Figure 1.
Knockout of Nrf2 in Gfap mutant or transgenic mice eliminates ARE (antioxidant response element) activation.
(A) Quantitation of brain transcripts from Nrf2 and its target gene Nqo1 for both GFAPTg and Gfap+/R236H mice (3 mos of age) shows reduced expression in Nrf2+/− background and further reduction in Nrf2−/−. (C, D) Histochemical staining for alkaline phosphatase activity in GFAPTg mice crossed with an ARE-alkaline phosphatase reporter line shows wide-spread activation of Nrf2 (C), whereas GFAPTg/Nrf2−/− mice show virtually no ARE-reporter activity (D; 6 wks of age). (B) Quantification of reporter activity in brains from either GFAPTg or GFAP+/R236H mice shows a marked reduction in Nrf2+/+ vs. Nrf2+/− mice and virtually no detectable activity in Nrf2−/− animals (3 mos of age). Error bars = standard deviation. For Gfap+/+ versus Gfap+/R236H or GFAPTg comparisons (all Nrf2+/+), significance is indicated with a (+) plus sign. For Nrf2+/+ vs Nrf2+/− or −/− comparisons, significance is indicated by an (*) asterisk (unpaired t-test: + or *p<0.05; ++ or **p<0.01; +++ or ***p<0.001, n≥3).
Table 1.
Primer sets for quantitative PCR.
Figure 2.
Astrocytes from Gfap +/R236H/Nrf2−/− mice exhibit Rosenthal fibers and appear reactive, but Gfap and stress markers are reduced.
(A) Rosenthal fibers are apparent as eosinophilic aggregates in H&E stained tissue (arrows, hippocampus CA1 shown) in Gfap+/R236H/Nrf2−/− mice. (B) Quantitation of Gfap transcript by QPCR and protein by ELISA shows reduced Gfap expression in Gfap+/R236H/Nrf2−/− versus Gfap+/R236H/Nrf2+/+ mice in hippocampus (8 wks of age, n≥4, t-test **p>0.01, ***p>0.001 comparing Gfap+/R236H/Nrf2+/+ with Gfap+/R236H/Nrf2−/−). (C) Compared with wild-type Gfap+/+/Nrf2+/+ mice, Gfap immunohistochemistry in hippocampus (gray stain, 3 mos of age) shows hypertrophic reactive astrocytes in to Gfap+/R236H/Nrf2+/+ and Gfap+/R236H/Nrf2−/− mice. (D-F) An alternative marker of reactive astrocytes, the endothelin-B receptor (Endbr1, red), is expressed in astrocytes (Gfap, green) from both Gfap+/R236H/Nrf2+/+ (E) and Gfap+/R236H/Nrf2−/− mice (F), compared with wild-type Gfap+/+/Nrf2+/+ (D) which show Endbr1 expression predominantly associated with blood vessels (hilus, 8 wks of age). Scale bar = 50 µm (shown in D, applies to all confocal images). (G) Hippocampal expression of stress related genes, including Nqo1, Cryab, and Cp, is reduced in Gfap+/R236H mice with the loss of Nrf2 (8 wks). Error bars indicate standard deviation (t-test **p<0.01, ***p<0.001; n = 4).
Figure 3.
Microglia are less reactive in Gfap+/R236H mice without Nrf2.
(A) Compared to wild-type mice, Gfap mutant mice show elevated Iba1 immunoreactivity in hippocampus either with or without Nrf2. (B) Western analysis of hippocampal protein shows reduced Iba1 expression in Gfap+/R236H/Nrf2−/− compared with Gfap+/R236H/Nrf2+/+ mice (8 wks), although levels remain elevated above wild-type (**p<0.01, unpaired t-test comparing Gfap+/R236H/Nrf2+/+ with Gfap+/R236H/Nrf2−/− mice, n = 4). Lanes 5 and 6 for Nrf2−/− samples are from a different region of the same gel as lanes 1–4 (Nrf2+/+ samples).
