Calcineurin activation contributes to Hcy-induced dephosphorylation and mitochondrial translocation of Bad.

<p>Cells were pretreated with 1 µM CsA or 10 µM BAPTA-AM for 30 min before treatment with 500 µM Hcy (<i>Hcy</i>) for 24 h. A, Total Bad and phospho-Bad levels were assessed by western blotting and densitometric analysis. B, Western blot analysis of Bad in mitochondrial (Mit) and cytoplasmic (Cyto) fractions from hippocampal neurons. C, Percentage of total Bad (cytoplasmic +mitochondrial fractions) localized to the mitochondrial fraction was determined by densitometric analysis of the data from B. Results are expressed as the mean ± S.E.M. of three experiments. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group.</p

Calcineurin activation and 14-3-3ε suppression together contribute to Hcy-induced apoptosis.

<p>Hippocampal neurons transduced with lentiviral 14-3-3ε for 48 h were then pretreated with 1 µM CsA for 30 min before treatment with 500 µM Hcy (<i>Hcy</i>) for 24 h. A, Total Bad and phospho-Bad levels were assessed by western blotting and densitometric analysis. B, Western blot analysis of Bad in mitochondrial (Mit) and cytoplasmic (Cyto) fractions from hippocampal neurons. C, Percentage of total Bad (cytoplasmic+mitochondrial fractions) localized in the mitochondrial fraction as determined by densitometric analysis of the data from B. D, The level of apoptosis was determined by counting the number of neurons with apoptotic nuclei. Four randomly selected fields of Hoechst-stained cells were counted and the average percentage of apoptotic cells per total number of cells was determined. E, Cleaved PARP was measured using western blotting. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group. ^Δ<i>P</i><0.05 vs. CsA group.</p

Hcy induces apoptosis and suppresses 14-3-3ε expression in cultured hippocampal neurons.

<p>A, Cultures were exposed for 24 h to either saline (<i>Con</i>) or 500 µM Hcy (<i>Hcy</i>) and were subsequently stained with the fluorescent DNA-binding dye Hoechst 33342 (<i>top</i>) or photographed under phase-contrast optics (<i>bottom</i>). Note the nuclear DNA condensation and fragmentation in neurons exposed to Hcy (arrow). Scale bar: 100 µm. B, Cultures were exposed to the indicated concentrations of Hcy for 24 h, and the percentages of neurons with apoptotic nuclei were quantified. Values are the mean ±S.E.M. of counts made in four to six cultures. C, Cells were treated as in B, and the levels of cleaved PARP were determined by western blotting. Top, representative western blots. Bottom, densitometric analysis of cleaved PARP normalized by β-actin (<i>n</i> = 3). D, 14-3-3ε mRNA and protein was measured by real-time quantitative PCR (<i>top</i>) and western blotting (<i>mid and bottom</i>), respectively. *<i>P</i><0.05 vs. control.</p

The effect of lentiviral 14-3-3ε transduction on apoptosis in hippocampal neurons.

<p>Hippocampal neurons transduced with lentiviral 14-3-3ε for 48 h were treated with Hcy for 24 h. A, 14-3-3ε mRNA and proteins were analyzed by real-time quantitative PCR or western blotting, respectively. B, The percentages of neurons with apoptotic nuclei (Hoechst staining) were quantified. C, Cleaved PARP was determined by western blotting. D, Total Bad and phospho-Bad levels were assessed by western blotting and densitometric analysis. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group.</p

Activation of calcineurin by Hcy and inhibition by specific blockers.

<p>A, Cultures were exposed to the indicated concentrations of Hcy (<i>Hcy</i>) for 24 h, and cellular calcineurin activity was then determined. B, Cells were pretreated with 1 µM CsA or 10 µM BAPTA-AM for 30 min before exposed to 500 µM Hcy (<i>Hcy</i>). Cellular calcineurin activity was measured at the indicated time periods after the addition of Hcy to the cells. Values represents the mean ± S.E.M. of four or more assays. C, Cells were pretreated with 1 µM CsA or 10 µM BAPTA-AM for 30 min before exposed to 500 µM Hcy (<i>Hcy</i>) for 24h. Calcineurin A (Calcineurin) protein level was determined by western blotting. Top, representative western blots. Bottom, densitometric analysis. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group.</p

Inhibition of Hcy-induced apoptosis by a calcineurin inhibitor or intracellular Ca²⁺ chelator.

<p>Cells were pretreated with 1 µM CsA or 10 µM BAPTA-AM for 30 min before treatment with 500 µM Hcy (<i>Hcy</i>) for 24 h. A, The level of apoptosis was determined by counting the number of neurons with apoptotic nuclei. Four randomly selected fields of Hoechst-stained cells were counted and the average percentage of apoptotic cells per total number of cells was determined. B, Cleaved PARP was studied by western blotting. Results are expressed as the mean ± S.E.M. of three experiments. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group.</p

Hypoxia-inducible factor targets in the brain were still highly upregulated following E3d HPC.

<p>The mRNA expression levels of selected HIF targets in the region supplied by the MCA after the last hypoxic exposure are shown. A single episode of HPC, E3d HPC and E6d HPC significantly increased the expression of EPO (A), VEGF (B), MCT4 (C) and GLUT-1 (D). There was no significant difference between HPC groups. Data are shown as mean ± SD; <i>n</i> = 6 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.01 compared to the control.</p

E3d HPC failed to provide protection against cerebral ischemia.

<p>Stroke volume was evaluated 72 hours after MCAO. E3d HPC failed to protect from ischemia 3 days after the last hypoxic exposure (A). Neurological deficits were also determined 72 hours after MCAO. Prominent improvements were observed 3 days after the last hypoxic exposure in the HPC and E6d HPC group (C, D). However, E3d HPC failed to ameliorate the neurological deficits induced by MCAO (C). Data are shown as mean ± SD; <i>n</i> = 6–8 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.01 compared to the control.</p

E3d HPC-induced reduction of extracellular adenosine levels were totally reversed by ENT1 inhibitor NBTI.

<p>After the last hypoxic exposure, animals received either CD73 inhibitor (AOPCP) or ENT1 inhibitor (NBTI) through the microdialysis infusate. AOPCP blocked the elevation of extracellular adenosine levels induced by HPC or E6d HPC (A), while NBTI totally reversed the E3d HPC-induced extracellular adenosine reduction (B). Data are shown as mean ± SD; <i>n</i> = 6 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.05 compared to the control.</p

E3d HPC reversed the upregulation of CD39 and CD73 and induced ENT-1 protein expression.

<p>Consistent with our mRNA data, a single episode of HPC and E6d HPC significantly increased the protein expression levels of CD39 (A) and CD73 (B) and had no effect on ENT-1 (C) expression. E3d HPC had the opposite effect, having no effect on CD39 (A) and CD73 (B) expression, but significantly increasing ENT-1 (C) protein expression. Representative Western blots are shown in panel D. Data are shown as mean ± SD; <i>n</i> = 6 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.05 compared to the control.</p