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. 2013 Jan;27(1):187-98.
doi: 10.1096/fj.12-208660. Epub 2012 Oct 4.

Attenuating astrocyte activation accelerates plaque pathogenesis in APP/PS1 mice

Andrew W Kraft  1 , Xiaoyan Hu, Hyejin Yoon, Ping Yan, Qingli Xiao, Yan Wang, So Chon Gil, Jennifer Brown, Ulrika Wilhelmsson, Jessica L Restivo, John R Cirrito, David M Holtzman, Jungsu Kim, Milos Pekny, Jin-Moo Lee
Affiliations

Attenuating astrocyte activation accelerates plaque pathogenesis in APP/PS1 mice

Andrew W Kraft et al. FASEB J. 2013 Jan.

Abstract

The accumulation of aggregated amyloid-β (Aβ) in amyloid plaques is a neuropathological hallmark of Alzheimer's disease (AD). Reactive astrocytes are intimately associated with amyloid plaques; however, their role in AD pathogenesis is unclear. We deleted the genes encoding two intermediate filament proteins required for astrocyte activation-glial fibrillary acid protein (Gfap) and vimentin (Vim)-in transgenic mice expressing mutant human amyloid precursor protein and presenilin-1 (APP/PS1). The gene deletions increased amyloid plaque load: APP/PS1 Gfap(-/-)Vim(-/-) mice had twice the plaque load of APP/PS1 Gfap(+/+)Vim(+/+) mice at 8 and 12 mo of age. APP expression and soluble and interstitial fluid Aβ levels were unchanged, suggesting that the deletions had no effect on APP processing or Aβ generation. Astrocyte morphology was markedly altered by the deletions: wild-type astrocytes had hypertrophied processes that surrounded and infiltrated plaques, whereas Gfap(-/-)Vim(-/-) astrocytes had little process hypertrophy and lacked contact with adjacent plaques. Moreover, Gfap and Vim gene deletion resulted in a marked increase in dystrophic neurites (2- to 3-fold higher than APP/PS1 Gfap(+/+)Vim(+/+) mice), even after normalization for amyloid load. These results suggest that astrocyte activation limits plaque growth and attenuates plaque-related dystrophic neurites. These activities may require intimate contact between astrocyte and plaque.

