Identifying Potential Novel Pathways andTherapeutic Targets of Major Depressive Disorderwith Functional GenomicsKevin Bao  ( kevinbao54@gmail.com )

BioScience Project

Research Article

Keywords: depression, major depression, major depressive disorder, ketamine, neuroplasticity,neuroin�ammation, glutamate, acetylcholine, protein interaction network

Posted Date: March 31st, 2021

DOI: https://doi.org/10.21203/rs.3.rs-374465/v1

License: This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License

Identifying Potential Novel Pathways and Therapeutic Targets of Major Depressive

Disorder with Functional Genomics

Kevin Bao1

1 BioScience Project, Wakefield, MA, 01880, USA

Abstract

Background: Major depressive disorder (MDD) is a debilitating illness and a leading cause of disability, but its pathophysiology remains to be completely elucidated. Resistance to traditional antidepressant treatment is highly prevalent within patient populations. Ketamine, a rapid-acting antidepressant that has shown success in treating resistant patients, is hypothesized to function by modulating excitatory and inhibitory neurotransmitters. This has led to a paradigm shift from the monoaminergic hypothesis, towards a glutamate and neuroplasticity hypothesis, though this picture may still be incomplete. The purpose of this study is to identify novel systems potentially implicated in MDD and suggest potential therapeutic mechanisms.

Methods: An integrative genomics and systems biology approach was used to identify genes and pathways that may be relevant to MDD. Starting with genes implicated in current, well-established paradigms, a correlation analysis was used to query publicly available microarray datasets to identify potential genes that may be relevant to MDD. Systems and pathways of interest were identified through functional enrichment. Through a manual review, genes of systems and pathways potentially relevant to MDD were used to generate a protein interaction network. Interesting genes identified from the network were functionally enriched and clustered to identify new high-level themes. Genes from the protein interaction network were then classified into sets based on different paradigms using functional annotations. Genes belonging to multiple sets – and thus being involved in multiple different paradigms relevant to MDD – were identified for future research.

Results: The highest-ranked enrichment cluster contained GABAeric signaling, retrograde endocannabinoid signaling, and morphine addiction. Other interesting pathways identified were T cell receptor signaling, cocaine addiction, and nicotine addiction. Other broad themes identified in other clusters were calcium, serotonin, the immune system, TGF beta, glutamate, telomeres, the guanine nucleotide exchange factor, the extracellular matrix, and DNA regulation. Potential genes of interest are listed in Table 3.

Conclusions: Throughout the study, interesting genes, groups of genes, and systems were identified manually and systematically, some of which validate existing literature and some give possible directions for future research. Further experiments are required to confirm causal links.

Introduction

Major depressive disorder (MDD) is a leading cause of disability worldwide, affecting over 264 million people [1] and 7.1% of adults in the United States [2]. Despite its prevalence, the pathophysiology of depression is still not well understood. The first major paradigm was the monoamine hypothesis, which proposed that lower levels of monoaminergic neurotransmitters – serotonin, dopamine, and norepinephrine – contribute to the pathogenesis of MDD. Traditional antidepressants, which include tricyclics, selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), and monoamine oxidase inhibitors (MAOIs), generally function to increase levels of these neurotransmitters.

Confirming the role of serotonin, the kynurenine pathway (KP) and its metabolites have also been a major interest of current research. The pathway metabolizes tryptophan, the precursor to serotonin, and produces quinolinic acid, an NMDA agonist that can induce excitotoxicity [3]. It is theorized that depressed patients have increased KP activity, resulting in less available tryptophan for serotonin synthesis, and consistent with this, a meta-analysis shows that depressed patients consistently have less tryptophan and kynurenic acid and higher levels of quinolinic acid than controls [4]. Naturally, this leads to questions about the upstream causes and downstream effects of KP dysregulation.

One potential upstream cause is the excess release of glucocorticoids. Tryptophan 2,3-dioxygenase controls the rate-limiting step of the KP and is induced by glucocorticoids [5]. Consistent with the psychopathogenesis of depression as a result of chronic stress, glucocorticoids are one of the major hormones released during the stress response. Another enzyme that controls the rate-limiting step of the KP is indoleamine-2,3-dioxygenase, which is induced by inflammatory factors [6,7].

The downstream effects of the KP point towards a role of the NMDA receptor in the biology of MDD, and more broadly neuroplasticity. One of the biggest shortcomings of the monoamine hypothesis is the long delay before traditional antidepressants produce therapeutic effects. Treatment-resistance to traditional antidepressants is also highly prevalent, suggesting that the monoaminergic hypothesis is incomplete. With the discovery of ketamine as an efficacious, rapid-acting antidepressant [8], the predominant paradigm in the current field of depression research has shifted towards a neuroplasticity hypothesis [9] and investigating the roles of glutamate and GABA. Ketamine’s mechanism of action is an ongoing area of research, but broadly it increases neuroplasticity by acting as an NMDAR antagonist – protecting against neurotoxicity – and inducing local synaptic protein synthesis [10]. However, this picture may not be entirely complete. Other NMDA antagonists have failed to produce antidepressant effects to the same extent as ketamine [108], which suggests that there are mechanisms unique to ketamine beyond modulation of glutamatergic and GABAergic systems.

The purpose of this study is to identify novel systems potentially implicated in the pathophysiology of depression as areas of future research and to propose possible therapeutic mechanisms based on molecular function.

Methodology

The following genes were chosen as representatives from the major current paradigms as a starting point:

SLC6A4, serotonin hypothesis – The serotonin transporter that mediates reuptake of serotonin from the synapse back into the presynaptic neuron. SSRIs inhibit this transporter to increase available synaptic serotonin levels.

HTR1A, serotonin hypothesis – Serotonin autoreceptor that is a part of negative feedback regulation of serotonin, preventing the release of serotonin in the presence of excess synaptic serotonin. Decreased HTR1A activity, through downregulation or desensitization, is associated with antidepressant effects [11].

TDO2, serotonin hypothesis - Tryptophan 2,3-dioxygenase, one of the enzymes of the rate-limiting step of the kynurenine pathway.

BDNF, neuroplasticity hypothesis – Brain-derived neurotropic factor, plays a major role in neuroplasticity and the formation of new synapses.

GRIN2B, glutamate hypothesis – Subunit for the NMDA receptor. This subunit in particular has been implicated in depression [12].

PTGS2, inflammation hypothesis – COX2, a major component of the inflammatory response and a target of NSAIDs. Celecoxib, a selective COX2 inhibitor and NSAID, may have antidepressant effects [13].

CRHR1 & CRHR2, HPA axis – CRH receptors that play an important role in mediating the stress response.

NR3C1, HPA axis – Glucocorticoid receptor that mediates transcription and downstream effects of glucocorticoids.

The Allen Human Brain Atlas [109] was used to find genesets correlating with the starting genes. The correlate and starting genes were enriched with DAVID 6.8 [110, 111] for KEGG pathways [112]. Manually, interesting pathways were identified from the literature, and the genes in these pathways along with the starting genes comprised the list of candidate genes for subsequent analysis. Candidate genes were visualized in networks with a variety of methods to determine connectivity and structure. STRING 11.0 [113] was used to create a protein-protein interaction (PPI) network that was further analyzed using Cytoscape 3.8.0 [114] and organized and filtered using MCODE 1.6.1 [115] and clusterMaker 1.3.1 [116]. EnrichmentMap 3.3.0 [117] was used

to construct a pathway interaction network with enriched pathways, and NetworkAnalyst 3.0 [118] was used to create a drug-protein interaction network with the candidate genes.

The candidate gene PPI networks were used to identify further interesting genes to add to the candidate gene list. This new candidate gene list was enriched again with DAVID to identify gene clusters.

Enrichment annotations were then used to group genes from a filtered PPI network into sets based on their involvement in different broad theories of depression: monoamine, neuroplasticity, immune system, neurotrophin, behavioral responses, glucocorticoid, DNA, cholinergic, and endocrine. UpSetR [119] was used to identify intersections between these gene sets.

Results

Identifying Potential Candidate Genes and Pathways

The Allen Human Brain Atlas was used to find genes that are expressed in correlation to the starting genes. Correlate genes with Pearson correlation coefficient ≥ .65 from all 9 genes were compiled into a gene list. Running the gene set through DAVID, the genes in the GAD disease class for depression from the set were identified and listed in Table 1. DAVID was also used to identify KEGG pathways enriched in the gene list, which are listed in Table 2.

Table 1

Term Count PValue Genes Pop Hits

Pop Total

Fold Enrichment Bonferroni Benjamini FDR

hsa04080:Neuroactive ligand-receptor interaction 48 8.16E-12

CSH1, MCHR1, AVPR2, MCHR2, GABRB3, THRB, GABRB1, TSHB, NR3C1, GABBR2, GHRHR, S1PR2, HRH1, PTGIR, GRIN2B, HRH2, GALR3, TAAR5, HTR1F, GPR156, GABRA2, PTGER2, PTH2R, GABRA4, RXFP1, GABRA3, GRIN1, GABRA5, GRIN2A, HTR4, GRIA3, NPY1R, NPY5R, P2RX5, CRHR1, GRM5, SSTR3, ADRB1, CHRM3, GRIA2, CHRM2, GRM7, CHRM1, TBXA2R, GHSR, NMBR, LHB, HTR2A 277 6879 3.010 2.007E-09 2.007E-09 1.057E-08

hsa04921:Oxytocin signaling pathway 28 8.07E-08 MEF2C, ADCY2, PTGS2, PPP3R1, CACNB1, KCNJ12, KCNJ3, PPP3CB, PLCB1, CAMK2A, PRKCA, CACNA2D1, CAMK1G, GNAO1, ROCK1, CACNG8, PRKAB2, PRKCG, CACNG3, CACNA2D3, PRKCB, KCNJ4, MAPK1, PLA2G4A, KCNJ6, CAMK1, CALM3, GUCY1B3 150 6879 3.243 1.985E-05 9.923E-06 1.045E-04

hsa04724:Glutamatergic synapse 24 8.70E-08 PRKCA, DLGAP1, GNAO1, ADCY2, GRIN1, GRIN2A, PPP3R1, PRKCG, GRIA3, HOMER1, SHANK2, KCNJ3, PRKCB, SLC17A7, GRM5, MAPK1, PLA2G4A, SLC1A2, GRIN2B, GRIA2, GRM7, PPP3CB, PLCB1, SLC1A1 114 6879 3.657 2.141E-05 7.138E-06 1.128E-04

hsa04723:Retrograde endocannabinoid signaling 22 1.87E-07

PRKCA, GABRA2, ADCY2, GNAO1, GABRB3, PTGS2, GABRA4, GABRA3, GABRB1, GABRA5, PRKCG, GRIA3, KCNJ3, PRKCB, SLC17A7, GRM5, MAPK1, NAPEPLD, KCNJ6, GRIA2, MAPK13, PLCB1 101 6879 3.784 4.602E-05 1.150E-05 2.424E-04

hsa05031:Amphetamine addiction 16 3.37E-06 PRKCA, STX1A, ARC, GRIN1, PPP3R1, GRIN2A, GRIA3, PRKCG, PRKCB, GRIN2B, GRIA2, PPP3CB, CALM3, CREB3L1, SLC18A1, CAMK2A 66 6879 4.211 8.278E-04 1.656E-04 4.362E-03

hsa04020:Calcium signaling pathway 27 9.55E-06 ADCY2, PPP3R1, ITPKA, ATP2B1, HRH1, HRH2, PPP3CB, NOS2, PLCB1, CAMK2A, PRKCA, SLC8A1, GRIN1, HTR4, GRIN2A, PRKCG, PRKCB, P2RX5, GRM5, ADRB1, CHRM3, CHRM2, CHRM1, TBXA2R, CALM3, CACNA1E, HTR2A 179 6879 2.620 2.348E-03 3.917E-04 1.238E-02

hsa05033:Nicotine addiction 12 9.91E-06 SLC17A7, GABRA2, GRIN2B, GRIA2, GABRB3, GABRA4, GABRA3, GABRB1, GRIN1, GABRA5, GRIN2A, GRIA3 40 6879 5.211 2.435E-03 3.483E-04 1.284E-02

hsa04720:Long-term potentiation 15 1.67E-05 PRKCA, GRIN1, CREBBP, PPP3R1, GRIN2A, PRKCG, PRKCB, GRM5, MAPK1, GRIA2, GRIN2B, PPP3CB, CALM3, PLCB1, CAMK2A 66 6879 3.948 4.107E-03 5.143E-04 2.168E-02

hsa04024:cAMP signaling pathway 28 2.10E-05 ADCY2, GABBR2, CNGB1, ATP2B1, BDNF, GRIN2B, CREB3L1, RAPGEF4, CAMK2A, HTR1F, PIK3R1, PTGER2, ROCK1, GRIN1, CREBBP, HTR4, GRIN2A, GRIA3, PDE4C, NPY1R, MAPK1, ADRB1, NPY, GRIA2, CHRM2, CHRM1, CALM3, GHSR 198 6879 2.457 5.146E-03 5.731E-04 2.717E-02

hsa04728:Dopaminergic synapse 21 3.57E-05 PRKCA, GNAO1, KIF5A, PPP2R5C, GRIN2A, PRKCG, GRIA3, KCNJ3, PRKCB, KCNJ6, GRIN2B, GRIA2, MAPK13, PPP3CB, CALM3, CREB3L1, SLC18A1, PLCB1, PPP2R2B, CAMK2A, PPP2R2C 128 6879 2.850 8.743E-03 8.778E-04 4.625E-02

hsa05032:Morphine addiction 17 5.03E-05 PRKCA, GABRA2, ADCY2, GNAO1, GABRA4, GABRB3, GABRA3, GABRB1, GABRA5, PRKCG, PDE4C, GABBR2, KCNJ3, PRKCB, KCNJ6, PDE2A, PDE8B 91 6879 3.245 1.231E-02 1.125E-03 6.521E-02

hsa04725:Cholinergic synapse 19 5.30E-05 PRKCA, ADCY2, GNAO1, PRKCG, KCNJ12, KCNJ3, PRKCB, KCNQ5, KCNJ4, MAPK1, KCNQ3, KCNJ6, CHRM3, CHRM2, CHRM1, CREB3L1, PLCB1, CAMK2A, PIK3R1 111 6879 2.973 1.295E-02 1.086E-03 6.864E-02

hsa04713:Circadian entrainment 17 8.63E-05 PRKCA, ADCY2, GNAO1, GRIN1, GRIN2A, GRIA3, PRKCG, KCNJ3, PRKCB, MAPK1, KCNJ6, GRIN2B, GRIA2, CALM3, GUCY1B3, PLCB1, CAMK2A 95 6879 3.109 2.101E-02 1.632E-03 1.118E-01

hsa04010:MAPK signaling pathway 30 2.57E-04 MEF2C, FGF8, PPP3R1, CACNB1, FGF13, BDNF, PAK2, SOS2, PPP3CB, FGF3, PRKCA, CACNA2D1, CACNG8, NLK, PRKCG, CACNG3, CACNA2D3, MECOM, PRKCB, MAPK1, PLA2G4A, DUSP2, RASGRF2, MAPK13, IKBKG, NTRK2, CACNA1E, DUSP8, DUSP7, DUSP6 253 6879 2.060 6.120E-02 4.500E-03 3.321E-01

hsa04261:Adrenergic signaling in cardiomyocytes 20 3.11E-04

PRKCA, CACNA2D1, ADCY2, CACNG8, PPP2R5C, CACNB1, CACNG3, CACNA2D3, TNNT2, ATP2B1, MAPK1, ADRB1, MAPK13, CALM3, CREB3L1, RAPGEF4, PLCB1, PPP2R2B, CAMK2A, PPP2R2C 138 6879 2.518 7.370E-02 5.091E-03 4.025E-01

hsa04726:Serotonergic synapse 17 5.48E-04 PRKCA, GNAO1, PTGS2, GABRB3, GABRB1, HTR4, PRKCG, KCNJ3, PRKCB, MAPK1, PLA2G4A, KCNJ6, SLC18A1, PLCB1, HTR3B, HTR1F, HTR2A 111 6879 2.660 1.261E-01 8.390E-03 7.076E-01

hsa04360:Axon guidance 18 8.84E-04 NGEF, ROCK1, EFNB3, PPP3R1, NTN4, NTNG2, LRRC4C, SLIT1, NCK2, SEMA5B, EPHA4, MAPK1, EPHB6, RND1, PAK2, SEMA4G, SEMA4C, PPP3CB 127 6879 2.462 1.956E-01 1.272E-02 1.140E+00

