The proposed studies will investigate nitric oxide (NO)-mediated mechanism of procaspase-9 activation that results in hypoxic neuronal death by transcription-independent and transcription-dependent pathways in the newborn. We will test the hypothesis that NO produced during hypoxia leads to increased phosphorylation at tyrosine 153 (Tyr153) of procaspase-9 and increased phosphorylation at tyrosine 24 (Tyr24) of apoptotic protease activating factor-1 (Apaf-1) resulting in tyrosine phosphorylation-dependent activation of procaspase-9.. We propose that tyrosine phosphorylation at specific residues during hypoxia leads to increased binding of procaspase-9 with Apaf-1 due to increased charge-charge interaction (increased number of H-bonds) between their prodomains and results in increased activation of procaspase-9. We propose that NO free radicals generated during hypoxia lead to inactivation of protein tyrosine phosphatases (PTP, SH-PTP-1 and SH-PTP-2) and result in increased activation of protein tyrosine kinases (PTK, EGFR kinase and Src kinase) and subsequently increased tyrosine phosphorylation of procaspase-9 and Apaf-1. The degree of cerebral tissue hypoxia will be determined by measurement of high energy phosphate compounds. Experimental protocols will be carried out on newborn piglets investigating: (1) the effect of hypoxia on Tyr153 phosphorylation of procaspase-9 and Tyr24 phosphorylation of Apaf-1, expression and activation of procaspase-9 and relate these to the degree of cerebral hypoxia; and perform these studies with the administration of highly selective nNOS inhibitors to demonstrate that the hypoxia-induced increased Tyr153 phosphorylation of procaspase-9, Tyr24 phosphorylation of Apaf-1, expression and increased activation of procaspase-9 are mediated by nNOS-derived NO; (2) the effect of inhibitors of EGFR kinase and Src kinase on increased phosphorylation of procaspase-9 at Tyr153 and Apaf-1 at Tyr24, activation of procaspase- 9 and procaspase-3, nuclear DNA fragmentation and immunocytochemical and morphological indices of neuronal death, to demonstrate that the mechanism of procaspase-9 activation that results in hypoxic neuronal death is dependent on EGFR and Src kinase activity; (3) the effect of tyrosine phosphorylation and dephosphorylation of procaspase-9 in vitro on caspase-9 activity to demonstrate that tyrosine phosphorylation regulates procaspase-9 activation; (4) the effect of NO donor (diethylamine NONOate) and peroxynitrite on the activity of SH-PTP-1 and SH-PTP-2 to demonstrate that NO-free radicals generated during hypoxia lead to inactivation of SH-PTP-1 and SH-PTP-2 resulting in increased activation of EGFR kinase and Src kinase and subsequently increased tyrosine phosphorylation of procaspase-9 and Apaf-1 by a protein tyrosine kinase-dependent mechanism. The proposed studies will establish a novel mechanism of NO-mediated EGFR kinase and Src kinasedependent tyrosine phosphorylation of procaspase-9 at Tyr153 and Apaf-1 at Tyr24 leading to increased activation of procaspase-9 that results in hypoxic neuronal death. The elucidation of NO-mediated tyrosine phosphorylation-dependent molecular mechanism of procaspase-9 activation during hypoxia will aid in the development of novel tyrosine-kinase inhibition based preventive strategies for hypoxia-induced brain dysfunction in the newborn.
