Biomedicine from Spain (CIBERSAM)] 00080 to EV and CIBERSAM (Spanish Ministry of Economy, with the author(s) at the time of review. Industry and Competitiveness, Institute of Health Carlos III). The proteomics work was supported by NIH grants R35 GM119536 and R01 NS098329 to JV. All authors declare Publisher’s note that they have no competing financial and/or non-financial interests. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product Acknowledgments that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 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