3.4 Analyses DE genes, canonical pathways, and upstream regulators
The data files after differential expression analyses have been deposited into NCBI’s Gene Expression Omnibus [67] and are accessible through GEO Series accession number GSE169296 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE169296). All DE genes due to stress identified in the nine examined brain subregions (ARC, VMH and PVN of the hypothalamus; CEA and BLA of the amygdala; CA1, CA3 and DG of the hippocampus; and OFC of the prefrontal cortex) are listed in Supplementary Table 1. Comparisons between female and male nonstress vs. stress canonical pathways and upstream regulators made using IPA are listed in Supplementary Tables 2 and 3, respectively.
3.4.1 Hypothalamus
ARC
Similar numbers of DE genes due to adolescent stress were revealed in the ARC of females (111 DE genes) and males (107 DE genes), with 14 common DE genes for both sexes (Fig. 4A; Suppl Tables 1.1 and 1.2). Among these common DE genes between males and females, Akap11_3, Atp6v0a1_2, Kcnma1_4, Lnpep_1, LOC108348175_4, and Wnk1_1 were commonly downregulated; Ank2_8, Avp_1, Birc6_1, and Zfhx4_1 were commonly upregulated; Ank2_10, Dock10_9, and Ece1_1 were downregulated in females but upregulated in males; while Robo1_2 was upregulated in females but downregulated in males (Fig. 4A).
Enriched canonical pathways in the ARC in females were “acetyl-CoA biosynthesis III from citrate”, “insulin secretion signaling”, “protein kinase A signaling”, “synaptogenesis signaling”, “tight junction signaling”, “UVB-induced MAPK signaling”, “xenobiotic metabolism CAR signaling”, and “xenobiotic metabolism PXR signaling”; while in males were “CCR3 signaling in eosinophils”, “CD27 signaling in lymphocytes”, “Cdc42 signaling”, “cellular effects of sildenafil”, “circadian rhythm signaling”, “citrulline biosynthesis”, “endocannabinoid neuronal synapse”, “eNOS signaling”, “γ-aminobutyric acid (GABA) receptor signaling”, “gap junction signaling”, “glutamate degration III”, “glutamate dependent acid resistance”, “gonadotropin-releasing hormone (GnRH) signaling”, “natural killer cell signaling”, “proline biosynthesis I”, “pyridoxal 5’-phosphate salvage”, “SAPK/JNK signaling”, “spermidine biosynthesis I”, and “TR/RXR activation” (Suppl Fig. 1; Suppl Table 2).
Upstream regulators in the ARC identified by IPA were CLOCK, CREB3L1, NHLH2, NR1H2, PER1, and THRB in both females and males; ASB4, CREB1, MYC, NKX2-1, POU3F1, and SIM1 in females only; and 27-hydroxycholesterol, ADCYAP1, AGRP, ARNTL, CARTPT, CNR1, corticosterone, CRH, CRHBP, CRHR1, CRY2, DIO3, DMD, DTNA, ethanol, FAAH, formaldehyde, HDAC4, histamine, HTT, KISS1, LEP, LHX1, LHX5, MAOA, MAPK8, MC4R, MCHR1, NEUROD2, NFKB1, NPY2R, NR1H3, NUCB2, PTHLH, REN, SLC16A2, and testosterone in males only (Suppl Table 3).
VMH
Eighty-four DE genes in females and 70 DE genes in males due to adolescent stress were revealed in the BLA (Fig. 4B; Suppl Tables 1.3 and 1.4). Eight DE genes were common between males and females, including Gad1_1, Gad2_1, and LOC103689999_1 commonly upregulated, Cers1_1, LOC108348096_1, and Myo9b_4 commonly downregulated, Ank2_10 that was downregulated in females but upregulated in males, and Slit1_2 that was upregulated in females but downregulated in males (Fig. 4B).
