Neurologic conditions including stroke, Alzheimer’s disease, Parkinson’s disease and Huntington’s disease are leading causes of death and long-term disability in the United States, and efforts to develop novel therapeutics for these conditions have historically had poor success in translating from bench to bedside. Hypoxia Inducible Factor-1alpha (HIF-1α) mediates a broad, evolutionarily conserved, endogenous adaptive program to hypoxia, and manipulation of components of the HIF pathway are neuroprotective in a number of human neurological diseases and experimental models. In this review, we discuss molecular components of one aspect of hypoxic adpatation in detail, and provide perspective on which targets within this pathway appear to be ripest for preventing and repairing neurodegeneration. Further, we highlight the role of HIF prolyl hydroxylases as emerging targets for the salutary effects of metal chelators on ferroptosis in vitro as well in animal models of neurological diseases.

SUMMARY The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2, we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). We show that Neh2 domain is sufficient for recognition, ubiquitination, and proteasomal degradation of Neh2-luciferase fusion protein. The Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time course of reporter activation. The reporter was used to screen the Spectrum library of 2000 biologically active compounds to identify activators of Nrf2. The most robust and yet nontoxic Nrf2 activators found—nordihydroguaiaretic acid, fisetin, and gedunin—induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism.

Mitochondrial dysfunction is a central feature of a number of acute and chronic neurodegenerative conditions, but clinically approved therapeutic interventions are only just emerging. Here we demonstrate the potential clinical utility of low molecular weight inhibitors of the hypoxia inducible factor prolyl-4-hydroxylases (HIF PHDs) in preventing mitochondrial toxin-induced cell death in mouse striatal neurons that express a ''knock-in'' mutant Huntingtin allele. Protection from 3-nitropropionic acid (3-NP, a complex II inhibitor)-induced toxicity by HIF PHD inhibition occurs without rescue of succinate dehydrogenase activity. Although HIF-1a mRNA is dramatically induced by mutant huntingtin, HIF-1a depletion by short interfering RNAs (siRNA) does not affect steady-state viability or protection from 3-NP-induced death by HIF PHD inhibitors in these cells. Moreover, 3-NP-induced complex II inhibition in control or mutant striatal neurons does not lead to activation of HIFdependent transcription. HIF PHD inhibition also protects cortical neurons from 3-NP-induced cytotoxicity. Protection of cortical neurons by HIF PHD inhibition correlates with enhanced VEGF but not PGC-1a gene expression. Together, these findings suggest that HIF PHD inhibitors are promising candidates for preventing cell death in conditions such as Huntington's disease and Alzheimer's disease that are associated with metabolic stress in the central nervous system. Antioxid. Redox Signal. 12, 435-443.

This review describes the catalytic mechanism, substrate specificity, and structural peculiarities of alpha-ketoglutarate dependent nonheme iron dioxygenases catalyzing prolyl hydroxylation of hypoxia-inducible factor (HIF). Distinct localization and regulation of three isoforms of HIF prolyl hydroxylases suggest their different roles in cells. The recent identification of novel substrates other than HIF, namely β2-adrenergic receptor and the large subunit of RNA polymerase II, places these enzymes in the focus of drug development efforts aimed at development of isoform-specific inhibitors. The challenges and prospects of designing isoform-specific inhibitors are discussed.

Hypoxia Inducible Factor (HIF) mediates a broad, conserved adaptive response to hypoxia, and the HIF pathway is a potential therapeutic target in cerebral ischemia. In this study, we investigated the mechanism by which in vitro ischemia (oxygen-glucose deprivation, OGD) affects canonical hypoxic HIF-1α stabilization. We validated the use of a reporter containing the oxygen dependent degradation domain of HIF-1α fused to firefly luciferase (ODD-luc) to quantitatively monitor distinct biochemical events leading to hypoxic HIF-1α expression or stabilization in a human neuroblastoma cell line (SH-SY5Y). When OGD was imposed following a 2 hour hypoxic stabilization of ODD-luc, the levels of the reporter were reduced, consistent with prior models proposing that OGD enhances HIF prolyl hydroxylase (PHD) activity. Surprisingly, PHD inhibitors and proteasome inhibitors do not stabilize ODD-luc in OGD. Further, OGD does not affect the half-life of ODD-luc protein following hypoxia, suggesting that OGD abrogates hypoxic HIF-1α induction by reducing HIF-1α synthesis rather than by enhancing its degradation. We observed ATP depletion under OGD versus hypoxia, and propose that ATP depletion enhances translational suppression, overcoming the selective synthesis of HIF concurrent with global decreases in protein synthesis in hypoxia. Taken together, these findings biochemically characterize a practical reporter for monitoring HIF-1α levels and support a novel model for HIF regulation in an in vitro model of human ischemia.

Homeostasis is the process by which cells adapt to stress and prevent or repair injury. Unique programs have evolved to sense and activate these homeostatic mechanisms and as such, homeostatic sensors may be potent therapeutic targets. The hypoxic response mediated by hypoxia inducible factor (HIF) downstream of oxygen sensing by HIF prolyl 4-hydroxylases (PHDs) has been well-studied, revealing cell-type specific regulation of HIF stability, activity, and transcriptional targets. HIF's paradoxical roles in nervous system development, physiology, and pathology arise from its complex roles in hypoxic adaptation and normoxic biology. Understanding how to engage the hypoxic response so as to recapitulate the protective mechanism of ischemic preconditioning is a high priority. Indeed, small molecules that activate the hypoxic response provide broad neuroprotection in several clinically relevant injury models. Screens for PHD inhibitors have identified novel therapeutics for neuroprotection that are ready to proceed to clinical trials for ischemic stroke. Better understanding the mechanisms of how to engage hypoxic adaption without altering development or physiology may identify additional novel therapeutic targets for diverse acute and chronic neuropathologies.

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