HDAC6 is a target for protection and regeneration following injury in the nervous system.

Central nervous system (CNS) trauma can result in tissue disruption, neuronal and axonal degeneration, and neurological dysfunction. The limited spontaneous CNS repair in adulthood and aging is often insufficient to overcome disability. Several investigations have demonstrated that targeting HDAC activity can protect neurons and glia and improve outcomes in CNS injury and disease models. However, the enthusiasm for pan-HDAC inhibition in treating neurological conditions is tempered by their toxicity toward a host of CNS cell types -a biological extension of their anticancer properties. Identification of the HDAC isoform, or isoforms, that specifically mediate the beneficial effects of pan-HDAC inhibition could overcome this concern. Here, we show that pan-HDAC inhibition not only promotes neuronal protection against oxidative stress, a common mediator of injury in many neurological conditions, but also promotes neurite growth on myelin-associated glycoprotein and chondroitin sulfate proteoglycan substrates. Real-time PCR revealed a robust and selective increase in HDAC6 expression due to injury in neurons. Accordingly, we have used pharmacological and genetic approaches to demonstrate that inhibition of HDAC6 can promote survival and regeneration of neurons. Consistent with a cytoplasmic localization, the biological effects of HDAC6 inhibition appear transcription-independent. Notably, we find that selective inhibition of HDAC6 avoids cell death associated with pan-HDAC inhibition. Together, these findings define HDAC6 as a potential nontoxic therapeutic target for ameliorating CNS injury characterized by oxidative stress-induced neurodegeneration and insufficient axonal regeneration.

Selective inhibition of hypoxia-inducible factor (HIF) prolyl-hydroxylase 1 mediates neuroprotection against normoxic oxidative death via HIF- and CREB-independent pathways.

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-1alpha and cAMP response element-binding protein (CREB). Yet we find that global hypoxia-inducible factor (HIF)-PHD inhibition protects neurons even when HIF-1alpha and CREB are directly suppressed. Moreover, two global HIF-PHD inhibitors continued to be neuroprotective even in the presence of diminished HIF-2alpha 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-1alpha and CREB; and that HIF-2alpha, not HIF-1alpha, regulates susceptibility to normoxic oxidative neuronal death.

Inhibition of prolyl hydroxylase protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity: model for the potential involvement of the hypoxia-inducible factor pathway in Parkinson disease.

Hypoxia-inducible factor (HIF) plays an important role in cell survival by regulating iron, antioxidant defense, and mitochondrial function. Pharmacological inhibitors of the iron-dependent enzyme class prolyl hydroxylases (PHD), which target alpha subunits of HIF proteins for degradation, have recently been demonstrated to alleviate neurodegeneration associated with stroke and hypoxic-ischemic injuries. Here we report that inhibition of PHD by 3,4-dihydroxybenzoate (DHB) protects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigral dopaminergic cell loss and up-regulates HIF-1alpha within these neurons. Elevations in mRNA and protein levels of HIF-dependent genes heme oxygenase-1 (Ho-1) and manganese superoxide dismutase (Mnsod) following DHB pretreatment alone are also maintained in the presence of MPTP. MPTP-induced reductions in ferroportin and elevations in nigral and striatal iron levels were reverted to levels comparable with that of untreated controls with DHB pretreatment. Reductions in pyruvate dehydrogenase mRNA and activity resulting from MPTP were also found to be attenuated by DHB. In vitro, the HIF pathway was activated in N27 cells grown at 3% oxygen treated with either PHD inhibitors or an iron chelator. Concordant with our in vivo data, the MPP(+)-elicited increase in total iron as well as decreases in cell viability were attenuated in the presence of DHB. Taken together, these data suggest that protection against MPTP neurotoxicity may be mediated by alterations in iron homeostasis and defense against oxidative stress and mitochondrial dysfunction brought about by cellular HIF-1alpha induction. This study provides novel data extending the possible therapeutic utility of HIF induction to a Parkinson disease model of neurodegeneration, which may prove beneficial not only in this disorder itself but also in other diseases associated with metal-induced oxidative stress.