hsa04911:Insulin secretion 14 1.02E-03 PRKCA, KCNMA1, STX1A, ADCY2, FFAR1, PRKCG, KCNMB1, PRKCB, CHRM3, KCNN1, CREB3L1, RAPGEF4, PLCB1, CAMK2A 85 6879 2.861 2.217E-01 1.383E-02 1.312E+00

hsa04750:Inflammatory mediator regulation of TRP channels 15 1.31E-03 PRKCA, PTGER2, ADCY2, PRKCG, PRKCB, PLA2G4A, HRH1, MAPK13, IL1RAP, CALM3, PLA2G6, PLCB1, CAMK2A, PIK3R1, HTR2A 98 6879 2.659 2.760E-01 1.686E-02 1.687E+00

hsa04730:Long-term depression 11 1.98E-03 CRHR1, PRKCA, MAPK1, PLA2G4A, GNAO1, GRIA2, GRIA3, GUCY1B3, PRKCG, PLCB1, PRKCB 60 6879 3.185 3.856E-01 2.406E-02 2.533E+00

hsa04727:GABAergic synapse 13 3.15E-03 PRKCA, GABRA2, GNAO1, ADCY2, GABRA4, GABRB3, GABRA3, GABRB1, GABRA5, PRKCG, GABBR2, PRKCB, KCNJ6 85 6879 2.657 5.403E-01 3.633E-02 4.011E+00

hsa04970:Salivary secretion 13 3.48E-03 PRKCA, ATP2B1, KCNMA1, ADCY2, ADRB1, CHRM3, AQP5, CALM3, GUCY1B3, PRKCG, TRPV6, PLCB1, PRKCB 86 6879 2.626 5.759E-01 3.824E-02 4.418E+00

hsa04022:cGMP-PKG signaling pathway 19 3.98E-03 KCNMA1, MEF2C, SLC8A1, MEF2A, ADCY2, ROCK1, PPP3R1, CNGB1, IRS1, KCNMB1, ATP2B1, MAPK1, ADRB1, PDE2A, PPP3CB, CALM3, CREB3L1, GUCY1B3, PLCB1 158 6879 2.089 6.247E-01 4.171E-02 5.031E+00

hsa04014:Ras signaling pathway 24 5.14E-03 PRKCA, FGF8, GRIN1, GRIN2A, PRKCG, FGF13, HGF, PRKCB, RASAL2, RASAL1, MAPK1, PLA2G4A, PAK2, GRIN2B, RASGRF2, ETS2, IKBKG, SOS2, CALM3, PLA2G6, SYNGAP1, SHC3, FGF3, PIK3R1 226 6879 1.845 7.183E-01 5.142E-02 6.456E+00

hsa05030:Cocaine addiction 9 6.23E-03 CDK5R1, BDNF, GRIN2B, GRIA2, GRIN1, GPSM1, GRIN2A, CREB3L1, SLC18A1 49 6879 3.191 7.850E-01 5.964E-02 7.778E+00

hsa04925:Aldosterone synthesis and secretion 12 6.23E-03 PRKCA, ADCY2, CAMK1G, PDE2A, STAR, CAMK1, CALM3, CREB3L1, PRKCG, PLCB1, CAMK2A, PRKCB 81 6879 2.574 7.852E-01 5.743E-02 7.781E+00

hsa04270:Vascular smooth muscle contraction 15 6.87E-03 PRKCA, KCNMA1, RAMP2, ADCY2, ROCK1, PRKCG, KCNMB1, PRKCB, MAPK1, PTGIR, PLA2G4A, PLA2G6, CALM3, GUCY1B3, PLCB1 117 6879 2.227 8.164E-01 6.085E-02 8.542E+00

hsa05014:Amyotrophic lateral sclerosis (ALS) 9 7.06E-03 SLC1A2, GRIN2B, GRIA2, MAPK13, GRIN1, PPP3CB, PPP3R1, GRIN2A, NEFM 50 6879 3.127 8.248E-01 6.031E-02 8.766E+00

hsa04370:VEGF signaling pathway 10 7.52E-03 PRKCA, MAPK1, PLA2G4A, PTGS2, MAPK13, PPP3CB, PPP3R1, PRKCG, PIK3R1, PRKCB 61 6879 2.848 8.440E-01 6.205E-02 9.321E+00

hsa04971:Gastric acid secretion 11 8.40E-03 PRKCA, ADCY2, CHRM3, HRH2, CALM3, PRKCG, GAST, PLCB1, CAMK2A, KCNK2, PRKCB 73 6879 2.618 8.745E-01 6.684E-02 1.035E+01

hsa04015:Rap1 signaling pathway 22 8.91E-03 PRKCA, FGF8, ADCY2, GNAO1, MAGI1, TLN2, GRIN1, GRIN2A, PRKCG, FGF13, HGF, RGS14, PRKCB, MAPK1, GRIN2B, LPAR5, MAPK13, CALM3, RAPGEF4, PLCB1, PIK3R1, FGF3 210 6879 1.820 8.894E-01 6.856E-02 1.095E+01

hsa04068:FoxO signaling pathway 16 9.61E-03 NLK, CREBBP, PRKAB2, SMAD3, HOMER1, IRS1, MAPK1, G6PC, CDKN1B, PLK2, CCND2, MAPK13, FOXG1, SOS2, AGAP2, PIK3R1 134 6879 2.074 9.069E-01 7.151E-02 1.176E+01

hsa04012:ErbB signaling pathway 12 1.06E-02 PRKCA, MAPK1, NCK2, CDKN1B, NRG3, PAK2, SOS2, PRKCG, SHC3, CAMK2A, PIK3R1, PRKCB 87 6879 2.396 9.271E-01 7.630E-02 1.289E+01

hsa05214:Glioma 10 1.13E-02 PRKCA, MAPK1, E2F3, SOS2, CALM3, PRKCG, SHC3, CAMK2A, PIK3R1, PRKCB 65 6879 2.672 9.395E-01 7.919E-02 1.374E+01

hsa04916:Melanogenesis 13 1.16E-02 PRKCA, MAPK1, TYRP1, GNAO1, ADCY2, CREBBP, CALM3, CREB3L1, PRKCG, PLCB1, WNT7A, CAMK2A, PRKCB 100 6879 2.258 9.437E-01 7.891E-02 1.406E+01

hsa04310:Wnt signaling pathway 16 1.24E-02 PRKCA, NLK, CREBBP, PPP3R1, PRKCG, PRKCB, DKK4, SOST, PRICKLE1, CCND2, PRICKLE2, PPP3CB, WIF1, PLCB1, WNT7A, CAMK2A 138 6879 2.014 9.539E-01 8.190E-02 1.496E+01

hsa04912:GnRH signaling pathway 12 1.46E-02 PRKCA, MAPK1, PLA2G4A, ADCY2, MAPK13, GNRH2, SOS2, CALM3, PLCB1, CAMK2A, LHB, PRKCB 91 6879 2.291 9.731E-01 9.312E-02 1.735E+01

hsa05142:Chagas disease (American trypanosomiasis) 13 1.56E-02 MAPK1, GNAO1, CD3E, MAPK13, IKBKG, SMAD3, NOS2, PLCB1, PPP2R2B, IFNGR2, PPP2R2C, PIK3R1, TLR9 104 6879 2.171 9.790E-01 9.669E-02 1.842E+01

hsa04611:Platelet activation 15 1.66E-02 ADCY2, ROCK1, TLN2, GP9, MAPK1, PTGIR, PLA2G4A, MAPK13, COL27A1, TBXA2R, FCER1G, GUCY1B3, PLCB1, COL24A1, PIK3R1 130 6879 2.004 9.839E-01 1.004E-01 1.954E+01

hsa04520:Adherens junction 10 1.96E-02 PTPRJ, MAPK1, NLK, WASF1, CREBBP, LMO7, SMAD3, ACTN1, WASL, CTNNA2 71 6879 2.447 9.922E-01 1.144E-01 2.258E+01

hsa04960:Aldosterone-regulated sodium reabsorption 7 2.25E-02 PRKCA, MAPK1, PRKCG, IRS1, SLC9A3R2, PIK3R1, PRKCB 39 6879 3.118 9.963E-01 1.276E-01 2.553E+01

hsa04915:Estrogen signaling pathway 12 2.59E-02 MAPK1, GNAO1, ADCY2, KCNJ6, SOS2, CALM3, CREB3L1, GABBR2, PLCB1, SHC3, KCNJ3, PIK3R1 99 6879 2.106 9.984E-01 1.424E-01 2.881E+01

hsa05200:Pathways in cancer 33 2.71E-02 E2F3, FGF8, ADCY2, PTGS2, ARNT2, FGF13, LAMB3, SOS2, CSF3R, RARA, NOS2, PLCB1, TRAF5, FGF3, PIK3R1, PRKCA, PTGER2, ROCK1, KLK3, CREBBP, SMAD3, PRKCG, HGF, BIRC3, MECOM, PRKCB, CTNNA2, DAPK1, MAPK1, CDKN1B, LPAR5, IKBKG, WNT7A 393 6879 1.459 9.989E-01 1.457E-01 3.000E+01

hsa04540:Gap junction 11 2.86E-02 PRKCA, GRM5, MAPK1, ADCY2, ADRB1, SOS2, GUCY1B3, PRKCG, PLCB1, PRKCB, HTR2A 88 6879 2.171 9.992E-01 1.497E-01 3.134E+01

hsa05231:Choline metabolism in cancer 12 2.95E-02 PRKCA, MAPK1, PLA2G4A, DGKB, SLC44A5, WASF1, SOS2, PRKCG, WASL, DGKI, PIK3R1, PRKCB 101 6879 2.064 9.994E-01 1.510E-01 3.216E+01

hsa04810:Regulation of actin cytoskeleton 20 3.22E-02 ARHGEF4, FGD1, FGF8, ROCK1, WASF1, DIAPH2, IQGAP3, ABI2, ACTN1, FGF13, MAPK1, PAK2, CHRM3, CHRM2, CHRM1, SOS2, MOS, WASL, FGF3, PIK3R1 210 6879 1.654 9.997E-01 1.605E-01 3.456E+01

hsa04062:Chemokine signaling pathway 18 3.80E-02 ADCY2, ROCK1, NFKBIB, CX3CL1, CCL24, CCR9, MAPK1, CCL22, PPBP, CXCL14, CCL21, IKBKG, SOS2, WASL, PLCB1, SHC3, XCR1, PIK3R1 186 6879 1.681 9.999E-01 1.837E-01 3.949E+01

hsa05143:African trypanosomiasis 6 3.83E-02 PRKCA, APOA1, PRKCG, PLCB1, PRKCB, TLR9 33 6879 3.158 9.999E-01 1.813E-01 3.970E+01

hsa04923:Regulation of lipolysis in adipocytes 8 3.99E-02 ADCY2, ADRB1, NPY, PTGS2, TSHB, NPY1R, IRS1, PIK3R1 56 6879 2.482 1.000E+00 1.848E-01 4.099E+01

hsa05223:Non-small cell lung cancer 8 3.99E-02 PRKCA, FHIT, MAPK1, E2F3, SOS2, PRKCG, PIK3R1, PRKCB 56 6879 2.482 1.000E+00 1.848E-01 4.099E+01

hsa04071:Sphingolipid signaling pathway 13 4.20E-02 PRKCA, S1PR2, MAPK1, ROCK1, MAPK13, PPP2R5C, FCER1G, PRKCG, PLCB1, PPP2R2B, PPP2R2C, PIK3R1, PRKCB 120 6879 1.882 1.000E+00 1.905E-01 4.268E+01

hsa00534:Glycosaminoglycan biosynthesis - heparan sulfate / heparin 5 4.57E-02 HS3ST3A1, HS3ST2, HS6ST3, EXTL1, HS2ST1 24 6879 3.619 1.000E+00 2.019E-01 4.545E+01

hsa04650:Natural killer cell mediated cytotoxicity 13 4.68E-02 PRKCA, PPP3R1, PRKCG, PRKCB, MAPK1, ULBP1, SOS2, PPP3CB, FCER1G, SHC3, SH2D1B, IFNGR2, PIK3R1 122 6879 1.851 1.000E+00 2.028E-01 4.624E+01

hsa04066:HIF-1 signaling pathway 11 4.79E-02 PRKCA, MAPK1, CDKN1B, CREBBP, PRKCG, NOS2, CAMK2A, IFNGR2, PIK3R1, NPPA, PRKCB 96 6879 1.990 1.000E+00 2.036E-01 4.704E+01

hsa05222:Small cell lung cancer 10 5.39E-02 FHIT, E2F3, LAMB3, CDKN1B, PTGS2, IKBKG, NOS2, BIRC3, TRAF5, PIK3R1 85 6879 2.044 1.000E+00 2.231E-01 5.123E+01

hsa04922:Glucagon signaling pathway 11 5.69E-02 CPT1C, G6PC, ADCY2, PRKAB2, CREBBP, PPP3CB, PPP3R1, CALM3, CREB3L1, PLCB1, CAMK2A 99 6879 1.930 1.000E+00 2.306E-01 5.321E+01

hsa04660:T cell receptor signaling pathway 11 6.02E-02 MAPK1, NCK2, PAK2, CD3E, MAPK13, NFKBIB, SOS2, IKBKG, PPP3CB, PPP3R1, PIK3R1 100 6879 1.911 1.000E+00 2.386E-01 5.526E+01

hsa05161:Hepatitis B 14 7.26E-02 PRKCA, EGR3, E2F3, EGR2, CREBBP, YWHAB, PRKCG, PRKCB, STAT6, MAPK1, CDKN1B, IKBKG, CREB3L1, PIK3R1 145 6879 1.677 1.000E+00 2.775E-01 6.232E+01

hsa04924:Renin secretion 8 7.27E-02 KCNMA1, PTGER2, ADRB1, PPP3CB, PPP3R1, CALM3, GUCY1B3, PLCB1 64 6879 2.171 1.000E+00 2.740E-01 6.240E+01

hsa05146:Amoebiasis 11 8.22E-02 PRKCA, ARG1, LAMB3, COL27A1, ACTN1, PRKCG, NOS2, PLCB1, COL24A1, PIK3R1, PRKCB 106 6879 1.803 1.000E+00 3.007E-01 6.710E+01

hsa04510:Focal adhesion 18 8.28E-02 PRKCA, ROCK1, TLN2, ACTN1, PRKCG, HGF, BIRC3, PRKCB, MAPK1, LAMB3, PAK2, CCND2, COL27A1, SOS2, COL6A3, SHC3, COL24A1, PIK3R1 206 6879 1.518 1.000E+00 2.983E-01 6.735E+01

hsa04722:Neurotrophin signaling pathway 12 8.29E-02 MAPK1, BDNF, MAPK13, NFKBIB, NTRK2, SOS2, CALM3, SHC3, CAMK2A, IRS1, PIK3R1, MATK 120 6879 1.737 1.000E+00 2.945E-01 6.741E+01

hsa04972:Pancreatic secretion 10 8.50E-02 PRKCA, ATP2B1, KCNMA1, ADCY2, CHRM3, PRKCG, PLCB1, RAB27B, PRKCB, CTRL 93 6879 1.868 1.000E+00 2.969E-01 6.835E+01

hsa04151:PI3K-Akt signaling pathway 27 9.10E-02 CSH1, FGF8, PPP2R5C, FGF13, LAMB3, COL27A1, SOS2, COL6A3, CSF3R, CREB3L1, PPP2R2B, PPP2R2C, FGF3, PIK3R1, PRKCA, YWHAB, HGF, IRS1, MAPK1, G6PC, CDKN1B, LPAR5, CCND2, CHRM2, CHRM1, IKBKG, COL24A1 345 6879 1.359 1.000E+00 3.109E-01 7.094E+01

hsa04664:Fc epsilon RI signaling pathway 8 9.37E-02 PRKCA, MAPK1, PLA2G4A, MAPK13, SOS2, FCER1G, PIK3R1, PRKCB 68 6879 2.044 1.000E+00 3.150E-01 7.207E+01

hsa04662:B cell receptor signaling pathway 8 9.95E-02 MAPK1, CR2, NFKBIB, SOS2, IKBKG, PPP3CB, PPP3R1, PIK3R1 69 6879 2.014 1.000E+00 3.274E-01 7.428E+01

Table 2

Key:

Yellow – Pathways that are involved in neuroplasticity

Blue – Pathways involved in other systems hypothesized to contribute to the pathophysiology of depression

Red – Pathways that are potentially involved in depression but have limited and/or dated literature backing them

Purple– Gene sets containing genes associated with depression from Table 1

Several notable pathways appear. Of course, the glutamatergic synapse is one of them.

The mechanism of NMDAR-mediated excitotoxicity is excessive calcium intake into the postsynaptic neuron, and this is done by increased mGluR1/5 activity. mGluR1/5 has also been shown to upregulate NMDARs [14]. A possible therapeutic mechanism would be inhibition of mGluR1/5.

