Song Her

Ischemia-induced changes in glucagon-like peptide-1 receptor and neuroprotective effect of its agonist, exendin-4, in experimental transient cerebral ischemia

Glucagon‐like peptide‐1 receptor (GLP‐1R) protects against neuronal damages in the brain. In the present study, ischemia‐induced changes in GLP‐1R immunoreactivity in the gerbil hippocampal CA1 region were evaluated after transient cerebral ischemia; in addition, the neuroprotective effect of the GLP‐1R agonist exendin‐4 (EX‐4) against ischemic damage was studied. GLP‐1R immunoreactivity and its protein levels in the ischemic CA1 region were highest at 1 day after ischemia/reperfusion (I/R). At 4 days after I/R, GLP‐1R immunoreactivity was hardly detected in CA1 pyramidal neurons, and its protein level was lowest. GLP‐1R protein level was increased again at 10 days after I/R, and GLP‐1R immunoreactivity was found in astrocytes and GABAergic interneurons. In addition, EX‐4 treatment attenuated ischemia‐induced hyperactivity, neuronal damage, and microglial activation in the ischemic CA1 region in a dose‐dependent manner. EX‐4 treatment also induced the elevation of GLP‐1R immunoreactivity and protein levels in the ischemic CA1 region. These results indicate that GLP‐1R is altered in the ischemic region after an ischemic insult and that EX‐4 protects against ischemia‐induced neuronal death possibly by increasing GLP‐1R expression and attenuating microglial activation against transient cerebral ischemic damage.

Stress-induced changes in primate prefrontal profiles of gene expression

Stressful experiences that consistently increase cortisol levels appear to alter the expression of hundreds of genes in prefrontal limbic brain regions. Here, we investigate this hypothesis in monkeys exposed to intermittent social stress-induced episodes of hypercortisolism or a no-stress control condition. Prefrontal profiles of gene expression compiled from Affymetrix microarray data for monkeys randomized to the no-stress condition were consistent with microarray results published for healthy humans. In monkeys exposed to intermittent social stress, more genes than expected by chance appeared to be differentially expressed in ventromedial prefrontal cortex compared to monkeys not exposed to adult social stress. Most of these stress responsive candidate genes were modestly downregulated, including ubiquitin conjugation enzymes and ligases involved in synaptic plasticity, cell cycle progression and nuclear receptor signaling. Social stress did not affect gene expression beyond that expected by chance in dorsolateral prefrontal cortex or prefrontal white matter. Thirty four of 48 comparisons chosen for verification by quantitative real-time polymerase chain reaction (qPCR) were consistent with the microarray-predicted result. Furthermore, qPCR and microarray data were highly correlated. These results provide new insights on the regulation of gene expression in a prefrontal corticolimbic region involved in the pathophysiology of stress and major depression. Comparisons between these data from monkeys and those for ventromedial prefrontal cortex in humans with a history of major depression may help to distinguish the molecular signature of stress from other confounding factors in human postmortem brain research.

Stress coping stimulates hippocampal neurogenesis in adult monkeys

Coping with intermittent social stress is an essential aspect of living in complex social environments. Coping tends to counteract the deleterious effects of stress and is thought to induce neuroadaptations in corticolimbic brain systems. Here we test this hypothesis in adult squirrel monkey males exposed to intermittent social separations and new pair formations. These manipulations simulate conditions that typically occur in male social associations because of competition for limited access to residency in mixed-sex groups. As evidence of coping, we previously confirmed that cortisol levels initially increase and then are restored to prestress levels within several days of each separation and new pair formation. Follow-up studies with exogenous cortisol further established that feedback regulation of the hypothalamic-pituitary-adrenal axis is not impaired. Now we report that exposure to intermittent social separations and new pair formations increased hippocampal neurogenesis in squirrel monkey males. Hippocampal neurogenesis in rodents contributes to spatial learning performance, and in monkeys we found that spatial learning was enhanced in conditions that increased hippocampal neurogenesis. Corresponding changes were discerned in the expression of genes involved in survival and integration of adult-born granule cells into hippocampal neural circuits. These findings support recent indications that stress coping stimulates hippocampal neurogenesis in adult rodents. Psychotherapies designed to promote stress coping potentially have similar effects in humans with major depression.

