Hypoxia inducible factor-1 (HIF-1) is a key regulator in hypoxia. It has been suggested to be a critical regulator of neurological outcomes following ischemic stroke, due to the functions of its downstream genes in cell death and cell survival. Yet, the mechanisms of HIF-1 induction and regulation in ischemic neurons remain largely unknown. Extensive research in cancer cells suggest that redox status plays a critical role in regulating HIF-1. Our preliminary results showed that redox status might control turning on and off HIF-1 expression in neurons under ischemic conditions. Moreover, our results suggest that HIF-1? protein degradation in hypoxia is possibly through proteasomal pathways activated by reactive oxygen species (ROS). We hypothesize that a reducing environment stabilizes HIF-1? protein in cerebral ischemia because elevated ROS may facilitate HIF- 1? protein degradation through proteasomal pathways, and that HIF-1? expression mediated by redox changes provides potential therapeutic intervention for stroke. This hypothesis will be tested using sequential complementary in vitro followed by in vivo ischemic paradigms. 1) We will test the hypothesis using a primary neuronal culture and a separated coculture of neurons of astrocytes under in vitro ischemic conditions. A causal relationship between redox status and HIF-1 expression will be established in neurons in vitro. 2) We will test the hypothesis with the rat middle cerebral artery occlusion model of ischemic stroke in vivo. We will study the intrinsic relationships between redox environment, HIF-1? expression, and neuronal injury in vivo by neuro-imaging (MRI, electron paramagnetic resonance imaging, two-photon microscopy), biochemical, cellular and molecular techniques. 3) We will test the hypothesis that elevated ROS levels are responsible for the degradation of HIF-1? protein in oxidizing environments under hypoxic conditions by activating proteasomal degradation pathways. These studies will provide a systematic investigation of redox changes, redox-mediated HIF-1? regulation, and neuronal injuries with in vitro and in vivo ischemic models. We believe that this research will determine how HIF-1 is turned on or off by redox status in brain ischemia. This research is expected to assist in developing effective therapies to ameliorate the cellular injuries in stroke patient through the identification of conditions that are optimal for HIF-1 mediated cell survival. Project Narrative This project is to determine how hypoxia inducible factor 1 (HIF-1) is turned on or off by redox status (oxidation and reduction balance in cells) in stroke. This research is expected to assist in developing effective therapies to ameliorate the cellular injuries in stroke patient through the identification of conditions that are optimal for HIF-1 mediated cell survival.
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