Sustained interleukin-1β overexpression exacerbates tau pathology despite reduced amyloid burden in an Alzheimer's mouse model

Abstract

Neuroinflammation is an important component of Alzheimer's disease (AD) pathogenesis and has been implicated in neurodegeneration. Interleukin-1 (IL-1), a potent inflammatory cytokine in the CNS, is chronically upregulated in human AD and believed to serve as part of a vicious inflammatory cycle that drives AD pathology. To further understand the role of IL-1β in AD pathogenesis, we used an inducible model of sustained IL-1β overexpression (IL-1β(XAT)) developed in our laboratory. The triple transgenic mouse model of AD, which develops plaques and tangles later in its life cycle, was bred with IL-1β(XAT) mice, and effects of IL-1β overexpression on AD pathology were assessed in F1 progeny. After 1 and 3 months of transgene expression, we found robust increases in tau phosphorylation despite an ∼70-80% reduction in amyloid load and fourfold to sixfold increase in plaque-associated microglia, as well as evidence of greater microglial activation at the site of inflammation. We also found evidence of increased p38 mitogen-activated protein kinase and glycogen synthase kinase-3β activity, which are believed to contribute to tau phosphorylation. Thus, neuroinflammation regulates amyloid and tau pathology in opposing ways, suggesting that it provides a link between amyloid accumulation and changes in tau and raising concerns about the use of immunomodulatory therapies in AD.

Figure 1.

Research design and chronic neuroinflammatory phenotype in 3xTgAD/IL-1β^XAT mice. A, The linear construct (∼10 kb) bears the murine GFAP promoter (mGFAP), a transcriptional stop signal flanked by LoxP sites, and the cDNA for hIL-1β gene fused to the signal sequence of mature hIL-1RA (ssIL-1β). After delivery of Cre, the transcriptional stop is excised out and the production of IL-1β is induced locally. B, 3xTgAD mice were bred to the IL-1β^XAT mice. Fifteen-month-old 3xTgAD/IL-1β^XAT and control littermates of the 3xTgAD genotype from the F1 generation were given a unilateral stereotactic injection of FIV–Cre in the subiculum. Groups of mice were killed 1 and 3 months after stereotactic surgery (at 16 and 18 months of age, respectively), and glial activation as well as amyloid and tau pathology were assessed. C, Murine IL-1β protein ELISA measurements. C, Contralateral; I, ipsilateral. Data were expressed as mean picograms mIL-1β per milligram protein ± SEM per group and analyzed by a one-way ANOVA test, followed by a Tukey's post hoc test. The level of mIL-1β in the ipsilateral hippocampus of 3xTgAD/IL-1β^XAT mice is significantly greater than all other groups (***p < 0.0001), but the three other groups are not significantly different from each other (n = 3–9 per group). One month after transgene activation, elevation in markers of glial activation was observed in the ipsilateral subiculum of 3xTgAD/IL-1β^XAT mice. D, Area fraction measurements of Iba1 immunohistochemical data. Data expressed as mean I/C ratio ± SEM per group and analyzed by unpaired Student's t test, ***p < 0.0001; n = 5–7 per group. E, Area fraction measurements of GFAP immunohistochemical data. Data expressed as mean I/C ratio ± SEM per group and analyzed by unpaired Student's t test, **p < 0.01; n = 3–4 per group. Representative confocal micrographs of the ipsilateral and contralateral subiculum immunostained with the microglial marker Iba1 (F) and the astrocytic marker GFAP (G) are shown. Scale bars, 30 μm. H, Representative photomicrographs from 16-month-old 3xTgAD/IL-1β^XAT mice immunostained with an antibody against V5, a viral epitope tag present in the injected FIV–Cre construct. Scale bars, 200 μm. I, Representative high-power photomicrograph from the same experiment demonstrating the presence of V5 in the nucleus of a GFAP-positive astrocyte in the ipsilateral but not the contralateral subiculum. Scale bars, 10 μm.

Figure 2.