Prolyl 4-hydroxylase activity-responsive transcription factors: from hydroxylation to gene expression and neuroprotection.

Most homeostatic processes including gene transcription occur as a result of deviations in physiological tone that threatens the survival of the organism. A prototypical homeostatic stress response includes changes in gene expression following alterations in oxygen, iron or 2-oxoglutarate levels. Each of these cofactors plays an important role in cellular metabolism. Accordingly, a family of enzymes known as the Prolyl 4-hydroxylase (PHD) enzymes are a group of dioxygenases that have evolved to sense changes in 2-oxoglutarate, oxygen and iron via changes in enzyme activity. Indeed, PHDs are a part of an established oxygen sensor system that regulates transcriptional regulation of hypoxia/stress-regulated genes and thus are an important component of events leading to cellular rescue from oxygen, iron or 2-oxoglutarate deprivations. The ability of PHD activity to regulate homeostatic responses to oxygen, iron or 2-oxoglutarate metabolism has led to the development of small molecule inhibitors of the PHDs as a strategy for activating or augmenting cellular stress responses. These small molecules are proving effective in preclinical models of stroke and Parkinson's disease. However the precise protective pathways engaged by PHD inhibition are only beginning to be defined. In the current review, we summarize the role of iron, 2-oxoglutarate and oxygen in the PHD catalyzed hydroxylation reaction and provide a brief discussion of some of the transcription factors that play an effective role in neuroprotection against oxidative stress as a result of changes in PHD activity.

Harnessing hypoxic adaptation to prevent, treat, and repair stroke.

The brain demands oxygen and glucose to fulfill its roles as the master regulator of body functions as diverse as bladder control and creative thinking. Chemical and electrical transmission in the nervous system is rapidly disrupted in stroke as a result of hypoxia and hypoglycemia. Despite being highly evolved in its architecture, the human brain appears to utilize phylogenetically conserved homeostatic strategies to combat hypoxia and ischemia. Specifically, several converging lines of inquiry have demonstrated that the transcription factor hypoxia-inducible factor-1 (HIF1-1) mediates the activation of a large cassette of genes involved in adaptation to hypoxia in surviving neurons after stroke. Accordingly, pharmacological or molecular approaches that engage hypoxic adaptation at the point of one of its sensors (e.g., inhibition of HIF prolyl 4 hydroxylases) leads to profound sparing of brain tissue and enhanced recovery of function. In this review, we discuss the potential mechanisms that could subserve protective and restorative effects of augmenting hypoxic adaptation in the brain. The strategy appears to involve HIF-dependent and HIF-independent pathways and more than 70 genes and proteins activated transcriptionally and post-transcriptionally that can act at cellular, local, and system levels to compensate for oxygen insufficiency. The breadth and depth of this homeostatic program offers a hopeful alternative to the current pessimism towards stroke therapeutics.

Antihelminthic benzimidazoles are novel HIF activators that prevent oxidative neuronal death via binding to tubulin.

AIMS: Pharmacological activation of the adaptive response to hypoxia is a therapeutic strategy of growing interest for neurological conditions, including stroke, Huntington's disease, and Parkinson's disease. We screened a drug library with known safety in humans using a hippocampal neuroblast line expressing a reporter of hypoxia-inducible factor (HIF)-dependent transcription. RESULTS: Our screen identified more than 40 compounds with the ability to induce hypoxia response element-driven luciferase activity as well or better than deferoxamine, a canonical activator of hypoxic adaptation. Among the chemical entities identified, the antihelminthic benzimidazoles represented one pharmacophore that appeared multiple times in our screen. Secondary assays confirmed that antihelminthics stabilized the transcriptional activator HIF-1α and induced expression of a known HIF target gene, p21(cip1/waf1), in post-mitotic cortical neurons. The on-target effect of these agents in stimulating hypoxic signaling was binding to free tubulin. Moreover, antihelminthic benzimidazoles also abrogated oxidative stress-induced death in vitro, and this on-target effect also involves binding to free tubulin. INNOVATION AND CONCLUSIONS: These studies demonstrate that tubulin-binding drugs can activate a component of the hypoxic adaptive response, specifically the stabilization of HIF-1α and its downstream targets. Tubulin-binding drugs, including antihelminthic benzimidazoles, also abrogate oxidative neuronal death in primary neurons. Given their safety in humans and known ability to penetrate into the central nervous system, antihelminthic benzimidazoles may be considered viable candidates for treating diseases associated with oxidative neuronal death, including stroke.

