Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in adaptation to hypoxic stress. Previous studies from our laboratory demonstrated that pharmacological activators of HIF-1 (e.g. deferoxamine, cobalt chloride) could also protect cultured primary neurons or an immortalized hippocampal neuroblast line (HT22) from oxidative stress-induced death. However, whether HIF-1 activation is sufficient to abrogate neuronal death resulting from oxidative stress or other hypoxia-independent death inducers remains unclear. To address this question we utilized a HIF-1␣ fusion protein that partially lacks the domain required for oxygen-dependent degradation of HIF-1␣ and that has a VP16 transcriptional activation domain from herpes simplex virus. HT22 cells were infected with a retrovirus encoding either the HIF-1␣-VP16 fusion protein or the activation domain of the VP16 protein alone as a control. Expression of HIF-1␣-VP16, but not VP16 alone, increased luciferase activity driven by a canonical hypoxia response element, increased mRNA of established HIF-1 target genes, and increased activity of one of these HIF-1 target genes. Unexpectedly, enhanced HIF-1 activity in HT22 cells enhanced sensitivity to oxidative death induced by glutathione depletion. Accordingly, suppression of HIF-1␣ expression using RNA interference prevented oxidative death. By contrast, HIF-1␣-VP16-expressing HT22 cells were more resistant to DNA damage (induced by camptothecin) or endoplasmic reticulum stress (induced by thapsigargin and tunicamycin) than were VP16-expressing cells, and suppression of HIF-1␣ expression using RNA interference rendered HT22 cells more sensitive to death induced by DNA damage or endoplasmic reticulum stress. Together, these data demonstrate that HIF-1 can mediate prodeath or prosurvival responses in the same cell type depending on the injury stimulus.

Oxidative stress contributes to tissue injury in conditions ranging from cardiovascular disease to stroke, spinal cord injury, neurodegeneration, and perhaps even aging. Yet the efficacy of antioxidants in human disease has been mixed at best. We need a better understanding of the mechanisms by which established antioxidants combat oxidative stress. Iron chelators are well established inhibitors of oxidative death in both neural and non-neural tissues, but their precise mechanism of action remains elusive. The prevailing but not completely substantiated view is that iron chelators prevent oxidative injury by suppressing Fenton chemistry and the formation of highly reactive hydroxyl radicals. Here, we show that iron chelation protects, rather unexpectedly, by inhibiting the hypoxia-inducible factor prolyl 4-hydroxylase isoform 1 (PHD1), an iron and 2-oxoglutarate-dependent dioxygenase. PHD1 and its isoforms 2 and 3 are best known for stabilizing transcriptional regulators involved in hypoxic adaptation, such as HIF-1␣ and cAMP response element-binding protein (CREB). Yet we find that global hypoxia-inducible factor (HIF)-PHD inhibition protects neurons even when HIF-1␣ and CREB are directly suppressed. Moreover, two global HIF-PHD inhibitors continued to be neuroprotective even in the presence of diminished HIF-2␣ levels, which itself increases neuronal susceptibility to oxidative stress. Finally, RNA interference to PHD1 but not isoforms PHD2 or PHD3 prevents oxidative death, independent of HIF activation. Together, these studies suggest that iron chelators can prevent normoxic oxidative neuronal death through selective inhibition of PHD1 but independent of HIF-1␣ and CREB; and that HIF-2␣, not HIF-1␣, regulates susceptibility to normoxic oxidative neuronal death.

Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases are a family of iron-and 2-oxoglutarate-dependent dioxygenases that negatively regulate the stability of several proteins that have established roles in adaptation to hypoxic or oxidative stress. These proteins include the transcriptional activators HIF-1␣ and HIF-2␣. The ability of the inhibitors of HIF prolyl 4-hydroxylases to stabilize proteins involved in adaptation in neurons and to prevent neuronal injury remains unclear. We reported that structurally diverse low molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases stabilize HIF-1␣ and up-regulate HIF-dependent target genes (e.g. enolase, p21 waf1/cip1 , vascular endothelial growth factor, or erythropoietin) in embryonic cortical neurons in vitro or in adult rat brains in vivo. We also showed that structurally diverse HIF prolyl 4-hydroxylase inhibitors prevent oxidative death in vitro and ischemic injury in vivo. Taken together these findings identified low molecular weight and peptide HIF prolyl 4-hydroxylase inhibitors as novel neurological therapeutics for stroke as well as other diseases associated with oxidative stress.Iron maintains a unique role in physiology via its ability to change readily its oxidation state in response to changes in its local environment. A general simplification of its primary function is that it mediates one-electron redox reactions. This chemical property of iron enables it to act as an essential component in several biological activities, including as a cofactor for enzymes such as tyrosine hydroxylase. Oxygen binding to biomolecules such as hemoglobin and myoglobin is also coordinated by iron. Indeed iron deficiency can lead to a host of disorders, including anemia and restless legs syndrome (1).Paradoxically, the biochemical properties that make iron beneficial in many biological processes appear to be a drawback when the balance between its accumulation/sequestration within cellular compartments and its release is disturbed in favor of iron accumulation (2). Indeed, iron overload is associated with several neurological conditions (3-5). For example, the iron content of nigral Lewy bodies is elevated in patients with Parkinson disease (6 -9). Alzheimer disease has also been found to be associated with an increase in the iron content of senile plaques (10 -15). Accumulation of mitochondrial iron has been shown to play a role in Friedrich ataxia (16,17). Similarly, changes in intracellular free iron levels have been observed in cerebral ischemia (18 -20). Direct evidence that disrupted iron homeostasis contributes to injury rather than simply being caused by it has been obtained by treatment with low molecular weight iron chelators or by overexpression of iron storage proteins. Small molecule iron chelators such as deferoxamine mesylate (DFO) 2 inhibit neuronal injury in rodent models of stroke (21), Parkinson disease (22), and multiple sclerosis (23). Moreover, DFO and some other metal chelators such as clioquinol have been shown to slow the progressi...

Studies of adaptive mechanisms to hypoxia led to the discovery of the transcription factor called hypoxia inducible factor (HIF). HIF is a ubiquitously expressed, heterodimeric transcription factor that regulates a cassette of genes that can provide compensation for hypoxia, metabolic compromise, and oxidative stress including erythropoietin, vascular endothelial growth factor, or glycolytic enzymes. Diseases associated with oxygen deprivation and consequent metabolic compromise such as stroke or Alzheimer's disease may result from inadequate engagement of adaptive signaling pathways that culminate in HIF activation. The discovery that HIF stability and activation are governed by a family of dioxygenases called HIF prolyl 4 hydroxylases (PHDs) identified a new target to augment the transcriptional activity of HIF and thus the adaptive machinery that governs neuroprotection. PHDs lose activity when cells are deprived of oxygen, iron or 2-oxoglutarate. Inhibition of PHD activity triggers the cellular homeostatic response to oxygen and glucose deprivation by stabilizing HIF and other proteins. Herein, we discuss the possible role of PHDs in regulation of both HIF-dependent and -independent cell survival pathways in the nervous system with particular attention to the co-substrate requirements for these enzymes. The emergence of neuroprotective therapies that modulate genes capable of combating metabolic compromise is an affirmation of elegant studies done by John Blass and colleagues over the past five decades implicating altered metabolism in neurodegeneration.