Enriched canonical pathways in the VMH of both females and males were “glutamate degration III”, “glutamate dependent acid resistance”, and “synaptic long-term depression”; in females only were “calcium signaling”, “GABA receptor signaling”, “neuroinflammation signaling”, and “TR/RXR activation”; while in males only were “aldosterone signaling in epithelial cells”, “antiproliferative role of somatostatin receptor 2”, “apelin cardiomyocyte singling pathway”, “axonal guidance signaling”, “breast cancer regulation by stathmin 1”, “D-myo-inositol(1,4,5)-trisphosphate biosynthesis”, “gap junction signaling”, “glycerol degradation I”, “glycerol-3-phosphate shuttle”, “G protein coupled receptor (GPCR)-mediated integration of enteroendocrine signaling”, “HIPPO signaling”, “insulin secretion signaling pathway”, “melatonin signaling”, “neuropathic pain signaling in dorsal horn neurons”, “neuroprotective role of THOP1 in Alzheimer’s Disease”, “phagosome formation, PPARα/RXRα activation”, “protein ubiquitination”, “role of NFAT in cardiac hypertrophy”, “UVA-induced MAPK signaling”, “Wnt/Ca ion”, and “α-adrenergic signaling” (Suppl Fig. 2; Suppl Table 2).
Upstream regulators in the VMH identified by IPA were CREB1, LHX 1, and LHX5 in both females and males; CLOCK, CREB3L1, ESR1, NFKB1, NR1H2, PER1, PPARA, and SIM1 in females only; and ADCYAP1, AR, ASCL1, ATOH1, ATXN7, β estradiol, CNR1, CPE, CSHL1, D-glucose, FEV, GHR, GHRH, GHRL, HTT, IGF1R, JAK2, levodopa, MAPT, MME, NPY, NTF3, PPARGC1A, TAC1, TNR, and VIP in males only (Suppl Table 3).
PVN
Fifty-seven DE genes in females and 68 DE genes in males due to adolescent stress were revealed in the BLA, with 7 common DE genes for both sexes (Fig. 4C; Suppl Tables 1.5 and 1.6). Among them, Ank2_2 and Ehmt2_4 were upregulated and Fry_3 was downregulated by stress in both females and males; while Mbp_3, Mog_1, Ogt_1, and Slit1_1 were downregulated in females but upregulated in males (Fig. 4C).
Canonical pathways enriched by adolescent stress in females only were “apelin cardiomyocyte signaling”, “calcium signaling”, “calcium-induced T lymphocyte apoptosis”, “cardiac hypertrophy signaling”, “CCR3 signaling in eosinophils”, “CCR5 signaling in macrophages”, “cellular effects of sildenafil”, “chemokine signaling”, “cholecystokinin/gastrin-mediated signaling”, “CRH signaling”, “CREB signaling in neuron”s, “dopamine-DARPP32 feedback in cAMP signaling”, “endocannabinoid neuronal synapse pathway”, “epithelial adherens junction signaling”, “estrogen receptor signaling”, “Fcγ receptor-mediated phagocytosis in macrophages and monocytes”, “gap junction signaling”, “glutamate receptor signaling”, “GnRH signaling”, “GP6 signaling”, “GPCR-mediated nutrient sensing in enteroendocrine cells”, “Gαq signaling”, “IL-15 production”, “insulin secretion signaling pathway”, “maturity onset diabetes of young (MODY) signaling”, “netrin signaling”, “nitric oxide signaling”, “nNOS signaling”, “opioid signaling”, “phospholipase C signaling”, “protein kinase A signaling”, “renin-angiotensin signaling”, “RhoA signaling”, “role of NFAT in cardiac hypertrophy”, “sperm motility”, “synaptic long-term depression”, “thrombin signaling”, and “α-adrenergic signaling”. Canonical pathways enriched by adolescent stress in male but not female PVN were “calcium transport I”, “glycerol degradation I”, and “glycerol-3-phosphate shuttle” (Suppl Fig. 3; Suppl Table 2).
