The long-term objective of our research program is to elucidate cellular and molecular mechanisms of brain injury induced by hypoxia in the newborn. The present study will focus on the mechanisms of procaspase-9 activation, a key step for initiating programmed cell death, during hypoxia. We hypothesize that during hypoxia, activation of procaspase-9 is due to increased expression and to post-translational modification of apoptotic proteins leading to an increased ratio of proapoptotic/antiapoptotic proteins (Bax/Bcl-2) in the cytosolic compartment. In addition, we hypothesize that excess Bax protein activates procaspase-9 through apoptotic protease activating factor-1 (Apaf-1). We propose that the hypoxia-induced increase in intranuclear Cainflux leading to increased expression of the proapoptotic proteins, Bax and Bad, is mediated through the increased activation of the Ca++-dependent kinase IV (CaM kinase IV) cascade, and results in phosphorylation of cyclic AMP-responsive element binding (CREB) protein at ser 133. In addition, we propose that hypoxia induces a modification of the affinity of Apaf-1 binding domains for ATP and cytochrome c favoring Apaf-1-mediated activation of procaspase-9. Using established techniques and the newborn piglet model, we will demonstrate that during hypoxia a) the increased expression of the proapoptotic protein Bax mediates activation of the apoptosome complex (procaspase-9-Apaf-1-Bcl-2/Bax) resulting in the activation of procaspase-9, b) the increased expression of proapoptotic proteins Bax and Bad is due to increased activation of the calcium-dependent CaM kinase IV cascade and increased phosphorylation of CREB in neuronal nuclei of newborn piglets, c) the hypoxia-induced modification of modulatory sites of the Apaf-1 molecule for ATP and cytochrome c and Bcl-2/Bax results in activation of procaspase-9 and d) by inhibiting nuclear Cainflux, inhibiting synthesis of proapoptotic proteins and inhibiting caspase-9 activity, we will prevent hypoxia-induced neuronal death in the newborn piglet brain. The proposed studies will establish a link between the hypoxia-induced increased expression of proapoptotic proteins and the modification of Apaf-1 binding domains, with activation of procaspase-9, a critical step that initiates the cascade of programmed neuronal death in the newborn brain.
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