Natalya A. Smirnova

Targeting Nrf2-Mediated Gene Transcription by Extremely Potent Synthetic Triterpenoids Attenuate Dopaminergic Neurotoxicity in the MPTP Mouse Model of Parkinson's Disease

UNLABELLED Although the etiology of Parkinsons disease (PD) remains unclear, ample empirical evidence suggests that oxidative stress is a major player in the development of PD and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity. Nuclear factor E2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that upregulates a battery of antioxidant response element (ARE)-driven antioxidative and cytoprotective genes that defend against oxidative stress. AIMS We evaluated whether the strategy of activation of Nrf2 and its downstream network of cytoprotective genes with small molecule synthetic triterpenoids (TP) attenuate MPTP-induced PD in mice. RESULTS We show that synthetic TP are thus far the most potent and direct activators of the Nrf2 pathway using a novel Neh2-luciferase reporter. They upregulate several cytoprotective genes, including those involved in glutathione biosynthesis in vitro. Oral administration of TP that were structurally modified to penetrate the brain-induced messenger RNA and protein levels for a battery of Nrf2-dependent cytoprotective genes reduced MPTP-induced oxidative stress and inflammation, and ameliorated dopaminergic neurotoxicity in mice. The neuroprotective effect of these TP against MPTP neurotoxicity was dependent on Nrf2, since treatment with TP in Nrf2 knockout mice failed to block against MPTP neurotoxicity and induce Nrf2-dependent cytoprotective genes. INNOVATION Extremely potent synthetic TP that are direct activators of the Nrf2 pathway block dopaminergic neurodegeneration in the MPTP mouse model of PD. CONCLUSION Our results indicate that activation of Nrf2/antioxidant response element (ARE) signaling by synthetic TP is directly associated with their neuroprotective effects against MPTP neurotoxicity and suggest that targeting the Nrf2/ARE pathway is a promising approach for therapeutic intervention in PD.

Controlled enzymatic production of astrocytic hydrogen peroxide protects neurons from oxidative stress via an Nrf2-independent pathway.

Neurons rely on their metabolic coupling with astrocytes to combat oxidative stress. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) appears important for astrocyte-dependent neuroprotection from oxidative insults. Indeed, Nrf2 activators are effective in stroke, Parkinson disease, and Huntington disease models. However, key endogenous signals that initiate adaptive neuroprotective cascades in astrocytes, including activation of Nrf2-mediated gene expression, remain unclear. Hydrogen peroxide (H2O2) plays an important role in cell signaling and is an attractive candidate mediator of adaptive responses in astrocytes. Here we determine (i) the significance of H2O2 in promoting astrocyte-dependent neuroprotection from oxidative stress, and (ii) the relevance of H2O2 in inducing astrocytic Nrf2 activation. To control the duration and level of cytoplasmic H2O2 production in astrocytes cocultured with neurons, we heterologously expressed the H2O2-producing enzyme Rhodotorula gracilis D-amino acid oxidase (rgDAAO) selectively in astrocytes. Exposure of rgDAAO-astrocytes to D-alanine lead to the concentration-dependent generation of H2O2. Seven hours of low-level H2O2 production (∼3.7 nmol·min·mg protein) in astrocytes protected neurons from oxidative stress, but higher levels (∼130 nmol·min·mg protein) were neurotoxic. Neuroprotection occurred without direct neuronal exposure to astrocyte-derived H2O2, suggesting a mechanism specific to astrocytic intracellular signaling. Nrf2 activation mimicked the effect of astrocytic H2O2 yet H2O2-induced protection was independent of Nrf2. Astrocytic protein tyrosine phosphatase inhibition also protected neurons from oxidative death, representing a plausible mechanism for H2O2-induced neuroprotection. These findings demonstrate the utility of rgDAAO for spatially and temporally controlling intracellular H2O2 concentrations to uncover unique astrocyte-dependent neuroprotective mechanisms.

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.

Development of Neh2-Luciferase Reporter and Its Application for High Throughput Screening and Real-Time Monitoring of Nrf2 Activators

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.

Inhibition of 12/15-lipoxygenase as therapeutic strategy to treat stroke.

