The Nrf2-ARE pathway: an indicator and modulator of oxidative stress in neurodegeneration

Abstract

Transcriptional activation of protective genes is mediated by a cis-acting element called the antioxidant responsive element (ARE). The transcription factor Nrf2 (NF-E2-related factor 2) binds to the ARE. Activation of this pathway protects cells from oxidative stress-induced cell death. Increased oxidative stress is associated with neuronal cell death during the pathogenesis of multiple chronic neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. We hypothesize that Nrf2-ARE activation is a novel neuroprotective pathway that confers resistance to a variety of oxidative, stress-related, neurodegenerative insults. In recent studies, primary neuronal cultures treated with chemical activators of the Nrf2-ARE pathway displayed significantly greater resistance to oxidative stress-induced neurotoxicity. Similar cultures generated from ARE-hPAP reporter mice demonstrated selective activation of the Nrf2-ARE pathway in astrocytes, suggesting that Nrf2 activation in astrocytes somehow confers resistance to naive neurons. Further, in chemical models of neurodegeneration, Nrf2 knockout mice are significantly more sensitive to mitochondrial complex I and II inhibitors. Combining these observations with the results implying that the astrocyte is central to Nrf2-ARE-mediated neuroprotection, we transplanted Nrf2-overexpressing astrocytes into the mouse striatum prior to lesioning with malonate. This procedure led to dramatic protection against malonate-induced neurotoxicity. Translating this to other chemical and genetic models of neurodegeneration will be discussed.

Fig. 1. Nrf2 KO Primary Cortical Neurons are Protected by Nrf2 Overexpressing Astrocytes

Mixed primary cortical cultures were prepared and treated with vehicle or 50 pM H₂O₂ (top panels). H₂O₂ killed virtually all neurons (beta-III tubulin labeled with Texas-Red in all panels). Cortical cultures were also infected with Ad-GFP or Ad-Nrf2 48hr before H₂O₂ treatement. Adenovirus preferentially infected astrocytes (GFP in green) and pretreatment with Ad-Nrf2 virus protects neurons from H₂O₂ insult (bottom right). Hoechst 33258 (blue). Reprinted from Journal of Neuroscience, Volume 24, Kraft et al., Nuclear factor E2-related factor 2-dependent antioxidant response element activation by tert-butylhydroquinone and sulforaphane occurring preferentially in astrocytes conditions neurons against oxidative insult, 1101-12, 2004 with permission from Journal of Neuroscience.

Fig. 2. Orchestrated regulation of Nrf2-dependent genes in astrocytes

Nrf2-dependent genes identified by oligonucleotide microarray analysis are depicted in shaded squares. Genes are categorized based on function and metabolic pathway. Genes are related to (A) glutathione homeostasis, (B) detoxification of H₂O₂ and iron homeostasis, and (C) NADPH homeostasis. Reprinted from Journal of Biological Chemistry, Volume 278, Lee et al., Identification of the NF-E2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis, 12029–12038, 2003 with permission from Journal of Biological Chemistry.

Fig. 3. Nrf2 activation and enhanced metabolic coupling between astrocytes and neurons

Primary cortical neuronal cultures were infected with Ad-GFP (50 MOI) prior to treatment with tBHQ. At this MOI the adenovirus selectively infected astrocytes allowing for FAC sorting to be performed on the cultures. Post-sort analysis of the isolated cell populations showed < 0.01% contamination on the neuronal GFP-negative pool with astrocytes and < 5% contamination of the astrocyte GFP-positive pool with neurons. RNA was isolated from the sorted pools and analyzed by microarray analysis (Kraft et al, 2004). A hypothetical diagram of some of the pathways changed with tBHQ treatment in the astrocytes (GFP-positive) and the neurons (GFP-negative) based upon 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 were also changed. Solid arrowheads indicate pathways with one or more enzymes upregulated by tBHQ treatment (eg. More than five enzymes converting glucose to pyruvate are increased by tBHQ in the astrocyte population). Hollow arrowheads on solid lines indicate known interactions that are not changed by tBHQ. Arrows with stippled lines indicate 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. Reprinted from Molecular Biology and Pharmacology of Tissue Repair, Volume 1302, Johnson et al., The Nrf2-ARE pathway: A potential therapeutic target for neurodegenerative diseases, 143–153, 2007 with permission from Elsevier.

Fig. 4. Transplantation of Nrf2 overexpressing astocytes protects against malonate lesioning

ARE-hPAP+ astrocytes were infected with Ad-GFP or Ad-Nrf2-GFP. GFP expression (green; A,B) and hPAP histochemistry (red stippling; B) were visualized to show the extent of infection (> 95% of cells) and activation Nrf2-ARE pathway by Nrf2 and not GFP. Astrocytes were lifted and transplanted in the striatum. Mice were lesioned 5 weeks post-transplant with malonate (n=4). Lesions were visualized by cresyl violet (C) and the volume was quantified (D). *p<0.05 compared to hemispheres receiving GFP-infected astrocytes. Reprinted from Proceeding of the National Academy of Sciences USA, Volume 102, Calkins et al., Protection from mitochondrial complex II inhibition in vitro and in vivo by Nrf2-mediated transcription, 244-9, 2005 with permission from the National Academy of Sciences USA.