Iron chelators are pluripotent neuronal anti-apoptotic agents which have been shown to slow clinical progression in Alzheimer's Disease and enhanced metabolic recovery in cerebral ischemia models. The precise mechanism(s) by which these agents exert their salutary effects remains unclear. In preliminary studies, we have found that structurally distinct iron chelators, deferoxamine mesylate, and mimosine prevent apoptosis induced by glutathione-depletion and oxidative stress in embryonic cortical neurons. We correlated the protective effects of iron chelators with their ability to increase protein expression and enzyme activity of two glycolytic enzymes, lactate dehydrogenase (LDH) and aldolase. The increase in glycolytic enzyme activity induced by iron chelators could be abrogated by actinomycin-D, an mRNA synthase inhibitor, suggesting that the induction is partly transcriptional. consistent with these findings, we demonstrated that iron chelators enhance DNA-binding of two transcription factor complexes, hypoxia- inducible factor-1 (HIF-1) and ATF-1/CREB, in cortical cultures as well as in H19-7 neuronal cell line. Finally, we have shown that message levels for LDH and erythropoietin, genes whose expression is known to be regulated by HIF-1, were elevated in cortical cultures. in response to iron chelator treatment. These preliminary results suggests the following overall hypothesis: iron chelators exert their pluripotent neuroprotective effects, in part by activating a signal transduction pathway that leads to increased expression of genes (e.g., glycolytic enzymes, the growth factor, erythropoietin, or the antioxidant, heme oxygenase) known to compensate for hypoxic or oxidative stress. We propose to test this hypothesis by: 1) determining whether iron chelators enhance heterodimeric HIV-1 (HIF-1 alpha + HIF-1 beta) and/or ATF-1/CREB DNA binding by decreasing peroxide levels; 2) determining the HIF-1 regulated genes induced by iron chelators in embryonic cortical neurons; 3) determining whether enforced expression of HIF-1alpha, HIF- 1beta, ATF-1, or CREB is sufficient for protection from oxidative stress-induced apoptosis; and 4) determining whether HIF-1 is necessary for protection from oxidative stress-induced apoptosis by iron chelators. These studies will define whether HIF-1 is a viable molecular target for therapy of neurological diseases that have been associated with oxidative stress and apoptosis including stroke, Alzheimer's Disease, Parkinson's Disease, and Fredrich's Ataxia. Furthermore, these studies also promise to further define mechanisms of protection by small molecule iron chelators.
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