The effect of soy isoflavones in brain development: the emerging role of multiple signaling pathways and future perspectives.

Soybean is a source of protein, fibers, and phytochemical isoflavones which are considered to have numerous health benefits for children and adulthood. On the other hand, isoflavones are widely known as phytoestrogens that exert their action via the estrogen signaling pathway. With this regard, isoflavones are also considered as endocrine-disrupting chemicals. Endogenous estrogen plays a crucial role in brain development through binding to estrogen receptors (ERs) or G protein-coupled estrogen receptors 1 (GPER1) and regulates morphogenesis, migration, functional maturation, and intracellular metabolism of neurons and glial cells. Soy isoflavones can also bind to ERs, GPER1, and, furthermore, other receptors to modulate their action. Therefore, soy isoflavone consumption may affect brain development during the pre-and post-natal periods. This review summarizes the current knowledge on the mechanisms of isoflavone action, particularly in the early stages of brain development by introducing representative human, and animal models, and in vitro studies, and discusses their beneficial and adverse impact on neurobehavior. As a conclusion, the soy product consumption during the pre-and post-natal periods under proper range of dose showed beneficial effects in neurobehavior development, including improvement of anxiety, aggression, hyperactive behavior, and cognition, whereas their adverse effect by taking higher doses cannot be excluded. We also present novel research lines to further assess the effect of soy isoflavone administration during brain development.

17ß-Estradiol (E2) Activates Matrix Mineralization through Genomic/Nongenomic Pathways in MC3T3-E1 Cells.

Estrogen plays an important role in osteoporosis prevention. We herein report the possible novel signaling pathway of 17ß-estradiol (E2) in the matrix mineralization of MC3T3-E1, an osteoblast-like cell line. In the culture media-containing stripped serum, in which small lipophilic molecules such as steroid hormones including E2 were depleted, matrix mineralization was significantly reduced. However, the E2 treatment induced this. The E2 effects were suppressed by ICI182,780, the estrogen receptor (ER)α, and the ERß antagonist, as well as their mRNA knockdown, whereas Raloxifene, an inhibitor of estrogen-induced transcription, and G15, a G-protein-coupled estrogen receptor (GPER) 1 inhibitor, had little or no effect. Furthermore, the E2-activated matrix mineralization was disrupted by PMA, a PKC activator, and SB202190, a p38 MAPK inhibitor, but not by wortmannin, a PI3K inhibitor. Matrix mineralization was also induced by the culture media from the E2-stimulated cell culture. This effect was hindered by PMA or heat treatment, but not by SB202190. These results indicate that E2 activates the p38 MAPK pathway via ERs independently from actions in the nucleus. Such activation may cause the secretion of certain signaling molecule(s), which inhibit the PKC pathway. Our study provides a novel pathway of E2 action that could be a therapeutic target to activate matrix mineralization under various diseases, including osteoporosis.

Isoflavones Mediate Dendritogenesis Mainly through Estrogen Receptor α.

The nuclear estrogen receptor (ER) and G-protein-coupled ER (GPER1) play a crucial role during brain development and are involved in dendrite and spine growth as well as synapse formation. Soybean isoflavones, such as genistein, daidzein, and S-equol, a daidzein metabolite, exert their action through ER and GPER1. However, the mechanisms of action of isoflavones on brain development, particularly during dendritogenesis and neuritogenesis, have not yet been extensively studied. We evaluated the effects of isoflavones using mouse primary cerebellar culture, astrocyte-enriched culture, Neuro-2A clonal cells, and co-culture with neurons and astrocytes. Soybean isoflavone-augmented estradiol mediated dendrite arborization in Purkinje cells. Such augmentation was suppressed by co-exposure with ICI 182,780, an antagonist for ERs, or G15, a selective GPER1 antagonist. The knockdown of nuclear ERs or GPER1 also significantly reduced the arborization of dendrites. Particularly, the knockdown of ERα showed the greatest effect. To further examine the specific molecular mechanism, we used Neuro-2A clonal cells. Isoflavones also induced neurite outgrowth of Neuro-2A cells. The knockdown of ERα most strongly reduced isoflavone-induced neurite outgrowth compared with ERß or GPER1 knockdown. The knockdown of ERα also reduced the mRNA levels of ER-responsive genes (i.e., Bdnf, Camk2b, Rbfox3, Tubb3, Syn1, Dlg4, and Syp). Furthermore, isoflavones increased ERα levels, but not ERß or GPER1 levels, in Neuro-2A cells. The co-culture study of Neuro-2A cells and astrocytes also showed an increase in isoflavone-induced neurite growth, and co-exposure with ICI 182,780 or G15 significantly reduced the effects. In addition, isoflavones increased astrocyte proliferation via ER and GPER1. These results indicate that ERα plays an essential role in isoflavone-induced neuritogenesis. However, GPER1 signaling is also necessary for astrocyte proliferation and astrocyte-neuron communication, which may lead to isoflavone-induced neuritogenesis.

