Proteomic Profiling Reveals Specific Molecular Hallmarks of the Pig Claustrum
https://ror.org/03ad39j10grid.5395.a0000 0004 1757 3729Department of Veterinary Sciences, University of Pisa, Pisa, Italy
https://ror.org/03ad39j10grid.5395.a0000 0004 1757 3729Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy
grid.412451.70000 0001 2181 4941Department of Medical, Oral and Biotechnological Sciences, University G. D’Annunzio of Chieti-Pescara, Chieti, Italy
Interuniversitary Consortium for Engineering and Medicine, COIIM, Campobasso, Italy
https://ror.org/03ad39j10grid.5395.a0000 0004 1757 3729Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy
https://ror.org/0005w8d69grid.5602.10000 0000 9745 6549School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, Italy
https://ror.org/03ad39j10grid.5395.a0000 0004 1757 3729Department of Pharmacy, University of Pisa, Pisa, Italy
https://ror.org/0005w8d69grid.5602.10000 0000 9745 6549School of Pharmacy, University of Camerino, Camerino, Italy
Abstract
The present study, employing a comparative proteomic approach, analyzes the protein profile of pig claustrum (CLA), putamen (PU), and insula (IN). Pig brain is an interesting model whose key translational features are its similarities with cortical and subcortical structures of human brain. A greater difference in protein spot expression was observed in CLA vs PU as compared to CLA vs IN. The deregulated proteins identified in CLA resulted to be deeply implicated in neurodegenerative (i.e., sirtuin 2, protein disulfide-isomerase 3, transketolase) and psychiatric (i.e., copine 3 and myelin basic protein) disorders in humans. Metascape analysis of differentially expressed proteins in CLA vs PU comparison suggested activation of the α-synuclein pathway and L1 recycling pathway corroborating the involvement of these anatomical structures in neurodegenerative diseases. The expression of calcium/calmodulin-dependent protein kinase and dihydropyrimidinase like 2, which are linked to these pathways, was validated using western blot analysis. Moreover, the protein data set of CLA vs PU comparison was analyzed by Ingenuity Pathways Analysis to obtain a prediction of most significant canonical pathways, upstream regulators, human diseases, and biological functions. Interestingly, inhibition of presenilin 1 (PSEN1) upstream regulator and activation of endocannabinoid neuronal synapse pathway were observed. In conclusion, this is the first study presenting an extensive proteomic analysis of pig CLA in comparison with adjacent areas, IN and PUT. These results reinforce the common origin of CLA and IN and suggest an interesting involvement of CLA in endocannabinoid circuitry, neurodegenerative, and psychiatric disorders in humans.
Supplementary Information
The online version contains supplementary material available at 10.1007/s12035-023-03347-2.
Introduction
Claustrum (CLA) is a thin sheet of gray matter located in the forebrain between the insula (IN) and the putamen (PU). Although its precise role remains a matter of debate, CLA is thought to be implicated in a variety of functions such as attention [1–3], impulsivity [4], regulation of sleep [5–7], and consciousness [8]. Moreover, a recent new hypothesis indicates CLA as a possible limbic–sensory–motor interface [9]. The pivotal role of CLA in these functions is supported by its extensive, reciprocal connectivity with the entire neocortex [10–13].
Recently, a wealth of data has been accumulated on the role of CLA in different neurological disorders. Changes in CLA morphology are described in Parkinson’s disease (PD), Alzheimer’s disease (AD), autism, schizophrenia, and depressive disorders. However, there is a lack of information regarding the involvement of CLA in these disorders, at molecular level [14].
CLA origin is another puzzling problem, and morphogenetic and neurochemical similarities led some authors to postulate a common origin for CLA and the insular cortex [15, 16]; on the other hand, a subcortical origin has also been reported [17–19]. Furthermore, according to the hybrid ontogeny theory, CLA is considered as an intermediary between the cortical plate and corpus striatum [17].
Advances on the knowledge of CLA role in different species have essentially been obtained using immunohistochemical, physiological, and behavioral methods [1, 3, 10–13, 20–23]; a different approach, employing a proteomic analysis, has been used to establish the anatomical definitions of rat CLA [24]. Recently, a single-cell integrating transcriptomic and circuit-level approach, advanced by Erwin et al. [25], identified two excitatory CLA neuron subtypes that are molecularly distinguishable from the adjacent cortex.
The position of this structure, encased as it is between the external and extreme capsule, renders measurements, characterizations, and manipulations difficult; this is particularly true for rodent CLA where the extreme capsule is not clearly defined [26]. Among mammals, the pig brain is an interesting model whose key translational features are its similarities with cortical and subcortical structures of human brain [27, 28].
Furthermore, the most caudal part of pig CLA is characterized by a wide enlargement which is well delineated and separated from the adjoining structures [29], and this allows for isolation and sampling of CLA, IN, and PU without mixing tissues from different structures.
Here, we used a proteomic approach to define the protein profile of pig CLA and compare it with those of IN and PU. We then sought to reveal specific molecular hallmarks of pig CLA to better understand its function and origin, as well as possible implications of our findings in relation to human neurological diseases.
Materials and Methods
Animals and Tissue Samples
The brains of eight adult pigs (Sus scrofa domesticus) were removed immediately after commercial slaughtering at a local abattoir (Desideri Luciano SPA, Via Abruzzi, 2, 56025 Pontedera PI, Tuscany, Italy). Animals were treated according to the European Regulation (CE1099/2009) concerning animal welfare during the commercial slaughtering process and were constantly monitored under mandatory official veterinary medical care. All the animals were in good body conditions and considered free of pathologies by the veterinary medical officer responsible for the health and hygiene of the slaughterhouse. The brains, extracted within 15 min of death, were cut into transverse blocks (0.5 cm thick) containing CLA, PU, and IN in their rostro-caudal extent. Tissues of the right hemisphere were fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) at pH 7.4 and processed for paraffin embedding. From the left hemisphere, specimens of CLA, PU, and IN (Fig. 1) were quickly extracted under a stereomicroscope, snap-frozen in liquid nitrogen, and stored at − 80 °C until use.
Protein Extractions and Proteomic Analysis
Specimens of CLA (n = 3), IN (n = 3), and PU (n = 3) were weighted, minced with scissors, and homogenized in 5 vol (w/V) of rehydration solution (7 M urea, 2 M thiourea, 4% 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS), 60 mM dithiothreitol (DTT) containing protease inhibitors (Merck KGaA, Darmstadt, Germany)) using a Teflon-glass homogenizer (10 strokes). The resulting homogenates were incubated for 1 h at room temperature (RT) with occasional stirring. Thereafter, samples were centrifuged at 17,000 g for 15 min at RT to eliminate insoluble materials. The protein content was measured by the RC/DC assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin as standard.