Phenylethanolamine N-methyltransferase gene expression: synergistic activation by Egr-1, AP-2 and the glucocorticoid receptor

The gene encoding the epinephrine synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT), is transcriptionally activated by Egr-1, AP-2, and the glucocorticoid receptor (GR). Stimulation by AP-2 requires its synergistic interaction with an activated GR. The present studies show that the GR also cooperates with Egr-1 or the combination of Egr-1 and AP-2 to activate the PNMT promoter. Together Egr-1, AP-2, and the GR can induce PNMT promoter-mediated luciferase reporter gene expression beyond the sum of their independent contributions as well as synergistically activate the endogenous PNMT gene leading to marked increases in PNMT mRNA. Examination of the effects of mutation of the AP-2 or Egr-1 binding sites on PNMT promoter activation by DEX and the factor binding to the remaining intact site or by all three transcriptional activators showed changes in luciferase reporter gene expression which suggest that DNA structure may be altered thereby reducing or enhancing synergistic activation. It also appears that the -165 bp Egr-1 site may not be critical for the synergism observed between Egr-1, AP-2 and the GR. When the glucocorticoid response element (GRE) within the PNMT promoter was mutated, PNMT promoter activation by Egr-1 and DEX, AP-2 and DEX or all three showed both inhibition and enhancement, even when the GRE was completely eliminated. These observations indicate that induction of PNMT gene transcription may occur either through GR interaction with other transcriptional proteins after binding to its cognate GRE or through direct protein-protein interaction in the absence of GRE binding. While the mechanisms by which Egr-1 and the GR and Egr-1, AP-2 and the GR function cooperatively to stimulate PNMT promoter activity remain to be elucidated, this synergistic stimulation of the PNMT promoter by these factors may provide important in vivo and in vitro regulatory control of the PNMT gene.

Phenylethanolamine N-methyltransferase gene expression. Sp1 and MAZ potential for tissue-specific expression.

PhenylethanolamineN-methyltransferase (PNMT) promoter-luciferase reporter gene constructs (pGL3RP863, pGL3RP444, and pGL3RP392) transfected into COS1, RS1, PC12, NIH/3T3, or Neuro2A cells showed the highest basal luciferase activity in the Neuro2A cells. DNase I footprinting with Neuro2A cell nuclear extract identified protected PNMT promoter regions spanning the –168/–165 and –48/–45 base pair Sp1/Egr-1 binding sites. Gel mobility shift assays and transient transfection assays using site-directed mutant PNMT promoter-luciferase reporter gene constructs indicated that the elevated basal luciferase activity in the Neuro2A cells was mediated by Sp-1. Furthermore, activation of the PNMT promoter by Sp1 depends on both its binding affinity for its cognate target sequences and its intracellular concentrations. When Sp1 levels were increased through an expression plasmid, luciferase reporter gene expression rose well beyond basal wild-type levels, even with either Sp1 binding element mutated. Finally, another transcription factor expressed in the Neuro2A cells competes with Sp1 by interacting with DNA sequences 3′ to the –48 base pair Sp1 site to prevent Sp1 binding and induction of the PNMT promoter. The DNA consensus sequence, Southwestern analysis, and gel mobility shift assays with antibodies identify MAZ as the competitive factor. These findings suggest that Sp1 may potentially contribute to the tissue-specific expression of the PNMT gene, with the competition between Sp1 and MAZ conferring additional tissue-specific control.

Glucocorticoid‐Dependent Action of Neural Crest Factor AP‐2: Stimulation of Phenylethanolamine N‐Methyltransferase Gene Expression

Abstract: AP‐2 is a vertebrate transcription factor expressed in neural crest cells and their derivative tissues, including the adrenal medulla, where epinephrine is produced. AP‐2 is shown to stimulate expression of the gene encoding the epinephrine biosynthetic enzyme phenylethanolamine N‐methyltransferase (PNMT). However, stimulation of the PNMT gene by AP‐2 requires glucocorticoids and appears to be mediated through the interaction of AP‐2 with activated type II glucocorticoid receptors. Mutation of AP‐2 and/or glucocorticoid receptor binding elements within the PNMT promoter disrupts the ability of AP‐2 and glucocorticoids to induce PNMT promoter activity. These findings suggest, in the case of PNMT, that AP‐2 stimulates gene expression through a novel glucocorticoid‐dependent mechanism.

Progesterone Pretreatment Enhances Serotonin-Stimulated BDNF Gene Expression in Rat C6 Glioma Cells Through Production of 5α-Reduced Neurosteroids

Tricyclic antidepressants and selective serotonin reuptake inhibitors are considered in theory to induce the outflow of neurotransmitters, norepinephrine, and serotonin from the synapses as a consequence of inhibiting their reuptake into the nerve terminals, resulting in the stimulation of glial cells surrounding the synapses in the brain. Then, we have investigated the direct actions of neurotransmitters on glial cell metabolism and function using rat C6 glioma cells as an in vitro model system and suggested that these neurotransmitters induce their differentiation probably through the production of 5α-reduced neurosteroids. On the other hand, the stimulation of the glioma cells with serotonin has been reported to enhance brain-derived neurotrophic factor (BDNF) gene expression, which may be closely related to the beneficial effects of antidepressant drugs. In the present study, to evaluate BDNF expression in differentiated glial cells, the glioma cells were pretreated with progesterone, and the effect of serotonin on BDNF messenger RNA levels in these cells was examined. Progesterone pretreatment enhanced the stimulatory action of serotonin on BDNF gene expression, and the enhancement of serotonin action observed in the cells pretreated with progesterone was almost completely abolished by finasteride, an inhibitor of the enzyme involved in the production of 5α-reduced neurosteroids. These findings propose the possibility that neurosteroid-mediated glial cell differentiation may result in the enhancement of serotonin-stimulated BDNF gene expression, which is considered to contribute to the survival, regeneration, and plasticity of neuronal cells in the brain, and hence, leading to the improvement of mood disorders and other symptoms in depressive patients.

