Luteolin Modulates Neural Stem Cells Fate Determination: In vitro Study on Human Neural Stem Cells, and in vivo Study on LPS-Induced Depression Mice Model

Luteolin is a natural flavone with neurotrophic effects observed on different neuronal cell lines. In the present study, we aimed to assess the effect of luteolin on hNSCs fate determination and the LPS-induced neuroinflammation in a mouse model of depression with astrocytogenesis defect. hNSCs were cultured in basal cell culture medium (control) or medium supplemented with luteolin or AICAR, a known inducer of astrogenesis. A whole-genome transcriptomic analysis showed that luteolin upregulated the expressions of genes related to neurotrophin, dopaminergic, hippo, and Wnt signaling pathways, and downregulated the genes involved in p53, TNF, FOXO, and Notch signaling pathways. We also found that astrocyte-specific gene GFAP, as well as other genes of the key signaling pathways involved in astrogenesis such as Wnt, BMP, and JAK-STAT pathways were upregulated in luteolin-treated hNSCs. On the other hand, neurogenesis and oligodendrogenesis-related genes, TUBB3, NEUROD 1 and 6, and MBP, were downregulated in luteolin-treated hNSCs. Furthermore, immunostaining showed that percentages of GFAP+ cells were significantly higher in luteolin- and AICAR-treated hNSCs compared to control hNSCs. Additionally, RT-qPCR results showed that luteolin upregulated the expressions of GFAP, BMP2, and STAT3, whereas the expression of TUBB3 remained unchanged. Next, we evaluated the effects of luteolin in LPS-induced mice model of depression that represents defects in astrocytogenesis. We found that oral administration of luteolin (10 mg/Kg) for eight consecutive days could decrease the immobility time on tail suspension test, a mouse behavioral test measuring depression-like behavior, and attenuate LPS-induced inflammatory responses by significantly decreasing IL-6 production in mice brain-derived astrocytes and serum, and TNFα and corticosterone levels in serum. Luteolin treatment also significantly increased mature BDNF, dopamine, and noradrenaline levels in the hypothalamus of LPS-induced depression mice. Though the behavioral effects of luteolin did not reach statistical significance, global gene expression analyses of mice hippocampus and brain-derived NSCs highlighted the modulatory effects of luteolin on different signaling pathways involved in the pathophysiology of depression. Altogether, our findings suggest an astrocytogenic potential of luteolin and its possible therapeutic benefits in neuroinflammatory and neurodegenerative diseases. However, further studies are required to identify the specific mechanism of action of luteolin.

LSM12 acts as a molecular adaptor for the recruitment of TWENTY-FOUR (TYF) to ATX2. The ATX2-LSM12-TYF complex thereby stimulates TYF-dependent translation of the rate-limiting clock gene period (per) to maintain 24 hr periodicity in circadian behaviors

MAP4K2
Mitogen-activated protein kinase kinase kinase kinase 2 -1.87 0.0153 The protein encoded by this gene is a member of the serine/threonine protein kinase family. This kinase can be activated by TNF-alpha, and has been shown to specifically activate MAP kinases. This kinase is also found to interact with TNF receptor-associated factor 2 (TRAF2), which is involved in the activation of MAP3K1/MEKK1.

IL1RL1
Interleukin 1 receptor-like 1 -1.83 0.0141 The protein encoded by this gene is a member of the interleukin 1 receptor family. Studies of the similar gene in mouse suggested that this receptor can be induced by proinflammatory stimuli, and may be involved in the function of helper T cells.

TAF15
TATA box binding protein associated factor 15 -1.82 0.0224 This gene encodes a member of the TET family of RNA-binding proteins. The encoded protein plays a role in RNA polymerase II gene transcription as a component of a distinct subset of multi-subunit transcription initiation factor TFIID complexes. Translocations involving this gene play a role in acute leukemia and extraskeletal myxoid chondrosarcoma, and mutations in this gene may play a role in amyotrophic lateral sclerosis

EXOC7
Exocyst complex component 7 -1.78 0.0063 The protein encoded by this gene is a component of the exocyst complex. The exocyst complex plays a critical role in vesicular trafficking and the secretory pathway by targeting post-Golgi vesicles to the plasma membrane. The encoded protein is required for assembly of the exocyst complex and docking of the complex to the plasma membrane. The encoded protein may also play a role in pre-mRNA splicing through interactions with pre-mRNA-processing factor 19. Functions as extracellular chaperone that prevents aggregation of non-native proteins. Prevents stress-induced aggregation of blood plasma proteins. Inhibits formation of amyloid fibrils by APP, APOC2, B2M, CALCA, CSN3, SNCA and aggregation-prone LYZ variants. When secreted, protects cells against apoptosis and against cytolysis by complement.

