The Nrf2/ARE pathway as a potential therapeutic target in neurodegenerative disease

Abstract

Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to induce expression of a variety of cytoprotective and detoxification genes. Several of the genes commonly regulated by Nrf2 have been implicated in protection from neurodegenerative conditions. Work from several laboratories has uncovered the potential for Nrf2-mediated transcription to protect from neurodegeneration resulting from mechanisms involving oxidative stress. For this reason, Nrf2 may be considered a therapeutic target for conditions that are known to involve free radical damage. Because common mechanisms of neurodegeneration, such as mitochondrial dysfunction and build-up of reactive oxygen species, are currently being uncovered, targeting Nrf2 may be valuable in combating conditions with variable causes and etiologies. Most effectively to target this protein in neurodegenerative conditions, a description of the involvement of Nrf2 and potential for neuroprotection must come from laboratory models. Herein, we review the current literature that suggests that Nrf2 may be a valuable therapeutic target for neurodegenerative disease, as well as experiments that illustrate potential mechanisms of protection.

FIG. 1.

Coordinated upregulation of cytoprotective genes occurs as a result of Nrf2 activation. Nrf2-dependent genes identified by microarray are listed in bold italics. These genes function together in the production and utilization of glutathione. Genes involved in cellular uptake of glutathione-constituent amino acids glycine and glutamate are increased. Additionally, the rate-limiting step of glutathione synthesis is the formation of the γ-glutamyl cysteine moiety by γ-glutamyl cysteine ligase. Transcription of both subunits of this enzyme is induced. Moreover, enzymes responsible for glutathione utilization, reduction, and conjugation are all represented in microarray data. Similarly, production and use of the common detoxification enzyme cofactor NADPH is stimulated. The pentose phosphate pathway is a common cellular mechanism whose two main functions are the production of reduction equivalents in the form of NADPH and the production of ribulose-5-phosphate for nucleotide and nucleic acid synthesis. In the case of Nrf2 activation, the pathway is focused on NADPH production, as the fructose-5-phosphate molecules are recycled back to glucose-6-phosphate by transaldolase and transketolase. NADPH also may be synthesized by malic enzyme oxidation of malate to pyruvate. NADPH is the major cofactor used in cellular reductions and is used especially frequently by detoxification enzymes like NQO1, P450s, and GSTs. Furthermore, Nrf2-dependent genes function together to detoxify superoxide, and meanwhile to prevent Fenton chemistry on the SOD-produced intermediate H₂O₂. This occurs by two methods. First, ferretin sequesters free iron by binding it in the ferric form. Second, catalase, thioredoxin, and peroxiredoxins reduce H₂O₂ to water. Thus, Nrf2-driven genes reduce superoxide to water and prevent production of hydroxyl radicals. [Modified from Lee et al., 2004 (34)].

FIG. 2.

Nrf2 deficiency leads to increase sensitivity to 3-NP in primary neurons and in vivo. Primary cortical cultures were prepared from Nrf2-deficient or wild-type embryos (E15). Cultures comprised ~90% neurons and 10% astrocytes (data not shown). After 5 days, in vitro cultures were treated with 3-NP at 0, 0.5, 1.0, or 2.0 mM for 48 h. Lactate dehydrogenase activity was measured in the media and in the remaining cells. Percentage lactate dehydrogenase in media is reflective of cell death. Nrf2-deficient cells were found to be more susceptible to 3-NP–induced toxicity than were wild-type cells (A). Mice were injected with 3-NP at 50 mg/kg every 12 h for seven doses, after which they were killed, and brains were harvested. Coronal sections containing striatum were stained with cresyl violet or fluorojade-B (B). Lesion volume was measured by using the Caveleiri estimator on fluorojade-B–stained sections (D). A gene–dose effect was observed in which Nrf2-deficient mice were much more susceptible to lesioning than were heterozygous or wild-type mice. Furthermore, before death, Nrf2-deficient mice were the only group that could not perform the rotarod task for 3 min at 5 rpm (C). *p < 0.05 compared with wild type; **p < 0.01 compared with wild type by Student's t test [Modified from Calkins et al., 2005 (7)]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 3.

