Mechanisms of expression of apoptotic protease activating factor-1 (Apaf-1) in nuclear, mitochondrial and cytosolic fractions of the cerebral cortex of newborn piglets

Apoptotic protease activating factor-1 (Apaf-1) is a critical regulator of apoptosis and a crucial part of the apoptosome that is assembled in response to several cellular stresses like hypoxia. We have previously shown that hypoxia results in increased influx of nuclear Ca2+ and increased expression of nuclear apoptotic proteins. The present study investigates that Apaf-1 is expressed during hypoxia in the cerebral cortex of newborn piglets and that administration of clonidine prevents the hypoxia induced increase expression of Apaf-1. Studies were conducted in 19 newborn piglets, 6 normoxic (Nx), 7 hypoxic (Hx FiO2 of 0.05-0.07 for 1 h) and 6 clonidine-treated hypoxic (Hx-Clo) piglets. Tissue hypoxia was confirmed biochemically by determining the levels of high energy phosphates ATP and phosphocreatine (PCr). Neuronal nuclei, mitochondrial membranes and cytosolic fractions were isolated and separated by 12% SDS-PAGE and probed with specific antibodies to Apaf-1. The expression of Apaf-1 in neuronal nuclei was 48.86 ± 5.27 in Nx, 108.43 ± 6.37 in Hx and 78.53 ± 7.00 in Hx-Clo. The Apaf-1 expression of in mitochondrial fraction was 72.73 ± 11.76 in Nx, 132.27 ± 16.15 in Hx and 85.17 ± 5.64 in Hx-Clo. Similarly, the expression of Apaf-1 in cytosolic fraction was 86.79 ± 6.97 in Nx, 193.95 ± 15.41 in Hx and 111.07 ± 7.91 in Hx-Clo. In summary, the results show that hypoxia results in increased expression of Apaf-1 proteins in neuronal nuclear, mitochondrial and cytosolic fractions. Administration of a high affinity Ca2+-ATPase, prevented the hypoxia induced increased expression of Apaf-1 protein, suggesting that the hypoxia-induced increased expression of Apaf-1 proteins is nuclear Ca2+-influx mediated. We conclude that cerebral hypoxia-induced increase in Apaf-1 protein will lead to increased activation of procaspase-9 to caspase-9 in the cytosolic compartment leading to a cascade of hypoxic neuronal death.