Blocking IL-1 signaling rescues cognition, attenuates tau pathology, and restores neuronal β-catenin pathway function in an Alzheimer's disease model

Abstract

Inflammation is a key pathological hallmark of Alzheimer's disease (AD), although its impact on disease progression and neurodegeneration remains an area of active investigation. Among numerous inflammatory cytokines associated with AD, IL-1β in particular has been implicated in playing a pathogenic role. In this study, we sought to investigate whether inhibition of IL-1β signaling provides disease-modifying benefits in an AD mouse model and, if so, by what molecular mechanisms. We report that chronic dosing of 3xTg-AD mice with an IL-1R blocking Ab significantly alters brain inflammatory responses, alleviates cognitive deficits, markedly attenuates tau pathology, and partly reduces certain fibrillar and oligomeric forms of amyloid-β. Alterations in inflammatory responses correspond to reduced NF-κB activity. Furthermore, inhibition of IL-1 signaling reduces the activity of several tau kinases in the brain, including cdk5/p25, GSK-3β, and p38-MAPK, and also reduces phosphorylated tau levels. We also detected a reduction in the astrocyte-derived cytokine, S100B, and in the extent of neuronal Wnt/β-catenin signaling in 3xTg-AD brains, and provided in vitro evidence that these changes may, in part, provide a mechanistic link between IL-1 signaling and GSK-3β activation. Taken together, our results suggest that the IL-1 signaling cascade may be involved in one of the key disease mechanisms for AD.

Figure 1. Administering an IL-1R blocking antibody rescues hippocampus-dependent cognitive impairments in the 3xTg-AD mice

(A) Acquisition curve during training of MWM is expressed as mean ± S.E.M. No significant difference was observed among the groups. (B) Escape latency and (C) number of crosses on platform location for 24-hr retention trial in MWM. Each bar is expressed as mean ± S.E.M., and *p<0.05 compared to control and IgG control groups. (D) The freezing index of CFC is expressed as mean ± S.E.M. *p<0.05 compared to control and IgG control groups. The number of mice tested: n=10 for control and IgG control, and n=8 for IL-1R blocking antibody treatment.

Figure 2. Blocking IL-1 signaling and effect on amyloid pathology in the 3xTg-AD mice

(A) Quantitative Aβ ELISA in detergent-soluble brain fraction and (B) detergent-insoluble (formic acid soluble) brain fraction. Each bar is expressed as mean ± S.E.M., and *p<0.05 compared to control and IgG control groups. (C) Representative immunohistochemical staining of amyloid plaque burden in the hippcampus and amygdala. Anti-Aβ₄₂ specific antibody detects aggregated Aβ-containing plaques (scale bar = 500 μm). (D) Amyloid burden in the hippocampus, sebiculum, entorhinal cortex and amygdala is expressed as a bar graph. ns stands for not significant. (E) Plaque count (over 20 μm in diameter) in 500 μm² subfield in the hippocampus, subiculum, entorhinal cortex and amygdala is expressed in the bar graph (mean ± S.E.M.). *p<0.05 compared to control, and ns stands for not significant. (F) Dot blot analysis of oligomeric Aβ species using antibody A11, and (G) antibody OC. Each bar is expressed as mean ± S.E.M., and *p<0.05 compared to control and IgG control groups. More dot blot data for A11 and OC are found in Suppl. Fig. 2B. (H) Immunoblot analysis of APP processing in the brain following the treatment. The densitometric analysis of C99 and C83 fragments was shown in the graph. Each bar is expressed as mean ± S.E.M. No statistical significance was obtained. The number of mice tested: n=10 for control and IgG control, and n=8 for IL-1R blocking antibody treatment.

Figure 3. Blocking IL-1 signaling decreases pro-inflammatory cytokines and enhances microglial phagocytosis

