Long-term Continuous CORT treatment decreases Flk1 protein levels <i>in vitro</i> and <i>in vivo</i>.

<p>(<i>A</i>) CORT (CORT; I µM) was applied to mouse primary cortical neurons at DIV 5. Flk1 protein levels were determined by western blotting analysis at 48 hand 72 h following CORT treatment. CON means DMSO treatment. Data represent mean±SE. (<i>n</i> = 6) expressed as fold change in Flk1 protein levels as compared to CON. β-actin is the loading control. *<i>P</i><0.05 (Bonferroni's test). (<i>B</i>) Flk1 protein levels in frontal cortex of mice treated with CORT or vehicle control (CON; 0.45% hydroxypropyl-β-cyclodextrin) for 7 weeks. Data represent mean±SE (<i>n</i> = 6–8) expressed as fold change in Flk1 protein levels as compared to CON. *<i>P</i><0.01 (t test).</p

Chronic CORT-induced Flk1 regulation is mediated through calcium.

<p>(<i>A</i>) Calcium chelator BAPTA-AM blocked CORT (CORT)-induced reduction in Flk1 protein levels. BAPTA-AM (50 µM) was applied 30 min before CORT (1 µM) treatment to cultured neurons at DIV 5. Cell lysates were collected at 48 h after CORT treatment and Flk1 protein levels were determined by western blot analysis. CON means DMSO treatment. Data represent mean±SE (<i>n</i> = 5) expressed as fold change in Flk1 protein levels as compared to CON. *<i>P</i><0.01 versus CON; #<i>P</i><0.01 versus CORT (Bonferroni's test). (<i>B</i>) Chronic CORT treatment increases NCS-1 protein levels in neurons. CORT (CORT; 1 µM) was applied to mouse primary cortical neurons at DIV 5. NCS-1 protein levels were determined by western blotting analysis at 48 h following CORT treatment. CON means DMSO treatment. Data represent mean±SE (<i>n</i> = 5) expressed as fold change in NCS-1 protein levels as compared to CON. *<i>P</i><0.01 (Bonferroni's test). (C) Chronic CORT treatment increases NCS-1 protein levels in mouse frontal cortex. NCS-1 protein levels in frontal cortex of mice treated with CORT (5 mg/kg) or vehicle control (CON; 0.45% hydroxypropyl-β-cyclodextrin) for 7 weeks were determined by western blot analysis. Data represent mean±SE (<i>n</i> = 6) expressed as fold change in NCS-1 protein levels as compared to CON. β-actin is the loading control.*<i>P</i><0.05 (Bonferroni's test).</p

GR downregulation is involved in chronic CORT-induced downregulation of Flk1.

<p>(A) GR downregulation following chronic CORT exposure in neurons. CORT (CORT; 1 µM) was applied to mouse primary cortical neurons at DIV 5. GR protein levels were determined by western blotting analysis at 48 h following CORT treatment. CON means DMSO treatment. Data represent mean±SE (<i>n</i> = 5) expressed as fold change in GR protein levels as compared to CON. *<i>P</i><0.05 (t test). (B) Chronic CORT treatment increases GR protein levels in mouse frontal cortex. GR protein levels in frontal cortex of mice treated with CORT (5 mg/kg) or vehicle control (CON; 0.45% hydroxypropyl-β-cyclodextrin) for 7 weeks were determined by western blot analysis. Data represent mean±SE (<i>n</i> = 5) expressed as fold change in GR protein levels as compared to CON. β-actin is the loading control.*<i>P</i><0.05 (<i>t</i> test). (C) RU486 (RU, a GR antagonist) blocked CORT-induced reduction in GR protein levels. RU (1 µM) was applied 30 min before CORT (1 µM) treatment to cultured neurons at DIV5. Cell lysates were collected at 48 h after CORT treatment and GR protein levels were determined by western blot analysis. CON means DMSO treatment. Data represent mean±SE (<i>n</i> = 5) expressed as fold change in GR protein levels as compared to CON. *<i>P</i><0.01 versus CON; #<i>P</i><0.01 versus CORT (Bonferroni's test). (D) RU486 (RU, a GR antagonist) blocked CORT-induced reduction in Flk1 protein levels. Data represent mean±SE (<i>n</i> = 5) expressed as fold change in Flk1 protein levels as compared to CON. *<i>P</i><0.01 versus CON; #<i>P</i><0.01 versus CORT (Bonferroni's test). (E) Western blot analysis of Flk1 protein expression after immunoprecipitation with GR antibody in lysates collected from DIV6 neurons. NoAb: no anti-GR antibody; total: 10% input from total cell lysates. (F) Western blot analysis of GR protein expression after immunoprecipitation with Flk1 antibody in lysates collected from DIV6 neurons. NoAb: no anti-Flk1 antibody. (G) Immunoprecipitation of Flk1 in cell lysates from corticosterone (CORT) or vehicle control (CON; DMSO) treated for 48 h. Western blotting was performed with anti-GR and anti-Flk1 antibodies. Data represent mean±SE (n = 4) expressed as fold change in GR protein levels (normalized to Flk1 protein levels) as compared to CON. *<i>P</i><0.05 versus CON (<i>t</i> test).</p

Demographic data for postmortem samples.

