Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 May 4:1144:192-201.
doi: 10.1016/j.brainres.2007.01.131. Epub 2007 Feb 7.

Nrf2-mediated protection against 6-hydroxydopamine

Rebekah J Jakel  1 , Jessica A Townsend, Andrew D Kraft, Jeffrey A Johnson
Affiliations

Nrf2-mediated protection against 6-hydroxydopamine

Rebekah J Jakel et al. Brain Res. .

Abstract

Parkinson's disease (PD) is a neurodegenerative movement disorder characterized by cell loss in the substantia nigra resulting in striatal dopamine depletion. Although the cause of sporadic PD is unknown, oxidative stress is thought to contribute to disease pathogenesis. One mechanism by which cells defend themselves against oxidative stress is through the transcriptional upregulation of cytoprotective genes. Under oxidative stress conditions, the transcription factor NF-E2-related factor (Nrf2) binds to the antioxidant response element (ARE) to induce antioxidant and phase II detoxification enzymes. Here we show that loss of Nrf2-mediated transcription exacerbates vulnerability to the neurotoxin 6-hydroxydopamine (6-OHDA) both in vitro and in vivo. We further demonstrate that activation of the Nrf2-ARE pathway by the known chemical inducer tert-butylhydroquinone can protect against 6-OHDA in vitro. Induction of this pathway by transplantation of astrocytes overexpressing Nrf2 can protect against 6-OHDA-induced damage in the living mouse. This suggests that the Nrf2-ARE pathway is a promising target for therapeutics aimed at reducing or preventing cell death in PD.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1. tBHQ induces ARE regulated genes in N27 cells and protects against 6-OHDA
N27 cells were transfected with ARE-luc/b-gal construct and exposed to tBHQ. As compared to vehicle control, tBHQ causes ARE activation (1μM, p=0.007; 3μM, p=1E-5; 10μM, p=1E-6; 30μM, p=4E-14; unpaired, 2-tailed t-tests; A). Note that maximal ARE activation occurs at approximately 10μM (A). N27 cells were exposed to tBHQ for 24 hours and harvested for RNA (B). RT-PCR reveals that treatment with tBHQ increases the amount of GSTmu3, NQO1, and HO-1 mRNA. β actin was used as a control for amount of RNA assayed. tBHQ protects against 6-OHDA in vitro (C). 6-OHDA causes a dose-dependent increase in cell death in N27 cells (as compared to 0μM 6-OHDA: 37.5μM, p=0.002; 75μM, p=0.0001; 150μM, p=0.00001). Pretreatment of N27 cells with 10μM tBHQ as compared to vehicle prior to exposure to 6-OHDA significantly attenuates cell death as measured by the MTS assay (37.5μM, p=0.0002; 75μM, p=0.003; 150μM, p=0.041). * significantly different from corresponding vehicle-treated control sample (p<0.05). # significantly different from corresponding sample in the absence of tBHQ (p<0.05).
FIGURE 2
FIGURE 2. Increased vulnerability of Nrf2−/− cells to 6-OHDA in vitro
Nrf2+/+ and −/− cells were exposed to 50μM 6-OHDA and assessed for TUNEL staining (A). The proportion of TUNEL+ cells per total cells was assessed for both genotypes (A and B). As compared to vehicle treatment, 6-OHDA increased cell death (Nrf2+/+, p=0.02; Nrf2−/−, p=0.00002; B). However, in the presence of 6-OHDA, there was significantly more apoptosis in Nrf2−/− cultures as compared to controls (p=0.003; B). A 6-OHDA dose-response curve also demonstrated increased vulnerability in null cultures as compared to wildtype cells (25μM, p=0.0041; 50μM, p=0.0212; 75μM, p=0.0395; C). Heterozygote cells were not significantly different than wildtype control cells (p>0.05; C). * significantly different from corresponding vehicle-treated control sample (p<0.05). # significantly different from corresponding sample in the absence of tBHQ (p<0.05).
FIGURE 3
FIGURE 3. Increased vulnerability of Nrf2−/− mice to 6-OHDA in vivo
6-OHDA was stereotactically injected into the striatum of wildtype and knockout Nrf2 mice with contralateral vehicle controls. After one week, mice were sacrificed and tissue was stained for THir (A). Nrf2 null mice demonstrated significantly larger lesions as revealed by THir and quantified by lesion volume analysis (A and B; p=0.0278). The 6-OHDA-induced reduction of THir in the SNpc was also more pronounced in the null mice as compared to the wildtypes (C). * significantly different as compared to Nrf2 wildtype animals (p<0.05).
FIGURE 4
FIGURE 4. Transplanted astrocytes
During the transplant surgeries, harvested astrocytes were kept on ice. Remaining cells (A) were plated on chamber slides overnight and stained for nuclei with Hoescht (blue), or anti-GFP for astrocytes (red). Infection with adenovirus expressing GFP can be seen (green, A). Unstained striatal sections shown above are from either Adeno-GFP or Adeno-Nrf2 transplanted animals and imaged for GFP fluorescence indicating surviving GFP-expressing astrocytes (B). Upon visual inspection, astrocytes survived equally well in both groups. Scale bar = 100μM.
FIGURE 5
FIGURE 5. Astrocytes expressing Adeno-Nrf2 significantly protect against 6-OHDA
Brain tissue collected from transplanted mice was stained for THir (A and B). Every third section was assessed for lesion area. Images taken at 1.25X magnification; scale bar = 1mm. Lesion volume was calculated for both hemispheres (C). Tranplantation of Adeno-GFP treated astrocytes resulted in a trend toward mild protection that did not reach statistical significance (p>0.05). Animals receiving astrocytes infected with Adeno-Nrf2 demonstrated significantly reduced lesion volume as compared to the contralateral striata (p=0.0326). * significantly different from contralateral vehicle-pretreated hemisphere (p<0.05).
See this image and copyright information in PMC

References

    1. Adams FS, La Rosa FG, Kumar S, Edwards-Prasad J, Kentroti S, Vernadakis A, Freed CR, Prasad KN. Characterization and transplantation of two neuronal cell lines with dopaminergic properties. Neurochem Res. 1996;21:619–27. - PubMed
    1. Alam ZI, Daniel SE, Lees AJ, Marsden DC, Jenner P, Halliwell B. A generalised increase in protein carbonyls in the brain in Parkinson’s but not incidental Lewy body disease. J Neurochem. 1997;69:1326–9. - PubMed
    1. Alam ZI, Jenner A, Daniel SE, Lees AJ, Cairns N, Marsden CD, Jenner P, Halliwell B. Oxidative DNA damage in the parkinsonian brain: an apparent selective increase in 8-hydroxyguanine levels in substantia nigra. J Neurochem. 1997;69:1196–203. - PubMed
    1. Cadet JL, Katz M, Jackson-Lewis V, Fahn S. Vitamin E attenuates the toxic effects of intrastriatal injection of 6-hydroxydopamine (6-OHDA) in rats: behavioral and biochemical evidence. Brain Res. 1989;476:10–5. - PubMed
    1. Castellani RJ, Perry G, Siedlak SL, Nunomura A, Shimohama S, Zhang J, Montine T, Sayre LM, Smith MA. Hydroxynonenal adducts indicate a role for lipid peroxidation in neocortical and brainstem Lewy bodies in humans. Neuroscience Letters. 2002;319:25–28. - PubMed

Publication types

MeSH terms

LinkOut - more resources