The role of AMPA receptors complicates the role of glutamate in neuroplasticity. Activation of AMPA receptors with glutamate actually increases BDNF release and neuroplasticity [15], and ketamine appears to increase prefrontal glutamate levels [16]. The disinhibition hypothesis states that ketamine’s mechanism of action is actually the result of NMDAR inhibition on GABAergic neurons, which results in less inhibition and indirectly results in the release of more glutamate that activates AMPA receptors. Both mechanisms are possibly at play, with ketamine increasing activation of “good” glutamate receptors while inhibiting “bad” ones. Following this idea, several mechanisms increase synaptic glutamate: inhibition of mGlur2/3 or mGlur4/8, inhibition of KCNJ3 channels (labeled GIRK in the picture), or inhibition of SLC1A7/2 channels (labeled EAAT in the picture).

Several downstream protein cascades of BDNF receptor binding, including Ras, MAPK, and PI3K-Akt pathways, were also enriched.

Akt signaling activates mTOR, which then induces synaptic protein synthesis and neuroplasticity. This pathway is hypothesized to be a part of ketamine’s mechanism of action [17,18]. Consistent with this idea and the neuroplasticity hypothesis, inhibiting mTOR should lessen ketamine’s efficacy, but results have been contradictory. Inhibiting mTOR with rapamycin completely blocked ketamine-induced synaptogenesis in rats [17], yet in depressed patients rapamycin actually prolonged ketamine’s antidepressant effects [19].

PI3K-Akt signaling is also activated by IL-2, IL-3, IL-4, IL-6, and IL-7, which also may be worth investigating as a connection to the immune system.

From Table 2, genes associated with depression that are in pathways of interest – gene sets highlighted in purple that also correspond to a highlighted pathway– along with the initial 9 genes make the list for candidate genes.

hsa 05033 :Nicotine addiction

GO:0016907 ~G-protein coupled acetylcholine receptor activity

hsa 05161 :Hepatitis B

hsa 04923 :Regulation of lipolysis in adipocytes

PIRSF000939 :dual specificity protein phosphatase (MAP kinase phosphatase )

hsa 05142 :Chagas disease (American trypanosomiasis )hsa 04810 :Regulation of actin cytoskeleton

h_nos 1Pathway :Nitric Oxide Signaling Pathway

SM00233 :PH

GO:0015467 ~G-protein activated inward rectifier potassium channel activity

SM00450 :RHOD

GO:0007187 ~G-protein coupled receptor signaling pathway, coupled to cyclic nucleotide

second messenger

GO:0035235 ~ionotropic glutamate receptor signaling pathway

hsa 04915 :Estrogen signaling pathwayhsa 04062 :Chemokine signaling pathway

hsa 04713 :Circadian entrainment

GO:0007215 ~glutamate receptor signaling pathway

GO:0032281 ~AMPA glutamate receptor complex

hsa 04071 :Sphingolipid signaling pathway

hsa 04068 :FoxO signaling pathway

GO:0007210 ~serotonin receptor signaling pathway

GO:0007214 ~gamma-aminobutyric acid signaling pathway

GO:0014069 ~postsynaptic density

GO:0007268 ~chemical synaptic transmission

hsa 04664 :Fc epsilon RI signaling pathway

hsa 04080 :Neuroactive ligand- receptor interaction

hsa 05214 :Glioma

hsa 04724 :Glutamatergic synapse

IPR011009 :Protein kinase- like domain

GO:0004683 ~calmodulin -dependent protein kinase activity

hsa 04066 :HIF-1 signaling pathway

GO:0048167 ~regulation of synaptic plasticity

hsa 04750 :Inflammatory mediator regulation of TRP channels

GO:0005234 ~extracellular -glutamate-gated ion channel activity

hsa 05223 :Non -small cell lung cancer

GO:0043005 ~neuron projectionGO:0060291 ~long-term synaptic potentiation

hsa 04020 :Calcium signaling pathway

hsa 05031 :Amphetamine addiction

GO:0005886 ~plasma membrane

GO:0000165 ~MAPK cascade

GO:0005829 ~cytosol

hsa 04670 :Leukocyte transendothelial migration

IPR006201 :Neurotransmitter -gated ion-channel

IPR017452 :GPCR,rhodopsin -like ,7TM

GO:0004970 ~ionotropic glutamate receptor activity

GO:0007190 ~activation of adenylate cyclase activity

hsa 04662 :B cell receptor signaling pathway

hsa 04012 :ErbB signaling pathway

IPR006028 :Gamma-aminobutyric acid A receptor

hsa 04971 :Gastric acid secretion

SM00918 :SM00918

hsa 04070 :Phosphatidylinositol signaling system

hsa 04916 :Melanogenesis

hsa 05220 :Chronic myeloid leukemia

GO:0051938 ~L-glutamate import

GO:0006813 ~potassium ion transport

GO:0030666 ~endocytic vesicle membrane

hsa 04151 :PI3K-Akt signaling pathway

hsa 05140 :Leishmaniasis

hsa 04510 :Focal adhesion

Potassium transport

GO:0061337 ~cardiac conduction

hsa 04114 :Oocyte meiosis

GO:0004890 ~GABA-A receptor activity

GO:1902711 ~GABA-A receptor complex

GO:0045907 ~positive regulation of vasoconstriction

GO:0005242 ~inward rectifier potassium channel activity

GO:0000187 ~activation of MAPK activityGO:0043198 ~dendritic shaft

GO:0007197 ~adenylate cyclase -inhibiting G-protein coupled acetylcholine receptor

signaling pathwayhsa 04720 :Long -term potentiation

hsa 04970 :Salivary secretion

GO:0032403 ~protein complex bindingGO:0007611 ~learning or memory

IPR001763 :Rhodanese -like domain

hsa 05145 :Toxoplasmosis

GO:0042493 ~response to drugGO:0016301 ~kinase activity

GO:0043547 ~positive regulation of GTPase activity

GO:0051378 ~serotonin binding

hsa 05231 :Choline metabolism in cancer

GO:0008503 ~benzodiazepine receptor activity

IPR008271 :Serine/threonine -protein kinase, active site

GO:0005246 ~calcium channel regulator activity

hsa 04014 :Ras signaling pathway

GO:0006816 ~calcium ion transporthsa 04022 :cGMP-PKG signaling pathway

GO:0034765 ~regulation of ion transmembrane transport

IPR001828 :Extracellular ligand- binding receptor

GO:0014066 ~regulation of phosphatidylinositol 3-kinase signaling

GO:0005245 ~voltage -gated calcium channel activity

IPR002231 :5-Hydroxytryptamine receptor family

hsa 05132 :Salmonella infection

hsa 04152 :AMPK signaling pathway

IPR006029 :Neurotransmitter -gated ion-channel transmembrane domain

GO:0005085 ~guanyl -nucleotide exchange factor activity

GO:0004993 ~G-protein coupled serotonin receptor activity

SM01381 :SM01381

hsa 04730 :Long -term depression

GO:0004698 ~calcium -dependent protein kinase C activity

GO:0007612 ~learning

GO:0000188 ~inactivation of MAPK activity

hsa 04960 :Aldosterone -regulated sodium reabsorption

IPR000995 :Muscarinic acetylcholine receptor family

hsa 04150 :mTOR signaling pathway

hsa 05218 :Melanoma

hsa 04010 :MAPK signaling pathway

hsa 05414 :Dilated cardiomyopathy

region of interest :H5 (pore- forming helix )

IPR020422 :Dual specificity phosphatase ,subgroup ,catalytic domain

IPR001849 :Pleckstrin homology domain

GO:0042177 ~negative regulation of protein catabolic process

IPR006202 :Neurotransmitter -gated ion-channel ligand -binding

GO:0005891 ~voltage -gated calcium channel complex

IPR016449 :Potassium channel, inwardly rectifying ,Kir

GO:0018105 ~peptidyl -serine phosphorylation

GO:1902476 ~chloride transmembrane transport

GO:0007613 ~memoryGO:0019233 ~sensory perception of pain

site:Role in the control of polyamine -mediated channel gating and in the blocking by

intracellular magnesium

IPR011993 :Pleckstrin homology -like domain

IPR019594 :Glutamate receptor, L-glutamate/glycine -binding

GO:0034707 ~chloride channel complex

hsa 04918 :Thyroid hormone synthesis

GO:0030425 ~dendrite

hsa 04660 :T cell receptor signaling pathwayGO:0005516 ~calmodulin binding

hsa 05030 :Cocaine addiction

hsa 04722 :Neurotrophin signaling pathway

IPR023152 :Ras GTPase -activating protein ,conserved site

hsa 04540 :Gap junction

hsa 04911 :Insulin secretion

IPR017441 :Protein kinase, ATP binding site

IPR001390 :Gamma-aminobutyric -acid A receptor ,alpha subunit

hsa 04917 :Prolactin signaling pathway

PIRSF000550 :protein kinase C ,alpha /beta/gamma types

hsa 04723 :Retrograde endocannabinoid signaling

IPR018000 :Neurotransmitter -gated ion-channel ,conserved site

hsa 04310 :Wnt signaling pathway

GO:0004674 ~protein serine /threonine kinase activity

GO:0007165 ~signal transduction

h_pgc 1aPathway :Regulation of PGC-1a

GO:0005622 ~intracellular

IPR000276 :G protein-coupled receptor , rhodopsin -like

GO:0007213 ~G-protein coupled acetylcholine receptor signaling pathway

GO:0006811 ~ion transport

IPR013518 :Potassium channel , inwardly rectifying ,Kir,cytoplasmic

GO:0005088 ~Ras guanyl -nucleotide exchange factor activity

hsa 04270 :Vascular smooth muscle contraction

SM00239 :C2

GO:0016595 ~glutamate binding

hsa 04912 :GnRH signaling pathway

GO:0004972 ~NMDA glutamate receptor activity

GO:0022851 ~GABA-gated chloride ion channel activity

GO:0006810 ~transport

GO:0070588 ~calcium ion transmembrane transport

GO:0008076 ~voltage -gated potassium channel complex

Serine/threonine- protein kinase

GO:0017146 ~NMDA selective glutamate receptor complex

SM00323 :RasGAP

hsa 05146 :Amoebiasis

hsa 04925 :Aldosterone synthesis and secretion

hsa 04520 :Adherens junction

hsa 05215 :Prostate cancer

GO:0005887 ~integral component of plasma membrane

hsa 04611 :Platelet activation

hsa 05131 :Shigellosis

GO:0008542 ~visual learning

hsa 04370 :VEGF signaling pathway

region of interest :Glutamate binding

IPR000008 :C2 calcium -dependent membrane targeting

hsa 05034 :Alcoholism

h_nfatPathway :NFAT and Hypertrophy of the heart (Transcription in the broken heart )

GO:0034220 ~ion transmembrane transport

hsa 04650 :Natural killer cell mediated cytotoxicity

GO:0032279 ~asymmetric synapse

hsa 04727 :GABAergic synapse

hsa 04728 :Dopaminergic synapse

hsa 04261 :Adrenergic signaling in cardiomyocytes

hsa 05014 :Amyotrophic lateral sclerosis (ALS)

hsa 04024 :cAMP signaling pathway

IPR024950 :Dual specificity phosphatase

IPR014375 :Protein kinase C ,alpha /beta/gamma types

IPR000719 :Protein kinase, catalytic domain

GO:0004871 ~signal transducer activity

GO:0017017 ~MAP kinase tyrosine /serine /threonine phosphatase activity

hsa 04725 :Cholinergic synapse

hsa 05200 :Pathways in cancer

IPR001320 :Ionotropic glutamate receptor

GO:0006468 ~protein phosphorylation

SM00220 :S_TKc

Potassium

hsa 04920 :Adipocytokine signaling pathwayGO:0043524 ~negative regulation of neuron apoptotic process

hsa 05032 :Morphine addiction

hsa 04921 :Oxytocin signaling pathway

region of interest :Calmodulin -binding

GO:0045211 ~postsynaptic membrane

IPR001936 :Ras GTPase -activating protein

hsa 04922 :Glucagon signaling pathway

GO:0005230 ~extracellular ligand- gated ion channel activity

IPR008343 :Mitogen-activated protein (MAP) kinase phosphatase

GO:0045202 ~synapse

hsa 04924 :Renin secretionhsa 05211 :Renal cell carcinoma

hsa 04015 :Rap 1 signaling pathway

GO:0007223 ~Wnt signaling pathway ,calcium modulating pathway

IPR001508 :NMDA receptor

SM00079 :PBPe

GO:0030054 ~cell junction

hsa 04666 :Fc gamma R-mediated phagocytosis

GO:2000601 ~positive regulation of Arp 2/3 complex -mediated actin nucleation

hsa 04726 :Serotonergic synapse

Receptor

hsa 04972 :Pancreatic secretion

All pathways in Table 2 were put into Cytoscape, and a network was constructed using EnrichmentMap. Connections are created between pathways if the similarity coefficient ≥ .63. Lighter-colored nodes represent a higher corresponding q-value with a cutoff value of .05.

Interaction Networks

Pathway Interaction Network

Protein Interaction Network

The initial 9 genes were used to build a protein-protein interaction network in STRING. The network includes first and second shell interactions and is based on experimental evidence using a combined score ≥ 0.40. The networks were further evaluated in Cytoscape. Subnetworks that connect the products of the initial 9 genes were identified using Cytohubba.

PPARGC1A

TDO2

SNCA

CREBBP

SMARCA2

GRIN2B

NCOA3

DLG3

SORT1

FGFR1

NCOR2

MAGI3

TANC1

MAPK3

NCOA4

KPNA2

CRH

RARA

CAMK2A

CRHR1

DPM1

PTGER1

STX1A

RAF1

FBXW7

UCN3

GRIN1

CAMK2D

MED25

PTPN11

DLG1

TRIM28

ARHGAP32

HSP90AB1

DLG2

BDNF

HSP90AA1

ELAVL1

TGFB1I1

CRHR2

SYNGAP1

EP300

NCOR1

UBE2I

HTR1A

MED1

UCN2

NRIP1

HSPA4

AKT1

JUN

MIB2

DLG4

CHD9

NR3C1

DAXX

CAPN1

GPM6B

PARK2

NTF4

STUB1

SMARCA4

GNAI3

UCN

CAV1

SLC6A4

PICK1

PTGS2

NCOA2

NTRK2

UBE2L3

ABHD16A

INADL

NUCB1

SUMO1

GRIP1

NTF3

SPTAN1

MDM2

ASMTL

NT5E

NCOA1

CAMK2G

HSPA1A

SNCG

FKBP4

COPS6

ACTN2

EGFR

FAM111B

UBE2L3

GRIN2B

UCN3

HTR1A

EGFR

CRHR2

ELAVL1

TDO2

AKT1

NR3C1RAF1

MAPK3

NTRK2MDM2

GRIP1

PICK1 PARK2

STX1A

NSF

SNCA

ACTB

CDC5L

NUCB1

CREBBP

FGFR1

JUN

PTPN11

CAMK2D

UBE2I

GNAI3

CAMK2A

DLG4

CAMK2G

HSPA1A

HSPA4

CRHR1

UCN

PTGS2

NAPB

BDNF

CRH

SLC6A4

Expanding the subnetwork to also include proteins that directly interact with the products of the initial 9 genes yields the network below.

Note how genes part of the serotonergic system (SLC6A4, HTR1A), glutamatergic system & neuroplasticity (BDNF, GRIN2B), neuroinflammation (PTGS2), and the stress response (NR3C1) are in the same subnetwork. This is not surprising because these are all systems in the

MED16

SMARCC2

HIST1H3C

HIST1H4H

SMARCD1

HIST1H3G

HIST1H4B

SMARCA2

HIST1H3I

MED4

ACTL6A

GPS1

HIST1H4K

CRHR2

HIST1H3H

HIST1H4L

CREBBP

COPS8

MED27

GRIN2B

HIST1H3F

HIST1H4I

DDB2

HIST1H3B

ZC3H13

HIST1H3A

COPS7A

HIST1H4JHIST2H4A

HIST1H3J

HTR1A

CRHR1

COPS6

MED14

NR3C1

COPS4

HIST1H4C

CUL4B

BPTF

MED15

UBC

MED29

BCL7B

H3F3A

MED28

BCL7A

UBB

COPS3

MED20

DET1

COPS2

MED21

CUL4A

SMARCD3

CCNC

MED30

MED7

BDNF

MED12

MED17

H3F3B

DCAF4

MED19

MED24

CDK8

COPS7B

MED1

MED11

HIST2H3A

SMARCD2

MED13

MED10

HIST2H3C

ESR1

TDO2

SMARCB1

HIST2H3D

COPS5

CDK19

SMARCC1

HIST1H3E

MED6

SLC6A4

SS18

ARID1B

HIST4H4

MED8

BCL7C

HIST1H2AK

MED18

HIST1H4D

SMARCE1

HIST1H2AM

HIST1H4A

HIST1H4F

UBA52

HIST1H2AG

HIST1H4E

RPS27A

HIST1H2AI

PTGS2

ARID1A

HIST1H2AL

HIST2H4B

SMARCA4

HIST1H3D

brain directly involved with the pathophysiology of depression. The HPA axis (CRH1, CRH2) – except for NR3C1, the glucocorticoid receptor – and the kynurenine pathway (TDO2) are not connected to the larger subnetwork. Again, this is not unexpected because the depressive effects of CRH and the metabolism of tryptophan are indirect.