Two-dimensional electrophoresis (2-DE) was performed essentially according to Ciregia et al. [30]. Briefly, isoelectrofocusing (IEF) was carried out using a pH 3–10 nonlinear (NL) gradient. Two hundred μg of proteins was filled up to 400 μL in rehydration solution containing 1% IPG buffer at pH 3–10 NL and 0.8% Pharmalyte. Immobiline DryStrip gels were rehydrated overnight in the sample and then transferred to the Ettan IPGphor II (GE Healthcare Europe, Uppsala, Sweden) apparatus. The second dimension (SDS-PAGE) was carried out by transferring the proteins to 12% polyacrylamide gels, and at the end of the second dimension, gels were stained with 1 μM bathophenanthroline disulfonate-bis(2,2′-bipyridine)[Ru(II)] tetrahydrate (RuBP) (Cyanagen Srl, Bo, Italy) staining [31]. Images were acquired using ImageQuant LAS 4010 (GE HealthCare) and analyzed by SameSpots (V4.1, TotalLab, Newcastle Upon Tyne, UK) software which generates 2-DE analyses which are robust and accurate. Briefly, the gels were aligned to place all spots in exactly the same location, and then, the spot detection produced a complete data set since all gels contain the same number of spots, each matched to its corresponding spot on all gels. After 2-DE gel alignment and subsequent spot detection, the software calculated background-corrected abundance, by determining the lowest intensity value of the image pixels outside [30, 31].
In-Gel Digestion and Mass Spectrometry
The gel pieces were digested as reported by Giusti et al. [32]. Samples were analyzed by LC-MS/MS as previously described [33] using a Proxeon EASY-nLC II (Thermo Fisher Scientific, Milan, Italy) chromatographic system coupled to a maXis HD UHR-TOF (Bruker Daltonics GmbH, Bremen, Germany) mass spectrometer. Briefly, peptides were loaded on the EASY-Column C18 trapping column (2 cm L, 100 μm ID, 5 μm ps; Thermo Fisher Scientific), and then separated on an Acclaim PepMap 100 C18 (25 cm L, 75 μm ID, 5 μm ps; Thermo Fisher Scientific) nanoscale chromatographic column at a flow rate of 300 nL/min and with a standard gradient from 3 to 35% of acetonitrile in 15 min. The mass spectrometer was equipped with a nanoESI spray source and operated in positive ion polarity and auto MS/MS mode (data-dependent acquisition (DDA)), using N2 as collision gas for collision-induced dissociation (CID) fragmentation. In-source reference lock mass (1221.9906 m/z) was acquired online throughout the runs.
Raw data were processed with DataAnalysis v. 4.2 to apply the lock mass calibration and then loaded in PEAKS Studio v7.5 software (Bioinformatic Solutions, Inc., Waterloo, Canada) using the “correct precursor only” option. The mass lists were searched against the NeXtProt database Sus scrofa domesticus (downloaded December 2018 and containing 42,184 entries). Carbamidomethylation of cysteines was selected as fixed modification, and oxidation of methionines, deamidation of asparagine and glutamine, as well as N terminus and lysine acetylation were set as variable modifications. Nonspecific cleavage was allowed to the one end of the peptides, with a maximum of 2 missed cleavages and 2 variable post-translational modifications (PTMs) per peptide; 10 ppm and 0.05 Da were set as the highest error mass tolerances for precursors and fragments, respectively; − 10logP threshold for peptide-spectrum matches (PSMs) was manually set from 15 to 35, in order to obtain a false discovery rate (FDR) value < 0.1% for both PSM and peptide sequences. For protein ID, the FDR value was < 0.1%.
Western Blot Analysis
Aliquots (5 μg of proteins) of protein extracts from different brain regions (CLA, n = 5; IN, n = 5; PU, n = 5) were mixed with Laemmli solution, resolved on 4–16% polyacrylamide gels (Mini-PROTEAN® Precast Gels, Bio-Rad, Hercules, CA, USA) using a Mini-PROTEAN Tetra Cell (Bio-Rad), and transferred onto 0.2-μm nitrocellulose membranes using a Trans-Blot Turbo transfer system (Bio-Rad) (Ciregia et al., 2013). Membranes were blocked in TBST (50 mM Tris [pH 7.5], 150 mM NaCl, and 0.1% Tween 20), supplemented with 3% non-fat dry milk for 1 h at room temperature, and subsequently probed with the following primary antibodies: a mouse monoclonal anti-calcium/calmodulin-dependent protein kinase II-α (CaMKII-α, dilution 1:1000, 6G9; Cell Signaling Technology, Inc., Danvers, MA, USA) and a rabbit monoclonal anti-dihydropyrimidinase like 2 (DPYL2, alias collapsin response mediator protein 2 (CRMP-2), D8L6V, dilution 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA) in TBST/blocking solution overnight at 4 °C. Membranes were then incubated with the secondary antibody for 1 h at room temperature: HRP-goat anti-rabbit (Enzo Life Sciences, Inc., NY, USA) and HRP-goat anti-mouse (PerkinElmer, Inc., MA, USA) secondary antibodies were used at 1:10,000 dilution. Immunoblots were developed using the enhanced chemiluminescence (ECL) detection system, the chemiluminescent images were acquired using LAS 4010 (GE HealthCare), and the immunoreactive specific bands were quantified using ImageQuant L software. To normalize the optical density (OD) of immunoreactive bands, the OD of whole proteins was measured and, immediately after the electroblot, membranes were stained with 1 μM RuBPS [34]. Differences of protein expression levels among different samples were assessed using a paired Student’s t test (p < 0.05).
Immunofluorescence
Immunofluorescence was performed on serial 5-μm sections using a mouse monoclonal anti-CaMKII-α (dilution 1:2000, 6G9; Cell Signaling Technology, Inc., Danvers, MA, USA) or a rabbit monoclonal anti-collapsin response mediator protein 2 (DPYL2 alias CRMP-2, dilution 1:200, D8L6V; Cell Signaling Technology, Inc., Danvers, MA, USA). Epitope retrieval was carried out at 120 °C in a pressure cooker for 5 min with a Tris/EDTA buffer, pH 9.0. Sections were blocked for 1 h with 5% normal horse serum (PK-7200, Vector Labs) in PBS and then incubated overnight at 4 °C in a solution of anti-CaMKII-α or anti-CRMP-2 in PBS containing 2% normal horse serum and 0.05% Triton X-100. Sections were then rinsed in PBS (3 × 10 min), incubated for 1 h at room temperature with DyLight 488 anti-mouse IgG (5 μg/mL, DI-2488; Vector Labs., Burlingame, CA, USA) or anti-rabbit IgG (5 μg/mL, DI-1088; Vector Labs., Burlingame, CA, USA). Finally, sections were washed with PBS and coverslipped with Vectashield medium containing 4′,6-diamidino-2-phenylindole (DAPI) (H-1500, Vector Labs). The specificity of immunohistochemical staining was tested using negative control sections, in which the primary or secondary antibody was replaced with PBS or non-immune serum. Under these conditions, nonspecific staining was absent.
Microphotographs were collected under a Nikon Ni-E light microscope (Nikon Instruments, Spa Calenzano, Florence, Italy), fully equipped for fluorescence acquisition, connected to a personal computer via Nikon digital image processing software (Digital Sight DS-U1, NIS-Elements BR 4.51.00 software). CLA and adjoining structures were identified according to a stereotaxic atlas [35].
Statistical Analysis and Bioinformatics
All experiments were performed at least in triplicate, and resulting values are expressed as mean ± standard error.
In 2-DE experiments, a comparison among the different brain areas was performed, and the significance of the differences of normalized volume for each spot was calculated by the software SameSpots including the analysis of variance (ANOVA) test. Therefore, the protein spots that exhibited ratio ≥ 1.2 or ≤ 0.83, p value ≤ 0.05, and q value ≤ 0.05 were taken into consideration for further identification by nanoLC-MS/MS. Volcano plot and statistical analysis on individual proteins was performed using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA). In western blot analysis, paired Student’s t test was used to compare differences among different brain areas, and differences with a p value < 0.05 were considered statistically significant.