Upstream regulators in the PVN identified by IPA were ASCL1 and FEV in both females and males; 5-hydroxytryptamine, ADCYAP1R1, ARNTL, ATOH1, CYFIP1, FEZF2, FYN, HDAC4, NFKB1, NOS1, PDYN, and SOD1 in females only; and ADCYAP1, AR, ATXN7, β estradiol, CARTPT, CLOCK, CNR1, CPE, CREB1, CREB3L1, CRHBP, CRHR1, CRY2, D-glucose, DICER1, ESR1, formaldehyde, histamine, HTT, JAK2, levodopa, MAOA, MAPT, MYC, NKX2-1, NPY, NR1H2, NUCB2, PER1, POU3F1, PPARA, PTHLH, SIM1, sucrose, TAC1, testosterone, TNR, and VIP in males only (Suppl Table 3).
3.4.2 Amygdala
BLA
Sixty-nine DE genes in females and 45 DE genes in males due to adolescent stress were revealed in the BLA, with 4 common DE genes for both sexes (Fig. 5A; Suppl Tables 1.7 and 1.8). Among them, LOC100360449_1 was upregulated by stress in both females and males; while Caskin1_2 was downregulated in females but upregulated in males, and Dgkz_2 and Sccpdh_1 were upregulated in females but downregulated in males (Fig. 5A).
Canonical pathways significantly enriched in the BLA in females were “kinetochore metaphase signaling” and “spermidine biosynthesis I”; while in males were “calcium signaling”, “DNA damage-induced 14-3-3σ signaling”, and “opioid signaling” (Suppl Fig. 4; Suppl Table 2). Upstream regulators were lysine methyltransferase 2A (KMT2A) and nuclear proto-oncogene (SKI) in males only (Suppl Table 3).
CEA
One hundrend eighty-eight and 108 DE genes due to adolescent stress were identified in the female and male CEA respectively, with 19 common DE genes (Fig. 5B; Suppl Tables 1.9 and 1.10). Among them, Astn2_1 and Prpf8_1 were downregulated by stress in both females and males; Inf2_1, Itpr1_4, Pcp4_1, Pde10a_1, Rasd2_1, and Scn4b_1 were upregulated by stress in both females and males; while B4galt6_1, Dgkz_2, Kit_1, and Smc3_1 were downregulated in females but upregulated in males, and Nptxr_1, Nrxn2_2, Slc17a7_2, Slit1_1, St6gal2_1, Sv2b_2 and Synj1_1 were upregulated in females but downregulated in males (Fig. 5B).
Canonical pathways significantly enriched by adolescent stress identified in the CEA of both females and males were “cAMP-mediated signaling”, “GPCR signaling”, “pregnenolone biosynthesis”, and “synaptic long-term potentiation”; in females only were “calcium-induced T lymphocyte apoptosis”, “cholecystokinin/gastrin-mediated signaling”, “CRH signaling”, “CREB signaling in neurons”, “Gαi signaling”, “opioid signaling”, and “NFAT in cardiac hypertrophy”; while in males only were “acetyl-CoA biosynthesis III from citrate”, “calcium signaling”, “gap junction signaling”, “glioblastoma multiforme signaling”, “histidine degradation VI”, “insulin receptor signaling”, “non-small cell lung cancer signaling”, “synaptogenesis signaling”, and “ubiquinol-10 biosynthesis” (Suppl Fig. 5, Suppl Table 2).
Upstream regulators in the CEA identified by IPA were HDAC4 and NFKB1 in both females and males; ELK1, FEV, KMT2A, MEF2D, SATB2, THRB, and TSHZ3 in females only; and MYC and POU3F1 in males only (Suppl Table 3).
3.4.3 Hippocampus
CA1
Fifty-five DE genes in females and 63 DE genes in males due to adolescent stress were revealed in the CA1, with 10 common DE genes (Fig. 6A; Suppl Tables 1.11 and 1.12). Among them, Kcnip2_3 was downregulated by stress in both females and males; LOC108348096_1 was upregulated by stress in both females and males; while Arpc2_8, Dcaf11_2, LOC103689945_1, Mt-atp8_1, Reln_1, Scn1b_3, and Tbl1x_2 were downregulated in females but upregulated in males, and B4galt6_1 was upregulated in females but downregulated in males (Fig. 6A).