Effects of Perfluorooctane Sulfonate on Cerebellar Cells via Inhibition of Type 2 Iodothyronine Deiodinase Activity.

Perfluorooctane sulfonate (PFOS) has been used in a wide variety of industrial and commercial products. The adverse effects of PFOS on the developing brain are becoming of a great concern. However, the molecular mechanisms of PFOS on brain development have not yet been clarified. We investigated the effect of early-life exposure to PFOS on brain development and the mechanism involved. We investigated the change in thyroid hormone (TH)-induced dendrite arborization of Purkinje cells in the primary culture of newborn rat cerebellum. We further examined the mechanism of PFOS on TH signaling by reporter gene assay, quantitative RT-PCR, and type 2 iodothyronine deiodinase (D2) assay. As low as 10-7 M PFOS suppressed thyroxine (T4)-, but not triiodothyronine (T3)-induced dendrite arborization of Purkinje cells. Reporter gene assay showed that PFOS did not affect TRα1- and TRß1-mediated transcription in CV-1 cells. RT-PCR showed that PFOS suppressed D2 mRNA expression in the absence of T4 in primary cerebellar cells. D2 activity was also suppressed by PFOS in C6 glioma-derived cells. These results indicate that early-life exposure of PFOS disrupts TH-mediated cerebellar development possibly through the disruption of D2 activity and/or mRNA expression, which may cause cerebellar dysfunction.

EID1 suppresses lipid accumulation by inhibiting the expression of GPDH in 3T3-L1 preadipocytes.

The imbalance between food intake and energy expenditure causes high accumulation of triglycerides in adipocytes. Obesity is related with the increased lipid accumulation in white adipose tissue, which is a major risk factor for the development of metabolic disorders, such as type 2 diabetes and cardiovascular disease. This study highlights the role of E1A-like inhibitor of differentiation 1 (EID1) in the modulation of adipogenesis through the downregulation of glycerol-3-phosphate dehydrogenase (GPDH), which is a key enzyme in the synthesis of triglycerides and is considered to be a marker of adipogenesis. By analyzing DNA microarray data, we found that when EID1 is overexpressed in preadipocytes (3T3-L1 cells) during adipocyte differentiation, EID1 inhibits lipid accumulation through the downregulation of GPDH. In contrast, EID1 is not involved in the regulation of intracellular glucose via the translocation of glucose transporter. A confocal image analysis showed that EID1 is located in the nucleus of preadipocytes in the form of speckles, which could be involved as a regulator of the transcriptional process. We further confirmed that EID1 is able to bind to the promoter sequence of GPDH in the nucleus. These findings provide a molecular explanation for the inhibitory effect of EID1 on lipid accumulation in adipocytes.

A Novel Mechanism of S-equol Action in Neurons and Astrocytes: The Possible Involvement of GPR30/GPER1.