High-fat diet-induced obesity increases lymphangiogenesis and lymph node metastasis in the B16F10 melanoma allograft model: roles of adipocytes and M2-macrophages.

To examine the effects of high‐fat diet (HFD) on melanoma progression, HFD‐fed C57BL/6N mice were subcutaneously injected with syngeneic B16F10 melanoma cells. At 3 weeks post‐injection, the tumors were resected; the mice were then sacrificed at 2 weeks post‐resection. HFD stimulated melanoma growth and lymph node (LN) metastasis as well as tumor and LN lymphangiogenesis. Lipid vacuoles in the tumor and M2‐macrophage (MΦ)s in the adipose and tumor tissues were increased in HFD‐fed mice. CCL19 and CCL21 contents were higher in LNs than in tumors. HFD increased both CCL19 and CCL21 levels in LNs and CCR7 in tumors. Adipose tissue‐conditioned media (CM) from HFD‐fed mice enhanced lymphangiogenesis, and mature adipocyte (MA)/M2‐MΦ co‐culture CM markedly stimulated the tube formation of lymphatic endothelial cell (LEC)s and B16F10 migration. Monocyte migration was moderately stimulated by B16F10 or MA CM, but tremendously stimulated by B16F10/M2‐MΦ co‐culture CM, which was enhanced by MA/B16F10/M2‐MΦ co‐culture CM. The co‐culture results revealed that MAs increased CCL2, M‐CSF and CCR7 mRNAs in B16F10s; vascular endothelial growth factor (VEGF)‐D mRNA in M2‐MΦs; and CCL19, CCL21 and VEGF receptor (VEGFR)3 mRNA in LECs. M2‐MΦs increased CCL2, M‐CSF and VEGF‐A mRNAs in B16F10s, whereas B16F10s increased VEGF‐C mRNAs in M2‐MΦs and VEGFR3 mRNA in LECs. These results indicate that in HFD‐fed mice, MA‐induced CCL2 and M‐CSF in tumor cells increase M2‐MΦs in tumor; the crosstalk between tumor cells and M2‐MΦs further increases cytokines and angiogenic and lymphangiogenic factors. Additionally, MA‐stimulated CCL19, CCL21/CCR7 axis contributes to increased LN metastasis in HFD‐fed mice.

Histone deacetylase inhibitors promote neurosteroid-mediated cell differentiation and enhance serotonin-stimulated brain-derived neurotrophic factor gene expression in rat C6 glioma cells

Progesterone treatment has previously been reported to promote the differentiation of glial cells probably through the production of 5α‐reduced neurosteroids, resulting in the enhancement of serotonin‐stimulated brain‐derived neurotrophic factor (BDNF) gene expression, which is considered to contribute to the survival, regeneration, and plasticity of neuronal cells in the brain and hence has been suggested to improve mood disorders and other symptoms in depressive patients. Based on these previous observations, the effects on glial cells of histone deacetylase (HDAC) inhibitors, which are known as agents promoting cell differentiation, were examined using rat C6 glioma cells as a model for in vitro studies. Consequently, trichostatin A (TSA), sodium butyrate (NaB), and valproic acid (VPA) stimulated glial fibrillary acidic protein (GFAP) gene expression, and their stimulatory effects on GFAP gene expression were inhibited by treatment of these cells with finasteride, an inhibitor of the enzyme producing 5α‐reduced neurosteroids. In addition, HDAC inhibitors enhanced serotonin‐stimulated BDNF gene expression, the enhancement of which could be abolished by the inhibition of 5α‐reduced neurosteroid production in the glioma cells. These results suggest that HDAC inhibitors may be able to promote the differentiation of rat C6 glioma cells through the production of 5α‐reduced neurosteroids, resulting in the enhancement of serotonin‐stimulated BDNF gene expression as a consequence of promoting their differentiation, indicating the possibility that differentiated glial cells may be implicated in preserving the integrity of neural networks as well as improving the function of neuronal cells in the brain.

Influence of serum-free culture conditions on steroid 5α-reductase mRNA expression in rat C6 glioma cells

Immunocytochemical studies previously showed that serum deprivation resulted in the appearance of steroid 5alpha-reductase (5alpha-R) in the cytoplasm of rat C6 glioma cells. To determine whether this increase in cytoplasmic 5alpha-R was due to changes in 5alpha-R gene expression, the effect of serum deprivation on 5alpha-R mRNA expression was examined. No significant change in the mRNA levels was observed in cells grown in serum-free culture medium. Therefore, the appearance of 5alpha-R immunoreactivity in the cell cytoplasm observed under serum-free conditions is probably not due to changes in 5alpha-R gene expression.