Overexpression of Nrf2 protects cerebral cortical neurons from ethanol-induced apoptotic death

Abstract

Ethanol (ETOH) can cause apoptotic death of neurons by depleting GSH with an associated increase in oxidative stress. The current study illustrates a means to overcome this ETOH-induced neurotoxicity by enhancing GSH through boosting Nrf2, a transcription factor that controls GSH homeostasis. ETOH treatment caused a significant increase in Nrf2 protein, transcript expression, Nrf2-DNA binding activity, and expression of its transcriptional target, NQO1, in primary cortical neuron (PCNs). However, this increase in Nrf2 did not maintain GSH levels in response to ETOH, and apoptotic death still occurred. To elucidate this phenomenon, we silenced Nrf2 in neurons and found that ETOH-induced GSH depletion and the increase in superoxide levels were exacerbated. Furthermore, Nrf2 knockdown resulted in significantly increased (P < 0.05) caspase 3 activity and apoptosis. Adenovirus-mediated overexpression of Nrf2 prevented ETOH-induced depletion of GSH from the medium and high GSH subpopulations and prevented ETOH-related apoptotic death. These studies illustrate the importance of Nrf2-dependent maintenance of GSH homeostasis in cerebral cortical neurons in the defense against oxidative stress and apoptotic death elicited by ETOH exposure.

Fig. 1.

Ethanol induces Nrf2 expression in a time-dependent manner in primary cortical neurons. PCNs were treated with ETOH (4 mg/ml) for the times indicated. A, Nrf2 protein expression was determined by immunoblot analyses and equal loading demonstrated by anti-GAPDH. Bottom, densitometric scanning analysis ratio of Nrf2 to GAPDH. Superscripts (1) and (2) in the densitometric plot represent the ratio intensity of 100- to 110- and 68-kDa products corrected to GAPDH, respectively. B, PCNs were transfected with either nontargeting scramble siRNA (Scr. siRNA) or a SMARTpool mix of four siNrf2 using siPORT amine. Cells were lysed 48 h after transfection, and immunoblots were analyzed with anti-Nrf2 and GAPDH. Bottom, densitometric scanning analysis ratio of 68-kDa Nrf2 to GAPDH. C, PCNs were treated with ETOH (4 mg/ml) for the times indicated and real-time qRT-PCR analysis for Nrf2 transcript expression normalized to GAPDH. D, cells were incubated with Act D (1 μg/ml) along with ETOH (4 mg/ml) for a given time in hours. Total RNA was then extracted and subjected to qRT-PCR for Nrf2 and GAPDH. A to C, results are the mean ± S.E.M. of experiments performed in triplicate. One-way ANOVA for A, C, and D and a t test for B was performed to establish statistical significance. *, P < 0.05 compared with untreated control; ns, not significant compared with untreated control; #, P < 0.05 compared with respective ETOH time treatment. Rel, relative.

Fig. 2.

Ethanol induces Nrf2/ARE binding activity in PCN nuclear extracts. A, nuclear protein obtained from controls and PCNs exposed to ETOH (4 mg/ml) for different time points was used to detect ARE-specific oligonucleotide-protein complexes by EMSA. B, supershift analysis was performed using Nrf2 antibody and the presence of Nrf2 in the ARE DNA-protein complex was assayed. Excess unlabeled and mutant oligonucleotide was used to determine specificity of the Nrf2/ARE binding. C, TransAM ELISA for Nrf2 was performed to confirm Nrf2 DNA binding activity in control and ETOH-treated PCN nuclear extracts (mean ± S.E.M., n = 3). Statistical significance was established by one-way ANOVA. D, a representative immunofluorescence photomicrograph (original magnification, 20×) from control and 30-min ETOH (4 mg/ml)-treated PCNs for Nrf2 nuclear localization. Anti-MAP2 was used as a neuronal marker, and the nucleus was identified by DAPI staining. Data are representative of three independent experiments. E, RT-PCR image and statistical analysis for NQO1 and GAPDH mRNA from PCNs treated with and without ETOH (4 mg/ml) (n = 4). *, P < 0.05 compared with untreated control; ns, not significant compared with untreated control. Con, control; Wt, wild-type.

Fig. 3.