S-equol is a major bacterial metabolite of the soy isoflavone daidzein. It is known to be a phytoestrogen that acts by binding to the nuclear estrogen receptors (ERs) that are expressed in various brain regions, including the cerebellum. However, the effects of S-equol on cerebellar development and function have not yet been extensively studied. In this study, the effects of S-equol were evaluated using a mouse primary cerebellar culture, Neuro-2A clonal cells, and an astrocyte-enriched culture. S-equol augmented the dendrite arborization of Purkinje cells induced by triiodothyronine (T3) and the neurite growth of Neuro-2A cell differentiation. Such augmentation was suppressed by G15, a selective G-protein coupled ER (GPR30) antagonist, and ICI 182,780, an antagonist for ERs in both cultures. On the other hand, in astrocytes, S-equol induced cell proliferation and cell migration with an increase in the phosphorylated extracellular-signal-regulated kinase 1/2 and F-actin rearrangements. Such effects were suppressed by G15, but not by ICI. These findings indicated that S-equol may enhanced cerebellar development by affecting both neurons and astrocytes through several signaling pathways, including GPR30 and ERs. We here report a novel mechanism of S-equol in cerebellar development that may provide a novel possibility to use S-equol supplementation during development.

The Effects of Gadolinium-Based Contrast Agents on the Cerebellum: from Basic Research to Neurological Practice and from Pregnancy to Adulthood.

Gadolinium (Gd)-based contrast agents (GBCAs) are used in magnetic resonance imaging (MRI) to increase the diagnostic yield. Current reports using animal models or human subjects have shown that GBCAs may be deposited in brain including the cerebellum. Although further studies may be required to clarify the toxicity of GBCAs, we should be more cautious to use these agents particularly in patients who more likely to have repeated enhanced MRI along their lifespan. In this editorial, current studies to clarify the toxicity of GBCAs in the cerebellum are introduced.

DNA Methylation in the Hypothalamic Feeding Center and Obesity.

Obesity rates have been increasing worldwide for decades, mainly due to environmental factors, such as diet, nutrition, and exercise. However, the molecular mechanisms through which environmental factors induce obesity remain unclear. Several mechanisms underlie the body's response to environmental factors, and one of the main mechanisms involves epigenetic modifications, such as DNA methylation. The pattern of DNA methylation is influenced by environmental factors, and altered DNA methylation patterns can affect gene expression profiles and phenotypes. DNA methylation may mediate the development of obesity caused by environmental factors. Similar to the factors governing obesity, DNA methylation is influenced by nutrients and metabolites. Notably, DNA methylation is associated with body size and weight programming. The DNA methylation levels of proopiomelanocortin (Pomc) and neuropeptide Y (Npy) in the hypothalamic feeding center, a key region controlling systemic energy balance, are affected by diet. Conditional knockout mouse studies of epigenetic enzymes have shown that DNA methylation in the hypothalamic feeding center plays an indispensable role in energy homeostasis. In this review, we discuss the role of DNA methylation in the hypothalamic feeding center as a potential mechanism underlying the development of obesity induced by environmental factors.

Gadolinium-based contrast agent accelerates the migration of astrocyte via integrin αvß3 signaling pathway.

Gadolinium (Gd)-based contrast agents (GBCAs) are chemicals injected intravenously during magnetic resonance imaging to enhance the diagnostic yield. Repeated use of GBCAs causes their deposition in the brain. Such deposition may affect various neuronal cells, including astrocytes. In this study, we examined the effect of GBCAs (Omniscan, Magnescope, Magnevist, and Gadovist) on astrocyte migration, which is critical for formation of neurons during development and maintaining brain homeostasis. All GBCAs increased cell migration and adhesion with increased actin remodelling. Knockdown of integrin αvß3 by RNAi or exposure to integrin αvß3 inhibitor reduced astrocyte migration. GBCAs increased phosphorylation of downstream factors of αvß3, such as FAK, ERK1/2, and Akt. The phosphorylation of all these factors were reduced by RNAi or integrin αvß3 inhibitor. GBCAs also increased the phosphorylation of their downstream factor, Rac1/cdc42, belonging to the RhoGTPases family. Coexposure to the selective RhoGTPases inhibitors, decreased the effects of GBCAs on cell migration. These findings indicate that GBCAs exert their action via integrin αvß3 to activate the signaling pathway, resulting in increased astrocyte migration. Thus, the findings of the study suggest that it is important to avoid the repeated use of GBCAs to prevent adverse side effects in the brain, particularly during development.