The list of genes obtained from proteins found differentially expressed in different comparisons was analyzed using Metascape [36]. Metascape utilizes the well-adopted hypergeometric test [37] and the Benjamini–Hochberg p value correction algorithm [38] to identify all ontology terms that contain a statistically greater number of genes in common with an input list than expected by chance. Metascape automatically clusters enriched terms into non-redundant groups and chooses the most significant (lowest p value) term within each cluster to represent the cluster in heat map representations. Moreover, given a list of proteins, it automatically extracts a protein interaction network formed by these candidates. Finally, a circos plot showing how genes from the input gene lists overlap among different brain areas was generated.
Proteins found differentially expressed in each comparison were functionally analyzed using the Ingenuity Pathways Analysis (IPA; Qiagen, Redwood City, USA; www.qiagen.com/ingenuity, Build version: 321501M, Content version: 21249400) with the aim to determine the predominant functional relationships among proteins based on known associations in the literature. A comparison of the different analyses was created, and the upstream regulators, molecular functions, and human diseases whose activity appears to change in a significant manner according to the activation z-score value were shown. Heat map was build using NG-CHM GUI 2.20.2 software [39].
Results
Proteomic Analysis
Two-dimensional electrophoresis was carried out to compare protein maps of different brain areas, and representative images of protein profile of CLA, IN, and PU are shown in Fig. 2A–C, respectively. Three different comparisons were performed: CLA vs IN, CLA vs PU, and IN vs PU. A greater difference of protein expression was observed between PU and the other two brain areas; in particular, 88 and 105 differentially expressed protein spots resulted from CLA vs PU and IN vs PU comparisons, respectively. On the contrary, minor significant differences in protein expression were observed in the comparison CLA vs IN. Volcano plots were constructed to graphically represent fold changes of protein expression (Fig. 3).
In Fig. 4, the circos plot shows how genes from the input gene lists obtained from different protein profile comparisons overlapped, and the protein divergence of CLA and IN when compared to PU placed both CLA and IN apart from PU with IN being the farthest. All spots showing an increase or decrease value ≥ 1.2 were subjected to nanoLC-ESI-MS/MS analysis and identified. Tables 1, 2, and 3 show the list of identified proteins together with their MW, pI, peptides and coverage values of MS/MS, ratio, and p values in three different comparisons. Twenty-one protein spots were identified in the CLA vs IN comparison of which only six proteins, Ras-related protein Rab-3A (RAB3A), ATP-citrate synthase (ACLY), methylcrotonyl-CoA carboxylase 1 (MCCC1), vesicle-fusing ATPase (NSF), copine 3 (CPN3), and myelin basic protein (MBP), resulted to be overexpressed in the CLA (Fig. 5).Spot # ID Protein name Gene Coverage Peptides Unic MW pI ANOVA p value Ratio CLA/PU 364 F1RST0 HSPH1 heat shock protein 70 family HSPH1 28 25 25 96,699 5.29 0.002 1.6 397 F1SUF2 Hexokinase HK1 36 33 24 83,569 6.96 0.016 1.4 407 F1RWX8 Ubiquitin-like modifier-activating enzyme 1 UBA1 15 16 16 117,757 5.55 0.00004 1.7 415 F1SDW6 Oxoglutarate dehydrogenase like OGDHL 26 28 21 115,227 6.39 0.026 1.7 471 F1RRW8 Dynamin 1 DNM1 39 43 10 97,328 7.97 0.037 1.9 476 F1RRW8 Dynamin 1 DNM1 39 38 11 97,328 7.97 0.012 1.5 490 F1SIH8 Transitional endoplasmic reticulum ATPase VCP 46 45 44 89,431 5.44 0.00004 1.4 505 F1SFG7 Dynamin-like GTPase, mitochondrial OPA1 33 25 25 78,120 8.07 0.034 1.9 637 F1RRS3 Vesicle-fusing ATPase NSF 7 5 5 83,585 6.52 0.007 1.7 655 F1SII4 Glycyl-tRNA synthetase GARS 19 18 18 83,260 7.02 0.02 1.9 665 F1RRS3 Vesicle-fusing ATPase NSF 10 7 7 83,500 6.52 0.004 2.3 665 I3LT90 Methylcrotonyl-CoA carboxylase 1 MCCC1 3 2 2 80,400 6.34 0.004 2.3 673 F1RRS3 Vesicle-fusing ATPase NSF 16 12 12 85,585 6.52 0.011 2 681 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 20 14 14 73,531 5.94 0.014 1.6 684 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 40 25 23 73,531 5.94 0.008 1.9 690 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 20 14 14 73,531 5.94 0.004 2.5 751 C3RZ98 Protein arginine N-methyltransferase 5 PRMT5 8 6 6 72,614 5.88 0.005 1.8 765 F1RS11 Syntaxin-binding protein 1 STXBP1 30 23 23 68,749 6.32 0.031 1.3 777 A8U4R4 Transketolase tkt 24 20 20 67,838 7.21 0.007 1.7 817 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 37 27 26 73,531 5.94 0.003 1.7 819 F1RS11 Syntaxin-binding protein 1 STXBP1 31 24 24 68,749 6.32 0.026 1.5 820 I3LBY0 Coatomer subunit delta ARCN1 14 7 7 57,250 5.69 0.008 1.6 824 I3LNG8 Stress-induced phosphoprotein 1 STIP1 25 17 17 47,879 6.36 0.00053 1.6 824 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 9 6 6 73,531 5.94 0.00053 1.6 826 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 37 30 23 73,531 5.94 0.01 1.5 827 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 45 27 23 73,531 5.94 0.04 1.8 831 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 44 57 53 73,531 5.94 0.00005 1.9 858 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 11 4 3 73,531 5.94 0.007 1.9 878 I3LN38 Collapsin response mediator protein 1 CRMP-1 26 13 11 58,144 6.47 0.025 1.6 878 F1RGA9 Coronin CORO1C 12 6 6 58,929 6.48 0.025 1.6 884 I3LR32 CCT-epsilon CCT5 33 23 23 54,518 5.57 0.002 1.4 901 I3LDA5 EH