MCODE was used to identify subnetworks that are highly connected. The top 4 subnetworks are displayed along with the initial 9 genes on the right. All 4 subnetworks are connected to the glucocorticoid receptor, which controls gene expression. All 4 subnetworks are distinct biological systems.

Of course, depression has genetic and epigenetic influences. NR3C1 is a nuclear transcription factor and likely mediates stress-induced epigenetic changes associated with depression, and specific histone genes are identified.

The COP9 signalosome complex is still not well understood and an area of current research. The tricyclic antidepressant imipramine upregulates its expression in rats [20].

SWI/SWF chromatin remodeling complexes control gene transcription and may be another mechanism of stress-induced epigenetic changes. They have been implicated in psychiatric diseases [21], including depression [22], and SMARCA3 may be a part of the mechanism of action of SSRIs [23].

The mediator complex is a downstream target of the estrogen receptor, which may be involved in postpartum depression.

MDM2

HSP90AA1

RPL3

NR3C1

RPL32

RPL17

RPL11

PELO

RPL29

SMARCA4

HSP90AB1

The 6th top MCODE subnetwork is composed of ribosomal genes. Dysfunction of ribosomal gene expression, possibly contributed to by dysfunctional glucocorticoid signaling, has shown to be associated with chronic social defeat [24].

STRING was also used to create a PPI network containing the list of candidate genes and first & second shell interactions. The network displayed below is a subnetwork that shows connections between the products of candidate genes as well as gene products that directly interact with them. The layout is arranged based on MCODE clusters using clusterMaker. Green colored nodes are candidate genes, and red colored nodes are the initial 9 genes. Red edges are connected to at least 1 initial gene. Darker edges are connections within MCODE clusters.

HTR1A

STAT5B

JUN

CCNE1

NCKAP1

PPP2R5C

CCT6A

CAMK2A

ROCK2

EGFR

HSP90AB1

RAF1

NEDD4L MAPT

HIST1H3I

CSH2 LMNB2

CKB

RYR1

ADD2

GUCY1A2

CNGB1

RUNX1

PAG1

SCN8A

KCNJ12

CSK

RXRG

CDK1

PDHB

DDX42

CCT3

KPNA2 RAB3C

RPS6KA4

TUBB4B

PTPN11

RAC3

GABPB1

HIST1H2AM

CSH1

MAPK3

PRKCA

FLT1

HOMER3

DCC

PDE6A

SH2B1

PRKCG

PTPN7

KCNJ4

CHD9

HSPA4

CCNB1

RANGAP1

CCNA1

TCP1

CKS1B

API5

SUMO1

IRS1

TPM3

PPIA

HIST2H2AB

CBLC

CRKL

GABBR1

KDR

DBN1

MEF2D

GUCY2D

HIST1H1B

DAPK3

CHN1

MECOM

BAD

PPARA

HIF1A

UBE2I MAP2K6

RPL17

NFKB1

CHERP

DSP PRKCE

ACTA1 FGF12

HIST1H2AG

REL

DUSP7 GABBR2

ABL2 ARHGAP12

SUGP1

GABPB2

SELL

PTPN6

SPEG

GRM7

NUCB1

PARK2

CCND3

AKT1

PPP2CB

FAS

NFKBIA

NCOR1

RBCK1

NTRK1

BRK1

KRAS

HIST1H2AI

MAP3K14

DUSP9

STIM2

RPS6KA3 ITPK1

TRPV4

DIAPH2

IRF3

RYR3

ITPKA

PLCB1

SPOP

HNF4A

CDK4

CCNB2

IL1R1

YWHAZ

ACTN2

SMARCC2

RNF31

CBY1

NCK2

NRAS

HIST1H3B

FKBP1B

IL33 ARHGEF4

SCML2

SOS2 NPR2

CNGA1

HRH2

RAPGEF4

ORAI3

HTR2A

SNCG

ESR1

GNB1

GSK3B

IRAK1

YWHAB

ABI1

HSP90AA1

NCOA3

CACNA 1C

CBLB

NCOR2

HIST1H3H

RYR2

IL1RL1 IL1R2

PITHD1

NPR1 ACTA2

PDE6B

TRA2A

RPS6KA6

ORAI2

PTGS2

IRS2

USP8

GNG2

RGS7 TNF

SMARCB1 MDM2

SPTAN1

CREBBP

CREB1

KCNQ3 DLGAP2

HIST1H2AL

SCN5A

NFKBIB

MYH2

PPP3CB

CD40

CDK5

GABRA5

TRAK2

MLXIPL

CAMK4

SLC8A1

PIAS1

BPTF

GNB4

LIMS1 MTOR

SMARCC1

GNAI2

OTUB1

NCOA1

NCOA2

NGF DLGAP3

HIST1H3JHIST1H4F

TTN

ARAF

CARD11

DCLRE1 C

GABRG2

GABRB3

RAPH1

NTF3

FLG

ADRB1

CAV1

RICTOR

CDC5L

RPTOR

MYD88

FAU

GNAI3

AP1B1

NFATC1 RAP1A

TAB3

WIPF1

PIK3CB

PPP2R2C

OBSCN

MET

GYG1

MYBL1 GABRA1

KCNJ6

PRKCD

FGF13

ATP2B3

CSF3R

KIF13B

NCOA4

ARPC3

CCND1 IFNAR1

UBA52

RPS6KA1

DHX8

YWHAH

DCUN1D1

SLC9A1 SCN2A

PIK3R2 CHEK2

GRIN2A HGF

STBD1

MEF2C

ENSG00000243667

ITGB1

PRKCB

KCNN1

ATP2B2

ARHGAP32

TGFB1I1

STUB1

SF3A2

CDKN1A

NEK2

RPL23A

MAPK1

PFKM

YWHAG

SMAD3

ABI2

SCN3A

HIST1H2AH ILKAP

PTPN4 CD3E

MYO1C

SHC4

RRAS2 UNC13A

KIAA1522

MYO5A

TDO2

PICK1

BCL6

HDAC 3

ARPC1B

RPL38

RIPK1

RPL5

APC

HNRNPK

SHARPIN

CITED2

H3F3A

HIST2H3D

HIST1H3G

CACNB 1

IL1RAP CD3D

CRHR2

SHANK1

GABRB2 UNC13B

MAPK10

ZFYVE16

ASMTL

PTGER1

MAP3K5

DAXX

ACTR2

PPP2CA

BCL2

PRKAA2

HIST1H3A

AXIN1

SKI

ZFYVE9

ACTR3B

CAMK2G

HIST1H2AK

CACNB 2

IL1B

HOMER1

ERBB2

ATP2B1

MOS NOS1

ACTG2

GABRA2

MAPKAPK5

GPM6B

INADL

RPS6KB1

FZR1

PPP2R1A

CASP8

PRKAB2

KHDRBS1

SMARCA4

PIK3R1

HRAS

CRTC2 PPP3CC

HIST1H3F

XPO6

RCAN1

HOMER2

KCNQ5

SHC2

SERTAD4

PHKG2

GABRB1

GABRA4

EEF2K

UCN

GAK

AR

CTNNB1

SHC1

FER

CYFIP1

ACTB

ARHGEF2

PIK3CA KCNN2

HIST2H3C

TAB1

HIST1H4A

HEATR2

RPS6KA2

RCAN3

RAC2

EPAS1

HRH1 C1orf116

IQCG

GABRA3

WWP2

SNCA

FKBP4

GAB1

IKBKG

GRB2

PPP2R1B

WASF1

DDX5

SMARCA2

KMT2A DAPK1

PPP2R2A

DAPK2

NEDD4 ATF1

NPPA ACTN3

SMAD9

SHC3

FGFR3

DLG3 GUCY2F

SLC18A1

NPY

MAGI3

NRIP1

MDM4

IKBKB PSMC5 CCND2

TUBA1B

EMC2

TUBB2A

VCL

ITGB3

NOS2

H3F3C

TPM1

BAIAP2L1

NPR3

KCNJ5 VR1

EPM2A

CACNA1 A

CACNA1 E

KCNH5

GPSM2

NPY1R

GRIP1

NUP153

RARA

CDC27

RPL32

IRAK4

TUBA1A

AURKA

NSF

PPARG

CD247

HIST3H3

H3F3B

ACTC1 KCNN4

FERMT2

KCNJ3

GNAO1

NHSL1

GRIA4

ORAI1

CACNA 2D2

RET

GRIA3

UCN3

MIB2

RXRB

ARPC4

RB1

CCT5

RGS14

TUBA4A

LMNA

MYLK DLG4

SOS1

WIPF2

KLC4

SGOL1

IDE

PTPRF RBL2

AMZ1

KCNQ2

KMT2B

CACNA 1B

PDE1B PLK2

UCN2 YAP1

PPARGC1A

CDK6

MAP3K7

CCT4

WASF2

DNM2

SPTBN1

CALM1

DLGAP1

PEA15

WASL

HIST1H4C

HIST1H4H

TRIP10

RAP1B GRIN2B

MARCKS

DLG2

LIMS3L

IL1RAPL2

PDGFRA

NLK

SORT1 UBE2L3

MED25

GNB2

TAB2

TLR3

PRKAB1

TUBA1C

PPP1CA

FOXO3

PXN

PRKAA1 SFN

NTF4

HIST1H4L

NCK1

FGFR1 ATP2B4

TCF7L2

DLG1

LIMS3

IL1RAPL1

CDC25B

SYNGAP1

TANC1 TIRAP

MYC

CCNA2

TRAF6

TRAF2

KLC1

MAPK14

HSPA5

SMAD2

CRHR1

PRKAG1

LRRK2

BDNF

HIST1H4D

DUSP6

CDKN1C

CAMKK1

RAC1

CACNA1 S

UBXN11

FLT4

CACNA1 F

CAMK1G

NT5E

CAPN1 MED1

CDKN1B

PCNA

CCT8

KLC2

CDC42

CHEK1

RELA

CRH

PRKAG2

CKS2

HIST4H4

HIST1H4E

MYB

BCL10

CREM

MAPK9

PLCG2

SH2B2

DLGAP5

CACNA1 D

DUSP2

FAM111B

FOS

STX1A

ACTR3

CCT2

SRC

RPL3

PAK1

IQGAP1

PPP2R5E

CALM3

RXRA

MAPKAPK2 MAPKAPK3

HIST2H4B

HIST1H2AA

SLK PFKFB2

KCNN3

PRR14

RCAN2

RUNX2

KCNQ4

TBXA2R

ABHD16A

PLCG1 COPS6

CTNNA1 PPP3CA

CCT7

MST4 HDAC 2

SMN1

INSR

TCF3

HIST1H3C

HIST2H3PS2

MAP2K2

HIST1H4J

ROCK1

SLC1A2

KCNJ9

MYLK2

PTPRR

ACTR3C

PAX5

LEMD3

GRM5

SLC6A4

ATF2

KAT2A

ARPC2

PPP3R1

TP53

RUVBL1

HDAC 1

COPS5

IGF1R

CALM2

LIMS2

HIST2H3A

SPDYA

HIST1H4B

RND3

KCNH1

CACNG2

MYBL2

FKBP1C

CAMK1D

GNAT1

CACNA 2D3

RASGRF2

DPM1

FBXW7 BUB1B

GNAI1

GNG3

FYN

TUBB

HTR3A

TUBB2B MEF2A

FKBP1A

CBL

WIPF3

NFKB2

HIST1H4K

MAP3K1

NOS3

GRIA2

FGFR4

ERBB4

NGFR

GNAT3

PIK3AP1

IQGAP3

STAT6

TPM2

CDC20

ACTN1

MAP2K1

MAVS

DIAPH3 ENAH

WDFY2

EP300

HIST2H4A

KIF5A

CDK3

PIK3R3

HIST1H4I

PIK3CD

CAMK1

DLGAP4SHANK2

CACNA 2D1

MAPK13

GNAT2

TRAF5

FGF3

ARRB2

ESR2

UBC

ACTN4

BRAF

GUCY1A3

RANBP2

SFPQ

TRIM28

ABL1

FRS2

HIST1H3E

PRKAG3 FNBP1L FGFR2

YWHAE

DUSP8 CACNB 3

GYS1

CACNG1

ENSG00000196689

PAX2

GATA1

AGAP2

STAT3

HMGB1 LAMTOR3

CCNE2

TCF12 GUCY1B3

CAMK2B

PKN2

VIM

RBM17 CRK

NTRK2

HIST1H3D

KCNQ1

STAT1

FGF8

CAMKK2

MAPK8 ARHGEF6

C19orf26

HFE

ZNF511

RASAL2

MAP3K4

FGD1

STAT5A

HSPA1A

CDK2

CYFIP2 TCF4

SMAD4

CAMK2D

ELAVL1

UBE2N

PAK2

TP53BP1

NR3C1 AKAP5

CHUK

PPP2R2B

TLR4

ERBB3

GRIN1 RGS16

ZNF114

NRM

ETS2

MATK

CDCA4

PLA2G4A

Network 1

Interesting genes from the MCODE subclusters were identified.

SHANK2 is a scaffolding protein in the postsynaptic density and may play a role in regulating mGluR activity. One proposed role of Shank proteins is to couple postsynaptic density to the endocytic zone, which recycles mGluR5 [25]. Triple knockdown of SHANK1/2/3 with concurrent mGluR5 agonism led to decreased downstream responses and lower spine mGluR5 levels, which as discussed earlier would decrease NMDAR levels. Confirming this, SHANK3 knockdown reduced NMDAR activity [26]. However, SHANK2 dysfunction has primarily been implicated in autism spectrum disorder and autistic-like social behavior – which may connect to the role of poor social behavior in the psychopathogenesis of depression – which is improved in mice by an NMDAR agonist that normalizes the hippocampal NMDA/AMPA ratio from a reduced value caused by SHANK2 knockout [27]. SHANK2 knockout mice also had lower hippocampal plasticity. Further complicating the role of SHANK2, a more recent study shows consistent increased hippocampal plasticity in SHANK2 knockout mice, though the knockout was achieved with exon 7 deletion (as opposed to exon 6 and 7 deletion in Won et al.) and NMDA/AMPA ratios were inconsistent [28]. SHANK2 knockout localized to Purkinje cells in mice increased repetitive and anxious behavior and impaired social behavior, probably due to decreased excitatory synapse activity brought about by the knockout [29, 30]. Deletion of exon 24 induced anhedonia and manic and reward-seeking behavior in mice that were treated with mood stabilizers in addition to social impairment [31]. Consistent with Won et al., a decreased NMDA/AMPA ratio was also observed, possibly due to an increase in NMDAR NR2A and NR2B (which is coded from GRIN2B) subunit levels. Thus, different SHANK2 mutations may have varying roles in multiple neuropsychiatric disorders, which may be an area of future research.

This does raise a question about the effects of ketamine on social behavior, and indeed ketamine does induce social-withdrawal in mice [32], which is reversed by a 5-HT7 agonist and memantine [33], which is also an NMDAR antagonist and further complicates the role of NMDARs in social behavior. No firm conclusions can be made on the effect of ketamine on social behavior on humans from animal model studies, but this does open an area for future research.

GRIA genes code for AMPAR subunits.

MAPK and MAPKK genes code for components of the ERK signaling pathway, a downstream target of BDNF that leads to protein synthesis and induces inflammation as part of the stress response [34]. This pathway seems to be essential for R-ketamine’s antidepressant effects, as opposed to S-ketamine which requires mTORC1 signaling [35, 36], as well as other traditional antidepressants [37]. Not only does inhibiting MAPK prevent antidepressant effects, but it also induces depressive symptoms, suggesting it has a role in the pathogenesis of depression. MAPKs are also involved in synaptic plasticity and neuroinflammation [38, 39]. The p38 MAPK-MK2 complex induces neuroinflammation and the release of proinflammatory cytokines. Activation of

NMDARs causes p38 MAPK-mediated internalization of certain AMPARs, which underlies long-term depression [40], and AMPAR downregulation caused by NMDAR stimulation may be a mechanism for the pathogenesis of depression consistent with the disinhibition hypothesis. Consistent with this, ketamine reduced hippocampal p38 MAPK levels [36]. ERK1/p44 MAPK levels were also increased with ketamine treatment, which upregulates certain AMAPARs [40], however ERK2/p42 MAPK also upregulates AMPARs but was decreased after ketamine treatment.