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Figures

Figure 1.
Figure 1.
Deletion of Gfap and Vim accelerates amyloid plaque pathogenesis. A–F) Brain sections from APP/PS1 Gfap+/+Vim+/+ and APP/PS1 Gfap−/−Vim−/− mice were immunostained with anti-Aβ antibodies (A) or stained with X-34 to label plaques (D). Gfap and Vim deletion increased Aβ-immunostained plaque load in the cerebral cortex (B) and hippocampus (C) at 8 and 12 mo of age, but there was no difference at 4 mo in either region. X-34-stained “compact” plaque load was also increased in cerebral cortex (E) and hippocampus (F) in APP/PS1 Gfap−/−Vim−/− mice. G) Size-frequency histogram of X-34-labeled amyloid plaques in 12-mo-old APP/PS1 Gfap+/+Vim+/+ (open bars) and APP/PS1 Gfap−/−Vim−/− (shaded bars) mice. APP/PS1 Gfap−/−Vim−/− mice had more plaques in every size category. Values are expressed as means ± se; n = 8–11 mice/group. *P < 0.05, **P < 0.01.
Figure 2.
Figure 2.
Insoluble Aβ is increased in aged APP/PS1 Gfap−/−Vim−/− mice. Cortices dissected from APP/PS1 Gfap+/+Vim+/+ and APP/PS1 Gfap−/−Vim−/− mice were extracted in PBS (A, C, E) and then in guanidine (B, D, F) at 4 (A, B), 8 (C, D), and 12 (E, F) mo of age. Consistent with plaque load data, Aβx-42 in guanidine extracts was increased in 8-mo-old APP/PS1 Gfap−/−Vim−/− mice, while Aβx-40 demonstrated a trend for increase (P=0.14; D). Similarly, in 12-mo-old mice, Aβx-40 was significantly higher in APP/PS1 Gfap−/−Vim−/− mice, while Aβx-42 was not (F). PBS-soluble fractions showed no difference between genotypes at all ages. n = 7–12 mice/group. *P < 0.05.
Figure 3.
Figure 3.
ISF Aβ is unchanged by Gfap and Vim deletion. In vivo microdialysis was performed in the hippocampi of APP/PS1 Gfap+/+Vim+/+ and APP/PS1 Gfap−/−Vim−/− mice. A) Steady-state levels of Aβ1-x were similar in mice of both genotypes. B) The half-life of Aβ was also similar, suggesting that the deletions did not alter the Aβ metabolism in these mice.
Figure 4.
Figure 4.
Gfap and Vim deletion exacerbates neuritic dystrophy in APP/PS1 mice. A–D) RTN-3-labeled dystrophic neurites were compared in the entorhinal cortex of 12-mo-old APP/PS1 Gfap+/+Vim+/+ mice (A, B) and APP/PS1 Gfap−/−Vim−/− mice (C, D). E, F) Deletion of Gfap and Vim increased the burden of RTN-3-labeled dystrophic neurites (E), even when normalized to plaque load (F). G) Individual plaques from mice of both genotypes were randomly imaged; plaque size (cross-sectional area) and RTN-3 burden (cross-sectional area) were measured for each plaque and plotted in a scatterplot, which demonstrates that the dystrophic neurite burden was greater in Gfap−/−Vim−/− mice across all plaque sizes. Linear regression for Gfap+/+Vim+/+ mice: y = 0.55x + 32.51, R2 = 0.77; and for Gfap−/−Vim−/− mice: y = 3.84x − 150.61, R2 = 0.10 (P<0.0001 for difference in slopes). RTN-3 immunostaining colabels silver-stained dystrophic neurites (see Supplemental Fig. S1). Scale bars = 150 μm (A, B); 20 μm (C, D). Values are expressed as means ± se; n = 5 mice/group. **P < 0.01.
Figure 5.
Figure 5.
Gfap/Vim deletion does not alter expression or processing of APP- or Aβ-degrading proteases. A) RNA extracted from cortex of 12-mo-old mice was subjected to real-time PCR to compare transcript levels for APP processing secretases, lipoprotein mediators, and Aβ-degrading enzymes (as indicated), and expressed as fold-change compared to APP/PS1 Gfap+/+ Vim+/+ mice. Of all transcripts examined, only APH-1a and MMP9 showed differences between genotypes. *P < 0.05. B–D) Protein extracted from cortex of mice was analyzed by Western blot with anti-APP (B); anti-apoE and anti-apoJ (C); and anti-MMP-2 and anti-MMP-9 (D; pMMP-2, pro-MMP-2; pMMP-9, pro-MMP-9) antibodies as indicated. There was no significant difference in expression of any protein examined. Values are means ± se; n = 4–7 mice/group. E) Representative blots comparing APP/PS1 Gfap+/+ Vim+/+ (left lane) to APP/PS1 Gfap−/− Vim−/− (right lane) mice for each of the proteins above.
Figure 6.
Figure 6.
Gfap−/−Vim−/− astrocytes surrounding plaques lack the reactive phenotype. A–D) Twelve-month-old APP Gfap+/+Vim+/+ mice (A, C) and APP/PS1 Gfap−/−Vim−/− mice (B, D) were injected with AAV-GFAP-GFP to label astrocytes (green) and euthanized 1 mo later. Plaques were labeled with X-34 (blue). Low-power images from APP Gfap+/+Vim+/+ (A) and APP/PS1 Gfap−/− Vim−/− mice (B) reveal a striking difference in cell morphology, which is better appreciated at high power (C, D). While wild-type astrocytes had hypertrophic proximal processes and intimate contact with plaques in APP mice (C), the Gfap−/−Vim−/− astrocytes lack both (D), and see Supplemental Videos S1 and S2. E-F) Astrocyte cell counts revealed no difference between genotypes (E); however, quantification of regions of overlap between astrocyte processes and X-34 staining shows a marked decrease in APP/PS1 Gfap−/−Vim−/− mice (F). Values are expressed as means ± se; n = 58–61 plaques in 3 mice/group. ****P < 0.0001. G, H) Astrocytes distant from plaques of both genotypes were indistinguishable. Scale bars = 50 μm (A, B); 20 μm (C, D, G, H).
Figure 7.
Figure 7.
Gfap and Vim deletion increase microglial abundance around plaques. A–D) Brain sections from 8-mo-old APP/PS1 Gfap+/+Vim+/+ (A, C) and APP/PS1 Gfap−/−Vim−/− (B, D) mice were immunostained with anti-Iba-1 antibodies (green) and X-34 (blue). E, F) Deletion of Gfap and Vim modestly increased the density of Iba-1-immunostained cells (E) but doubled the overlap between plaque and microglia, suggesting increased interaction (F); n = 3 mice/group. G) RT-PCR quantification of transcripts demonstrates increased expression of CD11b and Iba-1, consistent with the increased immunostaining. Astroglial transcripts (glutamine synthase, S100b) and inflammatory mediators (IL-1b, IL-6, IL-10, TNF-α, TGF-β, and iNOS) were unchanged; n = 4–7 mice/group. Values are expressed as means ± se. *P < 0.05. ***P < 0.001.
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