<p>Demographic data for postmortem samples.</p

Long-term Continuous CORT treatment increases VEGF protein levels <i>in vitro</i> and <i>in vivo</i>.

<p>(<i>A</i>) CORT (CORT; 1 µM) was applied to mouse primary cortical neurons at DIV 5. VEGF protein levels were determined by western blotting analysis at 48 hand 72 h following CORT treatment. CON means DMSO treatment. Data represent mean±SE (<i>n</i> = 6) expressed as fold change in VEGF protein levels as compared to CON. *<i>P</i><0.05 (Bonferroni's test). (<i>B</i>) VEGF protein levels in frontal cortex of mice treated with CORT (5 mg/kg) or vehicle control (CON; 0.45% hydroxypropyl-β-cyclodextrin) for 7 weeks were determined by western blot analysis. Data represent mean±SE (<i>n</i> = 5) expressed as fold change in VEGF protein levels as compared to CON. *<i>P</i><0.01 (t test). <i>(C)</i> VEGF protein levels in serum samples collected from mice treated with CORT (CORT; 5 mg/kg) or vehicle control (CON; 0.45% hydroxypropyl-β-cyclodextrin) for 7 weeks were analysed by ELISA. Data represent mean±SE (<i>n</i> = 5–6) expressed as fold change in VEGF protein levels as compared to CON. *<i>P</i><0.01 (t test).</p

Proposed model showing the effects of corticosterone on VEGF/Flk1 signaling pathway in mouse frontal cortex.

<p>The signaling events induced by corticosterone (CORT) are mediated through the Glucocorticoid receptor (GR). The reduction in Flk1 levels following long-term continuous CORT exposure results in the activation of PTEN, but inhibition of Akt and mTOR phosphorylation. The effects of CORT on Flk1 are mediated through calcium (Ca²⁺). CORT exposure results in increased levels of VEGF in cortex. The role of VEGF in Flk1 regulation (or vice versa) under CORT exposure remains unknown. Solid arrows represent activation, whereas dashed arrows represent inhibition of the pathways.</p

Effects of chronic corticosterone treatment on TrkB mRNA levels in the frontal cortex and hippocampus.

<p>CD-1 male mice were treated with corticosterone (CORT; 35 ug/ml/day) or vehicle (0.45% hydroxypropyl-β-cyclodextrin) for 7 weeks. TrkB mRNA levels were determined by qRT-PCR analysis. The level of TrkB mRNA was normalized to that of RPS3 RNA in the same sample. Values are expressed as fold change relative to vehicle-treated mice. Open and filled bars represent vehicle and corticosterone-treated groups, respectively. Error bars represent standard Error (SE) of n = 4 mice per group.</p

PI3K signaling is not involved in long-term continuous CORT-induced increases in VEGF protein levels.

<p>(<i>A</i>) VEGF protein levels were determined by western blotting analysis in neuronal cell lysates treated with LY294002 (LY; 20 µM) for 48 h. CON means DMSO treatment. Data represent mean±SE (<i>n</i> = 6) expressed as fold change in VEGF protein levels as compared to CON. *<i>P</i><0.05 (Bonferroni's test). (<i>B</i>) Pretreatment with LY did not prevent CORT-induced induction in VEGF protein levels in neurons. Cortical neurons at DIV 5 were treated with LY (20 µM) for 30 min followed by CORT (1 µM) exposure for 48 h. VEGF protein levels were determined in cell lysates by western blot analysis. CON means DMSO treatment. Data represent mean±SE (<i>n</i> = 6) expressed as fold change in VEGF protein levels as compared to CON. *<i>P</i><0.05 (Bonferroni's test).</p

Effects of cysteamine in chronic corticosterone-treated mice on proBDNF and mature BDNF (mBDNF) protein levels in the frontal cortex and hippocampus.

<p>CD-1 male mice were treated for 7 weeks with vehicle (0.45% hydroxypropyl-β-cyclodextrin) or corticosterone (CORT; 35 ug/ml) in the presence or absence of cysteamine (CYS; 150 mg/kg/day) during the last three weeks of corticosterone treatment. proBDNF and mBDNF protein levels were determined in the (A) frontal cortex and (B) hippocampus by Western blot analysis. The upper panels shows a representative autoradiogram of proBDNF and mBDNF and the lower panel represents the fold change in optical density values normalized to vehicle-treated controls. β-actin was used as a protein loading control. Values are mean ± SE (n = 6 mice per group). (C) BDNF protein levels as measured by ELISA in frontal cortex samples from mice treated with vehicle or cysteamine for 3 weeks as above. Data represent the fold change in BDNF protein levels (pg/mg protein) normalized to vehicle-treated controls. Values are mean ± SE (n = 5 mice per group).</p

Effects of chronic corticosterone treatment on TrkB protein levels in the frontal cortex.

<p>CD-1 male mice were treated with corticosterone (CORT; 35 ug/ml/day) or vehicle (0.45% hydroxypropyl-β-cyclodextrin) for (A) 3, (B) 5 or (C) 7 weeks. TrkB protein levels were determined by Western blot analysis. The upper panel shows a representative autoradiogram of TrkB and the lower panel represents the fold change in optical density values normalized to vehicle-treated controls. β-actin was used as a protein loading control. Values are mean ± SE (n = 5–6 mice per group). *p<0.05 versus vehicle.</p