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Biological pathways modulated by antipsychotics in the blood plasma of schizophrenia patients and their association to a clinical response Daniel Martins-de-Souza1,2, Fiorella A Solari3, Paul C Guest1, René P Zahedi3 and Johann Steiner4,5 Proteomics is a valuable tool to unravel molecular mechanisms involved in human disorders. Considering the mediocre effectiveness of antipsychotics, which are the main class of drug used to treat schizophrenia, we analyzed a cohort of 58 schizophrenia patients who had blood collected before and after 6 weeks of antipsychotic treatment using a shotgun mass spectrometry proteomic profiling approach. Our aim was to unravel molecular pathways involved with an effective drug response. The results showed that all patients had essentially the same biochemical pathways triggered Independent of the antipsychotic response outcome. However, we observed that these pathways were regulated in different directions in blood samples from those who responded well to antipsychotics, compared with those who had a poorer outcome. These data are novel, timely and may help to guide new research efforts in the design of new treatments or medications for schizophrenia based on biologically relevant pathways. npj Schizophrenia (2015) 1, Article number: 15050; doi:10.1038/npjschz.2015.50; published online 9 December 2015
Schizophrenia is the result of a combined dysfunction of genetic, biochemical and neurodevelopmental components that may be triggered by environmental factors. This cascade of events will most likely lead to disruption of cellular and tissue homeostasis, impairing the molecular pathways, which govern the cellular machinery throughout the body. Given the widespread nature of these effects, multiplex molecular approaches such as proteomics are required to provide new insights, as such methods can target hundreds of disease-relevant molecules, including those involved in the pathogenesis. In addition, by identifying specific proteins that are present at different steady-state levels in the disease state, proteomics can aid the discovery of molecular biomarker candidates.1 Although our understanding of the molecular basis of schizophrenia has evolved recently, there are still many factors that do not connect or are still unknown. Thus, identification of these factors and increasing our understanding of their impact on the disease would be an important step forward. In addition, no factors emerging from molecular studies have been translated into clinical use for schizophrenia studies to date, despite the urgent need for both disease- and medication-related biomarker candidates. This is of particular importance owing to the fact that schizophrenia is an incurable disorder, which demands continuous healthcare, thus generating lifelong suffering for the patients and substantial expenses on the health-care systems. In the US, treatment and management of schizophrenia was estimated at a cost of 62 billion dollars per year, in the year 2000.2 Although psychosocial interventions are available, disease management is mainly based on treatment with antipsychotic medications. However, ~ 40% of schizophrenia patients do not
respond properly to these medications and 460% end up abandoning treatment due to undesirable side effects.3 Consequently, the intellectual and cognitive capacities of the patients may worsen, making them incapable of functioning adequately in society, thereby producing a further socioeconomic burden. Consistent with our lack of understanding of disease pathophysiology, knowledge on the effects of medication on metabolism and other molecular pathways in the patients is scarce. With this in mind, we have employed a proteomic approach in an attempt to increase the understanding of the molecular pathways affected in response to currently used atypical antipsychotic medications. It was of particular interest to identify biomarker candidates associated with a positive response. The availability of such early-response biomarkers could eventually be used to help diminish the duration of poor response periods, thereby diminishing disease severity and improving the outcomes for the patients. The cohort studied in this investigation consisted of 58 acutely ill patients who received the atypical antipsychotic drugs olanzapine (n = 18), quetiapine (n = 14) or risperidone (n = 26) (Table 1). Blood samples were collected by intravenous puncture as previously described in the psychiatric Clinic of the University of Magdeburg (Germany).4 Citrate plasma samples were collected at baseline (T0), when patients were acutely ill and either antipsychotic-naive (n = 23) or antipsychotic-free for at least 6 weeks (n = 35), and after 6 weeks when all the subjects had been treated as inpatients (T6). Patients with other medical conditions such as type 2 diabetes mellitus, hypertension, cardiovascular or autoimmune diseases were excluded. In line with previous studies,5 the patients were separated into responders (n = 36) and
1 Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas, Brazil; 2UNICAMP’s Neurobiology Center, Campinas, Brazil; 3Leibniz Institut für Analytische Wissenschaften (ISAS), Dortmund, Germany; 4Department of Psychiatry and Psychotherapy, University of Magdeburg, Magdeburg, Germany and 5The Center for Behavioral Brain Sciences, Magdeburg, Germany. Correspondence: D Martins-de-Souza ([email protected]) Received 8 July 2015; revised 22 October 2015; accepted 28 October 2015
© 2015 Schizophrenia International Research Society/Nature Publishing Group
99 105 129 133 136 140 147 151 156 162 208 247 249 260 277 278 331 332 363 366 368 370 371 375 391 400 404 408 410 422 426 428 431 440 452 454 464 468 474 476 479 487 493 520 521 522 524 526 539 541 545 568 570 572 579 592 663 800
ID patient
Table 1.
7 28 22 17 24 25 31 26 17 20 38 25 22 21 26 37 31 23 27 28 34 26 35 18 25 19 29 15 12 28 26 20 24 33 14 22 25 19 28 17 24 37 28 16 27 17 21 26 24 16 28 21 26 16 14 38 23 16
PANSS P1-P7
17 32 29 34 24 26 31 9 16 16 18 25 41 11 12 29 9 29 25 16 20 9 14 30 11 27 10 13 26 41 23 10 26 12 20 16 18 18 14 17 14 7 29 24 17 7 13 38 27 7 12 30 12 7 18 25 11 15
PANSS N1-N7
37 63 45 49 46 58 55 40 28 37 35 55 41 31 38 58 41 39 41 40 61 39 43 50 51 54 40 52 47 45 46 60 42 45 36 48 37 48 51 37 44 62 51 38 50 40 40 71 58 30 35 43 45 25 34 43 22 30
PANSS G1-G16
T0 raw PANSS
61 123 101 106 104 115 126 79 64 76 109 111 107 76 83 137 92 95 100 93 132 81 104 101 97 108 97 87 91 121 105 94 95 104 80 94 94 88 99 76 91 115 116 90 102 67 79 138 112 53 84 100 89 51 75 122 59 65
PANSS total
0 21 15 10 17 18 24 19 10 13 31 18 15 14 19 30 24 16 20 21 27 19 28 11 18 12 22 8 5 21 19 13 17 26 7 15 18 12 21 10 17 30 21 9 20 10 14 19 17 9 21 14 19 9 7 31 16 9
PANSS P1-P7 10 25 22 27 17 19 24 2 9 9 11 18 34 4 5 22 2 22 18 9 13 2 7 23 4 20 3 6 19 34 16 3 19 5 13 9 11 11 7 10 7 0 22 17 10 0 6 31 20 0 5 23 5 0 11 18 4 8
PANSS N1-N7 21 47 29 33 30 42 39 24 12 21 19 39 25 15 22 42 25 23 25 24 45 23 27 34 35 38 24 36 31 29 30 44 26 29 20 32 21 32 35 21 28 46 35 22 34 24 24 55 42 14 19 27 29 9 18 27 6 14
PANSS G1-G16
T0 corrected PANSS
31 93 66 70 64 79 87 45 31 43 61 75 74 33 46 94 51 61 63 54 85 44 62 68 57 70 49 50 55 84 65 60 62 60 40 56 50 55 63 41 52 76 78 48 64 34 44 105 79 23 45 64 53 18 36 76 26 31
PANSS total 7 33 14 10 15 16 12 8 12 7 12 15 17 8 9 13 15 16 10 9 17 9 8 21 11 7 7 15 20 7 12 19 12 13 17 7 8 9 32 14 13 13 15 9 10 17 12 16 21 13 15 16 11 7 11 15 10 11
PANSS P1-P7 11 32 24 18 11 15 24 8 13 14 9 8 31 24 7 22 8 24 16 10 20 15 7 27 9 11 7 19 23 9 10 12 14 11 9 7 14 18 21 11 9 17 21 11 9 9 14 29 16 7 7 26 12 7 7 7 7 8
PANSS N1-N7 29 64 32 28 26 34 39 19 24 19 20 27 37 24 21 30 31 31 23 21 47 27 20 42 22 20 20 41 48 24 22 50 26 33 26 16 25 24 42 32 26 32 29 25 21 27 29 39 49 25 23 28 30 17 21 41 18 18
PANSS G1-G16
T6 raw PANSS
Patients demographics and PANSS score before and after treatment
50 141 74 59 55 72 82 38 54 43 44 54 90 59 42 71 61 75 52 43 91 54 38 97 45 41 40 82 98 43 47 89 55 65 65 33 50 54 105 64 52 70 68 49 43 57 58 87 94 56 55 73 56 34 42 68 38 40
PANSS total 0 26 7 3 8 9 5 1 5 0 5 8 10 1 2 6 8 9 3 2 10 2 1 14 4 0 0 8 13 0 5 12 5 6 10 0 1 2 25 7 6 6 8 2 3 10 5 9 14 6 8 9 4 0 4 8 3 4
PANSS P1-P7 4 25 17 11 4 8 17 1 6 7 2 1 24 17 0 15 1 17 9 3 13 8 0 20 2 4 0 12 16 2 3 5 7 4 2 0 7 11 14 4 2 10 14 4 2 2 7 22 9 0 0 19 5 0 0 0 0 1
PANSS N1-N7 13 48 16 12 10 18 23 3 8 3 4 11 21 8 5 14 15 15 7 5 31 11 4 26 6 4 4 25 32 8 6 34 10 17 10 0 9 8 26 16 10 16 13 9 5 11 13 23 33 9 7 12 14 1 5 25 2 2
PANSS G1-G16
T6 corrected PANSS
17 99 40 26 22 35 45 5 19 10 11 20 55 26 7 35 24 41 19 10 54 21 5 60 12 8 4 45 61 10 14 51 22 27 22 0 17 21 65 27 18 32 35 15 10 23 25 54 56 15 15 40 23 1 9 33 5 7
PANSS total 0.45 − 0.06 0.39 0.63 0.66 0.56 0.48 0.89 0.39 0.77 0.82 0.73 0.26 0.21 0.85 0.63 0.53 0.33 0.70 0.81 0.36 0.52 0.92 0.12 0.79 0.89 0.92 0.10 − 0.11 0.88 0.78 0.15 0.65 0.55 0.45 1.00 0.66 0.62 − 0.03 0.34 0.65 0.58 0.55 0.69 0.84 0.32 0.43 0.49 0.29 0.35 0.67 0.38 0.57 0.94 0.75 0.57 0.81 0.77