Involvement of integrin αvß3 in thyroid hormone-induced dendritogenesis.

Activation and/or modulation of the membrane-associated receptors plays a critical role in brain development. Thyroid hormone (TH) acts on both nuclear receptors (thyroid hormone receptor, TR) and membrane-associated receptors, particularly integrin αvß3 in neurons and glia. Integrin αvß3-mediated signal transduction mediates various cellular events during development including morphogenesis, migration, synaptogenesis, and intracellular metabolism. However, the involvement of integrin αvß3-mediated TH action during brain development remains poorly understood. Thus, we examined the integrin αvß3-mediated effects of TH (T3, T4, and rT3) in the neurons and astrocytes using primary cerebellar culture, astrocyte-enriched culture, Neuro-2A clonal cells, and co-culture of neurons and astrocytes. We found that TH augments dendrite arborization of cerebellar Purkinje cells. This augmentation was suppressed by knockdown of integrin αvß3, as well as TRα and TRß. A selective integrin αvß3 antagonist, LM609, was also found to suppress TH-induced arborization. However, whether this effect was a direct action of TH on Purkinje cells or due to indirect actions of other cells subset such as astrocytes was not clarified. To further study neuron-specific molecular mechanisms, we used Neuro-2A clonal cells and found TH also induces neurite growth. TH-induced neurite growth was reduced by co-exposure with LM609 or knockdown of TRα, but not TRß. Moreover, co-culture of Neuro-2A and astrocytes also increased TH-induced neurite growth, indicating astrocytes may be involved in neuritogenesis. TH increased the localization of synapsin-1 and F-actin in filopodia tips. TH exposure also increased phosphorylation of FAK, Akt, and ERK1/2. Phosphorylation was suppressed by co-exposure with LM609 and TRα knockdown. These results indicate that TRs and integrin αvß3 play essential roles in TH-induced dendritogenesis and neuritogenesis. Furthermore, astrocytes-neuron communication via TR-dependent and TR-independent signaling through membrane receptors and F-actin are required for TH-induced neuritogenesis.

The neurotoxic effect of lactational PFOS exposure on cerebellar functional development in male mice.

Recent studies showed a possible association between perfluorooctane sulfonate (PFOS) and developmental disabilities. We previously found the specific effects of PFOS exposure on learning and memory, however, its effect on the other developmental disabilities such as motor and social deficits remains unclear. We examined the effect of early lactational PFOS exposure on motor coordination, social activity, and anxiety in male mice. We orally administered a PFOS solution to dams from postnatal day 1-14. At 10 weeks old, we conducted a behavior test battery to evaluate motor performance, social activity, and anxiety, followed by electrophysiology and Western blot analysis. PFOS-exposed mice displayed impaired motor coordination. Whole-cell patch-clamp recordings from Purkinje cells revealed that the short-term and long-term plasticity at parallel fiber-Purkinje cell synapses are affected by PFOS exposure. Western blot analysis indicated that PFOS exposure increased syntaxin binding protein 1 (Munc18-1) and glutamate metabotropic receptor 1 (mGluR1) protein levels, which may be associated with the change in neurotransmitter release from parallel fibers and the level of long-term depression, respectively. The present study demonstrates that lactational PFOS exposure may have disrupted the pre- and postsynaptic plasticity at parallel fiber-Purkinje cell synapses, causing profound, long-lasting abnormal effects on the cerebellar function.

Effects of Gadolinium Deposits in the Cerebellum: Reviewing the Literature from In Vitro Laboratory Studies to In Vivo Human Investigations.

Gadolinium (Gd)-based contrast agents (GBCAs) are chemicals injected intravenously during magnetic resonance imaging (MRI) to enhance the diagnostic yield. The repeated use of GBCAs can cause their deposition in the brain, including the cerebellum. Such deposition may affect various cell subsets in the brain and consequently cause behavioral alterations due to neurotoxicity. Caution should thus be exercised in using these agents, particularly in patients who are more likely to have repeated enhanced MRIs during their lifespan. Further studies are required to clarify the toxicity of GBCAs, and potential mechanisms causing neurotoxicity have recently been reported. This review introduces the effects of GBCAs in the cerebellum obtained from in vitro and in vivo studies and considers the possible mechanisms of neurotoxicity involved.