Canonical pathways significantly enriched by adolescent stress in the CA1 in females but not males were “asparagine biosynthesis I”, “DNA double-strand break repair by homologous recombination”, “epithelial adherens junction signaling”, “hepatic fibrosis/hepatic stellate cell activation”, “hypoxia signaling in the cardiovascular system”, and “synaptogenesis signaling”; while in males but not females were “Fcγ receptor-mediated phagocytosis in macrophages and monocytes”, “Gα 12/13 signaling”, “Gαs signaling”, “netrin signaling”, “neuroprotective role of THOP1 in Alzheimer’s disease”, “nNOS signaling”, “protein kinase A signaling”, “RNK signaling in osteoclasts”, “reelin signaling in neurons”, “regulation of actin-based motility by Rho”, “RhoA signaling”, and “Rho family GTPases signaling” (Suppl Fig. 6A; Suppl Table 2).
Upstream regulators in the CEA identified by IPA were ASCL1, EMX2, and TBR1 in both females and males; ARNT2, ATXN1, HIF1A, JUN, NR3C1, and SUB1 in females only; and ATF3, ATOH1, FEV, HDAC5, LMO4, MEF2C, MYC, NFE2L2, and POU3F1 in males only (Suppl Table 3).
CA3
Nighty-nine and 63 DE genes by adolescent stress were identified in female and male CA3 respectively, with 9 common DE genes (Fig. 6B; Suppl Tables 1.13 and 1.14). Among them, Wfs1_1 was downregulated by stress in both females and males; Kcnip2_3 and Rn50_20_0060.5_2 were upregulated by stress in both females and males; while Ehmt2_3, Itpr1_1, NEWGENE_1304700_1, Ranbp2_2, and Ryr3_2 were downregulated in females but upregulated in males, and Slit1_2 was upregulated in females but downregulated in males (Fig. 6B).
Canonical pathways significantly enriched in the CA3 in both females and males were “fMLP signaling in neutrophils” and “insulin secretion signaling”; in females only were “14-3-3-mediated signaling”, “adrenomedullin signaling”, “aldosterone signaling in epithelial cells”, “apelin cardiomyocyte signaling”, “apelin endothelial signaling”, “axonal guidance signaling”, “calcium signaling”, “calcium-induced T lymphocyte apoptosis”, “cardiac hypertrophy signaling”, “CCR3 signaling in eosinophils”, “cellular effects of sildenafil”, “cholecystokinin/gastrin-mediated signaling”, “CRH signaling”, “CREB signaling in neurons”, “CXCR4 signaling”, “dopamine-DARPP32 feedback in cAMP signaling”, “endocannabinoid neuronal synapse”, “endothelin-1 signaling”, “eNOS signaling”, “estrogen receptor signaling”, “gap junction signaling”, “glioblastoma multiforme signaling”, “glutamate receptor signaling”, “GnRH signaling”, “GP6 signaling”, “GPCR-mediated integration of enteroendocrine signaling”, “GPCR-mediated nutrient sensing in enteroendocrine cells”, “growth hormone signaling”, “Gαq signaling”, “leptin signaling”, “melatonin signaling”, “neuropathic pain signaling in dorsal horn neurons”, “opioid signaling”, “P2Y purigenic receptor signaling”, “phagosome formation”, “phospholipase C signaling”, “prostanoid biosynthesis”, “protein kinase A signaling”, “renin-angiotensin signaling”, “NFAT in cardiac hypertrophy”, “sperm motility”, “spermidine biosynthesis I”, “synaptic long-term depression”, “synaptic long-term potentiation”, “synaptogenesis signaling”, and “α-adrenergic signaling”; while in males were “CD28 signaling in T helper cells”, “Cdc42 signaling”, “remodeling of epithelial adherens junctions”, and “TR/RXR activation” (Suppl Fig. 7; Suppl Table 2).
Upstream regulators in the CA3 identified by IPA were NFKB1 in both females and males; ELK1, FEV, HDAC4, KMT2A, MEF2D, SATB2, THRB, and TSHZ3 in females only; and ASCL1 in males only (Suppl Table 3).