Figure 4.
p62 in Gfap+/R236H mice colocalizes with Gfap aggregates and is reduced with loss of Nrf2.
(A) p62 immunohistochemistry in hippocampus from Gfap+/R236H/Nrf2+/+ mice shows astrocytic cells that are not apparent in Gfap+/+ mice. Gfap+/R236H/Nrf2−/− mice show similarly stained cells (3 mos of age). (B) Confocal microscopy with immunofluorescence for p62 (green) shows colocalization with Gfap aggregates (red) in both Nrf2+/+ and Nrf2−/− Gfap+/R236H mice (8 wks of age). (C) Western blot analysis of hippocampal protein shows increased p62 in Gfap mutant mice compared to wild-type, with Gfap+/R236H/Nrf2−/− mice having levels reduced to that of wild-type mice (8 wks of age, n≥4, t-test **p>0.01, comparing Gfap+/R236H/Nrf2+/+ with Gfap+/R236H/Nrf2−/−). Scale bar = 25 µm (shown in B, applies to both confocal images).
Figure 5.
Iron accumulation in astrocytes from Gfap mutants is reduced with loss of Nrf2.
(A) Modified Perl’s stain shows iron in small round cell bodies (brown) with oligodendrocyte morphology separate from Gfap immunostained astrocytes (blue/green) in Gfap+/+ olfactory bulb, whereas iron staining is prominent in astrocytes with reactive morphology in Gfap+/R236H mice and in some cases appears to localize with Gfap aggregates (3 mos of age). (B) Ferritin immunofluorescence shows occasional colocalization with Mac1 (hippocampus, 3 mos of age) suggesting some iron storage in microglia. (C-H) Iron histochemistry shows accumulation in astrocytes of both olfactory bulb (C-E) and hippocampus (F-H) in Gfap+/R236H mice (D, G) compared with Gfap+/+ brains (C, F). Loss of Nrf2 reduces iron staining in Gfap+/R236H/Nrf2−/− mice (E, H). (I-N) Immunostaining for ferritin (red) and Gfap (green) in olfactory bulb (I-K) and hippocampus (L-N) shows ferritin labeling in Gfap negative cells with oligodendrocyte morphology, especially in olfactory bulb (I) and sparse labeling in hippocampus (L) in wild-type mice at 8 wks of age. In Gfap+/R236H/Nrf2+/+ mice, ferritin colocalizes with Gfap in astrocytes in both olfactory bulb (J) and hippocampus (M). Gfap+/R236H/Nrf2−/− mice (K, N) show an intermediate pattern with less staining in Gfap positive cells, and in olfactory bulb (K), cells that appear to be oligodendrocytes. Scale bars = 25 µm (A); 50 µm (B, I).
Figure 6.
Ferritin transcripts and protein are differentially regulated.
(A) Transcripts for the ferritin heavy chain gene Fth1 are elevated in both olfactory bulb and hippocampus from Gfap+/R236H mice, and are reduced to wild-type levels with loss of Nrf2. (B) Fth1 protein levels, however remain unchanged in all genotypes. (8 wks of age, n = 4, t-test **p>0.01, ***p > 0.001 comparing Gfap+/R236H/Nrf2+/+ with Gfap+/R236H/Nrf2−/−).
Figure 7.
Loss of Nrf2 in a lethal model of AxD does not affect survival.
Double positive Gfap+/R236H/GFAPTg mice that die at approximately 25 days of age show no significant difference in survival with loss of Nrf2 (Gfap+/R236H/GFAPTg/Nrf2+/+, N = 15; Gfap+/R236H/GFAPTg/Nrf2−/−, N = 5, Log-rank (Mantel-Cox) Test). For GFAPTg/Nrf2−/− and Gfap+/R236H/Nrf2−/− mice, animals were euthanized at 6 wk and 3 mo time points for various experiments: for GFAPTg/Nrf2−/− (6 wks n = 20, 3 mos n = 12, 6 mos n = 2) for Gfap+/R236H/Nrf2−/− (6 wks n = 21, 3 mos n = 14, 6 mos n = 7).