SFSWAP
Modulates NF-kappa-B transcriptional activity. Following stress, promotes apoptosis. Inhibits apoptosis when associated with the mitochondrial membrane by interference with BAX-dependent release of cytochrome c into the cytoplasm. Following ER stress, suppresses stress-induced apoptosis by stabilizing mitochondrial membrane integrity through interaction with HSPA5. When secreted, acts as an important modulator during neuronal differentiation through interaction with STMN3. Plays a role in the clearance of immune complexes that arise during cell injury. Mediates the transfer of phospholipids and free cholesterol from triglyceride-rich lipoproteins into high-density lipoproteins as well as the exchange of phospholipids between triglyceride-rich lipoproteins themselves Facilitates the transfer of a spectrum of different lipid molecules, including sphingomyelin, phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, and phosphatidyl ethanolamine. Plays an important role in HDL remodeling which involves modulating the size and composition of HDL. Also plays a key role in the uptake of cholesterol from peripheral cells and tissues that is subsequently transported to the liver for degradation and excretion. Two distinct forms of PLTP exist in plasma: an active form that can transfer phosphatidylcholine from phospholipid vesicles to HDL, and an inactive form that lacks this capability Tuba1a tubulin, alpha 1A 4.27 7.67E-08 Tubulin is the major constituent of microtubules. It binds two moles of GTP, one at an exchangeable site on the beta chain and one at a non-exchangeable site on the alpha chain.
Gm1821; Ubb ubiquitin pseudogene; ubiquitin B 4.04 1.52E-05 It has distinct roles, such as in activation of protein kinases, and in signaling B2m beta-2 microglobulin 4 1.21E-08 Component of the class I major histocompatibility complex (MHC). Involved in the presentation of peptide antigens to the immune system. Involved in neural retina development; photoreceptor cell maintenance; and spermatogenesis. Localizes to several cellular components, including acrosomal membrane; photoreceptor inner segment; and photoreceptor outer segment. Is expressed in several structures, including brain and sensory organ.
*Gene function as reported in NCBI ( https://www.ncbi.nlm.nih.gov/guide/howto/find-func-gene/ ) Predicted to have heparin binding activity and serine-type endopeptidase activity. Involved in defense response to fungus; positive regulation of immune response; and response to bacterium. Predicted to localize to several cellular components, including cytoplasmic stress granule; plasma membrane; and secretory granule. Is expressed in liver.

Gpaa1
GPI anchor attachment protein 1 -1.45 0.0127 Predicted to contribute to GPI anchor binding activity, to be involved in attachment of GPI anchor to protein and proteincontaining complex assembly and, to localize to GPI-anchor transamidase complex and integral component of plasma membrane.

Crisp2
cysteine-rich secretory protein 2 -1.45 0.0153 Predicted to be involved in cell-cell adhesion, to localize to extracellular space.

Lcn2
lipocalin 2 -1.44 0.0002 Exhibits iron ion binding activity. Involved in several processes, including extrinsic apoptotic signaling pathway in absence of ligand; positive regulation of cold-induced thermogenesis; and siderophore transport. It is expressed in several structures, including genitourinary system; heart; liver; lung; and skeletal muscle.

Prmt3
protein arginine N-methyltransferase 3 -1.44 0.0164 Exhibits protein-arginine N-methyltransferase activity and ribosome binding activity. Involved in protein methylation. Localizes to cytosol. Is expressed in several structures, including central nervous system; genitourinary system; liver; sensory organ; and spleen.

Nfe2l3
nuclear factor, erythroid derived 2, like 3 -1.42 0.0133 Exhibits DNA-binding transcription repressor activity, RNA polymerase II-specific and RNA polymerase II cis-regulatory region sequence-specific DNA binding activity. Involved in negative regulation of transcription by RNA polymerase II. Predicted to localize to cytoplasm and nucleus. Is expressed in brain; forelimb bud; gonad; and ureter.