DG
Two hundred and five DE genes in females and 52 DE genes in males by adolescent stress were identified in the DG, with 4 common DE genes for both sexes (Fig. 6C; Suppl Tables 1.15 and 1.16). Among them, Atp6v0a1_2 and Dgkz_2 were downregulated by stress in both females and males; Caskin1_2 was upregulated by stress in both females and males; while Ptk2b_1 and Sptan1_2 were downregulated in females but upregulated in males (Fig. 6C).
Canonical pathways significantly enriched by adolescent stress in the DG of both females and males were “nNOS signaling in skeletal muscle cells”, “protein kinase A signaling”, and “T cell receptor signaling”; in females only were “actin cytoskeleton signaling”, “actin nucleation by ARP-WASP complex”, “agranulocyte adhesion and diapedesis”, “agrin interactions at neuromuscular junction”, “atherosclerosis signaling”, “B cell receptor signaling”, “CCR3 signaling in eosinophils”, “CD28 signaling in T helper cells”, “chemokine signaling”, “clathrin-mediated endocytosis signaling”, “ephrin A signaling”, “ERK/MAPK signaling”, “FAK signaling”, “fatty acid activation”, “fatty acid β oxidation I”, “Fcγ receptor-mediated phagocytosis in macrophages and monocytes”, “FXR/RXR activation”, “germ cell-sertoli cell junction signaling”, “glutamate receptor signaling”, “glutamine biosynthesis I”, “GP6 signaling”, “granulocyte adhesion and diapedesis”, “Gαq signaling”, “hepatic cholestasis”, “hepatic fibrosis/hepatic stellate cell activation”, “hepatic fibrosis signaling”, “H1F1α signaling”, “iCOS-iCOSL signaling in T helper cells”, “integrin-linked kinase (ILK) signaling”, “integrin signaling”, “iron homeostasis signaling”, “leukocyte extravasation signaling”, “LPS/IL-1mediated inhibition of RXR function”, “LXR/RXR activation”, “maturity onset diabetes of young (MODY) signaling”, “mitochondrial L-carnitine shuttle”, “natural killer cell signaling”, “oncostatin M signaling”, “onsteoarthritis”, “PAK signaling”, “paxillin signaling”, “PEDF signaling”, “phagosome formation”, “production of nitric oxide and reactive oxygen species in macrophages”, “PTEN signaling”, “Rac signaling”, “RANK signaling in osteoclasts”, “reelin signaling in neurons”, “regulation of actin-based motility by Rho”, “regulation of IL-2 expression in activated and anergic T lymphocytes”, “renal cell carcinoma signaling”, “RhoA signaling”, “RhoGDI signaling”, “osteoblasts, osteoclasts and chondrocytes in rheumatoid arthritis”, “semaphoring neuronal repulsive signaling”, “semaphoring signaling in neurons”, “Rho family GTPases signaling”, “spermidine biosynthesis I”, “sphingosine-1-phosphate signaling”, “STAT3”, “stearate biosynthesis I”, “synaptogenesis signaling pathway”, “Tec kinase signaling”, “tight junction signaling”, “TR/RXR activation”, “VEGF signaling”, and “γ-linolenate biosynthesis”; while in males only were “calcium signaling”, “CCR5 signaling in mancrophages”, “cholecystokinin/gastrin-mediated signaling”, “dilated cardiomyopathy signaling”, “epithelial adherens junction signaling”, “insulin secretion signaling”, “MSP-RON signaling in cancer cells”, “netrin signaling”, “nitric oxide signaling in the cardiovascular system”, “non-small cell lung cancer signaling”, “opioid signaling”, “renin-angiotensin signaling”, “selenocysteine biosynthesis II”, and “type II diabetes mellitus signaling” (Suppl Fig. 8; Suppl Table 2).
Upstream regulators in the DG identified by IPA were NFKB1 in both females and males; ASCL1, ATF3, EMX2, ESR1, HTT, NFE2L2, NFIX, NFKBIA, NR1H3, STAT3, and TBR1 in females only; and ADRB2, CACNA1D, FGF2, FKBP1B, FMR1, ITPR1, LMO4, and RARA in males only (Suppl Table 3).