Translation of ischemic preconditioning to the patient: prolyl hydroxylase inhibition and hypoxia inducible factor-1 as novel targets for stroke therapy.

Effective therapies for stroke must interdict multiple parallel and sequential pathophysiological events. A paradigm which offers insight into multivalent but thoughtfully coordinated protective programs is ischemic preconditioning. A central hypothesis of our group and others is that pharmacological agents that activate programs of gene expression normally induced by ischemic preconditioning will be effective agents for the prevention and treatment of stroke. Inhibitors of a class of enzymes, the hypoxia inducible factor-1 (HIF-1) prolyl hydroxylases stabilize the transcriptional activator HIF-1 and activate target genes involved in compensation for ischemia, including erythropoeitin (Epo) and vascular endothelial growth factor (VEGF). Here, we review evidence suggesting that the HIF-1 prolyl hyroxylases are inhibited during ischemic preconditioning and that pharmacological inhibitors of these enzymes are viable targets for stroke therapy.

Antioxidants, HIF prolyl hydroxylase inhibitors or short interfering RNAs to BNIP3 or PUMA, can prevent prodeath effects of the transcriptional activator, HIF-1alpha, in a mouse hippocampal neuronal line.

Hypoxia-inducible factor (HIF) is a transcriptional activator that promotes death or survival in neurons. The regulators and targets of HIF-1alpha-mediated death remain unclear. We found that prodeath effects of HIF-1 are not attributable to an imbalance in HIF-1alpha and HIF-1beta expression. Rather, the synergistic death caused by oxidative stress and by overexpression of an oxygen-resistant HIF-VP16 in neuroblasts was attributable to transcriptional upregulation of BH3-only prodeath proteins, PUMA or BNIP3. By contrast, overexpression of BNIP3 was not sufficient to potentiate oxidative death. As acidosis is known to activate BNIP3-mediated death, we examined other secondary stresses, such as oxidants or prolyl hydroxylase activity are necessary for exposing the prodeath functions of HIF in neurons. Antioxidants or prolyl hydroxylase inhibition prevented potentiation of death by HIF-1alpha. Together, these studies suggest that antioxidants and PHD inhibitors abrogate the ability of HIF-mediated transactivation of BH3-only proteins to potentiate oxidative death in normoxia. The findings offer strategies for minimizing the prodeath effects of HIF-1 in neurologic conditions associated with hypoxia and oxidative stress, such as stroke and spinal cord injury.

Small molecule activation of adaptive gene expression: tilorone or its analogs are novel potent activators of hypoxia inducible factor-1 that provide prophylaxis against stroke and spinal cord injury.

A major challenge for neurological therapeutics is the development of small molecule drugs that can activate a panoply of downstream pathways without toxicity. Over the past decade our group has shown that a family of enzymes that regulate posttranscriptional and transcriptional adaptive responses to hypoxia are viable targets for neuronal protection and repair. The family is a group of iron, oxygen, and 2-oxoglutarate-dependent dioxygenases, known as the HIF prolyl 4-hydroxylases (HIF PHDs). We have previously shown that pluripotent protection offered by iron chelators is mediated, in part, via the ability of these agents to inhibit the HIF PHDs. Our group and others have implicated the transcriptional activator HIF-1 in some of the salutary effects of iron chelation-induced PHD inhibition. While some iron chelators are currently employed in humans for conditions such as hemochromatosis, the diverse utilization of iron in physiological processes in the brain makes the development of HIF activators that do not bind iron a high priority. Here we report the development of a high throughput screen to develop novel HIF activators and/or PHD inhibitors for therapeutic use in the central nervous system (CNS). We show that tilorone, a low-molecular weight, antiviral, immunomodulatory agent is the most effective activator of the HIF pathway in a neuronal line. We also show that tilorone enhances HIF protein levels and increases the expression of downstream target genes independent of iron chelation and HIF PHD inhibition in vitro. We further demonstrate that tilorone can activate an HIF-regulated reporter gene in the CNS. These studies confirm that tilorone can penetrate the blood-brain barrier to activate HIF in the CNS. As expected from these findings, we show that tilorone provides effective prophylaxis against permanent ischemic stroke and traumatic spinal cord injury in male rodents. Altogether these findings identify tilorone as a novel and potent modulator of HIF-mediated gene expression in neurons with neuroprotective properties.