The Role of Ferrous Ion in the Effect of the Gadolinium-Based Contrast Agents (GBCA) on the Purkinje Cells Arborization: An In Vitro Study.

Gadolinium deposition in the brain has been observed in areas rich in iron, such as the dentate nucleus of the cerebellum. We investigated the role of Fe2+ in the effect of gadolinium-based contrast agents (GBCA) on thyroid hormone-mediated Purkinje cell dendritogenesis in a cerebellar primary culture. The study comprises the control group, Fe2+ group, GBCA groups (gadopentetate group or gadobutrol group), and GBCA+Fe2+ groups. Immunocytochemistry was performed with an anti-calbindin-28K (anti-CaBP28k) antibody, and the nucleus was stained with 4',6-diamidino-2-phenylindole (DAPI). The number of Purkinje cells and their arborization were evaluated with an analysis of variance with a post-hoc test. The number of Purkinje cells was similar to the control groups among all treated groups. There were no significant differences in dendrite arborization between the Fe2+ group and the control groups. The dendrite arborization was augmented in the gadopentetate and the gadobutrol groups when compared to the control group (p < 0.01, respectively). Fe2+ significantly increased the effect of gadopentetate on dendrite arborization (p < 0.01) but did not increase the effect of gadobutrol. These findings suggested that the chelate thermodynamic stability and Fe2+ may play important roles in attenuating the effect of GBCAs on the thyroid hormone-mediated dendritogenesis of Purkinje cells in in vitro settings.

Soy Isoflavones Accelerate Glial Cell Migration via GPER-Mediated Signal Transduction Pathway.

Soybean isoflavones, such as genistein, daidzein, and its metabolite, S-equol, are widely known as phytoestrogens. Their biological actions are thought to be exerted via the estrogen signal transduction pathway. Estrogens, such as 17ß-estradiol (E2), play a crucial role in the development and functional maintenance of the central nervous system. E2 bind to the nuclear estrogen receptor (ER) and regulates morphogenesis, migration, functional maturation, and intracellular metabolism of neurons and glial cells. In addition to binding to nuclear ER, E2 also binds to the G-protein-coupled estrogen receptor (GPER) and activates the nongenomic estrogen signaling pathway. Soybean isoflavones also bind to the ER and GPER. However, the effect of soybean isoflavone on brain development, particularly glial cell function, remains unclear. We examined the effects of soybean isoflavones using an astrocyte-enriched culture and astrocyte-derived C6 clonal cells. Isoflavones increased glial cell migration. This augmentation was suppressed by co-exposure with G15, a selective GPER antagonist, or knockdown of GPER expression using RNA interference. Isoflavones also activated actin cytoskeleton arrangement via increased actin polymerization and cortical actin, resulting in an increased number and length of filopodia. Isoflavones exposure increased the phosphorylation levels of FAK (Tyr397 and Tyr576/577), ERK1/2 (Thr202/Tyr204), Akt (Ser473), and Rac1/cdc42 (Ser71), and the expression levels of cortactin, paxillin and ERα. These effects were suppressed by knockdown of the GPER. Co-exposure of isoflavones to the selective RhoA inhibitor, rhosin, selective Cdc42 inhibitor, casin, or Rac1/Cdc42 inhibitor, ML-141, decreased the effects of isoflavones on cell migration. These findings indicate that soybean isoflavones exert their action via the GPER to activate the PI3K/FAK/Akt/RhoA/Rac1/Cdc42 signaling pathway, resulting in increased glial cell migration. Furthermore, in silico molecular docking studies to examine the binding mode of isoflavones to the GPER revealed the possibility that isoflavones bind directly to the GPER at the same position as E2, further confirming that the effects of the isoflavones are at least in part exerted via the GPER signal transduction pathway. The findings of the present study indicate that isoflavones may be an effective supplement to promote astrocyte migration in developing and/or injured adult brains.