Sustained IL-1β expression ameliorates amyloid load in 3xTgAD/IL-1β^XAT mice. A, Representative photomicrographs of contralateral and ipsilateral subicular sections from 3xTgAD and 3xTgAD/IL-1β^XAT mice immunostained with the anti-amyloid antibody 6E10 at 16 and 18 months. Scale bars, 100 μm. B, Quantification of 6E10 immunopositive amyloid plaque area fraction in the ipsilateral and contralateral subiculum of 3xTgAD and 3xTgAD/IL-1β^XAT mice at 16 and 18 months. Data are represented as the mean I/C ratio ± SEM for each group and was analyzed by a two-way ANOVA with Bonferroni's post hoc test; n = 3–5 mice per group. C–G, ELISA results obtained from whole ipsilateral and/or contralateral hippocampi of 3xTgAD and 3xTgAD/IL-1β^XAT mice at 16 months. Mean I/C ratios ± SEM of Aβ₄₀ and Aβ₄₂ in the guanidinium-HCl (Insoluble) fraction (C, D), Tper (Soluble) fraction (F, G), and Oligomeric Aβ in the soluble fraction (E) are shown. In all measures, 3xTgAD/IL-1β^XAT mice demonstrated a lower amyloid load in the ipsilateral subiculum/hippocampus at 16 and 18 months compared with 3xTgAD controls; n = 3–9 mice per group. Data were analyzed with unpaired Student's t tests; **p < 0.01, ***p < 0.0001.

Figure 3.

Sustained IL-1β expression does not alter expression of APP or its processing in 3xTgAD/IL-1β^XAT mice. Homogenates from ipsilateral and contralateral hippocampi of 16-month-old 3xTgAD and 3xTgAD/IL-1β^XAT mice were subjected to Western blot analysis with markers of APP expression and processing. Group means were not significantly different overall. A, Representative Western blot images of contralateral (C) and ipsilateral (I) hippocampi of 3xTgAD and 3xTgAD/IL-1β^XAT mice probed with antibodies against APP, BACE, β-CTF, and tubulin at 16 months. Molecular weights are expressed in kilodaltons. Mean I/C ratio ± SEM of relative band intensities per group (normalized to tubulin band intensities) is shown for APP (B), BACE (C), and β-CTF (D); n = 3–9 mice per group.

Figure 4.

Sustained IL-1β overexpression enhances plaque-associated microglia in 3xTgAD/IL-1β^XAT mice. Representative confocal micrographs of contralateral and ipsilateral subicular sections stained with Congo Red (fibrillar Aβ plaques), Iba-1 (microglia), and DAPI (nuclei) are shown. 3xTgAD/IL-1β^XAT mice showed more plaque-associated microglia at 16 months (B) and 18 months (C) compared with their 3xTgAD counterparts (A) in the ipsilateral subiculum. Scale bars, 30 μm. On quantification, 3xTgAD/IL-1β^XAT mice demonstrate approximately fourfold more plaque-associated microglia at 16 months and ∼6.5-fold more plaque associated microglia at 18 months in the ipsilateral subiculum compared with their 3xTgAD counterpart at 16 months (D). Data are shown as mean I/C ratio ± SEM of Iba1-positive cells per plaque; n = 3–4 mice per group. Data were analyzed with one-way ANOVA, followed by a Bonferroni's post hoc test; **p < 0.01. Quantitative results are shown for contralateral and ipsilateral hemispheres of 16-month-old 3xTgAD/IL-1β^XAT mice brain sections stained with 6E10 (amyloid plaques), CD68 (activated microglia), Iba1 (microglia), and DAPI (nuclei). The ipsilateral subiculum demonstrates a 4.6-fold increase in CD68 area when normalized to plaque area (E). Data were expressed as mean CD68/6E10 area Fraction within a 500 pixel diameter around the plaques ± SEM for contralateral (C) and ipsilateral (I) subiculum. Analyzed by a paired Student's t test, ***p < 0.001; n = 4 mice. The ipsilateral subiculum also demonstrates a 3.5-fold increase in CD68 area when normalized to Iba1 area (F). Data were expressed as mean CD68/Iba1 area fraction within a 500 pixel diameter around the plaques ± SEM for contralateral (C) and ipsilateral (I) subiculum. Analyzed by a paired Student's t test, *p < 0.05; n = 4 mice. G, Representative confocal micrographs from the experiment are shown. Scale bars, 20 μm.

Figure 5.