CALM1 is differentially expressed in depressed patients and is involved in dopamine signaling [41]. CaMK2 is activated by NMDAR-mediated calcium intake, which then activates Ras complexes which then activates MAPK signaling that upregulates surface AMPAR levels [40].

KCJN6 may play a role in rumination [42], which is a classic psychopathogenetic mechanism of depression.

NFkB is a transcription factor and a downstream target of PI3K-Akt signaling, and genes coding for NFkB subunits have been implicated in hippocampal synaptic plasticity. Knockout of NFKB1 (the p50 subunit) impaired hippocampal neurogenesis and cognition in mice [43]. On the other hand, chronic IKK-mediated activation of NFkB resulted in inflammation, neurodegeneration, and reduced BDNF levels in the hippocampus which was treated with deactivation of NFkB, which may suggest a therapeutic effect from the activation of NFKBI, including NFKBIB, and other agents inhibiting NFkB activity. This is consistent with the psychopathogensis of depression as a result of chronic stress. Modulators of NFkB signaling have promise as novel antidepressants. The gabapentinoids pregabalin and gabapentin promote hippocampal neurogenesis and prevent depressive symptoms through an NFkB-dependent mechanism [44]. L-acetylcarnitine (LAC) is also a therapeutic of interest that also interacts with NFkB. In rats, LAC had antidepressant effects within days and lasted for 2 weeks after withdrawal [45]. Depressed rats had lower prefrontal and hippocampal BDNF levels and lower depolarization-evoked glutamate released, which were both treated with LAC. LAC levels are also lower in depressed patients [46]. A meta-analysis in humans found LAC to be a significantly effective monotherapy for depression treatment and even had fewer adverse effects than traditional antidepressants [47]. The mechanism underlying LAC is complex. LAC acetylates NFkB p65, which then promotes transcription of mGluR2 receptors [46, 48]. mGluR2 receptors have been observed to be upregulated, and this is prevented with NFkB inhibition [46]. Although LAC does appear to be well-tolerated, LAC’s therapeutic effects are probably due to acute NFkB positive modulation, since chronic NFkB activation promoted depressive symptoms as discussed before; the effects of LAC treatment over a longer period should be further studied. In addition, upregulation of mGluR2 would cause depressive symptoms according to the disinhibition hypothesis. Previous studies have shown that elevated prefrontal mGluR2 levels are a characteristic of depressed patients [49] – which contrasts with Nasca et al., which observed downregulated prefrontal mGluR2 in depressed rats – and mGluR2 antagonism has

antidepressant effects [50, 51]. It is possible that LAC’s antidepressant effects occur through another mechanism, and its basic mechanism and its clinical effects are areas of further research.

SLC1A2 is the glutamate transporter responsible for the majority of glutamate reuptake [52].

CACNA1C is associated with several psychiatric disorders, including bipolar disorder, depression, and schizophrenia. Complete CACNA1C knockout impaired neurogenesis, but knockdown and a heterozygous genotype had antidepressant effects [53]. A CACNA1C SNP predicts the development of depression after threatening life events [54]. Voltage-gated calcium channels in general may be associated with psychiatric disorders [55,56].

Protein-Drug Interaction Network

N-BENZYL-4-[4-(3-CHLOROPHENYL )-1H-PYRAZOL-3-YL]-1H-PYRROLE-2-CARBOXAMIDE

Nimodipine

Flibanserin

Betahistine

Tripelennamine

Roflumilast

Estazolam

Metoclopramide

Pancuronium

Procyclidine

Nadolol

Hexane -1,6-Diol

Fluorometholone

Sertraline

Nepafenac

Sulfasalazine

GRIN2B

Primidone

Adenosine monophosphate

Thiocoumarin

N,N-DIMETHYL-4-(4-PHENYL-1H-PYRAZOL-3-YL)-1H-PYRROLE-2-CARBOXAMIDE

Ibutilide

Bifeprunox

Mepyramine

Olopatadine

Iloprost

Flumazenil

Flavoxate

Chlorprothixene

Oxyphencyclimine

Acebutolol

Ciclesonide

Amcinonide

Dopamine

Lornoxicam

Naproxen

(3R)-3-cyclopentyl -6-methyl-7-[(4-methylpiperazin -1-yl)sulfonyl ]-3,4-dihydro -2H-1,2-benzothiazine1,1-dioxide

Thiopental

PRKAB2

Ethylisothiourea

SB220025

CACNB1

Dapoxetine

Esmirtazapine

Epinastine

Ketotifen

Desflurane

Cyclopentolate

Aripiprazole

Trihexyphenidyl

Carvedilol

Paramethasone

Medrysone

Cocaine

Cimicoxib

Mefenamic acid

(3R)-3-cyclopentyl -7-[(4-methylpiperazin -1-yl)sulfonyl ]-3,4-dihydro -2H-1,2-benzothiazine

1,1-dioxide

Metharbital

L-Glutamic Acid

7-Nitroindazole

Purvalanol

Amperozide

Lysergic Acid Diethylamide

Levocetirizine Carbinoxamine

Dyphylline

Methyprylon

Cycrimine

Diphenidol

Clozapine

Amiodarone

Cortisone acetate

Alclometasone

Pseudoephedrine

Nimesulide

Etodolac

(3S)-3-cyclopentyl -6-methyl-7-[(4-methylpiperazin -1-yl)sulfonyl ]-3,4-dihydro -2H-1,2,4-benzothiadiazine

1,1-dioxide

Talbutal

L-Aspartic Acid

N-(3-(Aminomethyl )Benzyl)Acetamidine

Phosphonothreonine

Amisulpride

Thioproperazine

Cariprazine

Meclizine

Caffeine

Clonazepam

MethanthelineMivacurium

Terfenadine

Arbutamine

Desonide

Diflorasone

Minaprine

Licofelone

Flurbiprofen

2,3,6A,7,8,9-HEXAHYDRO-11H-[1,4]DIOXINO[2,3-G]PYRROLO[2,1-B][1,3]BENZOXAZIN-11-ONE

Butalbital

SLC1A1

3-Bromo-7-Nitroindazole

Olomoucine

Sertindole

Pipotiazine

Epicept NP-1

Risperidone

PDE4C

Ergoloid mesylate

Flupentixol

Quetiapine

Metixene

Isoprenaline

Budesonide

Flunisolide

Trimipramine

Niflumic Acid

Sulindac

N,N'-[biphenyl -4,4'-diyldi (2R)propane -2,1-diyl]dimethanesulfonamide

Butabarbital

Triflusal

4r-Fluoro-N6-Ethanimidoyl -L-Lysine

Arsenic trioxide

Lumateperone

Alverine

SO-101

Azatadine Pagoclone

Methoxyflurane

Biperiden

Ketamine

Ipratropium bromide

Salbutamol

Fluoxymesterone

NR3C1

Paroxetine

Prostaglandin G 2

Diclofenac

CX717

GRIA2

1-[4-(AMINOMETHYL)BENZOYL]-5'-FLUORO-1'H-SPIRO[PIPERIDINE-4,2'-QUINAZOLIN]-4'-AMINEN,N-dimethylarginine

MAPK1

MAP-0004

Acepromazine

OBE101

Histamine Phosphate

Zolpidem

Triazolam

Dicyclomine

Pilocarpine

Disopyramide

Pindolol

Prednicarbate

Vortioxetine

Verapamil

1-Phenylsulfonamide -3-Trifluoromethyl- 5-Parabromophenylpyrazole

Valdecoxib

Talampanel

Ifenprodil

4-(1H-IMIDAZOL-1-YL)PHENOL

Dexamethasone

HTR3B

BL-1020

Bromocriptine

HY10275

Astemizole

GABRA3

Oxazepam

Propantheline

Promethazine

Ziprasidone

Alprenolol

Fluocinonide

Butriptyline

Trazodone

Resveratrol

Fenoprofen

THIO-ATPA

Agmatine

1-(6-CYANO-3-PYRIDYLCARBONYL )-5',8'-DIFLUOROSPIRO[PIPERIDINE-4,2'(1'H)-QUINAZOLINE]-4'-AMINE

Miconazole

KCNJ3

Pimavanserin

Pergolide

Iloperidone

Hydroxyzine

Fospropofol

Diazepam

Clidinium

Oxybutynin

Succinylcholine

Dobutamine

Clobetasol propionate

Levomilnacipran

Mazindol

Flufenamic AcidPiroxicam

Aniracetam

Gavestinel

N-[2-(6-AMINO-4-METHYLPYRIDIN -2-YL)ETHYL]-4-CYANOBENZAMIDE

L-Citrulline

KCNJ6

APD791

Ondansetron

Tesmilifene

Loratadine

Ivermectin

Propofol

Pirenzepine

TolterodineCevimeline

Epinephrine

Methylprednisolone

Dexmethylphenidate

Fenfluramine

Etoricoxib

Rofecoxib

2-Amino-3-(3-Hydroxy-7,8-Dihydro -6h-Cyclohepta [D]-4-Isoxazolyl )Propionic

Acid

Ketobemidone

ETHYL4-[(4-METHYLPYRIDIN -2-YL)AMINO]PIPERIDINE-1-CARBOXYLATE

L-Arginine

Rufinamide

APD125

Apomorphine

Bepotastine

Triprolidine

Piperazine

Halazepam

Triflupromazine

Glycopyrronium

CHRM3

Bisoprolol

Rimexolone

Desvenlafaxine

Amoxapine

Tiaprofenic acid

Tolmetin

Willardiine

Acetylcysteine

N-[2-(4-AMINO-5,8-DIFLUORO-1,2-DIHYDROQUINAZOLIN-2-YL)ETHYL]-3-FURAMIDE

NOS2

ADX-48621

Fluspirilene

Lisuride

Flunarizine

Dexbrompheniramine

GABRB3

Isoflurane

Carbachol

Maprotiline

Bupranolol

Labetalol

Loteprednol

Tapentadol

Nortriptyline

Antipyrine

Celecoxib

Bromo-Willardiine

CNS-5161

ETHYL

4-[(4-CHLOROPYRIDIN -2-YL)AMINO]PIPERIDINE-1-CARBOXYLATE

ADX10059

Thiothixene

Buspirone

Clofedanol

Cetirizine

Zopiclone

Flurazepam

Ethopropazine

Brompheniramine

Bopindolol

Propranolol

Prednisolone

Mianserin

Dextromethorphan

Antrafenine

Lenalidomide

FG-9041

Milnacipran

4-(1,3-BENZODIOXOL-5-YLOXY)-2-[4-(1H-IMIDAZOL-1-YL)PHENOXY]-6-METHYLPYRIMIDINE

GRM5

Dimethyltryptamine

Pramipexole

Zuclopenthixol

Clemastine

GABRA5

Midazolam

Doxylamine

Tropicamide

2-HYDROXYMETHYL-6-OCTYLSULFANYL-TETRAHYDRO-PYRAN-3,4,5-TRIOL

Carteolol

Flurandrenolide

CRx-119

Duloxetine

Ginseng

Tenoxicam

(S)-ATPA

Cycloleucine

(3S)-1-(1,3-BENZODIOXOL-5-YLMETHYL)-3-[4-(1H-IMIDAZOL-1-YL)PHENOXY]PIPERIDINE

Prucalopride

Tegaserod

Ropinirole

Pheniramine

HRH1

Cinolazepam

Clorazepate

Buclizine

Cryptenamine

4-{[(2S)-3-(tert-butylamino )-2-hydroxypropyl ]oxy}-3H-indole -2-carbonitrile

Sotalol

Clocortolone

OPC-14523

Fluoxetine

Trisalicylate -choline

Ketorolac

(S)-2-Amino-3-(1,3,5,7-Pentahydro-2,4-Dioxo -Cyclopenta [E]Pyrimidin-1-Yl)

Proionic Acid

D-Serine

4-(1,3-BENZODIOXOL-5-YLOXY)-2-[4-(1H-IMIDAZOL-1-YL)PHENOXY]PYRIMIDINE

TD-2749

Mesoridazine

HTR1A

Phenindamine

Reserpine

Ketazolam

Chlordiazepoxide

Benzatropine

Propiomazine

Bufuralol

Phenylpropanolamine

Mifepristone

Bicifadine

Imipramine

Salsalate

Nabumetone

Quisqualate

Dcka, 5,7-Dichlorokynurenic Acid

(3R)-3-[(1,2,3,4-tetrahydroisoquinolin -7-yloxy)methyl]-2,3-dihydrothieno [2,3-f][1,4]oxazepin -5-amine

TD-5108

Cyclobenzaprine

Rizatriptan

Aceprometazine

SLC18A1

Fludiazepam

Alprazolam

Oxyphenonium

Benzquinamide

Droxidopa

Timolol

Hydrocortamate

Amineptine

Methylphenidate

Magnesium salicylate

Indomethacin

Fluoro-Willardiine

Dehydroepiandrosterone

(2S)-2-methyl-2,3-dihydrothieno [2,3-f][1,4]oxazepin -5-amine

Renzapride

Donepezil

Naratriptan

Paliperidone

Yohimbine

Clotiazepam

Eszopiclone

Trospium

Scopolamine

Asenapine

Norepinephrine

Mometasone

Zimelidine

Loxapine

Lumiracoxib

Acetaminophen

Iodo-Willardiine

Phenobarbital

4-({4-[(4-methoxypyridin -2-yl)amino]piperidin -1-yl}carbonyl )benzonitrile

IRS1

Piboserod

Thioridazine

Ergotamine

Alimemazine

Dofetilide

Gamma Hydroxybutyric Acid

Meprobamate CHRM1

Diphemanil Methylsulfate

Nebivolol

Atenolol

Hydrocortisone

Methamphetamine

Mirtazapine

Ibuprofen

Mesalazine

2-Amino-3-(5-Tert-Butyl-3-(Phosphonomethoxy )-4-Isoxazolyl )PropionicAcid

Orphenadrine

N-[2-(1,3-BENZODIOXOL-5-YL)ETHYL]-1-[2-(1H-IMIDAZOL-1-YL)-6-METHYLPYRIMIDIN-4-YL]-D-PROLINAMIDE

BDNF

HTR4

Cisapride

Sumatriptan

Bromodiphenhydramine

KCNJ12

Adinazolam

Clobazam

Umeclidinium

Homatropine Methylbromide

Dronedarone

Olanzapine

Fludrocortisone

4-Methoxyamphetamine

Protriptyline

Thalidomide

Aminosalicylic Acid

(S)-AMPA

Memantine

5-(4'-AMINO-1'-ETHYL-5',8'-DIFLUORO-1'H-SPIRO[PIPERIDINE-4,2'-QUINAZOLINE]-1-YLCARBONYL )PICOLINONITRILE

KCNQ5

Quinacrine

Remoxipride

Zolmitriptan

Cyclizine

Dimetindene

Lindane

Etomidate

Aclidinium

Nicardipine

Celiprolol

Metoprolol

Flumethasone

3,4-Methylenedioxymethamphetamine

Amitriptyline

Balsalazide

Dihomo-gamma-linolenic acid

(S)-Des-Me-Ampa

Gabapentin

ACCLAIM

PLA2G6

PLA2G4A

HTR2A

Methysergide

Diphenylpyraline

Metocurine

GABRB1

Topiramate

Methacholine

Atropine

Oxprenolol

Cabergoline

Prednisone

MMDA

Venlafaxine

Ketoprofen

PTGS2

Barbituric acid derivative

Acamprosate

KD7040

(1S,6BR,9AS,11R,11BR)-9A,11B-DIMETHYL-1-[(METHYLOXY)METHYL]-3,6,9-TRIOXO-1,6,6B,7,8,9,9A,10,11,11B-DECAHYDRO -3H-FURO[4,3,2-DE]INDENO[4,5-H][2]BENZOPYRAN-11-YL