Relative PANSS total improvement T0-T6 Bad responder Bad responder Bad responder Good responder Good responder Good responder Bad responder Good responder Bad responder Good responder Good responder Good responder Bad responder Bad responder Good responder Good responder Good responder Bad responder Good responder Good responder Bad responder Good responder Good responder Bad responder Good responder Good responder Good responder Bad responder Bad responder Good responder Good responder Bad responder Good responder Good responder Bad responder Good responder Good responder Good responder Bad responder Bad responder Good responder Good responder Good responder Good responder Good responder Bad responder Bad responder Bad responder Bad responder Bad responder Good responder Bad responder Good responder Good responder Good responder Good responder Good responder Good responder
All patients
Good response ⩾ 50% corrected PANSS total reduction
Yes No No Yes No No Yes No No Yes No No No No Yes No No Yes Yes Yes Yes Yes No Yes No No No No No No No Yes Yes No No Yes Yes No No No Yes Yes Yes No No Yes Yes Yes No No No No No Yes Yes No No No
First episode patient
Yes No No No No No Yes No No Yes No No No No Yes No No Yes Yes Yes Yes Yes No Yes No No No No No No No Yes Yes No No Yes Yes No No No Yes Yes Yes No No Yes Yes Yes No No No No No Yes Yes No No No
Drug naive
npj Schizophrenia (2015) 15050 11 7 0 0 32 15 0 0 1 11 7 0 0 0 0 10 0 0 0 3 34 0 10 3 0 0 14 21 15
6 2
7 0 0 2 0 9 7 19 1 0 9 23 0 0 0 0 0 6 0 32
0
0 15
Illness duration [years]
Quetiapie Quetiapie Risperidoe Olanzapie Risperidoe Risperidoe Olanzapie Olanzapie Risperidoe Risperidoe Olanzapie Risperidoe Risperidoe Olanzapie Risperidoe Risperidoe Olanzapie Risperidoe Risperidoe Olanzapie Risperidoe Risperidoe Risperidoe Quetiapie Quetiapie Quetiapie Olanzapie Quetiapie Olanzapie Risperidoe Risperidoe Quetiapie Olanzapie Olanzapie Risperidoe Risperidoe Risperidoe Risperidoe Quetiapie Quetiapie Olanzapie Quetiapie Risperidoe Quetiapie Olanzapie Quetiapie Olanzapie Olanzapie Quetiapie Risperidoe Olanzapie Risperidoe Risperidoe Quetiapie Olanzapie Olanzapie Risperidoe Risperidoe
Medication
F M F F F M M F M M M M M M F M F F M M M M F M F F M F F M M F M F M M F M M M F M M F M M F M M M F F M F F M M M
31 47 49 25 44 30 25 45 42 19 28 40 44 22 20 31 39 26 25 34 16 31 48 23 55 32 45 27 50 34 30 38 29 59 36 34 66 35 32 25 53 39 50 31 32 26 36 23 28 53 50 56 36 36 36 30 51 58
Gender Age
19.7 21.8 23.9 24.0 27.4 22.2 28.1 30.5 32.9 20.2 25.3 23.0 17.7 27.5 22.2 22.8 29.8 22.1 20.1 27.7 26.5 24.4 37.2 22.0 29.8 22.7 26.4 22.2 24.1 23.6 21.5 32.9 21.1 35.0 22.4 22.2 31.6 18.3 25.0 22.0 20.1 21.1 19.7 28.7 30.8 21.0 22.0 22.2 23.2 24.8 28.6 29.1 20.4 21.9 30.3 31.1 30.4 20.6
BMI
No Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes No Yes No Yes No No Yes No No Yes Yes Yes Yes Yes Yes Yes No No Yes Yes No Yes Yes Yes Yes Yes Yes No Yes Yes No Yes Yes Yes No Yes Yes Yes No No No
Smoking
Biological pathways modulated by antipsychotics D Martins-de-Souza et al
2
© 2015 Schizophrenia International Research Society/Nature Publishing Group
Biological pathways modulated by antipsychotics D Martins-de-Souza et al
3 Coronin-2A
T6-R T6-R T6-NR T6-NR
% of differentially expressed proteins
% of differentially expressed proteins
Loading control
Figure 1. (a) Biological processes represented by all 985 plasma proteins identified through the shotgun mass spectrometry profiling approach; (b) western blot validation of the mass spectrometry findings for coronin-2A, which showed increased levels in responders and decreased levels in non-responders; (c) Biological pathways affected by antipsychotics in responders (blue bars) and non-responders (red bars); (d) Percentage of proteins increased in responders (blue), decreased in responders (red), increased in non-responders (green) and decreased in non-responders (purple) in the same biological pathways shown in part (c). For (c and d) the y axis represents the percentage of differentially expressed proteins associated to those processes and a star indicates significant differences of the protein constituents (c) or directional changes (d) within a pathway.
non-responders (n = 22), with response defined as a 50% reduction of the corrected (subtraction of minimum scores representing ‘no symptoms’) total Positive and Negative Syndrome Scale (PANSS) scores. The percentage of responders was similar in the antipsychotic-naive (61%; 14/23) and antipsychotic-free (63%; 22/35) subgroups (Table 1). © 2015 Schizophrenia International Research Society/Nature Publishing Group
Blood samples were immediately centrifuged for 10 min at 2,000 × g and resulting supernatants divided in 250-μl aliquots, which were immediately frozen at − 80 °C. Protein concentrations were determined by Bradford assay. Next, the plasma samples were depleted of 14 high-abundant proteins using the MARS-14 immunodepletion system (Agilent; Wokingham, UK)6 as these npj Schizophrenia (2015) 15050
npj Schizophrenia (2015) 15050
SNX17_HUMAN
TGFI1_HUMAN
THS7A_HUMAN
LR16C_HUMAN
FA9_HUMAN HS71L_HUMAN ITIH3_HUMAN
RM03_HUMAN
AACT_HUMAN YE019_HUMAN
Q15036
O43294
Q9UPZ6
Q6F5E8
P00740 P34931 Q06033
P09001
P01011 A1L167
ABCAD_HUMAN ABCA13
ALBU_HUMAN APOA4_HUMAN VDAC1_HUMAN
COPA1_HUMAN
ECM1_HUMAN
ELMO1_HUMAN
NEBU_HUMAN
TBG1_HUMAN
CD5L_HUMAN CI117_HUMAN CCD71_HUMAN
CC74A_HUMAN
CENPP_HUMAN ADXL_HUMAN
YO003_HUMAN LIX1_HUMAN 5NT3L_HUMAN
Q86UQ4
P02768 P06727 P21796
Q9BXS0
Q16610
Q92556
P20929
P23258
O43866 Q5JU67 Q8IV32
Q96AQ1
Q6IPU0 Q6P4F2
Q6ZVN6 Q8N485 Q969T7
60 58 40
29 22
O15018 PDZD2_HUMAN PDZD2 Q7Z6W1 TMCO2_HUMAN TMCO2
38 22
31
201 47 26
27
85
46
435
28
123 1502 26
70
34 21 40 31
28 35
1317 71
28
154 45 299
39
30
39
44
40
33
35
41
HSD47 LIX1 NT5C3B
CENPP FDX1L
CCDC74A
CD5L C9orf117 CCDC71
TUBG1
NEB
ELMO1
ECM1
COL25A1
ALB APOA4 VDAC1
MYCD_HUMAN P73_HUMAN ZN215_HUMAN ABCB5_HUMAN
MYOCD TP73 ZNF215 ABCB5
MLH3 MYCBP2
SERPINA3 UBE2QL1
MRPL3
F9 HSPA1L ITIH3
RLTPR
THSD7A
TGFB1I1
SNX17
PRKDC
FARP2
Q8IZQ8 O15350 Q9UL58 Q2M3G0
Q9UHC1 MLH3_HUMAN O75592 MYCB2_HUMAN
FARP2_HUMAN
PRKDC_HUMAN
O94887
P78527
EFCAB6
CYR61
44,165
303,964 20,287
33,173 32,213 33,893
33,429 19,737
41,808
39,603 60,667 49,759
51,480
775,419
84,517
62,232
65,145
71,317 45,371 30,868
580,604
102,561 70,206 60,922 90,173
166,060 517,856
47,792 18,440
38,893
53,114 70,730 100,072
156,133
192,854
51,321
53,153
473,749
120,725
174,702
25.23 2.59
0.29 0.23 9.21
20.20 2.01
12.33
3.03 0.02 32.45
0.46
0.08
0.13
2.43
0.06
0.04 0.51 26.82
12.73
0.43 0.03 0.11 64.78
0.05 6.52
1.48 0.06
0.27
0.36 0.45 1.52
20.44
0.03
0.31
0.05
41.97
11.93
3.57
0.03
1.948
3.329
7.015
1.954
5.368
1.59 1.951 5.344 1.559
6.347
1.001 1.341
1.079
Up Up
4.449 1.144
Down 1.027 Down 2.73 Up 1.238
Up Up
Up
Up 5.29 Down 1.229 Up 4.607
Down 2.748
Down 7.593
Down 7.738
Up
Down 2.02
Down 1.8 Down 1.061 Up 1.167
Up
Down Down Down Up
Down 2.706 Up 5.641
Up 1.282 Down 4.273
Down 1.813
Down 6.308 Down 2.631 Up 1.254
Up
Down 1.223
Down 1.962
Down 3.201
Up
Up
Up
Down 1.779
Down 6.913
2 2
3 5 3
2 2
2
2 2 2
2
6
5
2
2
6 7 2
2
2 2 2 2
2 2
34 2
2
2 4 5
2
2
2
4
2
2
2
2
2
Biological process
Cell communication and signaling Protein CYR61 Cell communication and signaling EF-hand calcium-binding domainCell communication containing protein 6 and signaling FERM, RhoGEF and pleckstrin domain- Cell communication containing protein 2 and signaling DNA-dependent protein kinase Cell communication catalytic subunit and signaling Sorting nexin-17 Cell communication and signaling Transforming growth factor beta-1Cell communication induced transcript 1 protein and signaling Thrombospondin type-1 domainCell communication containing protein 7A and signaling Leucine-rich repeat-containing protein Cell communication 16C and signaling Coagulation factor IX Protein metabolism Heat shock 70 kDa protein 1-like Protein metabolism Inter-alpha-trypsin inhibitor heavy Protein metabolism chain H3 39S ribosomal protein L3, Protein metabolism mitochondrial Alpha-1-antichymotrypsin Protein metabolism Ubiquitin-conjugating enzyme E2QProtein metabolism like protein 1 DNA mismatch repair protein Mlh3 Reg. nucleic acid metab Probable E3 ubiquitin-protein ligase Reg. nucleic acid metab MYCBP2 Myocardin Reg. nucleic acid metab Tumor protein p73 Reg. nucleic acid metab Zinc finger protein 215 Reg. nucleic acid metab ATP-binding cassette sub-family B Transport member 5 ATP-binding cassette sub-family A Transport member 13 Serum albumin Transport Apolipoprotein A-IV Transport Voltage-dependent anion-selective Transport channel protein 1 Collagen alpha-1(XXV) chain Cell growth and maintenance Extracellular matrix protein 1 Cell growth and maintenance Engulfment and cell motility protein 1 Cell growth and maintenance Nebulin Cell growth and maintenance Tubulin gamma-1 chain Cell growth and maintenance CD5 antigen-like Immune response Uncharacterized protein C9orf117 Unknown Coiled-coil domain-containing protein Unknown 71 Coiled-coil domain-containing protein Unknown 74A Centromere protein P Unknown Adrenodoxin-like protein, Unknown mitochondrial Uncharacterized protein HSD47 Unknown Protein limb expression 1 homolog Unknown 7-methylguanosine phosphate-specific Unknown 5'-nucleotidase PDZ domain-containing protein 2 Unknown Transmembrane and coiled-coil Unknown domain-containing protein 2
Coronin-2A
Description
EFCB6_HUMAN
0.12
Number of peptides
CYR61_HUMAN
60,239
StDev
Q5THR3
35
Reg
O00622
CORO2A
Resp T0 × T6
COR2A_HUMAN
Mass
Q92828
Score
Entry name
Entry
Gene name
Differentially expressed proteins in responders before and after 6 weeks of antipsychotic treatment
Table 2.