domain containing 4 EHD4 11 7 3 42,252 6.32 0.002 2.4 918 F1RXD5 Copine 3 CPNE3 9 5 5 59,644 5.57 0.00011 1.7 933 F6QA08 Protein disulfide-isomerase PDIA3 45 28 28 56,859 5.93 0.00031 1.4 940 I3LR17 Coronin 1A CORO1A 27 16 16 43,411 6.12 0.008 2.3 964 F1RR02 Glial fibrillary acidic protein GFAP 47 26 26 49,437 5.65 0.00001 1.8 964 F1RMZ8 ATPase H+ transporting V1 subunit B2 ATP6V1B2 17 9 9 56,613 5.57 0.00001 1.8 971 I3LGA1 WD repeat domain 37 WDR37 11 5 5 54,116 6.95 0.032 1.8 1007 D0G0C8 Chaperonin containing TCP1, subunit 2 (beta) CCT2 27 16 16 57,444 6.09 0.036 1.8 1012 F1ST01 Selenium-binding protein 1 SELENBP1 29 17 17 52,534 6.54 0.016 1.9 1012 I3LKF3 Fascin FSCN1 26 14 14 53,234 8.19 0.016 1.9 1015 I3LKF3 Fascin FSCN1 34 16 16 53,234 8.19 0.016 1.7 1015 F1ST01 Selenium-binding protein 1 SELENBP1 9 3 3 52,534 6.54 0.016 1.7 1017 F1ST01 Selenium-binding protein 1 SELENBP1 10 5 5 52,534 6.54 0.021 1.9 1057 F1RR02 Glial fibrillary acidic protein GFAP 55 37 36 49,437 5.65 0.00008 2.2 1062 F1RR02 Glial fibrillary acidic protein GFAP 55 34 33 49,437 5.65 0.00029 2 1077 F1SEN2 Glutamate dehydrogenase 1, mitochondrial GLUD1 19 10 10 61,308 8.02 0.014 1.5 1131 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 17 10 9 42,639 6.61 0.01 1.5 1135 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 17 10 10 42,639 6.61 0.01 1.7 1137 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 19 11 2 42,639 6.61 0.001 1.9 1146 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 19 11 2 42,639 6.61 0.026 1.8 1146 I3LK59 2-Phospho-d-glycerate hydro-lyase ENO1 9 4 4 38,082 0.026 1.8 1153 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 23 12 2 42,639 6.61 0.013 1.5 1170 F1RUK8 Rab GDP dissociation inhibitor GDI2 52 24 21 50,327 5.78 0.009 1.5 1187 F1RR48 SH3 domain containing GRB2 like, endophilin B2 SH3GLB2 13 6 6 43,489 5.73 0.00039 1.6 1194 Q29387 Elongation factor 1-gamma EEF1G 31 21 21 49,624 6.16 0.019 2 1195 A6M928 Eukaryotic translation initiation factor 4A isoform 1 EIF4A1 22 9 3 46,154 5.32 0.002 2.8 1195 A6M930 Eukaryotic translation initiation factor 4A isoform 2 EIF4A2 21 9 3 46,502 5.33 0.002 2.8 1276 F1RK10 Succinate-CoA ligase (ADP-forming) subunit beta, mitochondrial SUCLA2 31 18 18 50,304 5.86 0.029 1.3 1315 F1SNE5 SH3 domain containing GRB2 like 2, endophilin A1 SH3GL2 37 17 17 37,614 5.26 0.00048 2.3 1336 K7GM43 Septin 5 SEPT5 26 13 13 43,787 6.55 0.006 1.8 1363 P46410 Glutamine synthetase GLUL 9 5 5 42,030 6.28 0.001 1.7 1376 F1RZB5 Tropomodulin 2 TMOD2 24 12 12 39,693 5.17 0.009 1.6 1414 F1RJ25 Fructose-bisphosphate aldolase ALDOC 47 25 25 39,377 6.21 0.02 1.6 1421 F1RL02 Mitogen-activated protein kinase MAPK1 39 15 14 37,974 6.56 0.042 1.6 1455 F1RF18 G protein subunit alpha o1 GNAO1 13 6 4 40,078 5.10 0.00037 1.4 1457 P00506 Aspartate aminotransferase, mitochondrial GOT2 10 6 6 47,436 9.14 0.002 2.1 1464 Q6QAQ1 Actin, cytoplasmic 1 ACTB 11 6 6 41,737 5.29 0.02 1.5 1479 Q9TV69 Trans-1,2-dihydrobenzene-1,2-diol dehydrogenase DHDH 27 10 10 36,527 6.34 0.003 1.6 1508 I3L8A1 NAD-dependent protein deacetylase SIRT2 20 10 9 35,964 7.47 0.001 2.1 1520 F1RIK3 Acyl-CoA thioesterase 7 ACOT7 8 4 4 40,189 8.06 0.003 1.6 1524 F1SEX0 Dimethylarginine dimethylaminohydrolase 1 DDAH1 25 5 5 20,779 5.36 0.002 1.8 1524 F1SUE3 Pyrophosphatase (inorganic) 1 PPA1 10 3 3 32,790 5.44 0.002 1.8 1542 F1RPC8 Crystallin mu CRYM 44 23 23 33,508 5.16 0.004 1.8 1580 P00336 l-Lactate dehydrogenase B chain LDHB 37 18 18 36,612 5.57 0.00026 1.5 1592 F2Z4Z8 G protein subunit beta 2 GNB2 23 8 7 33,758 5.60 0.00082 1.4 1627 I3LSK5 G protein subunit beta 1 GNB1 35 13 13 37,331 5.60 0.00038 2 1654 F1RM45 Apolipoprotein E APOE 20 7 7 36,665 5.92 0.006 2.1 1655 F1RM45 Apolipoprotein E APOE 12 4 4 36,665 5.92 0.002 1.6 1660 F1SGH5 Pyruvate dehydrogenase E1 component subunit beta PDHB 52 17 17 39,273 6.20 0.002 1.5 1660 F1RM45 Apolipoprotein E APOE 22 6 6 36,665 5.92 0.002 1.5 1757 Q06A94 Heterogeneous nuclear ribonucleoprotein A1 HNRNPA1L2 28 8 8 34,196 9.27 0.003 2.2 1826 P62258 14-3-3 protein epsilon YWHAE 69 38 38 29,174 4.63 0.007 1.4 2013 Q45FY6 Hypoxanthine-guanine phosphoribosyltransferase HPRT1 57 14 14 24,555 6.30 0.014 2.6 2029 Q6SEG5 Ubiquitin carboxyl-terminal hydrolase isozyme L1 UCHL1 66 38 37 24,859 5.22 0.00053 1.5 2100 Q06AU3 Ras-related protein Rab-3A RAB3A 15 3 3 24,970 4.85 0.006 0.45 2165 Q6SEG5 Ubiquitin carboxyl-terminal hydrolase isozyme L1 UCHL1 65 38 7 24,859 5.22 0.011 1.6 2165 Q5E946 Protein DJ-1 PARK7 56 16 16 20,035 6.84 0.011 1.6 2402 A8QW48 Beta-synuclein SNCB 25 5 4 14,115 4.46 0.002 1.6 2467 Q3I5G7 Alpha-synuclein SNCA 53 8 8 14,520 4.62 0.002 1.7 2497 Q8WNW3 Junction plakoglobin Jup 27 20 20 81,850 5.75 0.0003 2.4 Spot # ID Protein name Gene Coverage Peptides Unic MW pI ANOVA p value Ratio CLA/IN 345 F1S0N2 ATP-citrate synthase ACLY 21 11 11 57,558 7.12 0.025 2.1 397 F1SUF2 Hexokinase HK1 36 33 24 83,569 6.96 0.01 0.69 546 F1SCS1 DEAD-box helicase 1 DDX1 14 10 10 77,025 6.80 0.013 0.588 665 F1RRS3 Vesicle-fusing ATPase NSF 10 7 7 83,500 6.52 0.005 2.1 665 I3LT90 Methylcrotonyl-CoA carboxylase 1 MCCC1 3 2 2 80,400 6.34 0.005 2.1 918 F1RXD5 Copine 3 CPNE3 9 5 5 59,644 5.57 0.03 1.5 959 I3L7D3 Synapsin II SYN2 31 18 16 51,020 8.84 0.004 0.625 959 Q19PY3 tRNA-splicing ligase RtcB homolog RTCB 31 17 17 55,238 6.77 0.004 0.625 959 I3LK72 Acyl-CoA synthetase family member 3 ACSF3 27 13 13 48,212 8.71 0.004 0.625 959 F1SLF6 Chaperonin containing TCP1, subunit 7 (Eta) CCT7 24 14 14 59,471 6.74 0.004 0.625 960 I3L7D3 Synapsin II SYN2 12 6 6 51,020 8.84 0.045 0.625 960 F1SD97 Phenylalanyl-tRNA