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.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.This work was supported by National Institutes of Health National Institute on Alcohol Abuse and Alcoholism [Grant R01-AA010114] (to G.I.H.).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.073262.
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ABBREVIATIONS:
- FASD
- fetal alcohol spectrum disorder
- ARE
- antioxidant response element
- ETOH
- ethanol
- MEM
- minimal essential medium
- HS
- horse serum
- DAPI
- 4,6-diamidino-2-phenylindole
- FITC
- fluorescein isothiocyanate
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- MAP2
- microtubule-associated protein 2
- siRNA
- small interfering RNA
- ECL
- enhanced chemiluminescence
- ELISA
- enzyme-linked immunosorbent assay
- PCN
- primary cortical neuron
- DIV
- days in vitro
- Act D
- actinomycin D
- q
- quantitative
- RT
- reverse transcriptase
- PCR
- polymerase chain reaction
- Ad GFP
- adenovirus for green fluorescent protein
- Ad WT Nrf2
- adenovirus for wild-type Nrf2
- Ad DN Nrf2
- adenovirus for dominant-negative Nrf2
- FACS
- fluorescence-activated cell sorting
- MCB
- monochlorobimane
- HET
- hydroethidine/dihydroethidium
- PI
- propidium iodide
- GCLC
- γ-glutamylcysteine ligase
- PBS
- phosphate-buffered saline
- EMSA
- electrophoretic mobility shift assay
- PE
- phycoerythrin
- ANOVA
- analysis of variance
- siNrf2
- small interfering RNA against Nrf2
- DN
- dominant negative
- MG-132
- N-[(phenylmethoxy)carbonyl]-l-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-l-leucinamide
- GST
- glutathione transferase.
- Received April 26, 2011.
- Accepted August 26, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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