Unclassified Integral membrane protein
Unclassified Unclassified Unclassified
Unclassified Unclassified
Unclassified
Secreted polypeptide Unclassified Unclassified
Cytoskeletal protein
Cytoskeletal protein
Motor protein
Extracellular matrix protein
Extracellular matrix protein
Transport/cargo protein Transport/cargo protein Voltage-gated channel
Transport/cargo protein
DNA repair protein Transcription regulatory protein Transcription factor Transcription factor DNA binding protein Integral membrane protein
Protease inhibitor Unclassified
Ribosomal subunit
Coagulation factor Heat shock protein Protease inhibitor
Unclassified
Transcription regulatory protein Unclassified
Adapter molecule
Serine/threonine kinase
Cytoskeletal protein
Calcium binding protein
Extracellular matrix protein
Unclassified
Molecular class
Molecular function unknown Molecular function unknown
Molecular function unknown Molecular function unknown Molecular function unknown
Molecular function unknown Molecular function unknown
Molecular function unknown
Defense/immunity protein activity Molecular function unknown Molecular function unknown
Structural constituent of cytoskeleton
Structural constituent of cytoskeleton
Extracellular matrix structural constituent Extracellular matrix structural constituent Motor activity
Transporter activity Transporter activity Voltage-gated ion channel activity
Transporter activity
Transcription factor activity Transcription factor activity DNA binding Transporter activity
Protein binding Transcription regulator activity
Protease inhibitor activity Molecular function unknown
Structural constituent of ribosome
Extracellular matrix binding Heat shock protein activity Protease inhibitor activity
Molecular function unknown
Molecular function unknown
Protein serine/threonine kinase activity Receptor signaling complex scaffold activity Transcription regulator activity
Structural constituent of cytoskeleton
Extracellular matrix structural constituent Calcium ion binding
Molecular function unknown
Molecular function
Biological pathways modulated by antipsychotics D Martins-de-Souza et al
4
© 2015 Schizophrenia International Research Society/Nature Publishing Group
SEPT07_
SNX17
FARP2_HUMAN
MUC16_HUMAN
OR4KD_HUMAN
PRKX_HUMAN
RN111_HUMAN
SEPT7_HUMAN
SNX17_HUMAN
TEX14_HUMAN
Q8WXI7
Q8NH42
P51817
Q6ZNA4
Q16181
Q15036
Q8IWB6
THSD7A
MLH3 PARP2 DDX46
DNMT1 MYOCD PCBP2 ZC3H13
THS7A_HUMAN
Q9UHC1 MLH3_HUMAN Q9UGN5 PARP2_HUMAN Q7L014 DDX46_HUMAN
DNMT1_HUMAN MYCD_HUMAN PCBP2_HUMAN ZC3HD_HUMAN
ZFHX4_HUMAN ZN215_HUMAN ABCAD_HUMAN
ALBU_HUMAN APOA4_HUMAN APOB_HUMAN GOGA4_HUMAN S14L4_HUMAN SYN3_HUMAN VDAC1_HUMAN
Q9UPZ6
P26358 Q8IZQ8 Q15366 Q5T200
Q86UP3 Q9UL58 Q86UQ4
P02768 P06727 P04114 Q13439 Q9UDX3 O14994 P21796
TGFB1I1
© 2015 Schizophrenia International Research Society/Nature Publishing Group
ECM1
KIF17
KIF3A
MVP
MYBPC1
TUBG1
ECM1_HUMAN
KIF17_HUMAN
KIF3A_HUMAN
MVP_HUMAN
MYPC1_HUMAN
TBG1_HUMAN
SAMP_HUMAN CLPX_HUMAN
Q16610
Q9P2E2
Q9Y496
Q14764
Q00872
P23258
P02743 O76031
APCS CLPX
VOPP1
Q96AW1 ECOP_HUMAN
ALB APOA4 APOB GOLGA4 SEC14L4 SYN3 VDAC1
ZFHX4 ZNF215 ABCA13
WEE1
TGFI1_HUMAN
WEE1_HUMAN
O43294
P30291
TEX14
RNF111
PRKX
OR4K13
MUC16
FARP2
EFCAB6
O94887
CYR61
CYR61_HUMAN
EFCB6_HUMAN
O00622
Q5THR3
CHGA
2577 41
27
35
36
27
31
435
32
123 1,502 1,709 176 67 34 26
35 40 70
30 34 17 33
28 28 117
30
51
39
27
44
29
59
45
33
29
33
35
41
35
31
50,829
25,485 69,922
51,480
129,240
99,551
80,687
115,784
62,232
19,839
71,317 45,371 516,666 261,892 47,070 63,491 30,868
398,157 60,922 580,604
185,388 102,561 38,955 197,203
166,060 66,734 117,803
192,854
72,237
51,321
169,107
53,153
50,933
110,119
41,041
34,751
2,359,682
120,725
174,702
44,165
60,239
0.02
1.45 25.86
0.02
0.01
7.09
0.01
0.01
0.31
2.46
0.61 0.56 1.46 44.57 13.48 0.03 8.81
7.38 0.02 0.05
0.07 9.73 0.01 0.02
0.01 6.29 8.70
0.01
0.12
0.01
5.62
10.79
4.12
18.82
9.97
0.26
0.04
4.14
0.01
0.42
9.12
1.445
1.288
1.184
5.889
1.665
1.351
1.08 2.619 1.318 2.653
1.841
4.848 1.078 1.306 1.39 1.447 1.434 6.002
3.151
Up Up
9.908 2.463
Down 1.934
Down 1.213
Up
Down 1.08
Down 1.028
Down 5.743
Up
Down Down Up Up Up Down Up
Up 7.307 Down 9.007 Down 1.619
Down Up Down Down
Down 1.087 Up 1.04 Up 4.669
Down 1.223
Down 3.019
Down 4.21
Up
Up
Up
Up
Up
Down 4.954
Down 8.085
Up
Down 5.227
Down 1.137
1.259
Down 1.972 Up
9 3
2
2
4
2
2
2
2
6 7 17 15 3 2 2
2 2 2
2 2 2 3
2 2 11
2
4
2
2
3
2
4
4
3
3
2
2
2
2
3
nucleic nucleic nucleic nucleic
acid acid acid acid
metab metab metab metab
Transport Transport Transport Transport Transport Transport Transport
Reg. nucleic acid metab Reg. nucleic acid metab Transport
Reg. Reg. Reg. Reg.
Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Reg. nucleic acid metab Reg. nucleic acid metab Reg. nucleic acid metab
Biological process
Cell growth and maintenance Cell growth and maintenance Kinesin-like protein KIF17 Cell growth and maintenance Kinesin-like protein KIF3A Cell growth and maintenance Major vault protein Cell growth and maintenance Myosin-binding protein C, slow-type Cell growth and maintenance Tubulin gamma-1 chain Cell growth and maintenance Serum amyloid P-component Protein metabolism Protein metabolism
Thrombospondin type-1 domaincontaining protein 7A DNA mismatch repair protein Mlh3 Poly [ADP-ribose] polymerase 2 Probable ATP-dependent RNA helicase DDX46 DNA (cytosine-5)-methyltransferase 1 Myocardin Poly(rC)-binding protein 2 Zinc finger CCCH domain-containing protein 13 Zinc finger homeobox protein 4 Zinc finger protein 215 ATP-binding cassette sub-family A member 13 Serum albumin Apolipoprotein A-IV Apolipoprotein B-100 Golgin sub-family A member 4 SEC14-like protein 4 Synapsin-3 Voltage-dependent anion-selective channel protein 1 Vesicular, overexpressed in cancer, prosurvival protein 1 Extracellular matrix protein 1
Inactive serine/threonine-protein kinase TEX14 Transforming growth factor beta-1induced transcript 1 protein Wee1-like protein kinase
Sorting nexin-17
Septin-7
cAMP-dependent protein kinase catalytic subunit PRKX E3 ubiquitin-protein ligase Arkadia
Olfactory receptor 4K13
EF-hand calcium-binding domaincontaining protein 6 FERM, RhoGEF and pleckstrin domain-containing protein 2 Mucin-16
Protein CYR61
Coronin-2A
Chromogranin-A
Description
CORO2A
Number of peptides
COR2A_HUMAN
StDev
CMGA_HUMAN
Reg
P10645
Non Resp T0 × T6
Q92828
Mass
Gene name
Entry name
Entry
Score
Differentially expressed proteins in non-responders before and after 6 weeks of antipsychotic treatment
Table 3.