synthetase alpha subunit FARSA 18 10 10 57,628 7.85 0.045 0.625 1131 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 17 10 9 42,639 6.61 0.015 0.526 1137 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 19 11 2 42,639 6.61 0.021 0.476 1143 I3LK59 2-Phospho-d-glycerate hydro-lyase ENO1 34 13 10 38,082 6.43 0.029 0.435 1143 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 21 12 3 42,639 6.61 0.029 0.435 1154 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 29 15 4 42,639 6.61 0.008 0.664 1187 F1RR48 SH3 domain containing GRB2 like, endophilin B2 SH3GLB2 13 6 6 43,489 5.73 0.023 0.714 1464 Q6QAQ1 Actin, cytoplasmic 1 ACTB 11 6 6 41,737 5.29 0.034 0.714 1542 F1RPC8 Crystallin mu CRYM 44 23 23 33,508 5.16 0.003 0.667 1592 F2Z4Z8 G protein subunit beta 2 GNB2 23 8 7 33,758 5.60 0.042 0.833 1815 Q9GZU5 Nyctalopin NYX 2 2 2 52,000 9.10 0.026 0.588 1826 P62258 14-3-3 protein epsilon YWHAE 69 38 38 29,174 4.63 0.044 0.833 2100 Q06AU3 Ras-related protein Rab-3A RAB3A 15 3 3 24,970 4.85 0.018 2.7 2402 A8QW48 Beta-synuclein SNCB 25 5 4 14,115 4.46 0.02 0.769 2467 Q3I5G7 Alpha-synuclein SNCA 53 8 8 14,520 4.62 0.013 0.769 2487 P81558 Myelin basic protein MBP 27 4 4 18,486 11.28 0.00061 2.3 Spot # ID Protein name Gene Coverage Peptides Unic MW pI ANOVA p value Ratio IN/PU 332 F1SCV1 Gamma-tubulin complex component TUBGCP2 1 2 1 102,582 6.26 0.031 2.1 345 F1S0N2 ATP-citrate synthase ACLY 21 11 11 57,558 7.12 0.034 0.45 355 I3L8X6 Amphiphysin AMPH 19 15 15 71,840 4.56 0.006 2.2 364 F1RST0 Heat shock 110 kDa protein HSPH1 28 25 25 96,699 5.29 0.005 2 365 F1RST0 Heat shock 110 kDa protein HSPH1 17 15 15 96,699 5.29 0.001 2 394 F1SML4 Staphylococcal nuclease and Tudor domain containing 1 SND1 8 5 4 66,087 6.72 0.038 2 397 F1SUF2 Hexokinase HK1 36 33 24 83,569 6.96 0.00085 2.1 407 F1RWX8 Ubiquitin-like modifier-activating enzyme 1 UBA1 15 16 16 117,757 5.55 0.003 2.4 415 F1SDW6 Oxoglutarate dehydrogenase like OGDHL 26 28 21 115,227 6.39 0.032 2.2 455 F1RI39 Actinin alpha 4 ACTN4 27 20 20 101,837 5.23 0.002 2.5 471 F1RRW8 Dynamin 1 DNM1 39 43 10 97,328 7.97 0.011 2.6 476 F1RRW8 Dynamin 1 DNM1 39 38 11 97,328 7.97 0.001 2.1 490 F1SIH8 Transitional endoplasmic reticulum ATPase VCP 46 45 44 89,431 5.44 0.02 1.5 505 F1SFG7 OPA1, mitochondrial dynamin-like GTPase OPA1 33 25 25 78,120 8.07 0.01 2.9 546 F1SCS1 DEAD-box helicase 1 DDX1 14 10 10 77,025 6.80 0.00034 2.1 630 I3L8C5 Heat shock protein family A (Hsp70) member 12A HSPA12A 20 14 14 74,809 6.18 0.01 2 637 F1RRS3 Vesicle-fusing ATPase NSF 7 5 5 83,585 6.52 0.007 2.7 655 F1SII4 Glycyl-tRNA synthetase GARS 19 18 18 83,260 7.02 0.012 2.6 681 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 20 14 14 73,531 5.94 0.005 2.3 684 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 40 25 23 73,531 5.94 0.007 2.9 690 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 20 14 14 73,531 5.94 0.002 4.4 720 F1RPU0 Glycerol-3-phosphate dehydrogenase GPD2 38 34 34 80,921 6.54 0.022 2.1 724 P28491 Calreticulin CALR 36 23 23 48,288 4.32 0.01 1.5 751 C3RZ98 Protein arginine N-methyltransferase 5 PRMT5 8 6 6 72,614 5.88 0.01 1.5 765 F1RS11 Syntaxin-binding protein 1 STXBP1 30 23 23 68,749 6.32 0.002 1.4 799 O75083 WD repeat-containing protein 1 WDR1 16 11 2 66,194 6.17 0.001 1.7 817 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 37 27 26 73,531 5.94 0.002 1.9 819 F1RS11 Syntaxin-binding protein 1 STXBP1 31 24 24 68,749 6.32 0.002 1.9 820 I3LBY0 Coatomer subunit delta ARCN1 14 7 7 57,250 5.69 0.028 2.1 825 I3LNG8 Stress-induced phosphoprotein 1 STIP1 35 21 21 47,879 6.36 0.038 2.1 825 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 3 2 2 73,531 5.94 0.038 2.1 826 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 37 30 23 73,531 5.94 0.029 1.7 827 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 45 27 23 73,531 5.94 0.012 2.7 831 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 44 57 53 73,531 5.94 0.001 2.1 858 I3LJE2 Dihydropyrimidinase like 2 DPYSL2 11 4 3 73,531 5.94 0.014 2 878 I3LN38 Collapsin response mediator protein 1 CRMP-1 26 13 11 58,144 6.47 0.01 2.2 878 F1RGA9 Coronin CORO1C 12 6 6 58,929 6.48 0.01 2.2 884 I3LR32 CCT-epsilon CCT5 33 23 23 54,518 5.57 0.004 1.5 901 I3LDA5 EH domain containing 4 EHD4 11 7 3 42,252 6.32 0.007 3 933 E1CAJ5 Protein disulfide-isomerase PDIA3 45 28 28 56,859 5.93 0.049 1.4 940 I3LR17 Coronin 1A CORO1A 27 16 16 43,411 6.12 0.01 3.1 959 I3L7D3 Synapsin II SYN2 31 18 16 51,020 8.84 0.002 2.1 959 Q19PY3 tRNA-splicing ligase RtcB homolog RTCB 31 17 17 55,238 6.77 0.002 2.1 959 I3LK72 Acyl-CoA synthetase family member 3 ACSF3 27 13 13 48,212 8.71 0.002 2.1 959 F1SLF6 Chaperonin containing TCP1, subunit 7 (Eta) CCT7 24 14 14 59,471 6.74 0.002 2.1 960 I3L7D3 Synapsin II SYN2 12 6 6 51,020 8.84 0.046 2 960 F1SD97 Phenylalanyl-tRNA synthetase alpha subunit FARSA 18 10 10 57,628 7.85 0.046 2 964 F1RR02 Glial fibrillary acidic protein GFAP 47 26 26 49,437 5.65 0.001 1.8 964 F1RMZ8 ATPase H+ transporting V1 subunit B2 ATP6V1B2 17 9 9 56,613 5.57 0.001 1.8 971 I3LGA1 WD repeat domain 37 WDR37 11 5 5 54,116 6.95 0.033 2.2 1007 D0G0C8 Chaperonin containing TCP1, subunit 2 (beta) OS = Sus scrofa CCT2 27 16 16 57,444 6.09 0.036 2.2 1012 F1ST01 Selenium-binding protein 1 OS SELENBP1 29 17 17 52,534 6.54 0.034 2.1 1012 I3LKF3 Fascin FSCN1 26 14 14 53,234 8.19 0.034 2.1 1015 I3LKF3 Fascin FSCN1 34 16 16 53,234 8.19 0.024 1.9 1015 F1ST01 Selenium-binding protein 1 SELENBP1 9 3 3 52,534 6.54 0.024 1.9 1017 F1ST01 Selenium-binding protein 1 SELENBP1 10 5 5 52,534 6.54 0.012 2.3 1049 F1RR02 Glial fibrillary acidic protein GFAP 