Sustained overexpression of IL-1β leads to enhanced tau phosphorylation in 16-month-old 3xTgAD/IL-1β^XAT mice. Sections of the subiculum–CA1 junction of 16-month-old 3xTgAD and 3xTgAD/IL-1β^XAT brains were probed with antibodies against phospho-tau and total tau. Representative images of section stained with phospho-tau antibodies pT205 (A) and AT180 (B) show increased immunostaining in the ipsilateral hemispheres of 3xTgAD/IL-1β^XAT mice. HT7 immunostaining (C) failed to show any difference between the ipsilateral and contralateral hemispheres of either genotype. Scale bars, 100 μm. Insets show sections from the images as marked in the first panel of A magnified 2× digitally. Immunopositive area fractions were quantified in the entire field visualized (D, F, H) or in the stratum radiatum (E, G, I). All numerical data are represented as mean I/C ratio ± SEM per group. 3xTgAD/IL-1β^XAT mice demonstrate an approximate twofold increase in pT205 immunostaining (D, E) and an approximate fourfold increase in AT180 immunostaining (F, G) but not in HT7 immunostaining (H, I) in the ipsilateral subiculum; n = 4–6 mice per group; data were analyzed with an unpaired Student's t test. Lysates from ipsilateral (i) and contralateral (c) hippocampi of 16-month-old 3xTgAD and 3xTgAD/IL-1β^XAT mice were subjected to Western blot analysis with antibodies against phospho-tau and total tau. Representative images of Western blots with pT205, AT180, and PHF1, as well as total tau are shown (J). Molecular weights are expressed in kilodaltons. 3xTgAD/IL-1β^XAT mice demonstrate an approximate twofold increase in tau phosphorylation at pT205 (K), AT180 (L), and PHF1 (M) epitopes. All band intensities were normalized to total tau; n = 3–9 mice per group. Data were analyzed with unpaired Student's t tests; *p ≤ 0.05, **p < 0.01, ***p < 0.001.

Figure 6.

Sustained overexpression of IL-1β leads to enhanced tau phosphorylation in 18-month-old 3xTgAD/IL-1β^XAT mice. Sections of the subiculum–CA1 junction of 18-month-old 3xTgAD and 3xTgAD/IL-1β^XAT brains were probed with antibodies against phospho-tau and total tau. Representative images of section stained with the phospho-tau antibody pS396 (A) show increased immunostaining in the ipsilateral hemispheres of the 3xTgAD/IL-1β^XAT mice. HT7 immunostaining (B) failed to show any difference between the ipsilateral and contralateral hemispheres of either genotype. Scale bars, 100 μm. Insets show sections from the images as marked in the first of Fig. 5A magnified 2× digitally. C, D, Immunopositive area fractions were quantified in the entire field visualized. 3xTgAD/IL-1β^XAT mice demonstrate a an approximate twofold to threefold increase in pS396 immunostaining (C) but not in HT7 immunostaining (D). Numerical data are represented as mean I/C ratio ± SEM per group; n = 4–6 mice per group. Data were analyzed with an unpaired Student's t test; **p < 0.01.

Figure 7.

Sustained overexpression of IL-1β leads to activation of kinase pathways in 16-month-old 3xTgAD/IL-1β^XAT mice. Lysates from ipsilateral and contralateral hippocampi of 16-month-old 3xTgAD and 3xTgAD/IL-1β^XAT mice were subjected to Western blot analysis with markers of kinase activation. Representative image of Western blots (A) and densitometric quantification of the band intensities (B, C) demonstrated a decrease in pS9GSK3β in the ipsilateral hippocampus of the 3xTgAD/IL-1β^XAT mice suggesting an increase in the activity of GSK3β and an increase in phospho-p38MAPK suggesting enhanced activity of p38MAPK, but the steady-state levels of both enzymes were not changed between groups. Molecular weights are expressed in kilodaltons. Band intensities of phospho-epitopes were normalized to the steady-state levels of GSK3β and p38MAPK, respectively. Numerical data are represented as mean I/C ratio ± SEM per group; n = 3–9 per group. Data were analyzed with unpaired Student's t tests; *p ≤ 0.05, ***p < 0.001.

Figure 8.

Proposed role of IL-1β in our model. IL-1β transgene expression acts on the resident microglia and astrocytes, leading to their activation, which in turn triggers a positive feedback loop of IL-1β production and neuroinflammation. Microglial activation may aid in amyloid clearance. The local inflammatory milieu also leads to activation of kinase pathways that may directly or indirectly result in enhanced tau phosphorylation. Questions for future investigation are indicated.