Secreted polypeptide Protease
Cytoskeletal protein
Structural protein
Transport/cargo protein
Motor protein
Transcription regulatory protein Extracellular matrix protein Motor protein
Transport/cargo protein Transport/cargo protein Transport/cargo protein Transport/cargo protein Transport/cargo protein Transport/cargo protein Voltage-gated channel
DNA methyltransferase Transcription factor RNA binding protein Transcription regulatory protein Transcription factor DNA binding protein Transport/cargo protein
DNA repair protein DNA binding protein RNA helicase
Unclassified
Transcription regulatory protein Dual specificity kinase
Dual specificity kinase
Adapter molecule
Cell cycle control protein
Unclassified
Integral membrane protein G-protein coupled receptor Serine/threonine kinase
Cytoskeletal protein
Extracellular matrix protein Calcium binding protein
Unclassified
Secreted polypeptide
Molecular class
Structural constituent of cytoskeleton Binding Peptidase activity
Nucleocytoplasmic transporter activity Structural molecule activity
Motor activity
Extracellular matrix structural constituent Motor activity
Transcription regulator activity
Transporter activity Transporter activity Transporter activity Transporter activity Transporter activity Transporter activity Voltage-gated ion channel activity
Transcription factor activity DNA binding Transporter activity
DNA methyltransferase activity Transcription factor activity RNA binding Transcription regulator activity
Protein binding Catalytic activity Helicase activity
Protein threonine/tyrosine kinase activity Molecular function unknown
Receptor signaling complex scaffold activity Protein threonine/tyrosine kinase activity Transcription regulator activity
Protein binding
Protein serine/threonine kinase activity Molecular function unknown
G-protein coupled receptor activity
Structural constituent of cytoskeleton Molecular function unknown
Extracellular matrix structural constituent Calcium ion binding
Molecular function unknown
Defense/immunity protein activity
Molecular function
Biological pathways modulated by antipsychotics D Martins-de-Souza et al
5
npj Schizophrenia (2015) 15050
Biological pathways modulated by antipsychotics D Martins-de-Souza et al
Molecular function unknown
Molecular function unknown Molecular function unknown
Unknown
Molecular function unknown Unclassified
Unclassified Integral membrane protein Unclassified Unknown Unknown
Unknown
Molecular function unknown Molecular function unknown Unclassified Unclassified Unknown Unknown
Molecular function unknown
Molecular function unknown Unclassified
Unclassified Unknown
Unknown
Complement activity Molecular function unknown Structural molecule activity Molecular function unknown Molecular function unknown Molecular function unknown Complement protein Unclassified Structural protein Unclassified Unclassified Unclassified Immune response Immune response Energy Metabolism Energy Metabolism Unknown Unknown
Extracellular matrix binding Protease inhibitor activity Molecular function unknown Coagulation factor Coagulation factor Unclassified Protein metabolism Protein metabolism Protein metabolism
npj Schizophrenia (2015) 15050
7 3.745 Up 1.72 118 BRWD2_HUMAN WDR11 Q9BZH6
138,423
2 2
3 2.534 Up
Down 3.268 Up 1.541 0.09 6.46
26.80
25 22
75,614 20,287
40 NT5C3B
Q68D10 SPT2_HUMAN SPTY2D1 Q7Z6W1 TMCO2_HUMAN TMCO2
5NT3L_HUMAN Q969T7
33,893
2 2 Down 1.825 Up 2.584 0.02 15.84 67,189 69,112 25 24 FIGNL2 GDPD5 A6NMB9 FIGL2_HUMAN Q8WTR4 GDPD5_HUMAN
3
2 1.218
Down 2.308
Up 5.58
0.33 105,782
19,737 22
39
ADXL_HUMAN Q6P4F2
FDX1L
LFDH_HUMAN Q008S8
ECT2L
7 4 3 2 2 2 1.269 3.639 1.558 1.46 5.906 1.543 Up Up Up Down Up Down 1.57 6.28 4.82 0.01 29.92 0.02 84,583 151,563 3,843,119 559,165 60,667 49,759 995 36 44 25 47 26 CO2_HUMAN UTY_HUMAN TITIN_HUMAN K1109_HUMAN CI117_HUMAN CCD71_HUMAN P06681 O14607 Q8WZ42 Q2LD37 Q5JU67 Q8IV32
C2 UTY TTN KIAA1109 C9orf117 CCDC71
FA9_HUMAN PROS_HUMAN YE019_HUMAN P00740 P07225 A1L167
F9 PROS1 UBE2QL1
154 56 71
53,114 77,127 18,440
3.06 2.30 6.16
Up Up Up
3.201 1.065 2.05
2 2 2
ATP-dependent Clp protease ATPbinding subunit clpX-like, mitochondrial Coagulation factor IX Vitamin K-dependent protein S Ubiquitin-conjugating enzyme E2Qlike protein 1 Complement C2 Histone demethylase UTY Titin Uncharacterized protein KIAA1109 Uncharacterized protein C9orf117 Coiled-coil domain-containing protein 71 Epithelial cell-transforming sequence 2 oncogene-like Adrenodoxin-like protein, mitochondrial Putative fidgetin-like protein 2 Glycerophosphodiester phosphodiesterase domaincontaining protein 5 7-methylguanosine phosphatespecific 5'-nucleotidase Protein SPT2 homolog Transmembrane and coiled-coil domain-containing protein 2 WD repeat-containing protein 11
Entry name Entry
Table 3. (Continued )
Gene name
Score
Mass
Non Resp T0 × T6
Reg
StDev
Number of peptides
Description
Biological process
Molecular class
Molecular function
6 tend to obscure lower abundance proteins in proteomic studies. Flow-through fractions containing mostly low abundant proteins were treated successively with 5 mM dithiothreitol (30 min, room temperature) and 10 mM iodoacetamide (30 min, 60 °C in the dark) to block reactive sulfhydryl groups on the proteins. The proteins in the samples were then enzymatically digested using trypsin (Promega, Heidelberg) at a trypsin:protein ratio of 1:50 for 16 h at 37 °C. The peptides resulting from the trypsin digestion were analyzed using an Ultimate 3000 Rapid Separation Liquid Chromatography system (Dionex, Amsterdam, The Netherlands) coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific, Bremen, Germany). For the chromatography stage, 1 μg of peptide mixture from each sample was preconcentrated on a 100-μm trapping column (Acclaim C18 PepMap100, 100 μm × 2 cm, Thermo Scientific) at a flow rate of 20 μl/min in 0.1% trifluoroacetic acid. This was followed by separation on a 75-μm column (Acclaim C18 PepMap100, 75 μm × 50 cm, Thermo Scientific) using a binary gradient of solvents A (0.1% formic acid) and B (0.1% formic acid in 84% acetonitrile) ranging from 3/97% A/B to 58/42% A/B over 160 min at a flow rate of 250 nl/min. In order to minimize carry-over effects from previous sample runs, a wash program with high organic content was used prior to each sample injection.7 For the mass spectrometry stage, samples were measured in data dependent acquisition mode, survey scans were acquired in the Orbitrap at resolution 60,000 and the 10 most intense signals were fragmented in the ion trap using collision-induced dissociation (CID). The respective target values and maximum injection times were 106 ions and 100 ms for the MS stage, and 104 ions and 50 ms for the MS/MS stage. Precursor ions with charge states between +2 and +5 were selected for fragmentation, using a dynamic exclusion of 15 s. The resulting raw data were processed using an in-house version of the MASCOT search engine for protein identification and MASCOT Distiller for label-free spectral counting quantification. The cut-off criteria were set at a minimum of 2 peptides for identification and at least 5 MS/MS spectra for quantification. Differences in protein expression between T6 and T0 samples for each patient were determined using Student's t-test (P o0.05) with a false discovery rate set at 0.1. Differentially expressed proteins were classified according to their biological and molecular processes using the Human Protein Reference Database (http://www.hprd.org). For interpretation of the biological and biochemical significance of differentially expressed proteins, the UniProt identification codes of proteins with a T6/T0 ratio42:1 (P40.05) were uploaded into the Ingenuity Pathways Knowledgebase (IPKB). This software determines over-represented pathways by overlaying experimental protein data onto pre-existing biological pathway maps. The shotgun mass spectrometry profiling analysis resulted in identification of 18,227 peptides, corresponding to 985 proteins associated with 10 different biological processes (Figure 1a). Intriguingly, 25% of the proteins are of unknown function and are under investigation by our group. Samples results were divided in four groups. These were T0 responders (T0-R), T6 responders (T6-R), T0 non-responders (T0-NR) and T6 non-responders (T6-NR), independent of the antipsychotic employed. The comparison T6-R/T0-R resulted in identification of 41 differentially expressed proteins, which ranged across seven biological processes (Table 2). The comparison T6-NR/T0-NR identified 58 differentially expressed proteins, which covered eight biological processes (Table 3). One of the proteins (coronin-2A) was increased in responders and decreased in nonresponders. We evaluated the differential expression of coronin2A by western blot as described previously8 in two pools of the same samples analyzed by mass spectrometry to confirm this was not technically biased. The mass spectrometry finding was validated using a primary antibody against coronin-2A (Pierce, © 2015 Schizophrenia International Research Society/Nature Publishing Group
Biological pathways modulated by antipsychotics D Martins-de-Souza et al
PA5–30206) (Figure 1b). The blotting membrane was stained with Ponceau Red and used as loading control. In general, responders and non-responders showed similar effects on the same biochemical pathways after 6 weeks of antipsychotic treatment (Figure 1c). However, there were some differences as responders showed changes in almost twice the number of proteins involved in protein metabolism pathways, non-responders showed energy metabolism differences, which were not observed in the responders. Potentially the most important finding of this study was that antipsychotic treatment led to opposite directional changes in some of the component proteins in responders and non-responders, as described for coronin-2A above. For example, the responders showed a 4.1 to 1 ratio of decreased to increased proteins in the pathway ‘regulation of nucleic acids metabolism’, and this ratio was 2.0 to 1 in the non-responders (Figure 1c and d). A similar scenario was observed for altered proteins associated with ‘protein metabolism’. Although most of the protein components of this pathway showed a decrease in responders, all of the proteins of this same pathway were increased in non-responders. Proteins regulating nucleic acids metabolism modulated by antipsychotics may be pivotal to processes such as DNA methylation and demethylation as well as chromatin remodeling, which may balance gene expression in response to antipsychotics.9 The differences we observed here reinforce the notion that the success of antipsychotic medication may be dependent on individual genetic backgrounds. In this scenario, the absorption, transport, receptor binding, metabolism and excretion of antipsychotics will be distinct for each patient. This supports the concept of personalized medicine strategies using pharmacogenomic approaches as a means of maximizing the potential effects of medications.10 In this study, antipsychotic treatment also modulated proteins associated with the ‘metabolism of proteins’ pathway oppositely in responders and non-responders. This is most likely related to protein turnover effects and may explain some of the above differences in protein expression levels in the two groups, as described in a previous study of antipsychotic treatment response (Lester et al. 2012). The present results indicate that classifying responders and non-responders on the level of biochemical pathways rather than individual proteins may lead to a better understanding of the diverse effects of antipsychotics in schizophrenia patients. Investigation of individual proteins may not be as useful considering the diverse nature of human samples and the existence of redundant systems for the regulation of biochemical pathways in most biological systems. Nevertheless, several of these proteins such as coronin-2A are currently undergoing further analysis in our laboratory as potential antipsychotic treatment response biomarkers. Besides the added value to strategies of translational and personalized medicine, our results suggest new directions to be taken in the search for more effective treatments and the development of new medications for individuals suffering from schizophrenia.
© 2015 Schizophrenia International Research Society/Nature Publishing Group
7
ACKNOWLEDGMENTS We dedicate our efforts to psychiatric patients and their families and thank them for understanding the importance of science and for having made our research possible. DMS is funded by FAPESP (São Paulo Research Foundation, grants 2013/08711-3, 2013/25702-8 and 2014/10068-4).
CONTRIBUTIONS DMS and JS conceived the study. JS collected all samples from patients and clinical data. FAS and RPZ performed the mass spectrometry experiments. DMS obtained and analyzed the data and wrote the first version of the manuscript. FAS, RPZ and JS reviewed the data analysis and contributed significantly to manuscript writing. PCG edited the final version of the manuscript and contributed to data interpretation. All authors approved this version for publication.
COMPETING INTERESTS The authors declare no conflict of interest.
REFERENCES 1. Martins de Souza D. Proteomics tackling schizophrenia as a pathway disorder. Schizophr. Bull. 38, 1107–1108 (2012). 2. Jablensky A. Epidemiology of schizophrenia: the global burden of disease and disability. Eur. Arch. Psychiatry Clin. Neurosci. 250, 274–285 (2000). 3. Tandon R., Nasrallah H. A., Keshavan M. S. Schizophrenia, ‘Just the Facts’ 5 treatment and prevention Past, present, and future. Schizophr. Res. 122, 1–23 (2010). 4. Steiner, J. et al. Increased prevalence of diverse N-methyl-D-aspartate glutamate receptor antibodies in patients with an initial diagnosis of schizophrenia: specific relevance of IgG NR1a antibodies for distinction from N-methyl-D-aspartate glutamate receptor encephalitis. JAMA Psychiatry 70, 271–278 (2013). 5. Leucht, S., Davis, J. M., Engel, R. R., Kissling, W. & Kane, J. M. Definitions of response and remission in schizophrenia: recommendations for their use and their presentation. Acta Psychiatr Scand Suppl 119, 7–14 (2009). 6. Jaros, J. A., Guest, P. C., Bahn, S. & Martins de Souza, D. Affinity depletion of plasma and serum for mass spectrometry-based proteome analysis. Methods Mol. Biol. 1002, 1–11 (2013). 7. Burkhart, J. M., Schumbrutzki, C., Wortelkamp, S., Sickmann, A. & Zahedi, R. P. Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics. J. Proteomics 75, 1454–1462 (2012). 8. Guest, P. C. et al. MK-801 treatment affects glycolysis in oligodendrocytes more than in astrocytes and neuronal cells: insights for schizophrenia. Front. Cell Neurosci. 9, 180 (2015). 9. Guidotti, A. & Grayson, D. R. DNA methylation and demethylation as targets for antipsychotic therapy. Dialogues Clin Neurosci 16, 419–429 (2014). 10. Xu, Q. et al. Pharmacogenomics can improve antipsychotic treatment in schizophrenia. Front. Med. 7, 180–190 (2013).