(A) ELISA analysis of selected pro-inflammatory cytokines. Each bar is expressed as mean ± S.E.M. (n=10 for control and IgG control, and n=8 for anti-IL-1R treatment), and *p<0.05 compared to control and IgG control groups. (B) IL-1β levels are decreased in animals receiving the anti-IL-1R blocking antibody. Double immunofluorescent staining with IL-1β and microglia. Asterisks indicate amyloid plaques (scale bar = 10 μm). (C) Suppressing IL-1 signaling promotes the phagocytosis of Aβ by microglia. Representative double-immunofluorescent staining with Aβ (6E10) and microglia (Iba1) in the brain of anti-IL-1R-treated 3xTg-AD mice. Arrows indicate Aβ within microglial compartment (scale bar = 10 μm). (D) Double immunofluorescent staining of YM1 (green) and tomato lectin (red) around Aβ plaques in control and anti-IL-1R antibody-treated mice. Arrow indicates activated microglia with high YM1 expression. YM1 fluorescent intensity was measured and plotted in graph (mean ± S.E.M.). *p<0.05 or **p<0.01 compared to IgG control or control, respectively. (E) Immunoblots and densitometric analyses of Aβ degrading enzymes, insulin degrading enzyme (IDE) and neprilysin in the brain homogenates (n=10 for control and IgG control, n=8 for IL-1R blocking antibody treatment). No statistical significance is detected by densitometric analyses (mean ± S.E.M.).

Figure 4. Suppressing IL-1 signaling attenuates tau pathology in the 3xTg-AD mice

(A) Tau pathology is attenuated by blocking IL-1 signaling. Representative immunohostochemical staining with various tau antibodies; HT7 – total tau, AT8 – phosphorylated tau at Ser 199 and Ser 202, and AT100 – phosphorylated tau at Ser 212 and Thr 214. (B) Immunoblot and densitometric analyses of changes in the steady-state levels of phosphorylated tau in the brain. Each bar is expressed as mean ± S.E.M. (n=10 for control and IgG control, and n=8 for anti-IL-1R treatment), and *p<0.05 or **p<0.01 compared to control and IgG control groups. More immunoblot data for phospho-tau are found in Suppl. Fig. 2D. (C) Suppressing IL-1 signaling results in decreased activations of tau kinases. Immunoblot and densitometric analyses of the steady-state levels of GSK-3β, cdk5/p35/p25 and p38-MAPK. Each bar is expressed as mean ± S.E.M. (n = 10 for control and IgG control, and n=8 for anti-IL-1R treatment), and *p<0.05 or **p<0.01 compared to control and IgG control groups.

Figure 5. S100B and β-catenin signaling are altered in anti-IL-1R antibody-treated 3xTg-AD mice

(A) Immunoblot analysis of the steady-state levels of phospho-β-catenin at Ser 33/Ser 37/Thr 41. (B-E) Densitometric analysis of the intensity of immunoblots. Each bar is expressed as mean ± S.E.M. (n=6 for all groups), and *p<0.05 or **p<0.01 compared to corresponding group or ns for non-significant. (F) Double immunostaining with S100B (green) and GFAP (astrocyte marker, red) confirms that S100B is predominantly produced in astrocytes in the brain of 3xTg-AD mice. TOTO-3 (blue) is used to counter-stain nuclei. Scale bars - 20 μm (upper panels) and 10 μm (lower panels).

Figure 6. IL-1β triggers S100B-mediated alterations in β-catenin signaling in neurons

(A) Immunoblot and densitometric analysis (mean ± S.E.M.) of S100B in primary astrocytes exposed to recombinant IL-1β with or without anti-IL-1R blocking antibody for 24 hrs. *p<0.05 or **p<0.01 compared to control (n=6). GAPDH is used for a loading control. (B) Conditioned media from mouse primary astrocytes exposed to 0.5 ng/ml mouse recombinant IL-1β with or without 0.1 μg/ml anti-IL-1R blocking antibody for 24 hrs are used to treat SH-SY5Y cells for 24 hrs, and subseqent changes in β-catenin signaling cascades are detected by immunoblots. Densitometric analyses (mean ± S.E.M.) show a significant changes in nuclear translocation of β-catenin, cytosolic phospho-GSK-3β (at Ser 9) and phospho-Akt (at Ser 473) in co-treatment with anti-IL-1R blocking antibody (*p<0.05 or **p<0.01, n=6). GAPDH and nuclear matrix p84 are used to confirm no cross contamination between cytosolic and nuclear fractions, respectively. (C) SH-SY5Y cells are treated with various doses of purified S100B protein for 24 hrs, and changes in β-catenin and GSK-3β are examined by immunoblot analysis. Densitometric analysis of band intensity (mean ± S.E.M.) is expressed in a bar graph, and *p<0.05 or **p<0.01 compared to control (two separate experiments, n=4 per experiment).