43 23 23 49,437 5.65 0.003 2 1057 F1RR02 Glial fibrillary acidic protein GFAP 55 37 36 49,437 5.65 0.00049 2 1062 F1RR02 Glial fibrillary acidic protein GFAP 55 34 33 49,437 5.65 0.002 1.7 1064 F1SEN2 Glutamate dehydrogenase 1, mitochondrial GLUD1 26 16 16 61,608 8.02 0.012 2.2 1064 F1RUE3 Succinate-semialdehyde dehydrogenase ALDH5A1 11 6 6 57,784 8.61 0.012 2.2 1064 D2KPI8 Adenylosuccinate lyase ADSL 7 4 4 55,092 6.45 0.012 2.2 1077 F1SEN2 Glutamate dehydrogenase 1, mitochondrial GLUD1 19 10 10 61,308 8.02 0.001 1.8 1131 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 17 10 9 42,639 6.61 0.002 2.8 1137 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 19 11 2 42,639 6.61 0.002 4 1143 I3LK59 2-Phospho-d-glycerate hydro-lyase ENO1 34 13 10 38,082 6.43 0.002 3.9 1143 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 21 12 3 42,639 6.61 0.002 3.9 1146 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 19 11 2 42,639 6.61 0.002 3.3 1146 I3LK59 2-Phospho-d-glycerate hydro-lyase ENO1 9 4 4 38,082 0.002 3.3 1153 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 23 12 2 42,639 6.61 0.001 1.9 1154 I3LNG5 Calcium/calmodulin-dependent protein kinase CAMK2A 29 15 4 42,639 6.61 0.003 1.8 1170 F1RUK8 Rab GDP dissociation inhibitor GDI2 52 24 21 50,327 5.78 0.013 1.5 1187 F1RR48 SH3 domain containing GRB2 like, endophilin B2 SH3GLB2 13 6 6 43,489 5.73 0.00074 2.2 1194 Q29387 Elongation factor 1-gamma EEF1G 31 21 21 49,624 6.16 0.024 2.4 1195 A6M928 Eukaryotic translation initiation factor 4A isoform 1 EIF4A1 22 9 3 46,154 5.32 0.004 2.1 1195 A6M930 Eukaryotic translation initiation factor 4A isoform 2 EIF4A2 21 9 3 46,502 5.33 0.004 2.1 1244 F2Z5G5 ARP1 actin-related protein 1 homolog A ACTR1A 32 15 15 42,614 6.19 0.016 2.3 1244 F1RFI1 Elongation factor Tu TUFM 18 9 8 49,451 6.72 0.016 2.3 1276 F1RK10 Succinate-CoA ligase (ADP-forming) subunit beta, mitochondrial SUCLA2 31 18 18 50,304 5.86 0.007 1.6 1288 F1RHA0 2-Oxoisovalerate dehydrogenase subunit alpha TMEM91 16 6 6 47,020 6.87 0.019 2 1315 F1SNE5 SH3 domain containing GRB2 like 2, endophilin A1 SH3GL2 37 17 17 37,614 5.26 0.008 2.2 1336 K7GM43 Septin 5 SEPT5 26 13 13 43,787 6.55 0.012 2.7 1363 P46410 Glutamine synthetase GLUL 9 5 5 42,030 6.28 0.006 2.3 1376 F1RZB5 Tropomodulin 2 TMOD2 24 12 12 39,693 5.17 0.019 2 1409 F1SNE5 SH3 domain containing GRB2 like 2, endophilin A1 SH3GL2 19 11 11 37,614 5.26 0.00036 2.2 1413 F1S0R4 V-type proton ATPase subunit C ATP6V1C1 27 14 14 44,033 7.62 0.002 2.1 1414 F1RJ25 Fructose-bisphosphate aldolase ALDOC 47 25 25 39,377 6.21 0.00001 1.9 1421 F1RL02 Mitogen-activated protein kinase MAPK1 39 15 14 37,974 6.56 0.00001 1.9 1464 Q6QAQ1 Actin, cytoplasmic 1 ACTB 11 6 6 41,737 5.29 0.0007 2.1 1469 F1RF18 G protein subunit alpha o1 GNAO1 8 5 5 40,078 5.10 0.00065 2 1479 Q9TV69 Trans-1,2-dihydrobenzene-1,2-diol dehydrogenase DHDH 27 10 10 36,527 6.34 0.021 2.1 1487 F2Z5H6 V-type proton ATPase subunit ATP6V0D1 23 10 10 40,329 4.89 0.003 2.2 1520 F1RIK3 Acyl-CoA thioesterase 7 ACOT7 8 4 4 40,189 8.06 0.011 2.1 1524 F1SEX0 Dimethylarginine dimethylaminohydrolase 1 DDAH1 25 5 5 20,779 5.36 0.005 1.8 1524 F1SUE3 Pyrophosphatase (inorganic) 1 PPA1 10 3 3 32,790 5.44 0.005 1.8 1542 F1RPC8 Crystallin mu CRYM 44 23 23 33,508 5.16 0.00056 2.7 1569 F1RMB1 Phytanoyl-CoA 2-hydroxylase-interacting protein PHYHIP 9 4 4 38,539 6.70 0.001 2 1580 P00336 l-Lactate dehydrogenase B chain LDHB 37 18 18 36,612 5.57 0.005 1.8 1592 F2Z4Z8 G protein subunit beta 2 GNB2 23 8 7 33,758 5.60 0.00032 1.7 1614 K7GNZ3 NAC-A/B domain-containing protein NACA 9 2 2 23,384 4.52 0.003 2 1627 I3LSK5 G protein subunit beta 1 GNB1 35 13 13 37,331 5.60 0.00007 2 1645 C5H0C6 Ubiquitin thioesterase OTUB1 10 4 4 31,284 4.85 2.1 1654 F1RM45 Apolipoprotein E APOE 20 7 7 36,665 5.92 0.009 2.1 1655 F1RM45 Apolipoprotein E APOE 12 4 4 36,665 5.92 0.006 1.6 1660 F1SGH5 Pyruvate dehydrogenase E1 component subunit beta PDHB 52 17 17 39,273 6.20 0.007 1.7 1660 F1RM45 Apolipoprotein E APOE 22 6 6 36,665 5.92 0.007 1.7 1732 I3LRS8 Phosphatidylinositol transfer protein alpha PITPNA 50 19 19 31,820 6.71 0.019 3 1757 Q06A94 Heterogeneous nuclear ribonucleoprotein A1 HNRNPA1L2 28 8 8 34,196 9.27 0.032 2.3 1815 Q9GZU5 NYX_HUMAN nyctalopin NYX 2 2 2 52,000 9.10 0.008 2.1 1826 P62258 14-3-3 protein epsilon YWHAE 69 38 38 29,174 4.63 0.00034 1.7 1939 F1S8Y5 Phosphoglycerate mutase PGAM1 19 5 5 29,301 6.41 0.006 3.1 2013 Q45FY6 Hypoxanthine-guanine phosphoribosyltransferase HPRT1 57 14 14 24,555 6.30 0.007 4 2029 Q6SEG5 Ubiquitin carboxyl-terminal hydrolase isozyme L1 UCHL1 66 38 37 24,859 5.22 0.012 1.7 2087 I3L9H4 PITH domain containing 1 PITHD1 27 5 5 24,265 5.47 0.006 2 2100 Q06AU3 Ras-related protein Rab-3A RAB3A 15 3 3 24,970 4.85 0.004 0.175 2165 Q6SEG5 Ubiquitin carboxyl-terminal hydrolase isozyme L1 UCHL1 65 38 7 24,859 5.22 0.044 1.7 2165 Q5E946 Protein/nucleic acid deglycase DJ-1 PARK7 56 16 16 20,035 6.84 0.044 1.7 2402 A8QW48 Beta-synuclein SNCB 25 5 4 14,115 4.46 0.00041 2.1 2467 Q3I5G7 Alpha-synuclein SNCA 53 8 8 14,520 4.62 0.00003 2.3 2479 Q6DUB7 Stathmin STMN1 46 8 8 17,302 5.75 0.011 3.2 2487 P81558 Myelin basic protein MBP 27 4 4 18,486 11.28 0.006 0.38 2497 Q8WNW3 Junction plakoglobin Jup 27 20 20 81,850 5.75 0.00076 3.2
On the contrary, in CLA vs PU comparison, an increase of expression was observed for whole differentially expressed proteins except RAB3A, which showed a higher expression level in PU compared to CLA and IN (Fig. 5B).
