Phosphorylation of caspase-9 in the cytosolic fraction of the cerebral cortex of newborn piglets following hypoxia

We have previously shown that hypoxia leads to increased expression and increased activity of caspase-9 in the cerebral cortex of newborn piglets. Previous studies have demonstrated the importance of caspase-9 in the initiation of the apoptotic cascade, however, the mechanism of caspase-9 activation is not well understood. Experiments were conducted on newborn piglets 2-3 days of age that were anesthetized and mechanically ventilated. Hypoxia was induced by lowering the FiO₂ to 0.05-0.07× 1 h, and was confirmed biochemically by demonstrating decreased levels of ATP and PCr in the hypoxic groups in comparison with the normoxic group. The ATP level was 1.99 ± 0.66 in the hypoxic group versus 4.10 ± 0.19 in the normoxic group, P < 0.05, and the PCr value was 0.68 ± 0.14 in the hypoxic group, compared to 2.98 ± 0.39 in the normoxic group, P < 0.05. The cytosol of the neuronal nuclei from the cerebral cortex was probed with anti-phosphorylated Ser¹⁹⁶ caspase-9 antibody, using Western blot analysis. Protein bands were analyzed using image densitometry. In both the hypoxic and normoxic samples, protein bands were demonstrated just above the 50 kDa marker. Phosphorylated caspase-9 expression in OD × mm² was 43.85 ± 8.4 in the normoxic group and 67.6 ± 9.88 in the hypoxic group, P < 0.05. The results of this study demonstrate that caspase-9, a key protein in hypoxia induced apoptosis, is phosphorylated at the Ser¹⁹⁶ site during hypoxia. The results demonstrate that hypoxia results in a post-translational modification of caspase-9 at Ser¹⁹⁶, which may alter the activity of caspase-9 in the hypoxic newborn brain.