The purpose of this proposal is to elucidate hypoxia-induced Akt-related pathways induced in neuronal PC- 12 and RN46A neuronal cells by sustained hypoxia and by intermittent hypoxia, and to determine how these signaling pathways affect cell survival. We will test the hypothesis that hypoxia-induced interactions between different signaling molecules within the Akt signaling module, modulate tolerance or vulnerability to sustained and intermittent hypoxia in neuronal cells. We propose: (1) To identify, using proteomic approaches, members of the Akt signaling module in normoxic and hypoxic neuronal cells, and to identify differences in Akt-associated proteins during sustained and intermittent hypoxia; (2) To characterize protein-protein interactions within the Akt signaling modules; (3) To identify selective Akt - protein interactions within the signaling module that underlie neuronal survival to sustained and intermittent hypoxia; (4). To examine the effect of disruption of protein-protein interactions within the Akt signaling module on downstream genes involved in regulation of hypoxia-induced cellular apoptosis. We will use proteomic techniques, SDS-PAGE and MALDI-MS to identify the Akt-binding proteins co-immunoprecipitating with Akt in normoxic cells or cells exposed to sustained or intermittent hypoxia. Interactions of the components of the Akt signalosomes will be determined by TnT coupled transcription, translation, co-immunoprecipitation, and GST pull-down methods. Transformer kits will be used to make in frame and serial deletion mutants of Akt and its binding proteins in order to identify their Akt docking sites. Finally we will introduce into the cells TAT-fusion peptides corresponding to specific docking sites of targeted proteins binding to Akt signaling module, and disrupt their interaction with the Akt signaling complex to assess the effect of protein association/dissociation with the Akt signaling module, on cell survival to sustained and intermittent hypoxia. These studies will provide the groundwork for future intervention strategies aiming to prevent neuronal cell loss in diseases associated with intermittent and sustained hypoxia, such as sleep apnea and lung disease.