Hypoxia inducible factor prolyl 4-hydroxylase enzymes: center stage in the battle against hypoxia, metabolic compromise and oxidative stress.

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.

Proteasome inhibition protects HT22 neuronal cells from oxidative glutamate toxicity.

Oxidative stress caused by glutathione depletion after prolonged exposure to extracellular glutamate leads to a form of neuronal cell death that exhibits morphologically mixed features of both apoptosis and necrosis. However, specific downstream executioners involved in this form of cell death have yet to be identified. We report here that glutamate exposure does not activate caspase-3 in the HT22 neuronal cell line. Furthermore, no cytoprotection was achieved with either the pan-caspase inhibitor Z-VAD-fmk or the caspase-3-specific inhibitor DEVD-CHO. In contrast, inhibition of the proteasome by lactacystin protected both HT22 cells and rat primary neuronal cells against cell lysis. In parallel, oxidatively altered and ubiquitinated proteins accumulated in the mitochondrial fraction of cells after proteasome inhibition. These findings suggest that caspases can be decoupled from oxidative stress under some conditions, and implicate the ubiquitin/proteasome pathway in neuronal cell death caused by oxidative glutamate toxicity.

Hypoxia-inducible factor prolyl 4-hydroxylase inhibition. A target for neuroprotection in the central nervous system.

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-1alpha and HIF-2alpha. 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-1alpha 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.

The c-Raf inhibitor GW5074 provides neuroprotection in vitro and in an animal model of neurodegeneration through a MEK-ERK and Akt-independent mechanism.

Cerebellar granule neurons undergo apoptosis when switched from a medium containing high potassium (HK) to one that has low potassium (LK). LK-induced cell death is blocked by GW5074 [5-Iodo-3-[(3,5-dibromo-4-hydroxyphenyl) methylene]-2-indolinone], a synthetic drug that inhibits c-Raf activity in vitro. GW5074 has no direct effect on the activities of several apoptosis-associated kinases when assayed in vitro. In contrast to its effect in vitro, treatment of neurons with GW5074 causes c-Raf activation (when measured in vitro in the absence of the drug) and stimulates the Raf-MEK-ERK pathway. Treatment of neurons with GW5074 also leads to an increase in the activity of B-Raf, which is not inhibited by GW5074 in vitro at concentrations at which the drug exerts its neuroprotective effect. PD98059 and U0126, two distinct inhibitors of MEK, block the activation of ERK by GW5074 but have no effect on its ability to prevent cell death. Overexpression of a dominant-negative form of Akt does not reduce the efficacy of GW5074, demonstrating an Akt-independent mechanism of action. Neuroprotection is inhibited by SN-50, a specific inhibitor of nuclear factor-kappa B (NF-kappaB) and by the Ras inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS) implicating NF-kappaB and Ras in the neuroprotective signaling pathway activated by GW5074. In addition to preventing LK-induced apoptosis, treatment with GW5074 protects against the neurotoxic effects of MPP+ and methylmercury in cerebellar granule neurons, and glutathione depletion-induced oxidative stress in cortical neurons. Furthermore, GW5074 prevents neurodegeneration and improves behavioral outcome in an animal model of Huntington's disease. Given its neuroprotective effect on distinct types of cultured neurons, in response to different neurotoxic stimuli, and in an animal model of neurodegeneration, GW5074 could have therapeutic value against neurodegenerative pathologies in humans.