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npj Schizophrenia (2015) 15050
BRIEF COMMUNICATION
OPEN
Biological pathways modulated by antipsychotics in the blood plasma of schizophrenia patients and their association to a clinical response Daniel Martins-de-Souza1,2, Fiorella A Solari3, Paul C Guest1, René P Zahedi3 and Johann Steiner4,5 Proteomics is a valuable tool to unravel molecular mechanisms involved in human disorders. Considering the mediocre effectiveness of antipsychotics, which are the main class of drug used to treat schizophrenia, we analyzed a cohort of 58 schizophrenia patients who had blood collected before and after 6 weeks of antipsychotic treatment using a shotgun mass spectrometry proteomic profiling approach. Our aim was to unravel molecular pathways involved with an effective drug response. The results showed that all patients had essentially the same biochemical pathways triggered Independent of the antipsychotic response outcome. However, we observed that these pathways were regulated in different directions in blood samples from those who responded well to antipsychotics, compared with those who had a poorer outcome. These data are novel, timely and may help to guide new research efforts in the design of new treatments or medications for schizophrenia based on biologically relevant pathways. npj Schizophrenia (2015) 1, Article number: 15050; doi:10.1038/npjschz.2015.50; published online 9 December 2015
Schizophrenia is the result of a combined dysfunction of genetic, biochemical and neurodevelopmental components that may be triggered by environmental factors. This cascade of events will most likely lead to disruption of cellular and tissue homeostasis, impairing the molecular pathways, which govern the cellular machinery throughout the body. Given the widespread nature of these effects, multiplex molecular approaches such as proteomics are required to provide new insights, as such methods can target hundreds of disease-relevant molecules, including those involved in the pathogenesis. In addition, by identifying specific proteins that are present at different steady-state levels in the disease state, proteomics can aid the discovery of molecular biomarker candidates.1 Although our understanding of the molecular basis of schizophrenia has evolved recently, there are still many factors that do not connect or are still unknown. Thus, identification of these factors and increasing our understanding of their impact on the disease would be an important step forward. In addition, no factors emerging from molecular studies have been translated into clinical use for schizophrenia studies to date, despite the urgent need for both disease- and medication-related biomarker candidates. This is of particular importance owing to the fact that schizophrenia is an incurable disorder, which demands continuous healthcare, thus generating lifelong suffering for the patients and substantial expenses on the health-care systems. In the US, treatment and management of schizophrenia was estimated at a cost of 62 billion dollars per year, in the year 2000.2 Although psychosocial interventions are available, disease management is mainly based on treatment with antipsychotic medications. However, ~ 40% of schizophrenia patients do not
respond properly to these medications and 460% end up abandoning treatment due to undesirable side effects.3 Consequently, the intellectual and cognitive capacities of the patients may worsen, making them incapable of functioning adequately in society, thereby producing a further socioeconomic burden. Consistent with our lack of understanding of disease pathophysiology, knowledge on the effects of medication on metabolism and other molecular pathways in the patients is scarce. With this in mind, we have employed a proteomic approach in an attempt to increase the understanding of the molecular pathways affected in response to currently used atypical antipsychotic medications. It was of particular interest to identify biomarker candidates associated with a positive response. The availability of such early-response biomarkers could eventually be used to help diminish the duration of poor response periods, thereby diminishing disease severity and improving the outcomes for the patients. The cohort studied in this investigation consisted of 58 acutely ill patients who received the atypical antipsychotic drugs olanzapine (n = 18), quetiapine (n = 14) or risperidone (n = 26) (Table 1). Blood samples were collected by intravenous puncture as previously described in the psychiatric Clinic of the University of Magdeburg (Germany).4 Citrate plasma samples were collected at baseline (T0), when patients were acutely ill and either antipsychotic-naive (n = 23) or antipsychotic-free for at least 6 weeks (n = 35), and after 6 weeks when all the subjects had been treated as inpatients (T6). Patients with other medical conditions such as type 2 diabetes mellitus, hypertension, cardiovascular or autoimmune diseases were excluded. In line with previous studies,5 the patients were separated into responders (n = 36) and
1 Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas, Brazil; 2UNICAMP’s Neurobiology Center, Campinas, Brazil; 3Leibniz Institut für Analytische Wissenschaften (ISAS), Dortmund, Germany; 4Department of Psychiatry and Psychotherapy, University of Magdeburg, Magdeburg, Germany and 5The Center for Behavioral Brain Sciences, Magdeburg, Germany. Correspondence: D Martins-de-Souza ([email protected]) Received 8 July 2015; revised 22 October 2015; accepted 28 October 2015
© 2015 Schizophrenia International Research Society/Nature Publishing Group
99 105 129 133 136 140 147 151 156 162 208 247 249 260 277 278 331 332 363 366 368 370 371 375 391 400 404 408 410 422 426 428 431 440 452 454 464 468 474 476 479 487 493 520 521 522 524 526 539 541 545 568 570 572 579 592 663 800
ID patient
Table 1.
7 28 22 17 24 25 31 26 17 20 38 25 22 21 26 37 31 23 27 28 34 26 35 18 25 19 29 15 12 28 26 20 24 33 14 22 25 19 28 17 24 37 28 16 27 17 21 26 24 16 28 21 26 16 14 38 23 16
PANSS P1-P7
17 32 29 34 24 26 31 9 16 16 18 25 41 11 12 29 9 29 25 16 20 9 14 30 11 27 10 13 26 41 23 10 26 12 20 16 18 18 14 17 14 7 29 24 17 7 13 38 27 7 12 30 12 7 18 25 11 15
PANSS N1-N7
37 63 45 49 46 58 55 40 28 37 35 55 41 31 38 58 41 39 41 40 61 39 43 50 51 54 40 52 47 45 46 60 42 45 36 48 37 48 51 37 44 62 51 38 50 40 40 71 58 30 35 43 45 25 34 43 22 30
PANSS G1-G16
T0 raw PANSS
61 123 101 106 104 115 126 79 64 76 109 111 107 76 83 137 92 95 100 93 132 81 104 101 97 108 97 87 91 121 105 94 95 104 80 94 94 88 99 76 91 115 116 90 102 67 79 138 112 53 84 100 89 51 75 122 59 65
PANSS total
0 21 15 10 17 18 24 19 10 13 31 18 15 14 19 30 24 16 20 21 27 19 28 11 18 12 22 8 5 21 19 13 17 26 7 15 18 12 21 10 17 30 21 9 20 10 14 19 17 9 21 14 19 9 7 31 16 9
PANSS P1-P7 10 25 22 27 17 19 24 2 9 9 11 18 34 4 5 22 2 22 18 9 13 2 7 23 4 20 3 6 19 34 16 3 19 5 13 9 11 11 7 10 7 0 22 17 10 0 6 31 20 0 5 23 5 0 11 18 4 8
PANSS N1-N7 21 47 29 33 30 42 39 24 12 21 19 39 25 15 22 42 25 23 25 24 45 23 27 34 35 38 24 36 31 29 30 44 26 29 20 32 21 32 35 21 28 46 35 22 34 24 24 55 42 14 19 27 29 9 18 27 6 14
PANSS G1-G16
T0 corrected PANSS
31 93 66 70 64 79 87 45 31 43 61 75 74 33 46 94 51 61 63 54 85 44 62 68 57 70 49 50 55 84 65 60 62 60 40 56 50 55 63 41 52 76 78 48 64 34 44 105 79 23 45 64 53 18 36 76 26 31
PANSS total 7 33 14 10 15 16 12 8 12 7 12 15 17 8 9 13 15 16 10 9 17 9 8 21 11 7 7 15 20 7 12 19 12 13 17 7 8 9 32 14 13 13 15 9 10 17 12 16 21 13 15 16 11 7 11 15 10 11
PANSS P1-P7 11 32 24 18 11 15 24 8 13 14 9 8 31 24 7 22 8 24 16 10 20 15 7 27 9 11 7 19 23 9 10 12 14 11 9 7 14 18 21 11 9 17 21 11 9 9 14 29 16 7 7 26 12 7 7 7 7 8
PANSS N1-N7 29 64 32 28 26 34 39 19 24 19 20 27 37 24 21 30 31 31 23 21 47 27 20 42 22 20 20 41 48 24 22 50 26 33 26 16 25 24 42 32 26 32 29 25 21 27 29 39 49 25 23 28 30 17 21 41 18 18
PANSS G1-G16
T6 raw PANSS
Patients demographics and PANSS score before and after treatment
50 141 74 59 55 72 82 38 54 43 44 54 90 59 42 71 61 75 52 43 91 54 38 97 45 41 40 82 98 43 47 89 55 65 65 33 50 54 105 64 52 70 68 49 43 57 58 87 94 56 55 73 56 34 42 68 38 40
PANSS total 0 26 7 3 8 9 5 1 5 0 5 8 10 1 2 6 8 9 3 2 10 2 1 14 4 0 0 8 13 0 5 12 5 6 10 0 1 2 25 7 6 6 8 2 3 10 5 9 14 6 8 9 4 0 4 8 3 4
PANSS P1-P7 4 25 17 11 4 8 17 1 6 7 2 1 24 17 0 15 1 17 9 3 13 8 0 20 2 4 0 12 16 2 3 5 7 4 2 0 7 11 14 4 2 10 14 4 2 2 7 22 9 0 0 19 5 0 0 0 0 1
PANSS N1-N7 13 48 16 12 10 18 23 3 8 3 4 11 21 8 5 14 15 15 7 5 31 11 4 26 6 4 4 25 32 8 6 34 10 17 10 0 9 8 26 16 10 16 13 9 5 11 13 23 33 9 7 12 14 1 5 25 2 2
PANSS G1-G16
T6 corrected PANSS
17 99 40 26 22 35 45 5 19 10 11 20 55 26 7 35 24 41 19 10 54 21 5 60 12 8 4 45 61 10 14 51 22 27 22 0 17 21 65 27 18 32 35 15 10 23 25 54 56 15 15 40 23 1 9 33 5 7
PANSS total 0.45 − 0.06 0.39 0.63 0.66 0.56 0.48 0.89 0.39 0.77 0.82 0.73 0.26 0.21 0.85 0.63 0.53 0.33 0.70 0.81 0.36 0.52 0.92 0.12 0.79 0.89 0.92 0.10 − 0.11 0.88 0.78 0.15 0.65 0.55 0.45 1.00 0.66 0.62 − 0.03 0.34 0.65 0.58 0.55 0.69 0.84 0.32 0.43 0.49 0.29 0.35 0.67 0.38 0.57 0.94 0.75 0.57 0.81 0.77