An exclusive expression difference was observed for sirtuin 2 (SIRT2), protein disulfide-isomerase (PDIA3), transketolase (TKT), and aspartate aminotransferase, mitochondrial (GOT2) in CLA vs PU but not in IN vs PU (Fig. 5B). Normalized mean values of optical density of identified differentially expressed spots were analyzed using next-generation clustered heat map to generate a clustered heat map (Fig. 6) where we can appreciate the highest consistence of protein expression increases as red color in IN followed by CLA and then PU.
The biological meaning of the differentially quantified proteins in two different comparisons (CLA vs PU and CLA vs IN) was investigated by a general overview using Metascape [36] which depicts top enriched clusters and their enrichment patterns across multiple gene lists as a clustered heat map (Fig. 7A). The heat map is complemented by an enrichment network (Fig. 7B) where each network node represents a category of biological processes and/or pathways. In Fig. 7C, each network node is represented by a pie chart, where the sector size is proportional to the number of genes originated from each gene list. Some categories such as PD pathway (WP2371) and α-synuclein pathway (M275) were enriched exclusively in CLA vs PU comparison and likely represent processes associated with proteins differentially expressed between these areas. The network also shows that processes such as synaptic vesicle cycle, regulation of neuronal synaptic plasticity, and substantia nigra development were shared between both lists.
Proteins found differentially expressed in each different comparison were analyzed by IPA to discover the most enriched canonical pathways, possible upstream regulators, and downstream effects. Similarities between IN and CLA were evidenced by IPA results derived by comparing IN and CLA data with those of PU. In fact, the same most significant canonical pathways such as Parkinson’s signaling, synaptogenesis signaling, and insulin secretion signaling were generated. Figure 8 shows a coherent and comprehensible synopsis of IPA core analysis of CLA vs PU protein data set, so to obtain a quick overview of major biological themes and their relationships, the graphical summary includes a subset of the most significant canonical pathways, upstream regulators, diseases, and biological functions predicted by the analysis. Among the most significant regulators, inhibition of PSEN1 upstream regulator deserves to be underlined while activation of endocannabinoid neuronal synapse pathway is also worthy of attention.
Western Blot Analysis
The difference of protein expression observed by 2-DE among three different brain areas was validated in other 5 additional samples by western blot analysis. The expression level of two more representative proteins, CAMK2 and DPYL2/CRMP-2, was detected using specific antibodies. A single immunoreactive band at 48 kDa was detected for CAMK2 whereas a main band at molecular weight of 65 kDa and two minor bands at 70 kDa and 75 kDa were detected for DPYL2 in agreement with different isoforms of this protein in Sus scrofa. Probably, the isoform with the highest apparent molecular weight represents the neuronal isoform with a ubiquitous localization in dendrites, axons, and cellular bodies [40]. Immunoreactive bands were analyzed, and normalized values of OD were represented in violin plots (Fig. 9A, B). The violin plot of DPYL2 shows the sum of all three immunoreactive band ODs. The expression differences observed by western blot analysis confirmed the results obtained by 2-DE according to which CLA expressed half levels of both proteins compared to IN (the highest level) and PU.
Immunofluorescence
Immunofluorescent staining in pig CLA, PU, and IN revealed the presence of both CaMKII-α and DPYL2/CRMP-2 (Fig. 10). In the IN, CaMKII-α was mainly localized in neuron somas while DPYL2 labeling was mostly associated with neuropil surrounding negative cell bodies. A similar immunostaining pattern was observed in CLA where few DPYL2-positive somas were also found.
On the contrary, in PU, positivity to both CaMKII-α and DPYL2/CRMP-2 was localized in fibers while rare immunolabeled cell bodies were only seen for CaMKII-α.
Discussion
Over the past decades, because of its considerable resemblance to human anatomy and physiology, pig brain has been widely employed as a valuable model in biomedical studies [28]. Recently, the generation of genetically modified pig models of neurodegenerative disorders has been discussed [41]. In the present study, we carried out a comparative proteomic analysis of pig CLA, IN, and PU to reveal specific molecular hallmarks of CLA to clarify its role, origin, and possible implication in human neurological disorders.
Although the three areas showed superimposable 2-DE protein maps, a considerable divergence in protein expression level was observed between CLA and IN when compared to PU; in this context, the circos plot showed that CLA assumed an intermediate position with respect to both IN and PU.
The ontology of the claustro-insular complex is still subject to debate [15, 42], and homologies reaching birds and even reptiles have been put forward [43, 44]. According to Bruguier et al. [18], the claustral neuronal population is born first starting from the lateral pallium, then insular cells migrate radially through the CLA, occupying progressively more superficial positions; thus, IN development is linked to CLA. In line with this hypothesis, the minor differences of protein expression observed between CLA and IN seem to support their common origin in agreement with described morphogenetic and neurochemical similarities of these structures [15, 16].