Targeting newly identified damage pathways in the ischemic brain can help to circumvent the currently severe limitations of acute stroke therapy. Here we show that the activity of 12/15‐lipoxygenase was increased in the ischemic mouse brain, and 12/15‐lipoxygenase colocalized with a marker for oxidized lipids, MDA2. This colocalization was also detected in the brain of 2 human stroke patients, where it also coincided with increased apoptosis‐inducing factor. A novel inhibitor of 12/15‐lipoxygenase, LOXBlock‐1, protected neuronal HT22 cells against oxidative stress. In a mouse model of transient focal ischemia, the inhibitor reduced infarct sizes both 24 hours and 14 days poststroke, with improved behavioral parameters. Even when treatment was delayed until at least 4 hours after onset of ischemia, LOXBlock‐1 was protective. Furthermore, it reduced tissue plasminogen activator‐associated hemorrhage in a clot model of ischemia/reperfusion. This study establishes inhibition of 12/15‐lipoxygenase as a viable strategy for first‐line stroke treatment. Ann Neurol 2013

Distinct Nrf2 Signaling Mechanisms of Fumaric Acid Esters and Their Role in Neuroprotection against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Experimental Parkinson's-Like Disease.

A promising approach to neurotherapeutics involves activating the nuclear-factor-E2-related factor 2 (Nrf2)/antioxidant response element signaling, which regulates expression of antioxidant, anti-inflammatory, and cytoprotective genes. Tecfidera, a putative Nrf2 activator, is an oral formulation of dimethylfumarate (DMF) used to treat multiple sclerosis. We compared the effects of DMF and its bioactive metabolite monomethylfumarate (MMF) on Nrf2 signaling and their ability to block 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced experimental Parkinsons disease (PD). We show that in vitro DMF and MMF activate the Nrf2 pathway via S-alkylation of the Nrf2 inhibitor Keap1 and by causing nuclear exit of the Nrf2 repressor Bach1. Nrf2 activation by DMF but not MMF was associated with depletion of glutathione, decreased cell viability, and inhibition of mitochondrial oxygen consumption and glycolysis rates in a dose-dependent manner, whereas MMF increased these activities in vitro. However, both DMF and MMF upregulated mitochondrial biogenesis in vitro in an Nrf2-dependent manner. Despite the in vitro differences, both DMF and MMF exerted similar neuroprotective effects and blocked MPTP neurotoxicity in wild-type but not in Nrf2 null mice. Our data suggest that DMF and MMF exhibit neuroprotective effects against MPTP neurotoxicity because of their distinct Nrf2-mediated antioxidant, anti-inflammatory, and mitochondrial functional/biogenetic effects, but MMF does so without depleting glutathione and inhibiting mitochondrial and glycolytic functions. Given that oxidative damage, neuroinflammation, and mitochondrial dysfunction are all implicated in PD pathogenesis, our results provide preclinical evidence for the development of MMF rather than DMF as a novel PD therapeutic. SIGNIFICANCE STATEMENT Almost two centuries since its first description by James Parkinson, Parkinsons disease (PD) remains an incurable disease with limited symptomatic treatment. The current study provides preclinical evidence that a Food and Drug Administration-approved drug, dimethylfumarate (DMF), and its metabolite monomethylfumarate (MMF) can block nigrostriatal dopaminergic neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD. We elucidated mechanisms by which DMF and its active metabolite MMF activates the redox-sensitive transcription factor nuclear-factor-E2-related factor 2 (Nrf2) to upregulate antioxidant, anti-inflammatory, mitochondrial biosynthetic and cytoprotective genes to render neuroprotection via distinct S-alkylating properties and depletion of glutathione. Our data suggest that targeting Nrf2-mediated gene transcription using MMF rather than DMF is a promising approach to block oxidative stress, neuroinflammation, and mitochondrial dysfunction for therapeutic intervention in PD while minimizing side effects.

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 hydroxylases as targets for neuroprotection by "antioxidant" metal chelators: From ferroptosis to stroke.

Neurologic conditions including stroke, Alzheimer disease, Parkinson disease, and Huntington 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 (HIF)-1α mediates a broad, evolutionarily conserved, endogenous adaptive program to hypoxia, and manipulation of components of the HIF pathway is neuroprotective in a number of human neurological diseases and experimental models. In this review, we discuss molecular components of one aspect of hypoxic adaptation in detail and provide perspective on which targets within this pathway seem 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.

Catalytic Mechanism and Substrate Specificity of HIF Prolyl Hydroxylases

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.

In vitro ischemia suppresses hypoxic induction of hypoxia‐inducible factor‐1α by inhibition of synthesis and not enhanced degradation

Hypoxia‐inducible factor (HIF) mediates a broad, conserved adaptive response to hypoxia, and the HIF pathway is a potential therapeutic target in cerebral ischemia. This study 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 monitor quantitatively 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‐hr hypoxic stabilization of ODD‐luc, the levels of the reporter were reduced, consistent with prior models proposing that OGD enhances HIF prolylhydroxylase (PHD) activity. Surprisingly, PHD inhibitors and proteasome inhibitors do not stabilize ODD‐luc in OGD. Furthermore, 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 vs. 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.