ACETATE

Nilvadipine

Brexpiprazole

Eletriptan

Levocabastine

Doxacurium chloride

Thiocolchicoside

Temazepam

Fesoterodine

Cinnarizine

Clenbuterol

Isoetarine

Triamcinolone

Ephedra

Citalopram

Oxaprozin

GRM7

Barbital

Pethidine

5-Nitroindazole

SF1126

Amlodipine

Lurasidone

HTR1F

Emedastine

Diphenhydramine

Ganaxolone

Enflurane

ALKS 27

Anisotropine Methylbromide

Mephentermine

Bethanidine

Ulobetasol

Clomipramine

Tramadol

Bromfenac

Lithium

Hexobarbital

Secobarbital

7,8-Dihydrobiopterin

PIK3R1

CACNA2D3

Cinitapride

Bilastine

Mequitazine

Bethanechol Nitrazepam

Ethchlorvynol

Itopride

Tridihexethyl

Penbutolol

Betaxolol

Fluocinolone Acetonide

Dexfenfluramine

Phentermine

Meclofenamic acid

GRIA3

Heptabarbital

Pentobarbital

L-Thiocitrulline

4-[5-(4-FLUORO-PHENYL)-2-(4-METHANESULFINYL -PHENYL)-3H-IMIDAZOL-4-YL]-PYRIDINE

ICA-105665

Vilazodone

Acrivastine

Dimenhydrinate

Rocuronium

Quazepam

Lorazepam

Mepenzolate

Darifenacin

Practolol

Esmolol

Fluticasone Propionate

Escitalopram

Amphetamine

Salicylic acid

Halothane

Butethal

Atomoxetine

N-(4-(2-((3-Chlorophenylmethyl )Amino)Ethyl)Phenyl)- 2-Thiophecarboxamidine

[4-({5-(AMINOCARBONYL)-4-[(3-METHYLPHENYL )AMINO]PYRIMIDIN-2-YL}AMINO)PHENYL]ACETIC

ACID

Ezogabine

LI-301

Chlorcyclizine

Azelastine

Gallamine Triethiodide

Prazepam

GABRA4

Isopropamide

Chlorpromazine

Bevantolol

ADRB1

Desoximetasone

Desipramine

Fluvoxamine

Suprofen

Glycine

Aprobarbital

GRIN1

N-Omega-Hydroxy-L-Arginine

(1aR,8S,13S,14S,15aR)-5,13,14-trihydroxy -3-methoxy-8-methyl-8,9,13,14,15,15a-hexahydro -6H-oxireno [k][2]benzoxacyclotetradecine -6,12(1aH)-dione

KCNQ3

MN-305

Isothipendyl

Desloratadine

CHRM2

Bromazepam

ACP-104

Solifenacin

Methylscopolamine bromide

Pirbuterol

Fluticasone furoate

Betamethasone

Nefazodone

SLC6A4

Diflunisal

GRIN2A

Amobarbital

Icosapent

6-Nitroindazole

5-(2-PHENYLPYRAZOLO [1,5-A]PYRIDIN-3-YL)-1H-PYRAZOLO[3,4-C]PYRIDAZIN-3-AMINE

NTRK2

SLV 308

Chloropyramine

Fexofenadine

Ibudilast

Flunitrazepam

NGX267

Tiotropium

Cyproheptadine

Fenoterol

Ulipristal

Spironolactone

Doxepin

Firocoxib

Carprofen

Tenocyclidine

Quinidine barbiturate

Alpha-Linolenic Acid

N-Omega-Propyl -L-Arginine

(3R,5Z,8S,9S,11E)-8,9,16-TRIHYDROXY-14-METHOXY-3-METHYL-3,4,9,10-TETRAHYDRO-1H-2-BENZOXACYCLOTETRADECINE -1,7(8H)-DIONE

Mibefradil

Rotigotine

Antazoline

Methdilazine

AN2728

Glutethimide

ArecolineMethotrimeprazine

Hyoscyamine

Metipranolol

Difluprednate

Beclomethasone dipropionate

Chlorphenamine

Pomalidomide

Meloxicam

Felbamate

Ethanol

SLC8A1

S-Ethylisothiourea

(S)-N-(1-(3-CHLORO-4-FLUOROPHENYL )-2-HYDROXYETHYL)-4-(4-(3-CHLOROPHENYL )-1H-PYRAZOL-3-YL)-1H-PYRROLE-2-CARBOXAMIDE

Magnesium Sulfate

Lesopitron

Alcaftadine

Tolazoline

Piclamilast

Sevoflurane

Molindone

Pipecuronium

Promazine

Levobunolol

ORG-34517

Megestrol acetate

Sibutramine

Parecoxib

Phenylbutazone

Haloperidol

Methylphenobarbital

Acetylsalicylic acid

Aminoguanidine

The list of candidate genes was put into NetworkAnalyst to create a network of protein-drug interactions using the DrugBank database. The largest subnetwork was visualized in Cytoscape.

Key:

Red nodes – Candidate genes

Light blue nodes – Drugs

Purple node & edges– Ketamine

Green nodes & edges– SSRIs

Orange nodes & edges – SNRIs

Dark blue node & edges – Tricyclics

No MAOIs were found.

Note how traditional antidepressants act on proteins of multiple pathways, not just monoaminergic systems. These interactions show other potential mechanisms that may contribute to these drugs’ antidepressant effects. They also provide evidence that the products of the candidate genes are possibly implicated in the pathophysiology of depression.

Quite nicely and without intention, ketamine is at the center of the network. Though ketamine is hypothesized to act primarily on the glutamatergic system to produce antidepressant effects, its mechanism of action may involve other systems as well, including the serotonergic system [57].

Other smaller subnetworks are shown below.

Enrichment Analysis

Functional Annotation Clustering

The genes identified from Network 1 and their isoforms are

SHANK1 SHANK2 GRIA2 GRIA3 GRIA4 MAPKAPK2 MAPKAPK3 MAP2K2 MAP3K14 MAPK3 MAP3K1 MAPK8 MAPK9 MAPK13 MAPK10 MAPK3K4

MAPKAPK5 MAP3K5 MAP3K7 MAP2K1 MAP2K6 MAPK1 MAPK14 CALM1 CALM3 CALM2 CAMK2G CAMKK2 CAMK1 CAMKK1 CAMK1D CAMK4

CAMK1G CAMK2B CAMK2D CAMK2A KCNJ5 KCNJ3 KCNJ9 KCNJ6 KCNJ12 KCNJ4 CACNA1C CACNB1 CACNB2 CACNG2 CACNB3 CACNA1A

CACNA1S CACNA2D1 CACNG1 CACNA1E CACNA2D2 CACNA1B CACNA1F CACNA1D CACNA2D3 NFKB2 NFKBIB NFKB1 NFKBIA SLC1A2

The gene list above was combined with the candidate gene list. This combined gene list was run through DAVID for functional annotation clustering, and the resulting clusters are shown in the table below.

Several of these clusters have clear themes and highlight interesting molecular interactions and properties. The highest ranked cluster contains the following systems.

As previously discussed, GABAergnic neurons are implicated through the disinhibition hypothesis. Some other methods of reducing GABA release are inhibiting CACNA1A/B (labeled VGCC in the picture) or activating GABBR1/2 autoreceptors (labeled GABAB in the picture).

Endocannabinoids released by a local increase in calcium in postsynaptic dendritic spines inhibit further release of GABA and glutamate from the presynaptic neuron by binding to CB1 receptors. The endocannabinoid system is highly expressed in the hippocampus, a brain region implicated in depression, and is involved in the stress response [58]. CB1 receptors are present on both GABAergic and glutamatergic neurons, and CB1 receptor activation can lead to less release of both types of neurotransmitters downstream through depolarization-induced inhibition of excitation (DSE) and inhibition (DSI). According to the disinhibition hypothesis, this may produce conflicting effects with regards to depression treatment. High-affinity CB1R antagonists and agonists – since DSE and DSI are transient events – are both worth investigating for therapeutic effects, especially if they can selectively target different neuron types. Endocannabinoids may also play various roles in the synaptic plasticity of various brain regions [59].

Although morphine addiction may not appear directly relevant to depression, this pathway provides an interesting insight into the regulation of dopaminergic neurons with GABA. Additionally, acute morphine produces a similar effect as ketamine by inhibiting GABA receptors, which increases dopamine signaling for morphine – which may also occur with ketamine. This may also partially explain the potential for ketamine to become addictive, albeit to a lesser extent than morphine. Tianeptine, an atypical depressant, is a mu-opioid receptor (labeled MOR on the picture) agonist, similar to morphine. Interestingly, the mu-opioid receptor antagonist naltrexone has been shown to lessen the antidepressant effects of ketamine [60]. All of this suggests that the opioid system is involved in the pathophysiology of depression and the mechanism of action of ketamine. Buprenorphine, a partial mu-receptor agonist, has been shown to rapidly treat suicidal ideation in a randomized control trial [61]. Less potent opioids like buprenorphine in a controlled setting may have use as a last-line option for severe treatment-resistant depression. Regardless, the opioid system has potential as a novel target for depression treatment.

Clusters 8 and 11 have a theme of the immune response, and cluster 11 also includes neurotransmitter systems. T-cell signaling is also a reoccurring theme.

Several IL2 related functional annotations are in cluster 11, although they do not meet the threshold for statistical significance. IL2 is an anti-inflammatory cytokine, and it induces ITK which dephosphorylates the transcription factor that leads to the immune response, including the release of proinflammatory cytokines and TNF. IL2 may be a novel therapeutic that reduces the release of proinflammatory cytokines, particularly IL17 from Th17 cells, and by stimulating T regulator cells that control inflammation [62].

Cocaine addiction is in cluster 12. While cocaine is obviously not a practical therapeutic agent, the involved pathways reveal some interesting molecular interactions.

In contrast to the hippocampus, the NAc of depressed patients is hypertrophic and has increased plasticity, which seems to be reversed by ketamine [63]. Stress increases VTA mesolimbic signaling which was treated with fluoxetine [64], thus BDNF release in the NAc is also increased and explains the increased plasticity. Ketamine reduces proBDNF – the precursor to BDNF – levels in the NAc (in contrast to the hippocampus, where it is increased) [36], which may explain ketamine’s therapeutic mechanism in the NAc. IKK levels were increased in the NAc, which mediated increased plasticity by increasing spine formation [66]. Depressive symptoms seem to be a result of increased plasticity due to BDNF-TrkB signaling rather than mesolimbic dopamine signaling in and of itself [65].

Exposure to chronic social stress resulted in increased cocaine self-administration in rats [120, 121]. Social defeat that caused cocaine-seeking behavior also led to increased GluR1 AMPAR subunit levels, indicative of increased plasticity, and the NMDAR antagonist dizocilpine reduced cocaine self-administration after chronic social defeat stress. However, not all of the data reached a statistically significant p-value. From a cognitive-behavioral perspective, reducing maladaptive reward-seeking behavior in response to stress may also contribute to ketamine’s antidepressant effects as an NMDAR antagonist.

From the diagram, activation of mGlur2/3 results in less production of BDNF in the NAc. However with regards to reducing depressive symptoms, activating mGlur2/3 would produce theoretically conflicting effects in glutamatergic synapses. DRD1 inhibition and DRD2 activation would also decrease BDNF production in this context.

Clusters 3 and 10 contain nicotine addiction. This pathway shows another way that GABAergic and glutamatergic neurons are regulated, through nicotinic acetylcholine receptor (nAChR).

A cholinergic hypothesis of depression is not new and supported by many lines of evidence [67]. It was developed when acetylcholinesterase inhibitors were observed to induce depressive symptoms [67, 68].

Consistent with the psychopathogenesis of depression, acute changes in acetylcholine levels due to acute stress can produce adaptive behaviors, but chronic changes can be responsible for maladaptive behaviors [70]. Interestingly, depressive symptoms in mice induced by an acetylcholinesterase inhibitor could even be reversed by nAChR antagonists and fluoxetine, which also increased acetylcholinesterase activity [71]. Also, increased acetylcholine signaling in the hippocampus particularly seems to contribute to depressive symptoms. Alpha-7 receptor agonists resulted in increased glutamate release and induced long-term potentiation in the hippocampus [72]. Clinical studies have shown that transdermal nicotine has antidepressant effects, notably also in older patients with late-life depression which is characterized by poor antidepressant response [73, 74], which is possibly due to the chronic desensitization of alpha-4 beta-2 nAChRs. Bupropion, a drug that supports smoking cessation, also functions as an antidepressant. It is an antagonist of alpha-4 beta-2 nAChRs [75], which possibly contributes to its antidepressant effects by reducing GABA inhibition of dopaminergic neurons. In postmortem subjects, beta-2 subunits were significantly less available in depressed subjects – possibly due to higher levels of acetylcholine – with the availability of the receptors significantly correlating with severity of depression [69].

However, a model of alpha-4 beta-2 nAChR stimulation inducing depression completely contradicts the previous NAc plasticity model. Decreasing alpha-4 beta-2 activity, whether through antagonism or desensitization, reduces inhibitory neuron activity and increases dopamine release, which increases NAc plasticity downstream. However, there is evidence that alpha7 nAChR stimulation induces depressive symptoms [76] and cytisine, a selective alpha-4 beta-2 partial agonist, which is consistent with the NAc plasticity model [77]. However, there is contradictory evidence that alpha7 nAChR knockout induced depressive symptoms and increased NAc synaptogenesis and BDNF levels [78]. It is clear that this system is complex and has some involvement in the pathophysiology of depression, but more research is needed before a consistent model can be created. Alpha-7 receptors are also present on macrophages, and when triggered inhibit the release of proinflammatory cytokines [79].

Addictive drugs, including morphine, nicotine, and cocaine decrease GABA inhibition of dopamine [80]. Acute stress also appears to decrease GABA inhibition through the glucocorticoid system, which is consistent with the NAc plasticity model. Annotations involving GABA are also clustered with nicotine addiction.

Consistent with the NAc plasticity model, chronic stress elevates the VTA AMPAR/NMDAR ratio and alpha-7 activation increases susceptibility to social stress [81]. In addition, chronic nicotine decreased subsequence VTA dopamine signaling in response to acute IV nicotine. This supports chronic nicotine patches as a treatment, if this result is due to globally decreased VTA dopamine signaling – which would lead to decreased NAc plasticity downstream. However, chronic nicotine also led to increased social avoidance.

Benzodiazepine receptor activity is also in the same cluster as nicotine addiction, which might also suggest another connection [82].

There are other clusters with consistent themes, which may or may not be involved in the pathophysiology of depression, and they were identified manually.

Clusters 4, 5, 20, and 30 involve calcium signaling.

Cluster 23 involves serotonin.

Cluster 25 involves the immune system.

Cluster 26 involves TGF beta.

Cluster 36 involves telomere regulation. Depressed individuals appear to have shorter telomere length [83], and telomere length predicts SSRI response [84, 85].

Cluster 37 involves guanine nucleotide exchange factor.

Cluster 41 involves the extracellular matrix.

Cluster 45 involves DNA regulation.

Set Intersection

Every gene from the unfiltered PPI network from the end of part 2.II was put into DAVID and annotated with Gene Ontology terms. A python script was used to find certain keywords and genes associated with those keywords. In the table below, genes are grouped into sets based on which theory of depression they fall under, with the keyword used in the second row.

Using UpSetR, the intersection of the sets was plotted, and intersections were ordered by increasing intersection degree. Genes belonging to multiple sets are functionally linked to multiple theories of depression and are the most interesting.

Monoamine Hypothesis Neuroplasticity Immune SystemNeurotrophin Behavorial Responses

Glucocorticoid DNA Cholinergic

Serotonin DopamineNorepinephrine Glutamate GABA Plasticity Dendri Potentiation Depression

Neurogenesis

Neuron death Immun Cytokine Inflam T cell T cell [cont.] B cell neurotro fear

response to stress

social behavior

Glucocorticoid Riboso Telomere Chromatin DNA DNA [cont.] DNA [cont.] DNA [cont.] DNA [cont.]

Transcription

Transcription [cont.]

Transcription [cont.]

Transcription [cont.]

Transcription [cont.]

Transcription [cont.]