In general, we found PU to show a lower level of differentially expressed proteins except for Rab3A that showed a very significant high level of expression. Traditionally, PU is involved in different functions such as learning and motor control, reward, cognitive functioning, and addiction [45–49]; moreover, it appears to be correlated with a broad spectrum of movement disorders including PD, and Huntington’s disease (HD) as well as psychiatric diseases such as schizophrenia or obsessive–compulsive disorder (OCD) [50–55]. Rab proteins are small GTPases involved in all stages of vesicular transport and membrane fusion in mammalian cells, and Rab3 isoforms (Rab3A, Rab3B, Rab3C, and Rab3D) are expressed almost exclusively in neurons and secretory cells and are mainly located at synaptic membranes regulating Ca2+-dependent neurotransmitter release [56]. Rab3A has been indicated as one of physiological substrates of leucine-rich repeat kinase 2 (LRRK2) whose increased activity is related to PD pathogenesis [57]. In light of the functions ascribed to Rab3A, the high expression of Rab3A in pig PU adds molecular evidence in support of the potential role of this brain area in PD pathogenesis as prompted by different approaches such as shape analysis [58], and it is worth noting that Rab3A expression was also elevated in CLA with a value significantly higher than in IN, suggesting a possible common role of PU and CLA in neurological disorders.
Exclusive expression differences were observed for SIRT2, PDIA3, TKT, and GOT2 in CLA vs PU. SIRT2 belongs to mammalian sirtuin family that consists of seven members (SIRT1–SIRT7) with diverse functions depending upon substrates, distinct subcellular localization, and expression patterns [59]. SIRT2 is localized in the cytoplasm of both neurons and oligodendrocytes [60], and the well-known substrate of SIRT2 is α-tubulin, an important component of microtubule cytoskeleton whose acetylation by SIRT2 is linked to brain aging and neurological disorders [61] such as AD [62, 63] and PD [64, 65]. Similarly, PDIA3 is indicated as potentially involved in neurodegeneration processes. The protein disulfide-isomerases (PDIs) are generally localized to the endoplasmic reticulum (ER) where they mediate thiol–disulfide interchanges, which is a critical process during post-translational protein folding [66], and PDIA3 is markedly upregulated in most common neurodegenerative diseases, highlighting ER as an emerging driver of neurodegeneration [67, 68]. Also, TKT, a multifunctional protein in the non-oxidative branch of the pentose phosphate pathway, seems to be related to some neurological disorders such as AD, PD, and Wernicke-Korsakoff syndrome, and reduced levels of TKT have been found in the substantia nigra of PD patients [69].
CLA neurodegeneration and dysfunction are described in patients affected by AD or PD [70, 71]. Aggregation of misfolded proteins is a determinant in many neurodegenerative diseases such as frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), PD, AD, and HD [72], and amyloid β (Aβ) deposits and neurofibrillary tangles have been described in the CLA of AD patients [73, 74]. In addition, α-synuclein and Aβ lesions have been also found in the CLA of cases affected by PD or dementia with Lewy bodies (DLB) [75] while claustral degeneration has been reported in familial Alzheimer’s disease [71]. In the present study, Metascape analysis of CLA vs PU suggested the activation of α-synuclein and PD pathways, corroborating the implication of these structures in the abovementioned diseases. Two other interesting pathways evidenced by Metascape analysis were the neuron projection development pathway and L1 recycling pathway; the first is involved in axonal growth while the second plays a role in clathrin-coated vesicle trafficking. The endocytic recycling pathway seems to be implicated in the aggregation, toxicity, and secretion of α-synuclein, whose misfolding is common in several neurodegenerative diseases [76]. Among CLA differentially expressed proteins involved in these pathways (Supplementary Table 1), we also found DPYSL2 and CAMK2, and the expression of both proteins was validated by western blot analysis while immunofluorescence revealed their distribution and cellular localization in the three cerebral structures. CAMK2 plays a key role in the redistribution of α-synuclein during neurotransmitter release [77] and can interact and potentially alter α-synuclein conformation [78] whereas DPYSL2 has a function in neuronal development and polarity, in cell migration and endocytosis, and is implicated in neurological disorders like CAMK2 [79].
Another interesting protein overexpressed in CLA compared to PU is aspartate aminotransferase 2 (GOT2), a pyridoxal 5′-phosphate (PLP)-dependent enzyme that exists as cytosolic (GOT1) and intramitochondrial (GOT2). GOT2 deficiency is a mitochondriopathy that is reported to be implicated in treatable metabolic epilepsies [80]. In both humans and rodents, CLA seems to be a potential locus for generating epileptiform activity during kindling [81], and GOT2 overexpression found in pig CLA strengthens the assumption that depicts this structure as involved in seizure generation.
Our findings revealed that copine 3 (CPNE3) and MBP were overexpressed in CLA compared to IN. Copines are calcium-dependent phospholipid-binding proteins involved in membrane-trafficking phenomena and protein–protein interactions [82]. A recent study reported that CPNE3 interacts with anxiety to affect working memory (WM) [83]. WM is a cognitive ability that allows one to hold and manipulate information and is foundational to the organization of goal-directed behavior [84]. Goll et al. [2] have proposed that CLA has the capacity to focus attention, an essential function for goal-direct behavior; besides, White et al. [3] have provided data describing CLA as an anatomical and functional substrate that may underlie functions, such as executive attention or WM. Thus, such reported findings support our data regarding CPNE3 and, taken together, may indicate the CLA as a possible site of mutual influences between anxiety and working memory.
In psychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder, WM impairment is reported [83], and in this context, our data showing an overexpression of MBP in CLA are quite interesting. MBP is important in maintaining the structure of the myelin sheath [85]. Myelin dysfunction produces abnormal connectivity of neural networks and is considered one of the main factors implicated in schizophrenia pathogenesis [86, 87]. Different studies have shown structural differences in the CLA of schizophrenic patients experiencing delusions [88, 89].
Last but not the least, protein data set analysis of CLA vs PU comparison by IPA produced an intriguing result highlighting the activation of endocannabinoid neuronal synapse pathway. The endocannabinoid system (ECS) is a widespread neuromodulator network involved in central nervous system development and plays a major role in tuning many cognitive and physiological processes [90]. The activation of endocannabinoid neuronal synapse pathway revealed by IPA analysis supports previous immunohistochemical studies demonstrating the presence of cannabinoid receptor 1 (CB1) and two endogenous cannabinoid-degrading enzymes (MGL and FAAH) in pig and dog CLA [91, 92]. Taken together, the whole findings provide a neuroanatomical support for a possible neuromodulator role of endocannabinoids within CLA circuitry and may reinforce the postulate depicting CLA and ECS as involved in neurodegenerative diseases [70, 93].
Conclusion
In conclusion, the comparative study of the proteomic profile of pig CLA highlighted the overexpression of specific proteins deeply implicated in both neurodegenerative (e.g., PD, AD, and HD) and psychiatric disorders in humans, pointing out the translational significance of the investigation. In this context, the present findings may contribute to better understand the molecular involvement of CLA in the pathogenetic mechanisms of these diseases. Furthermore, the minor differences of protein expression observed between CLA and IN strengthen the hypothesis of their common origin.
Untitled section
Supplementary Information
Funding
Open access funding provided by Università di Pisa within the CRUI-CARE Agreement. This work was supported by the University of Pisa.
Data Availability
All data generated or analyzed during this study are included in this published article.
Declarations
Ethics Approval and Consent to Participate
Not applicable. Our samples were from animals from a local abattoir.
Consent for Publication
All authors have read and approved the submission of the manuscript.
Competing Interests
The authors declare competing interests.