The crucial role of vitamin C and its transporter (SVCT2) in bone marrow stromal cell autophagy and apoptosis

AbstractVitamin C is an antioxidant that plays a vital role in various biological processes including bone formation. Previously, we reported that vitamin C is transported into bone marrow stromal cells (BMSCs) through the sodium dependent Vitamin C Transporter 2 (SVCT2) and this transporter plays an important role in osteogenic differentiation. Furthermore, this transporter is regulated by oxidative stress. To date, however, the exact role of vitamin C and its transporter (SVCT2) in ROS regulated autophagy and apoptosis in BMSCs is poorly understood. In the present study, we observed that oxidative stress decreased survival of BMSCs in a dose-dependent manner and induced growth arrest in the G1 phase of the cell cycle. These effects were accompanied by the induction of autophagy, confirmed by P62 and LC3B protein level and punctate GFP-LC3B distribution. The supplementation of vitamin C significantly rescued the BMSCs from oxidative stress by regulating autophagy. Knockdown of the SVCT2 transporter in BMSCs synergistically decreased cell survival even under low oxidative stress conditions. Also, supplementing vitamin C failed to rescue cells from stress. Our results reveal that the SVCT2 transporter plays a vital role in the mechanism of BMSC survival under stress conditions. Altogether, this study has given new insight into the role of the SVCT2 transporter in oxidative stress related autophagy and apoptosis in BMSCs

Cysteamine Attenuates the Decreases in TrkB Protein Levels and the Anxiety/Depression-Like Behaviors in Mice Induced by Corticosterone Treatment

OBJECTIVE: Stress and glucocorticoid hormones, which are released into the circulation following stressful experiences, have been shown to contribute significantly to the manifestation of anxiety-like behaviors observed in many neuropsychiatric disorders. Brain-derived neurotrophic factor (BDNF) signaling through its receptor TrkB plays an important role in stress-mediated changes in structural as well as functional neuroplasticity. Studies designed to elucidate the mechanisms whereby TrkB signaling is regulated in chronic stress might provide valuable information for the development of new therapeutic strategies for several stress-related psychiatric disorders. MATERIALS AND METHODS: We examined the potential of cysteamine, a neuroprotective compound to attenuate anxiety and depression like behaviors in a mouse model of anxiety/depression induced by chronic corticosterone exposure. RESULTS: Cysteamine administration (150 mg/kg/day, through drinking water) for 21 days significantly ameliorated chronic corticosterone-induced decreases in TrkB protein levels in frontal cortex and hippocampus. Furthermore, cysteamine treatment reversed the anxiety and depression like behavioral abnormalities induced by chronic corticosterone treatment. Finally, mice deficient in TrkB, showed a reduced response to cysteamine in behavioral tests, suggesting that TrkB signaling plays an important role in the antidepressant effects of cysteamine. CONCLUSIONS: The animal studies described here highlight the potential use of cysteamine as a novel therapeutic strategy for glucocorticoid-related symptoms of psychiatric disorders

Long-Term Continuous Corticosterone Treatment Decreases VEGF Receptor-2 Expression in Frontal Cortex

Objective: Stress and increased glucocorticoid levels are associated with many neuropsychiatric disorders including schizophrenia and depression. Recently, the role of vascular endothelial factor receptor-2 (VEGFR2/Flk1) signaling has been implicated in stress-mediated neuroplasticity. However, the mechanism of regulation of VEGF/Flk1 signaling under longterm continuous glucocorticoid exposure has not been elucidated. Material and Methods: We examined the possible effects of long-term continuous glucocorticoid exposure on VEGF/Flk1 signaling in cultured cortical neurons in vitro, mouse frontal cortex in vivo, and in post mortem human prefrontal cortex of both control and schizophrenia subjects. Results: We found that long-term continuous exposure to corticosterone (CORT, a natural glucocorticoid) reduced Flk1 protein levels both in vitro and in vivo. CORT treatment resulted in alterations in signaling molecules downstream to Flk1 such as PTEN, Akt and mTOR. We demonstrated that CORT-induced changes in Flk1 levels are mediated through glucocorticoid receptor (GR) and calcium. A significant reduction in Flk1-GR interaction was observed following CORT exposure. Interestingly, VEGF levels were increased in cortex, but decreased in serum following CORT treatment. Moreover, significant reductions in Flk1 and GR protein levels were found in postmortem prefrontal cortex samples from schizophrenia subjects. Conclusions: The alterations in VEGF/Flk1 signaling following long-term continuous CORT exposure represents a molecula

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

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

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

Effects of cysteamine in chronic corticosterone-treated mice in the Open Field test.

<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 the corticosterone treatment. (A) Mean total of the time-spent in the center for the entire session, (B) the ambulatory counts for each 5 min period, (C) the total ambulatory distance and (D) the ambulatory distance in the center over total. Values plotted are mean ± SEM (n = 8–10 per group). Bonferroni's post hoc test. *p<0.05 versus vehicle and ^#p<0.05 versus CORT.</p