Relative PANSS total improvement T0-T6 Bad responder Bad responder Bad responder Good responder Good responder Good responder Bad responder Good responder Bad responder Good responder Good responder Good responder Bad responder Bad responder Good responder Good responder Good responder Bad responder Good responder Good responder Bad responder Good responder Good responder Bad responder Good responder Good responder Good responder Bad responder Bad responder Good responder Good responder Bad responder Good responder Good responder Bad responder Good responder Good responder Good responder Bad responder Bad responder Good responder Good responder Good responder Good responder Good responder Bad responder Bad responder Bad responder Bad responder Bad responder Good responder Bad responder Good responder Good responder Good responder Good responder Good responder Good responder
All patients
Good response ⩾ 50% corrected PANSS total reduction
Yes No No Yes No No Yes No No Yes No No No No Yes No No Yes Yes Yes Yes Yes No Yes No No No No No No No Yes Yes No No Yes Yes No No No Yes Yes Yes No No Yes Yes Yes No No No No No Yes Yes No No No
First episode patient
Yes No No No No No Yes No No Yes No No No No Yes No No Yes Yes Yes Yes Yes No Yes No No No No No No No Yes Yes No No Yes Yes No No No Yes Yes Yes No No Yes Yes Yes No No No No No Yes Yes No No No
Drug naive
npj Schizophrenia (2015) 15050 11 7 0 0 32 15 0 0 1 11 7 0 0 0 0 10 0 0 0 3 34 0 10 3 0 0 14 21 15
6 2
7 0 0 2 0 9 7 19 1 0 9 23 0 0 0 0 0 6 0 32
0
0 15
Illness duration [years]
Quetiapie Quetiapie Risperidoe Olanzapie Risperidoe Risperidoe Olanzapie Olanzapie Risperidoe Risperidoe Olanzapie Risperidoe Risperidoe Olanzapie Risperidoe Risperidoe Olanzapie Risperidoe Risperidoe Olanzapie Risperidoe Risperidoe Risperidoe Quetiapie Quetiapie Quetiapie Olanzapie Quetiapie Olanzapie Risperidoe Risperidoe Quetiapie Olanzapie Olanzapie Risperidoe Risperidoe Risperidoe Risperidoe Quetiapie Quetiapie Olanzapie Quetiapie Risperidoe Quetiapie Olanzapie Quetiapie Olanzapie Olanzapie Quetiapie Risperidoe Olanzapie Risperidoe Risperidoe Quetiapie Olanzapie Olanzapie Risperidoe Risperidoe
Medication
F M F F F M M F M M M M M M F M F F M M M M F M F F M F F M M F M F M M F M M M F M M F M M F M M M F F M F F M M M
31 47 49 25 44 30 25 45 42 19 28 40 44 22 20 31 39 26 25 34 16 31 48 23 55 32 45 27 50 34 30 38 29 59 36 34 66 35 32 25 53 39 50 31 32 26 36 23 28 53 50 56 36 36 36 30 51 58
Gender Age
19.7 21.8 23.9 24.0 27.4 22.2 28.1 30.5 32.9 20.2 25.3 23.0 17.7 27.5 22.2 22.8 29.8 22.1 20.1 27.7 26.5 24.4 37.2 22.0 29.8 22.7 26.4 22.2 24.1 23.6 21.5 32.9 21.1 35.0 22.4 22.2 31.6 18.3 25.0 22.0 20.1 21.1 19.7 28.7 30.8 21.0 22.0 22.2 23.2 24.8 28.6 29.1 20.4 21.9 30.3 31.1 30.4 20.6
BMI
No Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes No Yes No Yes No No Yes No No Yes Yes Yes Yes Yes Yes Yes No No Yes Yes No Yes Yes Yes Yes Yes Yes No Yes Yes No Yes Yes Yes No Yes Yes Yes No No No
Smoking
Biological pathways modulated by antipsychotics D Martins-de-Souza et al
2
© 2015 Schizophrenia International Research Society/Nature Publishing Group
Biological pathways modulated by antipsychotics D Martins-de-Souza et al
3 Coronin-2A
T6-R T6-R T6-NR T6-NR
% of differentially expressed proteins
% of differentially expressed proteins
Loading control
Figure 1. (a) Biological processes represented by all 985 plasma proteins identified through the shotgun mass spectrometry profiling approach; (b) western blot validation of the mass spectrometry findings for coronin-2A, which showed increased levels in responders and decreased levels in non-responders; (c) Biological pathways affected by antipsychotics in responders (blue bars) and non-responders (red bars); (d) Percentage of proteins increased in responders (blue), decreased in responders (red), increased in non-responders (green) and decreased in non-responders (purple) in the same biological pathways shown in part (c). For (c and d) the y axis represents the percentage of differentially expressed proteins associated to those processes and a star indicates significant differences of the protein constituents (c) or directional changes (d) within a pathway.
non-responders (n = 22), with response defined as a 50% reduction of the corrected (subtraction of minimum scores representing ‘no symptoms’) total Positive and Negative Syndrome Scale (PANSS) scores. The percentage of responders was similar in the antipsychotic-naive (61%; 14/23) and antipsychotic-free (63%; 22/35) subgroups (Table 1). © 2015 Schizophrenia International Research Society/Nature Publishing Group
Blood samples were immediately centrifuged for 10 min at 2,000 × g and resulting supernatants divided in 250-μl aliquots, which were immediately frozen at − 80 °C. Protein concentrations were determined by Bradford assay. Next, the plasma samples were depleted of 14 high-abundant proteins using the MARS-14 immunodepletion system (Agilent; Wokingham, UK)6 as these npj Schizophrenia (2015) 15050
npj Schizophrenia (2015) 15050
SNX17_HUMAN
TGFI1_HUMAN
THS7A_HUMAN
LR16C_HUMAN
FA9_HUMAN HS71L_HUMAN ITIH3_HUMAN
RM03_HUMAN
AACT_HUMAN YE019_HUMAN
Q15036
O43294
Q9UPZ6
Q6F5E8
P00740 P34931 Q06033
P09001
P01011 A1L167
ABCAD_HUMAN ABCA13
ALBU_HUMAN APOA4_HUMAN VDAC1_HUMAN
COPA1_HUMAN
ECM1_HUMAN
ELMO1_HUMAN
NEBU_HUMAN
TBG1_HUMAN
CD5L_HUMAN CI117_HUMAN CCD71_HUMAN
CC74A_HUMAN
CENPP_HUMAN ADXL_HUMAN
YO003_HUMAN LIX1_HUMAN 5NT3L_HUMAN
Q86UQ4
P02768 P06727 P21796
Q9BXS0
Q16610
Q92556
P20929
P23258
O43866 Q5JU67 Q8IV32
Q96AQ1
Q6IPU0 Q6P4F2
Q6ZVN6 Q8N485 Q969T7
60 58 40
29 22
O15018 PDZD2_HUMAN PDZD2 Q7Z6W1 TMCO2_HUMAN TMCO2
38 22
31
201 47 26
27
85
46
435
28
123 1502 26
70
34 21 40 31
28 35
1317 71
28
154 45 299
39
30
39
44
40
33
35
41
HSD47 LIX1 NT5C3B
CENPP FDX1L
CCDC74A
CD5L C9orf117 CCDC71
TUBG1
NEB
ELMO1
ECM1
COL25A1
ALB APOA4 VDAC1
MYCD_HUMAN P73_HUMAN ZN215_HUMAN ABCB5_HUMAN
MYOCD TP73 ZNF215 ABCB5
MLH3 MYCBP2
SERPINA3 UBE2QL1
MRPL3
F9 HSPA1L ITIH3
RLTPR
THSD7A
TGFB1I1
SNX17
PRKDC
FARP2
Q8IZQ8 O15350 Q9UL58 Q2M3G0
Q9UHC1 MLH3_HUMAN O75592 MYCB2_HUMAN
FARP2_HUMAN
PRKDC_HUMAN
O94887
P78527
EFCAB6
CYR61
44,165
303,964 20,287
33,173 32,213 33,893
33,429 19,737
41,808
39,603 60,667 49,759
51,480
775,419
84,517
62,232
65,145
71,317 45,371 30,868
580,604
102,561 70,206 60,922 90,173
166,060 517,856
47,792 18,440
38,893
53,114 70,730 100,072
156,133
192,854
51,321
53,153
473,749
120,725
174,702
25.23 2.59
0.29 0.23 9.21
20.20 2.01
12.33
3.03 0.02 32.45
0.46
0.08
0.13
2.43
0.06
0.04 0.51 26.82
12.73
0.43 0.03 0.11 64.78
0.05 6.52
1.48 0.06
0.27
0.36 0.45 1.52
20.44
0.03
0.31
0.05
41.97
11.93
3.57
0.03
1.948
3.329
7.015
1.954
5.368
1.59 1.951 5.344 1.559
6.347
1.001 1.341
1.079
Up Up
4.449 1.144
Down 1.027 Down 2.73 Up 1.238
Up Up
Up
Up 5.29 Down 1.229 Up 4.607
Down 2.748
Down 7.593
Down 7.738
Up
Down 2.02
Down 1.8 Down 1.061 Up 1.167
Up
Down Down Down Up
Down 2.706 Up 5.641
Up 1.282 Down 4.273
Down 1.813
Down 6.308 Down 2.631 Up 1.254
Up
Down 1.223
Down 1.962
Down 3.201
Up
Up
Up
Down 1.779
Down 6.913
2 2
3 5 3
2 2
2
2 2 2
2
6
5
2
2
6 7 2
2
2 2 2 2
2 2
34 2
2
2 4 5
2
2
2
4
2
2
2
2
2
Biological process
Cell communication and signaling Protein CYR61 Cell communication and signaling EF-hand calcium-binding domainCell communication containing protein 6 and signaling FERM, RhoGEF and pleckstrin domain- Cell communication containing protein 2 and signaling DNA-dependent protein kinase Cell communication catalytic subunit and signaling Sorting nexin-17 Cell communication and signaling Transforming growth factor beta-1Cell communication induced transcript 1 protein and signaling Thrombospondin type-1 domainCell communication containing protein 7A and signaling Leucine-rich repeat-containing protein Cell communication 16C and signaling Coagulation factor IX Protein metabolism Heat shock 70 kDa protein 1-like Protein metabolism Inter-alpha-trypsin inhibitor heavy Protein metabolism chain H3 39S ribosomal protein L3, Protein metabolism mitochondrial Alpha-1-antichymotrypsin Protein metabolism Ubiquitin-conjugating enzyme E2QProtein metabolism like protein 1 DNA mismatch repair protein Mlh3 Reg. nucleic acid metab Probable E3 ubiquitin-protein ligase Reg. nucleic acid metab MYCBP2 Myocardin Reg. nucleic acid metab Tumor protein p73 Reg. nucleic acid metab Zinc finger protein 215 Reg. nucleic acid metab ATP-binding cassette sub-family B Transport member 5 ATP-binding cassette sub-family A Transport member 13 Serum albumin Transport Apolipoprotein A-IV Transport Voltage-dependent anion-selective Transport channel protein 1 Collagen alpha-1(XXV) chain Cell growth and maintenance Extracellular matrix protein 1 Cell growth and maintenance Engulfment and cell motility protein 1 Cell growth and maintenance Nebulin Cell growth and maintenance Tubulin gamma-1 chain Cell growth and maintenance CD5 antigen-like Immune response Uncharacterized protein C9orf117 Unknown Coiled-coil domain-containing protein Unknown 71 Coiled-coil domain-containing protein Unknown 74A Centromere protein P Unknown Adrenodoxin-like protein, Unknown mitochondrial Uncharacterized protein HSD47 Unknown Protein limb expression 1 homolog Unknown 7-methylguanosine phosphate-specific Unknown 5'-nucleotidase PDZ domain-containing protein 2 Unknown Transmembrane and coiled-coil Unknown domain-containing protein 2
Coronin-2A
Description
EFCB6_HUMAN
0.12
Number of peptides
CYR61_HUMAN
60,239
StDev
Q5THR3
35
Reg
O00622
CORO2A
Resp T0 × T6
COR2A_HUMAN
Mass
Q92828
Score
Entry name
Entry
Gene name
Differentially expressed proteins in responders before and after 6 weeks of antipsychotic treatment
Table 2.
