NF-E2-related Factor-2 Mediates Neuroprotection against Mitochondrial Complex I Inhibitors and Increased Concentrations of Intracellular Calcium in Primary Cortical Neurons

Andy Y Shih,Jong-Min Lee,Timothy H Murphy,Jeffrey A Johnson

doi:10.1074/jbc.m305204200

Abstract

NF-E2-related factor-2 (Nrf2) regulates the gene expression of phase II detoxification enzymes and antioxidant proteins through an enhancer sequence referred to as the antioxidant-responsive element (ARE). In this study, we demonstrate that Nrf2 protects neurons in mixed primary neuronal cultures containing both astrocytes ( approximately 10%) and neurons ( approximately 90%) through coordinate up-regulation of ARE-driven genes. Nrf2-/- neurons in this mixed culture system were more sensitive to mitochondrial toxin (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine or rotenone)-induced apoptosis compared with Nrf2+/+ neurons. To understand the underlying mechanism of this observed differential sensitivity, we compared the gene expression profiles using oligonucleotide microarrays. Microarray data showed that Nrf2+/+neuronal cultures had higher expression levels of genes encoding detoxification enzymes, antioxidant proteins, calcium homeostasis proteins, growth factors, neuron-specific proteins, and signaling molecules compared with Nrf2-/- neuronal cultures. As predicted from the microarray data, Nrf2-/- neurons were indeed more vulnerable to the cytotoxic effects of ionomycin- and 2,5-di-(t-butyl)-1,4-hydroquinone-induced increases in intracellular calcium. Finally, adenoviral vector-mediated overexpression of Nrf2 recovered ARE-driven gene expression in Nrf2-/- neuronal cultures and rescued Nrf2-/- neurons from rotenone- or ionomycin-induced cell death. Taken together, these findings suggest that Nrf2 plays an important role in protecting neurons from toxic insult.

Highlights

NF-E2-related factor-2 (Nrf2) regulates the gene expression of phase II detoxification enzymes and antioxidant proteins through an enhancer sequence referred to as the antioxidant-responsive element (ARE)
Adenoviral vector-mediated overexpression of Nrf2 recovered ARE-driven gene expression in Nrf2؊/؊ neuronal cultures and rescued Nrf2؊/؊ neurons from rotenone- or ionomycin-induced cell death. These findings suggest that Nrf2 plays an important role in protecting neurons from toxic insult
We have shown that ARE-driven gene expression is dependent on Nrf2 in primary neuronal cultures and that Nrf2Ϫ/Ϫ neurons are more sensitive to mitochondrial toxin-mediated apoptosis

Summary

EXPERIMENTAL PROCEDURES

Primary Neuronal Culture—Nrf2ϩ/Ϫ mice were bred with Nrf2ϩ/Ϫ mice, and primary neuronal cultures were prepared individually. The Nrf genotype of each culture was determined by a PCR-based method as described previously [11]. Over 90% of the cells in the cultures (both Nrf2Ϫ/Ϫ and Nrf2ϩ/ϩ) were neurons as determined by immunostaining of the astrocyte-specific marker glial fibrillary acidic protein (GFAP; Dako Corp.) and the neuron-specific markers microtubule-associated protein-2 (MAP2; Chemicon International, Inc.) and ␤III-tubulin (Promega) (data not shown). NQO1 Activity—NQO1 enzymatic activity was determined by a colorimetric method for whole cell extracts [34] and by histochemistry for fixed cultures as described previously [35]. 10 –20% of the neurons and virtually 100% of the astrocytes were infected with viral vectors as determined by GFP fluorescence and MAP2 and GFAP staining

RESULTS

DISCUSSION

Prolyl endopeptidase