Prenatal ethanol exposure increases Nrf2 expression and activation in fetal brain cortices. Pregnant rats (Sprague-Dawley) at embryonic day 16 were administered ETOH (4 g/kg b.wt.) or isocaloric dextrose by gastric intubation at 12-h intervals for 2 days. At embryonic day 18 brain cortex from embryos were dissected and processed for Nrf2 protein expression by immunoblotting (A, n = 6), Nrf2 mRNA expression by qRT-PCR (B, n = 6). A, bottom, densitometric scanning ratio of Nrf2/GAPDH intensities. A and B, Student's t test (paired) was performed to determine the significance of treatment. Nuclear and cytosolic extracts were used for immunoblot analyses of Nrf2 to determine localization (C, mean ± S.E.M., n = 5), and nuclear extracts were used in TransAM ELISA for Nrf2 activity determination (D, mean ± S.E.M., n = 3). Statistical significance was established by one-way ANOVA. E, RT-PCR image and statistical analysis for NQO1 and GAPDH mRNA from control and ETOH-treated fetal brain cortices (n = 4). *, P < 0.05 compared with isocaloric dextrose-treated control; ns, not significant compared with isocaloric dextrose-treated animals. Rel, relative; Wt, wild-type.

Fig. 4.

siRNA-mediated Nrf2 knockdown exacerbates ethanol-induced oxidative stress and neuronal death. A, PCNs were transfected with either nontargeting scramble siRNA or a SMARTpool mix of four siNrf2s using siPORT amine. Twenty-four hours after transfection of scrambled siRNA or siNrf2, the cells were treated with or without ETOH (4 mg/ml) for an additional 24 h. Protein extracts were then immunoblot-analyzed with anti-Nrf2 and GAPDH. B, live cells were loaded with MCB and cells were fluorometrically analyzed for GSH content, and data are presented as relative fluorescence units (RFU) (mean ± S.E.M., n = 6). C, live cells were loaded with HET followed by FACS for ethidium positivity (measure of superoxide) to indicate level of oxidative stress (mean ± S.E.M., n = 6). D, cell extracts were used in the Caspase-Glo 3/7 assay and caspase 3/7 activity was determined as luminescence units (mean ± S.E.M., n = 6). E, live cells were stained using annexin V-FITC conjugate/PI, and FACS was used to measure apoptotic cell death (mean ± S.E.M., n = 6). One-way ANOVA was performed to establish statistical significance. *, @, P < 0.05 compared with scramble untreated control; #, P < 0.05 compared with siNrf2.

Fig. 5.

Adenovirus-mediated overexpression of Nrf2 contained ethanol-induced oxidative stress and neuronal death. A and B, PCNs (4DIV) in serum containing medium were infected with adenovirus encoding Nrf2 cDNA (A) or DN Nrf2 (B) at 200 MOI. Twenty-four and 48 h after infection, cells were processed for protein and immunoblot-analyzed for Nrf2 overexpression and normalized with anti-tubulin/GAPDH expression. Adenovirus encoding GFP cDNA was used as the control virus in this study. C to J, PCNs were infected with either Ad GFP/Ad Nrf2/Ad DN Nr2 for 24 h at 4DIV and followed by 24 h of ETOH (4 mg/ml) treatment. C, total RNA was extracted and subjected to qRT-PCR for GCLC, an Nrf2 transcriptional target and a constitutively expressed gene, GAPDH. Relative (Rel.) quantification of GCLC/GAPDH transcripts is illustrated (mean ± S.E.M., n = 6). D, neurons infected and treated as in C were subjected to flow cytometric determination of MCB staining to measure cellular GSH. A representative experiment is shown (D) wherein neuronal population were divided into low (orange), medium (green), and high GSH (blue) containing cells based on differential MCB staining. E, percentage of cells positive for MCB derived from high, medium, and low GSH populations are represented in blue, green, and orange graphs, respectively (mean ± S.E.M., n = 6). F, a representative FACS diagram, portraying the effect of Nrf2 on ethanol-induced superoxide generation measured in terms of oxidation of HET into the fluorescent product ethidium. Red vertical bars were introduced to visualize shift in peaks. G, estimation of effector caspase 3/7 activity in neuronal extracts obtained from the aforementioned treatment by the Caspase-Glo assay (mean ± S.E.M., n = 4). H, neurons were stained with annexin V-PE conjugate/7-amino-actinomycin and read immediately by flow cytometry to measure the extent of apoptosis (mean ± S.E.M., n = 6). One-way ANOVA was performed to establish statistical significance. *, @ P < 0.05 versus Ad GFP control; #, P < 0.05 versus Ad GFP + ETOH; ns, not significant.