Map6
microtubule-associated protein 6 -1.42 0.0152 Exhibits microtubule binding activity. Predicted to be involved in several processes, including axonal transport of mitochondrion; generation of neurons; and lysosome localization. Predicted to localize to several cellular components, including cis-Golgi network; cytoplasmic vesicle; and perinuclear region of cytoplasm. It is expressed in cranial ganglion; dorsal root ganglion; and sensory organ.

Supplementary table 9. Top 10 upregulated genes in the hippocampus of LPS-induced depression mice treated with luteolin (LPS+L) compared to untreated LPS-induced depression mice (LPS).
Gene symbol Gene title Fold change p-value Gene function* Fmod fibromodulin -5.05 0.0053 Encodes fibromodulin protein belonging to the family of small interstitial proteoglycans. Owing to the interaction with type I and type II collagen fibrils and in vitro inhibition of fibrillogenesis, the encoded protein may play a role in the assembly of extracellular matrix. It may also regulate TGF-beta activities by sequestering TGF-beta into the extracellular matrix.
Ttr transthyretin -3.61 0.0061 This gene encodes a carrier protein responsible for the transport of thyroid hormones and retinol.
Pigz phosphatidylinositol glycan anchor biosynthesis, class Z -3.38 5.85E-06 The glycosylphosphatidylinositol (GPI) anchor is a glycolipid found on many blood cells that serves to anchor proteins to the cell surface.
Gfap glial fibrillary acidic protein -3.11 3.01E-05 Encodes the GFAP protein, a structural constituent of cytoskeleton. Involved in several processes, including long-term synaptic potentiation; and neurogenesis. Localizes to several cellular components, including astrocyte end-foot; cell body; and intermediate filament.

Kbtbd11
kelch repeat and BTB (POZ) domain containing 11 -3.07 0.0011 Is expressed in several structures, including central nervous system; early tubule; ganglia; lung; and sensory organ.

Tmsb15b2; Tmsb15l
thymosin beta 15b2; thymosin beta 15b like -2.95 0.0044 Exhibits actin monomer binding activity. Involved in negative regulation of stress fiber assembly and sequestering of actin monomers. Localizes to filamentous actin and stress fiber.
Ptgds prostaglandin D2 synthase (brain) -2.89 1.01E-05 It is a protein coding gene. Exhibits prostaglandin-D synthase activity and retinoid binding activity. Involved in prostaglandin biosynthetic process; and regulation of circadian sleep/wake cycle, sleep. Localizes to extracellular region. Is expressed in several structures, including nervous system; and sensory organ. Exhibits palmitoyl-CoA 9-desaturase activity and stearoyl-CoA 9-desaturase activity. Involved in monounsaturated fatty acid biosynthetic process. Localizes to endoplasmic reticulum. Is expressed in several structures, including central nervous system, and sensory organ.

Camk2n1
calcium/calmodulin-dependent protein kinase II inhibitor 1 2.98 5.46E-08 Exhibits protein kinase inhibitor activity. Predicted to be involved in several processes, including negative regulation of cellular protein metabolic process; positive regulation of insulin secretion involved in cellular response to glucose stimulus; and positive regulation of systemic arterial blood pressure. Localizes to postsynaptic density. Is expressed in several structures, including brain.

Tgtp1; Tgtp2
T cell specific GTPase 1; T cell specific GTPase 2 1.98 0.0007 Predicted to be involved in cellular response to interferon-beta and defense response. Predicted to localize to endoplasmic reticulum membrane. Is expressed in several structures, including genitourinary system; hemolymphoid system; integumental system; nervous system; and nose.

Erdr1
erythroid differentiation regulator 1 2.26 6.01E-10 It is a protein coding gene involved in negative regulation of cell migration; negative regulation of cell population proliferation; and somatic stem cell population maintenance. It is expressed in several structures, including central nervous system and retina.

H2-K1
histocompatibility 2, K1, K region 1.96 6.90E-09 Exhibits MHC class I protein binding activity and peptide antigen binding activity. Involved in several processes, including antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, TAP-dependent; antigen processing and presentation of exogenous peptide antigen via MHC class I; and positive regulation of T cell mediated cytotoxicity. Localizes to external side of plasma membrane. Is expressed in several structures, including brain.