A Possible Novel Mechanism of Action of Genistein and Daidzein for Activating Thyroid Hormone Receptor-Mediated Transcription.

Thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily that regulate their target genes for controlling organ development and functional maintenance. Soybean isoflavones, especially genistein and daidzein, modulate various hormone-mediated pathways. However, their effects on TRs have not yet been extensively studied. In this study, the effects of these isoflavones on TR action were evaluated using transient transfection-based reporter gene assays and molecular docking studies. Genistein and daidzein augmented T3-liganded TR-mediated transcription in a concentration-dependent manner. In the mammalian 2-hybrid study, these isoflavones augmented the recruitment of steroid receptor coactivator-1 and nuclear corepressor to liganded or unliganded TRs. Using a series of mutant TRs, we also showed that the activation function-2 domain of TRs was responsible for the augmentation by these isoflavones. CV-1 cells had expressed TRα, TRß1, and ERα mRNAs. However, neither the overexpression nor the knocking down of ERα altered the augmentation of TR action by isoflavones, indicating that the effects of isoflavones are exerted through their direct action on TRs. In silico molecular docking studies showed that genistein and daidzein can directly bind to the TR-ligand-binding domain. These findings indicate that the augmentation of the TR-mediated transcription by genistein and daidzein is due to their direct binding to TR-ligand-binding domain to induce the recruitment of steroid receptor coactivator-1. Our study reports a novel mode of action of soybean isoflavones on TR function. The biological effects and the relevance of these isoflavones to human health may be partially attributable to the activation of thyroid hormone signaling.

Effects of Gadolinium-Based Contrast Agents on Thyroid Hormone Receptor Action and Thyroid Hormone-Induced Cerebellar Purkinje Cell Morphogenesis.

Gadolinium (Gd)-based contrast agents (GBCAs) are used in diagnostic imaging to enhance the quality of magnetic resonance imaging or angiography. After intravenous injection, GBCAs can accumulate in the brain. Thyroid hormones (THs) are critical for the development and functional maintenance of the central nervous system. TH actions in brain are mainly exerted through nuclear TH receptors (TRs). We examined the effects of GBCAs on TR-mediated transcription in CV-1 cells using transient transfection-based reporter assay and TH-mediated cerebellar Purkinje cell morphogenesis in primary culture. We also measured the cellular accumulation and viability of Gd after representative GBCA treatments in cultured CV-1 cells. Both linear (Gd-diethylene triamine pentaacetic acid-bis methyl acid, Gd-DTPA-BMA) and macrocyclic (Gd-tetraazacyclododecane tetraacetic acid, Gd-DOTA) GBCAs were accumulated without inducing cell death in CV-1 cells. By contrast, Gd chloride (GdCl3) treatment induced approximately 100 times higher Gd accumulation and significantly reduced the number of cells. Low doses of Gd-DTPA-BMA (10(-8) to 10(-6)M) augmented TR-mediated transcription, but the transcription was suppressed at higher dose (10(-5) to 10(-4)M), with decreased ß-galactosidase activity indicating cellular toxicity. TR-mediated transcription was not altered by Gd-DOTA or GdCl3, but the latter induced a significant reduction in ß-galactosidase activity at high doses, indicating cellular toxicity. In cerebellar cultures, the dendrite arborization of Purkinje cells induced by 10(-9)M T4 was augmented by low-dose Gd-DTPA-BMA (10(-7)M) but was suppressed by higher dose (10(-5)M). Such augmentation by low-dose Gd-DTPA-BMA was not observed with 10(-9)M T3, probably because of the greater dendrite arborization by T3; however, the arborization by T3 was suppressed by a higher dose of Gd-DTPA-BMA (10(-5)M) as seen in T4 treatment. The effect of Gd-DOTA on dendrite arborization was much weaker than that of the other compounds. These results indicate that exposure to specific GBCAs may, at least in part, cause toxic effects in the brain by disrupting the action of THs on TRs. The toxic effects of GBCAs may depend on the chemical structure of GBCA and the dose. Thus, it is very important to choose appropriate GBCAs for imaging to prevent adverse side effects.