Unclassified Integral membrane protein
Unclassified Unclassified Unclassified
Unclassified Unclassified
Unclassified
Secreted polypeptide Unclassified Unclassified
Cytoskeletal protein
Cytoskeletal protein
Motor protein
Extracellular matrix protein
Extracellular matrix protein
Transport/cargo protein Transport/cargo protein Voltage-gated channel
Transport/cargo protein
DNA repair protein Transcription regulatory protein Transcription factor Transcription factor DNA binding protein Integral membrane protein
Protease inhibitor Unclassified
Ribosomal subunit
Coagulation factor Heat shock protein Protease inhibitor
Unclassified
Transcription regulatory protein Unclassified
Adapter molecule
Serine/threonine kinase
Cytoskeletal protein
Calcium binding protein
Extracellular matrix protein
Unclassified
Molecular class
Molecular function unknown Molecular function unknown
Molecular function unknown Molecular function unknown Molecular function unknown
Molecular function unknown Molecular function unknown
Molecular function unknown
Defense/immunity protein activity Molecular function unknown Molecular function unknown
Structural constituent of cytoskeleton
Structural constituent of cytoskeleton
Extracellular matrix structural constituent Extracellular matrix structural constituent Motor activity
Transporter activity Transporter activity Voltage-gated ion channel activity
Transporter activity
Transcription factor activity Transcription factor activity DNA binding Transporter activity
Protein binding Transcription regulator activity
Protease inhibitor activity Molecular function unknown
Structural constituent of ribosome
Extracellular matrix binding Heat shock protein activity Protease inhibitor activity
Molecular function unknown
Molecular function unknown
Protein serine/threonine kinase activity Receptor signaling complex scaffold activity Transcription regulator activity
Structural constituent of cytoskeleton
Extracellular matrix structural constituent Calcium ion binding
Molecular function unknown
Molecular function
Biological pathways modulated by antipsychotics D Martins-de-Souza et al
4
© 2015 Schizophrenia International Research Society/Nature Publishing Group
SEPT07_
SNX17
FARP2_HUMAN
MUC16_HUMAN
OR4KD_HUMAN
PRKX_HUMAN
RN111_HUMAN
SEPT7_HUMAN
SNX17_HUMAN
TEX14_HUMAN
Q8WXI7
Q8NH42
P51817
Q6ZNA4
Q16181
Q15036
Q8IWB6
THSD7A
MLH3 PARP2 DDX46
DNMT1 MYOCD PCBP2 ZC3H13
THS7A_HUMAN
Q9UHC1 MLH3_HUMAN Q9UGN5 PARP2_HUMAN Q7L014 DDX46_HUMAN
DNMT1_HUMAN MYCD_HUMAN PCBP2_HUMAN ZC3HD_HUMAN
ZFHX4_HUMAN ZN215_HUMAN ABCAD_HUMAN
ALBU_HUMAN APOA4_HUMAN APOB_HUMAN GOGA4_HUMAN S14L4_HUMAN SYN3_HUMAN VDAC1_HUMAN
Q9UPZ6
P26358 Q8IZQ8 Q15366 Q5T200
Q86UP3 Q9UL58 Q86UQ4
P02768 P06727 P04114 Q13439 Q9UDX3 O14994 P21796
TGFB1I1
© 2015 Schizophrenia International Research Society/Nature Publishing Group
ECM1
KIF17
KIF3A
MVP
MYBPC1
TUBG1
ECM1_HUMAN
KIF17_HUMAN
KIF3A_HUMAN
MVP_HUMAN
MYPC1_HUMAN
TBG1_HUMAN
SAMP_HUMAN CLPX_HUMAN
Q16610
Q9P2E2
Q9Y496
Q14764
Q00872
P23258
P02743 O76031
APCS CLPX
VOPP1
Q96AW1 ECOP_HUMAN
ALB APOA4 APOB GOLGA4 SEC14L4 SYN3 VDAC1
ZFHX4 ZNF215 ABCA13
WEE1
TGFI1_HUMAN
WEE1_HUMAN
O43294
P30291
TEX14
RNF111
PRKX
OR4K13
MUC16
FARP2
EFCAB6
O94887
CYR61
CYR61_HUMAN
EFCB6_HUMAN
O00622
Q5THR3
CHGA
2577 41
27
35
36
27
31
435
32
123 1,502 1,709 176 67 34 26
35 40 70
30 34 17 33
28 28 117
30
51
39
27
44
29
59
45
33
29
33
35
41
35
31
50,829
25,485 69,922
51,480
129,240
99,551
80,687
115,784
62,232
19,839
71,317 45,371 516,666 261,892 47,070 63,491 30,868
398,157 60,922 580,604
185,388 102,561 38,955 197,203
166,060 66,734 117,803
192,854
72,237
51,321
169,107
53,153
50,933
110,119
41,041
34,751
2,359,682
120,725
174,702
44,165
60,239
0.02
1.45 25.86
0.02
0.01
7.09
0.01
0.01
0.31
2.46
0.61 0.56 1.46 44.57 13.48 0.03 8.81
7.38 0.02 0.05
0.07 9.73 0.01 0.02
0.01 6.29 8.70
0.01
0.12
0.01
5.62
10.79
4.12
18.82
9.97
0.26
0.04
4.14
0.01
0.42
9.12
1.445
1.288
1.184
5.889
1.665
1.351
1.08 2.619 1.318 2.653
1.841
4.848 1.078 1.306 1.39 1.447 1.434 6.002
3.151
Up Up
9.908 2.463
Down 1.934
Down 1.213
Up
Down 1.08
Down 1.028
Down 5.743
Up
Down Down Up Up Up Down Up
Up 7.307 Down 9.007 Down 1.619
Down Up Down Down
Down 1.087 Up 1.04 Up 4.669
Down 1.223
Down 3.019
Down 4.21
Up
Up
Up
Up
Up
Down 4.954
Down 8.085
Up
Down 5.227
Down 1.137
1.259
Down 1.972 Up
9 3
2
2
4
2
2
2
2
6 7 17 15 3 2 2
2 2 2
2 2 2 3
2 2 11
2
4
2
2
3
2
4
4
3
3
2
2
2
2
3
nucleic nucleic nucleic nucleic
acid acid acid acid
metab metab metab metab
Transport Transport Transport Transport Transport Transport Transport
Reg. nucleic acid metab Reg. nucleic acid metab Transport
Reg. Reg. Reg. Reg.
Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Cell communication and signaling Reg. nucleic acid metab Reg. nucleic acid metab Reg. nucleic acid metab
Biological process
Cell growth and maintenance Cell growth and maintenance Kinesin-like protein KIF17 Cell growth and maintenance Kinesin-like protein KIF3A Cell growth and maintenance Major vault protein Cell growth and maintenance Myosin-binding protein C, slow-type Cell growth and maintenance Tubulin gamma-1 chain Cell growth and maintenance Serum amyloid P-component Protein metabolism Protein metabolism
Thrombospondin type-1 domaincontaining protein 7A DNA mismatch repair protein Mlh3 Poly [ADP-ribose] polymerase 2 Probable ATP-dependent RNA helicase DDX46 DNA (cytosine-5)-methyltransferase 1 Myocardin Poly(rC)-binding protein 2 Zinc finger CCCH domain-containing protein 13 Zinc finger homeobox protein 4 Zinc finger protein 215 ATP-binding cassette sub-family A member 13 Serum albumin Apolipoprotein A-IV Apolipoprotein B-100 Golgin sub-family A member 4 SEC14-like protein 4 Synapsin-3 Voltage-dependent anion-selective channel protein 1 Vesicular, overexpressed in cancer, prosurvival protein 1 Extracellular matrix protein 1
Inactive serine/threonine-protein kinase TEX14 Transforming growth factor beta-1induced transcript 1 protein Wee1-like protein kinase
Sorting nexin-17
Septin-7
cAMP-dependent protein kinase catalytic subunit PRKX E3 ubiquitin-protein ligase Arkadia
Olfactory receptor 4K13
EF-hand calcium-binding domaincontaining protein 6 FERM, RhoGEF and pleckstrin domain-containing protein 2 Mucin-16
Protein CYR61
Coronin-2A
Chromogranin-A
Description
CORO2A
Number of peptides
COR2A_HUMAN
StDev
CMGA_HUMAN
Reg
P10645
Non Resp T0 × T6
Q92828
Mass
Gene name
Entry name
Entry
Score
Differentially expressed proteins in non-responders before and after 6 weeks of antipsychotic treatment
Table 3.
