Nrf2 regulates expression of many genes that function to alleviate oxidative stress. Astrocyte-specific overexpression of Nrf2 lessens pathology in models of Huntington’s disease, ALS, and Parkinson’s disease (Calkins et al., 2005
; Jakel et al., 2007
; Vargas et al., 2008
; Chen et al., 2009
). Loss of Nrf2 exacerbates disease and injury and increases inflammation (Calkins et al., 2005
; Shih et al., 2005
; Kraft et al., 2006
). These beneficial effects of Nrf2 are likely due to its ability to increase glutathione levels and relieve oxidative stress. Here, we show that astrocyte-specific overexpression of Nrf2 is beneficial in a mouse model of Alexander disease, but this is probably not due to effects on glutathione.
Mice overexpressing human wild-type GFAP, a severe model of Alexander disease, have increased ARE activity, increased NQO1 activity, and increased transcript levels of several genes regulated by Nrf2 (Hagemann et al., 2005
). R236H/+ mice also have an elevated antioxidant response, with ARE activity being most prominent in regions with Rosenthal fibers (Hagemann et al., 2006
). Here, we show the antioxidant response in R236H/+ mice is increased as measured by Nrf2
transcripts and ARE-hPAP activity and extend the previous results by quantifying Nrf2 activity in multiple brain regions.
Nrf2 overexpression consistently decreased Gfap
transcripts, GFAP protein, and Rosenthal fibers in R236H/+mice, especially so in olfactory bulb. Why the effects in olfactory bulb are so robust is not clear, but may relate to heterogeneity among brain regions in terms of astrocyte numbers and morphology as well as properties such as gene expression, composition of neurotransmitter and transporters, and response to injury (Emsley and Macklis, 2006
) (for review, see Zhang and Barres, 2010
). The effects of Nrf2 overexpression on GFAP levels in Gfap
mice also varied by brain region—GFAP protein decreased in olfactory bulb, brainstem, and cervical spinal cord, increased in hippocampus, and did not change in cerebral cortex. One possible explanation for these region-specific effects is variable activity of the 2.2 kb GFAP promoter used to direct expression of Nrf2, and indeed Nrf2 transcripts were highest in the olfactory bulb. However, other downstream indicators of Nrf2 activity (hPAP activity and Nqo1
transcripts) were not correspondingly increased in olfactory bulb, and promoter activity does not account for the GFAP increase found in hippocampus. Nrf2 is known to heterodimerize with other binding partners, such as the small Maf proteins (Itoh et al., 1997
; Motohashi et al., 2002
) to initiate transcription of its target genes, and perhaps expression of these transcription cofactors varies by brain region.
Regional variability of Nrf2 effects on GFAP may also explain why GFAP was not previously identified as being regulated by Nrf2. One set of studies used astrocyte cultures derived from cerebral cortex (Lee et al., 2003
; Shih et al., 2003
; Kraft et al., 2004
), and our results show that Nrf2 overexpression has no effect on GFAP in cortex of mice that are wild type at the Gfap
locus. Similarly, we detected no decrease in Gfap
mRNA in GFAP-Nrf2 cervical spinal cord, confirming results from lumbar spinal cord of the lower-expressing GFAP-Nrf2.4 mice (Vargas et al., 2008
). While our results do show that Nrf2 affects GFAP expression in several regions of the CNS, possibly due to effects on transcription, transcript stability, and/or protein turnover, they do not address whether this is a direct or indirect effect.
Depletion of glutathione is thought to contribute to pathology in other neurodegenerative diseases, such as Parkinson’s disease (for review, see Martin and Teismann, 2009
), but surprisingly we found no evidence that lower glutathione levels contribute to the pathogenesis of Alexander disease. Glutathione levels are unchanged at the whole-brain level in R236H/+ mice and are even increased in olfactory bulb where there is considerable pathology. In addition, the ratio of reduced to oxidized glutathione (GSH/GSSG), for which lower values are often interpreted as indicators of oxidative stress, was instead increased at the whole-brain level of R236H/+ mice, and unchanged in olfactory bulb. These results suggest either that the brains of Alexander disease mice do not have oxidative stress or that the endogenous Nrf2 response is able to keep glutathione homeostasis intact. Whether glutathione homeostasis is maintained or enhanced specifically in astrocytes cannot be determined from our data. However, global deletion of the Gclm
gene, which is thought to cause depletion of glutathione in all cell types, did not worsen the phenotype of the R236H/+ mice. Together, these data support the notion that glutathione depletion is not a significant factor in the pathogenesis of Alexander disease.
Whether the beneficial effects of Nrf2 overexpression are mediated by increased glutathione is a more difficult question. Nrf2 does regulate the expression of several genes involved in glutathione synthesis and homeostasis, and the protective effects of Nrf2 in other neurodegenerative disease and injury models corresponded with increases in glutathione (Kraft et al., 2004
; Vargas et al., 2008
; Calkins et al., 2010b
). However, in the present study, GFAP-Nrf2 mice showed no change in total glutathione in olfactory bulb, where Nrf2 overexpression had its most dramatic effects for decreasing GFAP and Rosenthal fibers. Since primary astrocyte cultures prepared from spinal cord of GFAP-Nrf2.4 mice do exhibit increases in both intracellular glutathione and glutathione secretion (Vargas et al., 2008
), it remains a formal possibility that such changes occur in olfactory bulb but are offset by changes in other cell types. An alternative possibility is that the protective effect of Nrf2 on GFAP levels may be mediated through its effects on other aspects of the antioxidant response, such as iron regulation or other detoxifying enzymes (e.g., superoxide dismutase or catalase).
R236H/+mice are theoretically an ideal model for Alexander disease since they are a knock-in of a point mutation commonly found in severe early-onset cases of human Alexander disease, and they replicate several features of the disease: elevated GFAP, gliosis, Rosenthal fibers, and both stress and immune responses (Hagemann et al., 2006
). However, the disease phenotype in these mice is not as severe as early-onset cases in humans, in that they have neither a shortened life span nor an apparent defect in myelin (Hagemann et al., 2006
). Behavioral studies currently underway show preliminary indications of deficits in hippocampal-dependent spatial learning (T. L. Hagemann, personal communication), and it will be interesting to examine the effects of Nrf2 overexpression on these phenotypes as well.
In conclusion, we show that astrocyte-specific overexpression of Nrf2 is sufficient to decrease GFAP transcripts, protein, and Rosenthal fibers in a mouse model of Alexander disease. Drugs to increase Nrf2 expression or activity might prove valuable for treating Alexander disease and possibly other diseases in which a decrease in gliosis may be beneficial. Since the Nrf2 transgene begins to be expressed during development, we can only conclude that it prevents an increase in GFAP and Rosenthal fibers, and future studies can test whether Nrf2 is sufficient to reverse high levels of GFAP and Rosenthal fibers. Nrf2 activity-inducing drugs, such as sulforaphane, cystamine, carnosic acid, and curcumin, have been used to treat mice in models of 3-NP/Huntington’s disease, ischemia, and Alzheimer’s disease with promising results (Lim et al., 2001
; Satoh et al., 2008
; Calkins et al., 2010a
), but whether these drugs can cross the blood– brain barrier in Alexander disease will need to be determined. Given the utility of increased Nrf2 expression in the R236H/+ mice, further studies will need to be directed at finding drugs that can control Nrf2 activity in the brain.