Acetylcholine Nicotine

HTR1A HTR1APPARGC1A HTR2A KRAS ATP2B2 ABL1 BRAF SHANK2 BCL6 ABL1 ABL1 APC NT5E PDPK1 ITGB1 ABL1 PDPK1 HTR1A ATP2B4 GNG8 ARID1A DDX3X DCLRE1C HIST1H2AL PDPK1 SMAD2 DPF2 MED21 PTPN11 PDPK1 RRAGC CSTF3 IRF3 PAX5 RPS2 HTR3A BAD

HTR2A HTR2A ADRB1 NAPA GABRA1 HRAS ARF4 SHANK2 GRIA1 COPS2 AKT1 ABL2 ACTR2 AKT1 HTR2A ITGB3 BCL10 RAF1 BCL2 BCLAF1 HRAS BAD HBS1L POLD1HIST1H2AM XRN2 SMAD3 DPF3 MED22 RAC1 XRN2 ARHGEF2 CHUK IL1B PARK2 RPS21 GNAI2 BCL2

ATP2B2 ABL1 CRH NAPB GABRB2 KRAS ARID1B CREB1 PLK2 ETV6 BAD BCL10 ACTR3 ABCF1 ABL1 IFNAR1 BCL6 SOS1 ADRB1 ERRFI1 KRAS BCL2 KPNB1 POLD3 HIST1H3A NT5E SMAD4 DNM2 MED23 RGS12 ABL1 RUVBL1 CUL2 IL1RAP PRDX5 RPS23 GNB1 GNAT3

CRH GNAI3 EPAS1PPARGC1A GABRG2 CAMK1 BTBD3 CRH PICK1 ARHGEF2 BAG5 BCL6 BCL10 BCL6 AKT1 IL1B BAD SRC CRH RBM4 SHANK1 FAS MDN1 H3F3A HIST1H3B ABL1 SMAD9 EGLN1 MED24 RGS14 ARF4 RUVBL2 CUL4A IRAK1 PPARA RPS25 CACNA1A RELA

GRIN2A GNAO1 PARK2 SHANK1 PRKCE CAMK2A DCC GRIN2A PTK2B SNW1 CBL BCL2 BCL6 CD40 AKT2 ITCH BCL2 BDNF GABRA5 AHSA1 SHANK2 KRAS PELO H3F3B HIST1H3C AKT1 SNW1 EPAS1 MED28 RCAN1 AKT1 S100A1 CUL4B IL33 PPARG RPS26 PLA2G4A CREB1

GPM6B GNB1 STX1A SHANK2 RAC1 CAMK2B FYN MAPK1 STXBP1 SMARCE1 DCC CD247 BAD FAS BRAF KPNA3 CD40 EEF2K GRM7 EGFR ANXA7 STUB1 RPL10L LEMD3 HIST1H3D APC SUPT3H EGFR MED29 RFC3 ARID1A SEH1L CCNA1 ILF2 PICALM RPS27 PLCB1 HDAC2

NOS1 GNG2 SNCA SHC3 RAC3 CDC20 GATA1 NTRK2 YAP1PPARGC1A CD3D BCL2 FOS BCL10 KIF13B DCLRE1C EIF4A3 MEF2C GRM7 DLG4 CRH RPL11 NEK2 HIST1H3E ARID1A SUPT7L EGF MED30 RFC4 ARID1B SIN3A CCNA2 ILF3 PIK3R1 RPS27A RGS10 NTRK1

PDE1B SIN3A CREB1 STXBP1 CDC42 IQGAP1 PLK2 CTNNB1 REL CD3E FER REL BCL6 LAT EP300 NGFR RPS6KB1 HSP90AA1 GRIN1 HSPD1 RPL12 NSMCE2 HIST1H3F ARID1B SRC EREG MED4 RFC5 ARID2 SKI CCNC ITCH PIK3R2 RPS28 RGS8 NFKB1

SLC18A1 WNT1 CACNG2 CDK5 NCK2 PTK2B GRIN2A TRAF2 CD40 FKBP1A RELA BAD MTOR FAS NGF UCN HSP90AB1 MTOR HMGB1 RPL14 RAD50 HIST1H3G ARID2 SS18 ERBB2 MED6 RPA1 ATP2B4 SMAD2 CCND1 KAT2A PLCB1 RPS3 PPARA

SLC6A4 ARRB2 CACNA1A DLG4 SHANK1 RGS14 KDM1A CREB1 DDX3X FOS RICTOR BCL2 MED1 SH2B2 NTRK1 HSP90B1 PPP3CB NEDD4 RPL23 SRC HIST1H3H BTAF1 STUB1 ERBB4 MDC1 RET AGAP2 SMAD3 CCNE1 KAT8 PLK1 RPS3A TNF

SNCA CTNNB1 CPLX1 DBN1 TRAF6 SNAP25 NOS1 CTNNB1 DCLRE1C GNAO1 SEH1L CD247 MAPK1 SOS1 NTRK2 MTOR SLC6A4 NR3C1 RPL23A UPF1 HIST1H3I BCL10 SENP2 ESR1 MTA2 RARA BTAF1 SMAD4 CDK1 KDM1A PCBP1 RPS4X

CRH CRH GRIN1 ABI1 SNCA RGS14 CHGA FAU IQGAP1 SHARPIN CD3D MAPK14 TIRAP PTPN11 MAPK1 UCN PTGES3 RPL24 AURKB HIST1H3J BCL6 SMARCC1 ESR2 MCM10 RARB BCL10 SMAD9 CDK2 KMT2A PBRM1 RPS5

CDK5 CDK5 GRM5 BATF3 VAMP2 SPEN CDK5 FER JUN SMAD3 CD3E MAPK3 AURKB SORT1 MAPK13 PTGS2 RPL26 CTNNB1 HIST1H4A BCL7A SMARCC2 ESRRA MAVS RARG BCL6 SNW1 CDK5 KMT2B KCTD1 RPS6KA1

DNM2 DAPK1 HRH1 CACNA1A DAXX FKBP1A KRAS TIRAP CRKL MAP3K14 CASP8 MAPK8 RPS6KB1 RPL3 CCT2 HIST1H4B BAD SMARCA1 ESRRB MAPK1 RXRA BCL7A SUPT3H CDK8 MATR3 PRPF19 RPS6KA3

FGF20 DLG4 HRH2 CAMK1 EGLN1 FYN NFKBIA CAMK1D CRK MAP3K4 CARD11 MAPK9 TNF RPL34 CCT3 HIST1H4C BCLAF1 SMARCA2 ESRRG MAPK10 RXRB BCLAF1 SUPT7L CDK9 MTOR PRPF6 RPS6KA4

FGF8 EGFR ITPKA CAMK1D GSK3B FAS REL CAMK4 CITED2 MAP3K7 CDKN1A MAPKAPK2 YWHAH RPL35A CCT4 HIST1H4D BCL2 SMARCA4 EEF1A1 MAPK13 RXRG CNOT1 SRC CDKN1A MED1 PGR RPS6KA6

FLOT1 GRIA1 KMT2A CAMK2A IKBKG HRAS RELA CTNNBIP1 DDX39B MEF2C HSPD1 MAPKAPK3 UBE2L3 RPL38 CCT5 HIST1H4E CNOT1 SMARCA5 EIF2AK2 MAPK14 RPL6 CD3D SS18 CDKN1B MED10 PCNA RPS6

GRIN2A GRIA2 MEF2C CAMK2B LRRK2 NCK2 RACGAP1 CHUK EP300 NEDD4 HIF1A MEF2C UCN RPL5 CCT6A HIST1H4F CKS1B SMARCB1 FGF2 MAPK3 RPS27A SMARCD3 SENP1 CDKN1C MED11 PSMC2 RPS7

GSK3B GRIA3 NOLC1 CAMK4 LRRK2 ORAI1 ROCK2 CRH FER NFKB1 IRS2 MYH6 RPL6 CCT7 HIST1H4H CKS2 SMARCD2 FGF23 MAPK8 RPS3 CD40 SENP2 DAPK3 MED12L PSMC3 RPS8

HDAC2 GRIA4 PLK2 CDC20 MTOR OTUB1 SEH1L CDK19 FKBP1A NR1D1 ITGB1 MYH7 RPL7 CCT8 HIST1H4I CDKN2AIP SMARCD3 FGFR1 MAPK9 RPS6KA1 CKS1B SMARCC1 DAXX MED12 PSMC5 RPS9

HIF1A GRIN1 PTGS2 CDC42 MAP3K5 REL SH2B2 EGFR FKBP1B NR2F2 MAPK1 NPPA RPL7A FEN1 HIST1H4J COPS2 SMARCE1 FGFR4 MAP2K1 RPS6KA3 CKS2 SMARCC2 DIDO1 MED13L PSMC6 RPSA

LRRK2 GRIN2A PPP3CB CFL1 PARK2 ARHGEF2 SRC ESR1 FYN NUP153 MEF2C NOS1 RPS10 HNRNPC HIST1H4K COPS3SMARCAD1 FZR1 MAP2K2 RPS6KA4 CDKN2AIP SMARCA1 DRG1 MED13 PIAS1 RPLP0

NPR1 GRIN2B RAC1 CDK5 PPARA SIN3A TRAF2 EIF2AK2 FAS PAK1 NTRK1 PRKAA2 RPS14 HNRNPU HIST1H4L COPS4 SYF2 FEN1 MAP2K6 RPS6KA6 COPS2 SMARCA2 DLG1 MED14 PIAS2 RPLP1

PARK2 GRM5 RGS14 DLG4 PICALM SMAD3 TRAF6 GBP5 HRAS PAK2 NFATC2 PPP3CA RPS15 HIST1H3A HIST2H2AB COPS5 SAP130 FOXO3 MAP3K7 RNF2 COPS3 SMARCA4 DPF2 MED15 PIAS3 RPLP2

PDE1B GRM7 RARA DISC1 RRAS2 SRC ANLN HSPD1 LIMS1 PAX2 PIK3CD PTK2B RPS16 HIST1H3B HIST2H3A COPS6 SUPV3L1 GTF3C2 MAPKAPK2 RBX1 COPS4 SMARCA5 DPF3 MED16 PIAS4 RNF2

PTGS2 HOMER1 SYNGAP1 DBN1 STAT3 TIRAP AURKA HGF MAD1L1 PARVA PIK3R1 RGS14 RPS17 HIST1H3C HIST2H3C COPS7A TARDBP GRIP1 MORF4L1 RUNX1 COPS5 SMARCB1 DNM2 MED17 PRKAA1 RNF31

RAC1 HOMER2 SNCA EEF2K SLC9A1 TRAF2 AURKB HMGB1 NCK1 PPIA PLCG2 RPS6KA1 RPS18 HIST1H3D HIST2H3D COPS7B TAF15 GRB2 MEF2A RUNX2 COPS6 SMARCD1 EGLN1 MED18 PRKAA2 RBX1

RGS8 HOMER3 YWHAH EIF4G2 SNCA TRAF6 CSK HRH1 NCK2 PPARG PRKCB RPS6KA3 RPS19 HIST1H3E HIST2H4A COPS8 THOC5 HSPA8 MEF2C SCML2 COPS7A SMARCD2 EPAS1 MED19 PRKCB RUNX1

SLC6A4 LRRK2 YWHAG GRIN1 UBB CSK CAMK4 HIF1A NKAP PIK3CA PRKCD STIP1 RPS27 HIST1H3F HIST2H4B CREBBP TRAF2 HLTF MEF2D STAT1 COPS7B SMARCD3 EGFR MED20 PKN2 RUNX2

SNCA MEF2C UNC13A GRIP1 UCN CACNA1C CALM1 IKBKB NFKBIA PIK3CB PRKDC UCN2 RPS28 HIST1H3G HIST4H4 CRTC2 TRAF5 HELLS NELFB STAT3 COPS8 SMARCE1 EGF MED21 PRKDC SRRM1

SNCG NTRK1 UNC13B HMGB1 CAMK4 CALM2 IKBKG NIPBL PIK3CD PTK2B RPS5 HIST1H3H HDAC1 CBL TRAF6 HGF NLK STAT5A CREBBPSMARCAD1 EREG MED22 PPP2CA SRSF7

NTRK2 HDAC2 CAPZA1 CALM3 IL1BPPARGC1A PIK3R1 PTPN6 RPS6 HIST1H3I HDAC2 CITED2 TATDN1 HNF4A NEDD4 STAT6 CRTC2 SAP130 ERBB2 MED23 PPP2R1A SCML2

NR1H4 ITPKA CARD11 CARD11 IL1RAP RBCK1 PIK3R2 STAT5B RPS7 HIST1H3J HDAC3 DBF4B UPF1 HNRNPK NBN SUMO1 CRK TARDBP ERBB4 MED24 PPP3CA STAT1

PARK2 ILK CTNNBL1 CAV1 IL1RL1 RORC PLCB1 TLR4 RPSA HIST1H4A LRWD1 DDX17 WWP1 HNRNPU NOS1 SPDYA CBL TAF15 ESR1 MED25 PPP3CB STAT3

PLCB1 ITGB1 CHGA CDC25B IL1R1 RELA PLCG1 TCF3 RPLP0 HIST1H4B KAT2A DDX3X WWP2 HMGB1 NONO SPEN CITED2 TAB1 ESR2 MED27 PPP3R1 STAT5A

PCNA IL1RAPL1 C2 CDC42 IL1R2 RICTOR PLCG2 HIST1H4C KDM1A DDX5 WASL HIRA NOTCH4 SFPQ DDX17 TAB2 ESRRA MED28 RIPK1 STAT5B

PTGS2 IL1RAP CHUK CHGA IL33 SKP1 PAG1 HIST1H4D KMT2B DDX39A WNT1 HIST1H3A NFIC SRA1 DDX3X TAB3 ESRRB MED29 RGS12 STAT6

PTK2B LRRK2 CRHR1 CSF3R ITCH SH2B2 KCNN4 HIST1H4E MED25 DDX39B XAB2 HIST1H3B NFKB1 STOML2 DDX41 THOC5 ESRRG MED30 RGS14 SNRPD3

SLC1A2 MTOR DLG1 CDK9 LAT SMAD3 PTGS2 HIST1H4F MTA2 DMAP1 YEATS2 HIST1H3C NFKB2 SFN DDX5 TIAL1 EEF1A1 MED31 RCAN1 SNRPE

SNAP25 MAP1B EREG DAPK3 MAPK13 SMAD4 PSMC2 HIST1H4H MORF4L1 POLD1 YAP1 HIST1H3D NFATC1 SMC1A DDX21 TIRAP EIF2AK2 MED4 RPTOR SNRPG

SYT1 MAPT EIF2AK2 DIAPH2 MAPK14 SOS1 PSMC3 HIST1H4I NCOR1 POLD3 ZFP42 HIST1H3E NFATC2 SMC3 DDX39A TRAF2 EIF4A3 MED6 RFC3 SNRPB

STX1A MEF2A GBP5 DLG1 MAPKAPK2 SRC PSMC5 HIST1H4J NR3C1 DNAJA1 ZPR1 HIST1H3F NCOA1 SMCHD1 DDX39B TRAF5 EXOSC10 MED7 RFC4 SUMO1

STXBP1 MEF2C HSPD1 EREG MYD88 SENP2 PSMC6 HIST1H4K PLK1 EP300 ACTL6A HIST1H3G NCOA2 SMN1 DMAP1 TRAF6 FGF1 MED8 RFC5 SUMO2