Secreted polypeptide Protease
Cytoskeletal protein
Structural protein
Transport/cargo protein
Motor protein
Transcription regulatory protein Extracellular matrix protein Motor protein
Transport/cargo protein Transport/cargo protein Transport/cargo protein Transport/cargo protein Transport/cargo protein Transport/cargo protein Voltage-gated channel
DNA methyltransferase Transcription factor RNA binding protein Transcription regulatory protein Transcription factor DNA binding protein Transport/cargo protein
DNA repair protein DNA binding protein RNA helicase
Unclassified
Transcription regulatory protein Dual specificity kinase
Dual specificity kinase
Adapter molecule
Cell cycle control protein
Unclassified
Integral membrane protein G-protein coupled receptor Serine/threonine kinase
Cytoskeletal protein
Extracellular matrix protein Calcium binding protein
Unclassified
Secreted polypeptide
Molecular class
Structural constituent of cytoskeleton Binding Peptidase activity
Nucleocytoplasmic transporter activity Structural molecule activity
Motor activity
Extracellular matrix structural constituent Motor activity
Transcription regulator activity
Transporter activity Transporter activity Transporter activity Transporter activity Transporter activity Transporter activity Voltage-gated ion channel activity
Transcription factor activity DNA binding Transporter activity
DNA methyltransferase activity Transcription factor activity RNA binding Transcription regulator activity
Protein binding Catalytic activity Helicase activity
Protein threonine/tyrosine kinase activity Molecular function unknown
Receptor signaling complex scaffold activity Protein threonine/tyrosine kinase activity Transcription regulator activity
Protein binding
Protein serine/threonine kinase activity Molecular function unknown
G-protein coupled receptor activity
Structural constituent of cytoskeleton Molecular function unknown
Extracellular matrix structural constituent Calcium ion binding
Molecular function unknown
Defense/immunity protein activity
Molecular function
Biological pathways modulated by antipsychotics D Martins-de-Souza et al
5
npj Schizophrenia (2015) 15050
Biological pathways modulated by antipsychotics D Martins-de-Souza et al
Molecular function unknown
Molecular function unknown Molecular function unknown
Unknown
Molecular function unknown Unclassified
Unclassified Integral membrane protein Unclassified Unknown Unknown
Unknown
Molecular function unknown Molecular function unknown Unclassified Unclassified Unknown Unknown
Molecular function unknown
Molecular function unknown Unclassified
Unclassified Unknown
Unknown
Complement activity Molecular function unknown Structural molecule activity Molecular function unknown Molecular function unknown Molecular function unknown Complement protein Unclassified Structural protein Unclassified Unclassified Unclassified Immune response Immune response Energy Metabolism Energy Metabolism Unknown Unknown
Extracellular matrix binding Protease inhibitor activity Molecular function unknown Coagulation factor Coagulation factor Unclassified Protein metabolism Protein metabolism Protein metabolism
npj Schizophrenia (2015) 15050
7 3.745 Up 1.72 118 BRWD2_HUMAN WDR11 Q9BZH6
138,423
2 2
3 2.534 Up
Down 3.268 Up 1.541 0.09 6.46
26.80
25 22
75,614 20,287
40 NT5C3B
Q68D10 SPT2_HUMAN SPTY2D1 Q7Z6W1 TMCO2_HUMAN TMCO2
5NT3L_HUMAN Q969T7
33,893
2 2 Down 1.825 Up 2.584 0.02 15.84 67,189 69,112 25 24 FIGNL2 GDPD5 A6NMB9 FIGL2_HUMAN Q8WTR4 GDPD5_HUMAN
3
2 1.218
Down 2.308
Up 5.58
0.33 105,782
19,737 22
39
ADXL_HUMAN Q6P4F2
FDX1L
LFDH_HUMAN Q008S8
ECT2L
7 4 3 2 2 2 1.269 3.639 1.558 1.46 5.906 1.543 Up Up Up Down Up Down 1.57 6.28 4.82 0.01 29.92 0.02 84,583 151,563 3,843,119 559,165 60,667 49,759 995 36 44 25 47 26 CO2_HUMAN UTY_HUMAN TITIN_HUMAN K1109_HUMAN CI117_HUMAN CCD71_HUMAN P06681 O14607 Q8WZ42 Q2LD37 Q5JU67 Q8IV32
C2 UTY TTN KIAA1109 C9orf117 CCDC71
FA9_HUMAN PROS_HUMAN YE019_HUMAN P00740 P07225 A1L167
F9 PROS1 UBE2QL1
154 56 71
53,114 77,127 18,440
3.06 2.30 6.16
Up Up Up
3.201 1.065 2.05
2 2 2
ATP-dependent Clp protease ATPbinding subunit clpX-like, mitochondrial Coagulation factor IX Vitamin K-dependent protein S Ubiquitin-conjugating enzyme E2Qlike protein 1 Complement C2 Histone demethylase UTY Titin Uncharacterized protein KIAA1109 Uncharacterized protein C9orf117 Coiled-coil domain-containing protein 71 Epithelial cell-transforming sequence 2 oncogene-like Adrenodoxin-like protein, mitochondrial Putative fidgetin-like protein 2 Glycerophosphodiester phosphodiesterase domaincontaining protein 5 7-methylguanosine phosphatespecific 5'-nucleotidase Protein SPT2 homolog Transmembrane and coiled-coil domain-containing protein 2 WD repeat-containing protein 11
Entry name Entry
Table 3. (Continued )
Gene name
Score
Mass
Non Resp T0 × T6
Reg
StDev
Number of peptides
Description
Biological process
Molecular class
Molecular function
6 tend to obscure lower abundance proteins in proteomic studies. Flow-through fractions containing mostly low abundant proteins were treated successively with 5 mM dithiothreitol (30 min, room temperature) and 10 mM iodoacetamide (30 min, 60 °C in the dark) to block reactive sulfhydryl groups on the proteins. The proteins in the samples were then enzymatically digested using trypsin (Promega, Heidelberg) at a trypsin:protein ratio of 1:50 for 16 h at 37 °C. The peptides resulting from the trypsin digestion were analyzed using an Ultimate 3000 Rapid Separation Liquid Chromatography system (Dionex, Amsterdam, The Netherlands) coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific, Bremen, Germany). For the chromatography stage, 1 μg of peptide mixture from each sample was preconcentrated on a 100-μm trapping column (Acclaim C18 PepMap100, 100 μm × 2 cm, Thermo Scientific) at a flow rate of 20 μl/min in 0.1% trifluoroacetic acid. This was followed by separation on a 75-μm column (Acclaim C18 PepMap100, 75 μm × 50 cm, Thermo Scientific) using a binary gradient of solvents A (0.1% formic acid) and B (0.1% formic acid in 84% acetonitrile) ranging from 3/97% A/B to 58/42% A/B over 160 min at a flow rate of 250 nl/min. In order to minimize carry-over effects from previous sample runs, a wash program with high organic content was used prior to each sample injection.7 For the mass spectrometry stage, samples were measured in data dependent acquisition mode, survey scans were acquired in the Orbitrap at resolution 60,000 and the 10 most intense signals were fragmented in the ion trap using collision-induced dissociation (CID). The respective target values and maximum injection times were 106 ions and 100 ms for the MS stage, and 104 ions and 50 ms for the MS/MS stage. Precursor ions with charge states between +2 and +5 were selected for fragmentation, using a dynamic exclusion of 15 s. The resulting raw data were processed using an in-house version of the MASCOT search engine for protein identification and MASCOT Distiller for label-free spectral counting quantification. The cut-off criteria were set at a minimum of 2 peptides for identification and at least 5 MS/MS spectra for quantification. Differences in protein expression between T6 and T0 samples for each patient were determined using Student's t-test (P o0.05) with a false discovery rate set at 0.1. Differentially expressed proteins were classified according to their biological and molecular processes using the Human Protein Reference Database (http://www.hprd.org). For interpretation of the biological and biochemical significance of differentially expressed proteins, the UniProt identification codes of proteins with a T6/T0 ratio42:1 (P40.05) were uploaded into the Ingenuity Pathways Knowledgebase (IPKB). This software determines over-represented pathways by overlaying experimental protein data onto pre-existing biological pathway maps. The shotgun mass spectrometry profiling analysis resulted in identification of 18,227 peptides, corresponding to 985 proteins associated with 10 different biological processes (Figure 1a). Intriguingly, 25% of the proteins are of unknown function and are under investigation by our group. Samples results were divided in four groups. These were T0 responders (T0-R), T6 responders (T6-R), T0 non-responders (T0-NR) and T6 non-responders (T6-NR), independent of the antipsychotic employed. The comparison T6-R/T0-R resulted in identification of 41 differentially expressed proteins, which ranged across seven biological processes (Table 2). The comparison T6-NR/T0-NR identified 58 differentially expressed proteins, which covered eight biological processes (Table 3). One of the proteins (coronin-2A) was increased in responders and decreased in nonresponders. We evaluated the differential expression of coronin2A by western blot as described previously8 in two pools of the same samples analyzed by mass spectrometry to confirm this was not technically biased. The mass spectrometry finding was validated using a primary antibody against coronin-2A (Pierce, © 2015 Schizophrenia International Research Society/Nature Publishing Group
Biological pathways modulated by antipsychotics D Martins-de-Souza et al
PA5–30206) (Figure 1b). The blotting membrane was stained with Ponceau Red and used as loading control. In general, responders and non-responders showed similar effects on the same biochemical pathways after 6 weeks of antipsychotic treatment (Figure 1c). However, there were some differences as responders showed changes in almost twice the number of proteins involved in protein metabolism pathways, non-responders showed energy metabolism differences, which were not observed in the responders. Potentially the most important finding of this study was that antipsychotic treatment led to opposite directional changes in some of the component proteins in responders and non-responders, as described for coronin-2A above. For example, the responders showed a 4.1 to 1 ratio of decreased to increased proteins in the pathway ‘regulation of nucleic acids metabolism’, and this ratio was 2.0 to 1 in the non-responders (Figure 1c and d). A similar scenario was observed for altered proteins associated with ‘protein metabolism’. Although most of the protein components of this pathway showed a decrease in responders, all of the proteins of this same pathway were increased in non-responders. Proteins regulating nucleic acids metabolism modulated by antipsychotics may be pivotal to processes such as DNA methylation and demethylation as well as chromatin remodeling, which may balance gene expression in response to antipsychotics.9 The differences we observed here reinforce the notion that the success of antipsychotic medication may be dependent on individual genetic backgrounds. In this scenario, the absorption, transport, receptor binding, metabolism and excretion of antipsychotics will be distinct for each patient. This supports the concept of personalized medicine strategies using pharmacogenomic approaches as a means of maximizing the potential effects of medications.10 In this study, antipsychotic treatment also modulated proteins associated with the ‘metabolism of proteins’ pathway oppositely in responders and non-responders. This is most likely related to protein turnover effects and may explain some of the above differences in protein expression levels in the two groups, as described in a previous study of antipsychotic treatment response (Lester et al. 2012). The present results indicate that classifying responders and non-responders on the level of biochemical pathways rather than individual proteins may lead to a better understanding of the diverse effects of antipsychotics in schizophrenia patients. Investigation of individual proteins may not be as useful considering the diverse nature of human samples and the existence of redundant systems for the regulation of biochemical pathways in most biological systems. Nevertheless, several of these proteins such as coronin-2A are currently undergoing further analysis in our laboratory as potential antipsychotic treatment response biomarkers. Besides the added value to strategies of translational and personalized medicine, our results suggest new directions to be taken in the search for more effective treatments and the development of new medications for individuals suffering from schizophrenia.
© 2015 Schizophrenia International Research Society/Nature Publishing Group
7
ACKNOWLEDGMENTS We dedicate our efforts to psychiatric patients and their families and thank them for understanding the importance of science and for having made our research possible. DMS is funded by FAPESP (São Paulo Research Foundation, grants 2013/08711-3, 2013/25702-8 and 2014/10068-4).
CONTRIBUTIONS DMS and JS conceived the study. JS collected all samples from patients and clinical data. FAS and RPZ performed the mass spectrometry experiments. DMS obtained and analyzed the data and wrote the first version of the manuscript. FAS, RPZ and JS reviewed the data analysis and contributed significantly to manuscript writing. PCG edited the final version of the manuscript and contributed to data interpretation. All authors approved this version for publication.
COMPETING INTERESTS The authors declare no conflict of interest.
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npj Schizophrenia (2015) 15050