3.4.4 OFC of prefrontal cortex
Fifty-four DE genes in females and 96 DE genes in males due to adolescent stress were identified in the OFC, with 8 common DE genes in both females and males (Fig. 7; Suppl Tables 1.17 and 1.18). Among them, Ank2_2 and Ryr3_1 were upregulated and Btg2_1, Ece1_1, Gabbr1_1, Ptpn13_2, and Taok3_1 were downregulated by stress in both females and males; while Macf1_4 was upregulated in females but downregulated in males (Fig. 7)
Noteworthy canonical pathways significant in both males and females were “calcium signaling”, “netrin signaling”, “nNOS signaling in skeletal muscle cells”, and “protein kinase A signaling”; in females only were “bladder cancer signaling”, “CCR3 signaling in eosinophils”, “Cdc42 signaling”, “cell cycle: G1/S checkpoint regulation”, “citrulline biosynthesis”, “cyclins and cell cycle regulation”, “hereditary breast cancer signaling”, “HIPPO signaling”, “mismatch repair in eukaryotes”, “non-small cell lung cancer signaling”, “phospholipase C signaling”, “proline biosynthesis I”, “regulation of actin-based motility by Rho”, “renin-angiotensin signaling”, “role of BRCA1 in DNA damage response”, and “superpathway of citrulline metabolism”; and in males only were “alanine biosynthesis III”, “cAMP-mediated signaling”, “cellular effects of sildenafil”, “cholecystokinin/gastrin-mediated signaling”, “endocannabinoid neuronal synapse pathway”, “endothelin-1 signaling”, “ERK/MAPK signaling”, “GPCR signaling”, “GABA receptor signaling”, “molybdenum cofactor biosynthesis”, “neuroinflammation signaling”, “prostanoid biosynthesis”, “RhoA signaling”, “Rho family GTPases signaling”, and “synaptic long-term depression” (Suppl Fig. 9; Suppl Table 2).
Out of all of the brain regions studied, the OFC had the most top upstream regulators (Suppl Table 2). Upstream regulators in the OFC identified by IPA were BCL2L1 and dopamine in both females and males; ACHF, BCKDK, BDNF, BHLHE40, BMP2, CRHR1, CRTC2, CUL3, DMD, DTNA, EIF2AK4, HNRNPR, KLHL17, mannitol, mir-130, mir-320, NEUROD1, NFE2L2, PAX2, SGK1, SNTA1, and TNFAIP3 in females only; and ABCD1, ABCD2, acetic acid, ADAM19, ADCYAP1R1, ADIPOR2, ADORA2A, AFP, ALB, allopregnanolone, ANXA2, APEX1, ARHGAP21, ATF2, B4GALT6, BACH1, BDKRB2, β estradiol, CACNA1C, CALCA, CCL5, CD209, CD28, CD83, CHMP2B, CREB1, CREM, cyclic AMP, DCN, DOCK8, DUSP4, E2F1, EGF, EIF4EBP1, EIF4EBP2, ELF4, ELK1, ELK4, ERG, F2, FEV, formaldehyde, GAB1, ganglioside, ganglioside GD1a, ganglioside GM1, GATAD2A, GLI3, GPR37L1, GRIN3A, GUSB, HDAC4, HTRA1, ID2, ID3, IFNG, IKBKE, IL1RN, KISS1R, KNG1, LAMC1, levodopa, L-glutamic acid, LHX2, LRPAP1, MAP2K7, MAPK3, MAPT, MARK2, MECP2, MED12, MEF2C, MEF2D, mir-26, MMP1, MMP3, MST1, MSX1, MSX2, NEK6, NEK7, NFKB1, NGF, NPPA, phospholipid, PLA2G10, PLA2G4A, PLCE1, PNKD, PRDX2, progesterone, PTGS1, quinolinic acid, RARG, RCAN1, RPS6KA4, RPS6KA5, RTN4R, S1PR1, SAMSN1, SASH1, SEPTIN5, SHANK3, SNCA, SOD1, SRF, TCF12, TET1, THEM4, TIA1, TLR2, TSHZ3, TYRO3, VDR, VEGFD, VIPR2, WNT5A, WWP2, ZAP70, and ZBTB16 in males only (Suppl Table 3).