SNCA NEDD4 HMGB1 EIF2AK2 NOV TAB2 PSMD1 HIST1H4L PBRM1 ETS2 ACTL6B HIST1H3H NCOA3 SMN2 POLD1 TRADD FGF2 MTA2 RPA1 SLC9A1

UNC13A NEDD4L HRH2 ESPL1 NGFR THOC5 PSMD11 HIST2H4A PPP3CA FBXW7 ATF1 HIST1H3I NCOA4 TTC5 POLD3 WWP1 FGF23 MAVS RET SPEN

UNC13B NFKB2 IKBKB FLOT1 NOS2 TRAF2 PSMD12 HIST2H4B TDG FANCI ATF2 HIST1H3J NCOR2 TXNIP EP300 WWP2 FGF9 MAPK1 RARA SFPQ

UCN PAK2 IKBKG GBP5 NFKB1 TRAF6 PSMD13 HIST3H3 TRIM28 FOS ATF7 HIST1H4A NRIP1 TXN ETS2 WASL FGFR1 MAPK10 RARB SRA1

VAMP2 PAK4 IGF1R HSPD1 NFKB2 THY1 PSMD2 HIST4H4 TP53 GPS1 ADNP HIST1H4B NR1D1 TDG ETV6 WNT1 FGFR2 MAPK13 RARG SMN1

PARK2 ITGB1 HFE NR1H3 WDFY2 PSMD3 MAPK1 TP63 GABPB1 AR HIST1H4C NR1H2 TMPO FAU XAB2 FOXO3 MAPK14 RXRA SMN2

PICALM IL1B IRF3 NR1H4 WWP1 PIAS1 MAPK3 MOS GABPB2 ASF1A HIST1H4D NR1H3 THRAP3 FER YEATS2 GTF3C2 MAPK3 RXRB SNCA

PAFAH1B1 IL1RAP IL1B PRDX5 WWP2 PRKDC MAP2K7 MYC GATA1 ARRB2 HIST1H4E NR1H4 TRIP4 FOS YAP1 GTF2I MAPK8 RXRG TTC5

PLK2 IRAK1 IL1RAPL2 PPARA WNT1 PPP3CA MAP3K4 GATAD2A AURKA HIST1H4F NR1I2 TOP2B GPS1 ZFP42 GRIN1 MAPK9 RPL10A TXNIP

PICK1 IRAK4 IRAK1 PPARG ARPC2 PPP3CB MAPKAPK5 H3F3A AXIN1 HIST1H4H NR1I3 TCF3 GABPB1 ZPR1 GRIP1 MAP2K1 RPL11 TXN

PPP3CA IL1RL1 IRAK4 PIK3CD ACTN4 PTK2 NBN H3F3B BZW1 HIST1H4I NR2F2 TCF4 GABPB2 ACTL6A GSK3B MAP2K2 RPL12 TDG

RAC1 IL1R1 IL1R2 PIK3AP1 ATF2 PTPN11 PCNA HRAS BPTF HIST1H4J NR3C1 TCF7L2 GATA1 ACTL6B HSPA1A MAP2K6 RPL13 TMPO

SYNGAP1 IL1R2 KIF4A PARP4 ADRB1 PTPN6 PTGES3 JUN BAZ1A HIST1H4K NR5A1 TBL1XR1 GATAD2A ACTN1 HSPA1B MAP3K7 RPL13A THRAP3

SYT1 IL33 MAPK14 PTGER1 AXIN1 RAC1 PRKDC KHDRBS1 BAZ1B HIST1H4L NR5A2 TBL1X HRAS ACTN2 HSPA5 MORF4L1 RPL14 TRIP13

SYT2 ILF2 MAP3K7 PTGS2 CSK RAC2 RFC3 LIMS1 BRD8 HIST2H3A NACC1 TGFB1I1 JUN ACTN4 HSPA8 MYD88 RPL15 TRIP4

TANC1 ITCH MAPKAPK2 PRKCD CREB1 RAC3 RFC4 MDM2 CREB1 HIST2H3C ORC2 TRIM28 KHDRBS1 ATF1 HSPH1 MEF2A RPL17 TLR3

TLR3 LAT MAPKAPK3 RAC1 CACNB3 RIPK1 RFC5 MDM4 CREB5 HIST2H3D ORC4 TRIM33 KRAS ATF2 HLTF MEF2C RPL18 TLR4

TLR4 MATK MYD88 RPS19 CACNA1F RGS2 RPA1 MECOM CREM HIST2H4A ORC5 TNF LIMS1 ATF7 HELLS MEF2D RPL18A TRAK2

YWHAH MAVS NOS2 RPS6KA4 CAMK4 RET TCP1 MLXIPL CDH1 HIST2H4B PAX2 TP53BP1 MDM2 ADNP HGF NPPA RPL19 TCF12

UNC13A MAP3K14 NFKB2 STAT5B CASP8 RPL22 MYC MYBL1 CAMK4 HIST4H4 PAX5 TP53 MDM4 AR HNF4A NELFB RPL21 TCF3

MAP3K5 NFATC2 TBXA2R CARD11 RPS27A MYB CAMKK2 HDAC1 PARK2 TP63 MECOM ASF1AHNRNPA2B1 NLK RPL22 TCF4

MYD88 PIK3CD TLR3 CTNNB1 RPS6 MYSM1 CALM1 HDAC2 PPIA TP73 MET ARRB2 HNRNPK NGFR RPL23 TCF7L2

MEF2C PAFAH1B1 TLR4 CAV1 RPSA NKAP CALM2 HDAC3 PPARA YWHAB MLXIPL AURKB HMGB1 NEDD4 RPL23A TBL1XR1

NGFR PLK1 TRPV4 CDC42 RNF31 NFKBIA CALM3 HIF1A PPARG YWHAH MYBL1 AXIN1 HIRA NEDD4L RPL24 TBL1X

NEDD4 PRKCE TNF CBY1 RUNX2 NFKBIB CASP2 IKBKB PICALM YWHAQ MYBL2 BATF3 HIST1H1B NTRK1 RPL26 TGFB1I1

NPY PKN2 UCN CHGA SPPL3 NEK2 CTNNB1 IKBKG PIK3R1 UBA52 MYB BZW1 HIST1H4A NOS1 RPL27 TRPV4

NOS2 PPP3CB CHUK STAT1 NIPBL CTNNBIP1 IGF1R PLCB1 UBB MYSM1 BPTF HIST1H4B NONO RPL27A TRIM28

NFKB1 RARA CUL1 STAT5B OTUB1 CTNND1 INSR PEA15 UBC NCK1 BAZ1A HIST1H4C NOTCH4 RPL29 TRIM33

NFKB2 RPS6KA4 CCNB2 SORT1 PHF10 CDC42 INTS3 PDGFRA UCHL5 NCK2 BAZ1B HIST1H4D NCBP1 RPL3 TNF

NR1H4 STAT1 CCND1 SART1 PHF5A CDC5L ILK PHLDA3 UBE2D3 NKAP BRD8 HIST1H4E NCBP2 RPL30 TP53BP1

PAX5 STAT3 CCND3 STOML2 PITHD1 CENPJ IFNAR1 PLK1 UBE2E2 NFKBIA CREB1 HIST1H4F NFIC RPL31 TP53

PPARG SPTBN1 CDK6 SNAP23PPARGC1A CHEK1 IRF3 PLK2 UBE2I NFKBIB CREB5 HIST1H4H NFKB1 RPL32 TP63

PIK3CD TLR3 DLG1 STXBP2 RAD18 CHEK2 IL1B PARP4 UBE2L3 NIPBL CREM HIST1H4I NFKB2 RPL34 TP73

PAG1 TLR4 DNM2 TCP1 RAD50 CBY1 IRAK1 PBRM1 UBE2N PHF10 CDH1 HIST1H4J NFATC1 RPL35 YWHAB

PCBP2 TNF ERBB2 TCF7L2 RAD54B CHD1L IL33 KCTD1 UBE2V1 PHF5A CARF HIST1H4K NFATC2 RPL35A YWHAH

KCNN4 USP8 ERBB4 TBL1XR1 RALY CHD8 ILF2 PRPF19 UBE2V2PPARGC1A CAMK1 HIST1H4L NCOA1 RPL36 YWHAQ

PRKCB EXOC2 TRPV4 RORC CHD9 ILF3 PGR USP34 RAD54B CAMK1D HIST2H3A NCOA2 RPL37A UBA52

PRKCD EXOC7 TRIM28 RBBP5 CUL4A KPNA2 PCNA UCN RALY CAMK2A HIST2H3C NCOA3 RPL38 UBB

PRKCE FERMT2 TNF RBBP7 CUL4B KPNB1 PTGES3 MYC RANBP2 CAMK2D HIST2H3D NCOA4 RPL39 UBC

PRKDC FN1 UBA52 RB1 CCNA2 LRWD1 PSMC5 VHL RBCK1 CAMK4 HIST2H4A NCOR1 RPL4 UCHL5

PTK2B FLOT2 UBB RBL2 CCNB1 KAT2A PIAS1 ZNF114 RORC CAMKK2 HIST2H4B NCOR2 RPL5 UBE2D1

PTK2 GRIP1 UBC RELA CCND1 KAT8 PIAS2 ZNF511 RBBP5 CARD11 HIST4H4 NRIP1 RPL6 UBE2D2

PTPN6 GSK3B UBE2D1 RCOR1 CCNE1 KDM1A PIAS3 ZNF526 RBBP7 CTNNB1 HDAC1 NR1D1 RPL7 UBE2D3

RPL39 GRB2 UBE2D2 RBMS1 CCNE2 KMT2A PIAS4 ZNF638 RB1 CTNNBIP1 HDAC2 NR1H2 RPL7A UBE2I

RPS27A HSP90AB1 UBE2N RNPS1 CDK1 KMT2B PICK1 ZNF653 RBL2 CTNND1 HDAC3 NR1H3 RPL8 UBE2L3

RPS6 HSPA1A UBE2V1 POLR1C CDK2 MTOR PRKAA1 REL CAV1 HIF1A NR1H4 RPL9 UBE2N

SELL HSPA1B MYC POLR1E CDK3 MED1 PRKAA2 RELA CDC37 IPO9 NR1I2 RPS10 UBE2V1

STAT6 HSPD1 VAMP2 POLR2A CDK5 MED11 PRKCB RCOR1 CDC5L IVNS1ABP NR1I3 RPS11 UBE3A

TLR3 HSPH1 POLR2E CDK9 MED12L PRKCD RBMX CDC73 IKBKB NR2F2 RPS12 USP34

TLR4 HFE POLR2F CDKN1A MED12 PKN2 RNPS1 CHEK1 IKBKG NR3C1 RPS13 USP9X

TCF12 HMGB1 RRAGC CDKN1B MED13L PRKDC POLR1C CHEK2 INSR NR5A1 RPS14 UCN

TCF3 HIF1A RUVBL1 CDKN1C MED13 PPP2CA POLR1E CBY1 INTS1 NR5A2 RPS15 MYC

TRIM28 IKBKB RUVBL2 DDB2 MED14 PPP2R5C POLR2A CHD8 INTS3 NUP153 RPS15A VHL

TNF IKBKG SH2B1 DAPK3 MED16 PPP2R1A POLR2E CHD9 INTS5 NUP50 RPS16 ZNF114

UBA52 IDE SHC1 DAXX MED17 PPP3CA POLR2F CPSF2 INTS6 NACC1 RPS17 ZNF511

UBB INSR SIN3A DIDO1 MED19 PPP3CB RWDD3 CPSF7 ILK ORC2 RPS18 ZNF526

UBC ILK SKI DRG1 MED20 PTK2B RAF1 CSTF1 IFNAR1 PAX2 RPS19 ZNF638

ZNF653

Endocrine

Corticotro Thyroid Endocrin

CRHR1 BRAF AKT1

CRHR2 CRKL KRAS

CRH GATA1

FGF2PPARGC1A

FGF8 RAF1

UCN SMAD3

FGF2

FGF23

FGF8

FGFR1

MED1

MED13

MAPK1

MAPK3

MAP2K1

MEF2C

NCOA1

RPLP0

Table 3

For brevity, an overview will only be done on the genes with evidence supporting a connection to depression from the 6, 5, and 4-degree intersections.

Urocortin (UCN) is a high-level regulator of the stress response and within the same family as CRH. It promotes anxious behavior and decreases appetite [86], which is a symptom of depression, though its wide-reaching regulatory roles makes it an unlikely practical therapeutic target.

PGC-1 alpha (PPARGC1A) regulates mitochondrial biogenesis and gene expression. Activation of PGC-1 alpha through exercise redirects the kynurenine pathway towards the production of kynurenic acid, which is an NMDA antagonist [87]. Downregulated PGC-1 alpha results in oxidative stress, inflammation, and NFkB activation, and TNF decreases PGC-1 alpha expression [88].

TrkA (NTRK1) is the receptor for nerve growth factor and related to TrkB, the receptor for BDNF. The tricyclic antidepressant amitriptyline is a TrkA/B agonist [89].

MEF2C is a transcription factor. In mice, increased activity results in increased dendritic spine density and complexity in several brain regions, including the hippocampus [90].

In mice, decreasing NAc RAC1 expression induces social defeat behavior and anhedonia, which is reversed by upregulation [91].

ABL1 is a proto-oncogene for gastric cancer. Depression-induced oxidative stress exacerbates the prognosis of cancer patients through ABL1-involved pathways [92].

Beta-catenin (CTNNB1) is a key player in the canonical Wnt pathway, and WNT1 is a ligand initiating the pathway. Beta-catenin promotes transcription of certain genes, and direct nucleic beta-catenin insertion into the NAc promotes stress-resilience and produces antidepressant effects [93]. D2 dopamine receptor activation promotes beta-catenin mediated transcription [94]. In the hippocampus, canonical Wnt signaling has a positive role in neurogenesis [95]. Glutamate activation of NMDARs results in calpain-dependent cleavage of beta-catenin in the hippocampus [96], and cleaved beta-catenin resulted in greater transcription activity, which is paradoxical considering ketamine’s antidepressant effects and mechanism as an NMDAR antagonist. However, the study was conducted with a focus on cancer rather than on neuronal cells, and

Genes belonging to 6 groups

UCN KRAS CRH

Genes belonging to 5 groups

PPARGC1A NTRK1 MAPK1 MEF2C BAD BCL2

Genes belonging to 4 groups

PTGS2 RAC1 ABL1 CTNNB1 GSK3B NOS1 HDAC2 PTK2B PPP3CB PPP3CA EGFR MTOR HRAS NEDD4 HMGB1 PPARA PLCB1 CREB1 AKT1 TNF

Genes

belonging to

3 groups

HTR2A CDK5 SNCA PARK2 ADRB1 WNT1 DNM2 HIF1A SIN3A RARA UBB TRAF6 NFKB2 ARHGEF2 CAMK4 CAMK1D NR1H4 TRAF2 TLR4 ILK

REL GRIP1 IKBKG STAT3 CDC42 NCK2 IL1RAP BCL6 TLR3 BRAF GRIN1 RGS14 YWHAH GATA1 PTPN11 SRC NGFR PDPK1 MAPKAPK2 MAPK13

RELA NFKB1 SMAD3 MED1 MAPK3

extranuclear interactions of beta-catenin may also be present [97]. Overall, it appears that increasing beta-catenin signaling produces antidepressant effects, and Wnt ligands may produce therapeutic effects.

GSK-3b is also involved in canonical Wnt signaling. GSK-3b ubiquitinates beta-catenin for proteolysis, and cleaved beta-catenin is resistant to this degradation [96]. Consistent with the hypothesis of beta-catenin having therapeutic effects, overactive GSK-3b is associated with depressive symptoms [98]. Also, GSK-3b mediates maladaptive NAc plasticity [99], and knockdown prevents depressive symptoms. GSK-3b is also inhibited lithium, and ketamine, which is necessary for its antidepressant effects [18]. Akt signaling leads to mTOR-mediated inhibition of GSK-3b, which may explain the role of mTORC1 in the pathophysiology of depression. GSK-3b inhibitors may be a novel antidepressant.

NOS1 codes for nitric oxide synthase 1. NO modulates norepinephrine, serotonin, dopamine, and glutamate, and inhibition of NOS prevented neurotoxicity resulting from NMDA agonism [100]. Many conventional and atypical antidepressants interact with NO pathways.

HDAC2 codes for histone deacetylase 2. Depression is associated with increased acetylation of histone H3 and decreased HDAC2 expression in the NAc, and HDAC2 also mediates hippocampal plasticity [101]. Direct insertion of HDAC has antidepressant effects and reverses the effects of chronic defeat stress. Interestingly, the study also implicates genes involved in the effects of cocaine, as previously enriched.

Patients with lung cancer with EGFR mutations had higher TNF-alpha levels, which is normally associated with depression, but paradoxically tended to have lower depression severity [102].

Maternal immune activation during pregnancy leads to inflammation, which results in decreased NEDD4 activity that leads to decreased neurogenesis and synapse formation and is a risk factor for depression [103].

HMGB1 is a chromatin protein that aids in transcription. Upregulated HMGB1 is associated with increased presence and activity of kynurenine pathway enzymes [104].

PPAR-alpha (PPARA) is a transcription factor. PEA, an endogenous PPAR-alpha agonist, has antidepressant effects [105, 106]. PPARs may also be involved in the endocannabinoid system [107].

Conclusion

The purpose of this was to identify novel systems underlying the pathophysiology of depression as research directions and potential therapeutic targets. First, starting representative genes were selected from the major current paradigms of depression: the serotonin hypothesis (and kynurenine pathway), glutamate hypothesis, neuroplasticity, inflammation, the HPA axis, and the stress response. Using publicly available microarray datasets on Allen Brain Atlas, gene

correlates were identified and filtered for genes related to depression using DAVID to make a gene candidate list. Creating a protein interaction network from the starting genes, subnetworks originating from the glucocorticoid receptor coded by NR3C1 were identified based on high interconnectivity. These subnetworks had themes of histones, the COP9 signalosome, SWI/SNF chromatin remodeling complexes, the estrogen receptor, and ribosomes. A drug-protein interaction network was created to identify potential therapeutic agents for further investigation and to find interactions between currently established antidepressants with candidate genes. Ketamine was at the center of the network and connected to many of the candidate genes, thereby validating this methodology. Creating a protein interaction network with the candidate genes and organizing for highly interconnected subclusters, SHANK2, GRIA2/3/4, MAPK & MAPKK genes, CALM & CAMK genes, KCJN6, CACN genes, NFKB genes, and SLC1A2 were identified as genes of interest. The candidate genes and the genes in interesting clusters identified from the network were enriched with DAVID. The most notable enrichment clusters had themes of GABAergic regulation, endocannabinoid signaling, opioid signaling, cholinergic signaling, and T-cell and antibody signaling. Potential pathogenesis and therapeutic mechanisms involving these systems were proposed based on KEGG pathway illustrations and molecular interactions. Using Gene Ontology annotations, every gene from the candidate protein interaction network was classified into groups based on current theories of depression. Genes belonging to multiple groups are highly interesting and were identified. These genes may be the subject of future research and novel paradigms.

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Conflict of Interest

The author declares no conflict of interest.