ARE activation after mitochondrial complex II inhibitor toxicity in cell culture and striatum. Primary cortical cultures were prepared from ARE-hPAP embryos (E15). Cultures comprised ~90% neurons and 10% astrocytes (data not shown). After 5 days, in vitro cultures were treated with vehicle (A, left) or 3-NP at 5.0 mM (A, right) for 48 h. hPAP histochemistry was evaluated by using the alkaline phosphatase substrate vector red, and cells were co-stained with GFAP (green) and Hoescht's (blue) (A). Vector red staining colocalized with surviving GFAP-labeled cells in the 3-NP–treated culture. Mice were injected intrastriatally with malonate (0.5 μmol). Coronal sections containing striatum were stained with BCIP/NBT to evaluate hPAP activity and counterstained with nuclear fast red (B, left). In serial sections, GFAP was visualized with immunohistochemical staining (B, right). ARE-hPAP activity occurred surrounding the lesion 48 h after toxin administration. The staining area also contains GFAP-immunoreactive astrocytes. [Modified from Calkins et al., 2005 (7)]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 4.

Nrf2-KO primary cortical neurons are protected by Nrf2-overexpressing astrocytes. Mixed primary cortical cultures were prepared and treated with vehicle or 50 μM H₂O₂ (top panels). H₂O₂ killed virtually all neurons (A and C, β-III tubulin labeled with Texas red in all panels). Cortical cultures were also infected with Ad-GFP or Ad-Nrf2 48 h before H₂O₂ treatment. Only infection with Ad-Nrf2 induced expression of the hPAP reporter construct (B) Adenovirus preferentially infected astrocytes (GFP in green), and pretreatment with Ad-Nrf2 virus protects neurons from H₂O₂ insult (C, bottom right). Hoechst 33258 (blue). *p < 0.05 compared with 0 MOI by Student's t test [Reproduced from Kraft et al., 2004, with permission (30)]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 5.(tion)

Nrf2-overexpressing astroycte grafts protect from malonate lesions. Astrocyte cultures were grown from P0 or P1 ARE-hPAP reporter pups and transduced with either Ad-GFP or Ad-Nrf2. Forty-eight hours later, hPAP activity was visualized with vector red alkaline phosphatase substrate, and cultures were immunohistochemically stained with GFAP. Ad-GFP–infected astrocytes did not exhibit hPAP activity, whereas those infected with Ad-Nrf2 did (A). Astrocytes treated as such were transplanted into striatum, and after 5 weeks, the grafted mice were lesioned with malonate. Those hemispheres receiving Ad-Nrf2-infected astrocytes were significantly protected from malonate as compared with those receiving Ad-GFP–infected astrocytes (B), as measured by cresyl violet staining (C). *p < 0.05 compared with Ad-GFP hemispheres by Student's t test [Modified from Calkins et al., 2005 (7)]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 6.

Nrf2 activation and enhanced metabolic coupling between astrocytes and neurons. Primary cortical neuronal cultures were infected with Ad-GFP (50 MOI) before treatment with tBHQ. Cells were lifted, sorted, and RNA was isolated from the sorted pools and analyzed with microarray analysis [Kraft et al., 2004 (32)]. A hypothetical diagram of some of the pathways changed with tBHQ treatment in the astrocytes (GFP-positive) and the neurons (GFP-negative) based on microarray analysis is shown above. The protective cellular shield reflects genes that are consistently changed with activation of Nrf2 to combat oxidative stress and toxicants. The majority of these genes change in the astrocyte. Other genes involved in energy metabolism also were changed. Solid arrowheads, pathways with one or more enzymes upregulated by tBHQ treatment (e.g., more than five enzymes converting glucose to pyruvate are increased by tBHQ in the astrocyte population). Hollow arrowheads on solid lines, known interactions that are not changed by tBHQ. Arrows with stippled lines, a proposed interaction between neurons and glia that is not yet proven. Files containing all of the microarray data are available for download at http://www.pharmacy.wisc.edu/facstaff/sciences/JohnsonGroup/microdata.cfm [Reproduced from Johnson et al. (27), with permission].