NLRP3 is activated in Alzheimer's disease and contributes to pathology in APP/PS1 mice

Abstract

Alzheimer's disease is the world's most common dementing illness. Deposition of amyloid-β peptide drives cerebral neuroinflammation by activating microglia. Indeed, amyloid-β activation of the NLRP3 inflammasome in microglia is fundamental for interleukin-1β maturation and subsequent inflammatory events. However, it remains unknown whether NLRP3 activation contributes to Alzheimer's disease in vivo. Here we demonstrate strongly enhanced active caspase-1 expression in human mild cognitive impairment and brains with Alzheimer's disease, suggesting a role for the inflammasome in this neurodegenerative disease. Nlrp3(-/-) or Casp1(-/-) mice carrying mutations associated with familial Alzheimer's disease were largely protected from loss of spatial memory and other sequelae associated with Alzheimer's disease, and demonstrated reduced brain caspase-1 and interleukin-1β activation as well as enhanced amyloid-β clearance. Furthermore, NLRP3 inflammasome deficiency skewed microglial cells to an M2 phenotype and resulted in the decreased deposition of amyloid-β in the APP/PS1 model of Alzheimer's disease. These results show an important role for the NLRP3/caspase-1 axis in the pathogenesis of Alzheimer's disease, and suggest that NLRP3 inflammasome inhibition represents a new therapeutic intervention for the disease.

Fig. 1. Protective effects of NLRP3 gene deficiency in APP/PS1 mice on memory and behaviour

(a) Western blot (WB) and quantification of cleaved caspase-1 in brain lysates from frontal cortex (FC) and hippocampus (HC) of Alzheimer’s patients (AD, n=12) and controls (Ctrl, n=8) (mean±SEM, Student´s t-test, * p<0.05, ** p<0.01; + is positive control). (b) WB of cleaved caspase-1 and quantification in mice at 16 months (n=5, mean±SEM, ANOVA, Tukey´s (post hoc) test, ** p<0.01). (c) Parenchymal IL-1β in mouse brains from (B) (n=5, mean±SEM, ANOVA, Tukey´s test, * p<0.05, ** p<0.01). (d) Immunohistochemistry of microglia from APP/PS1 mice for Iba1 (red) and Asc (green). Bar= 10 µm. (e) Morris Water Maze analysis as distance travelled (cm) and integrated distance (AUC) for WT (n=16), NLRP3^−/− (n=12), APP/PS1 (n=14) and APP/PS1/NLRP3^−/− (n=15) mice (mean ±SEM, ANOVA, Tukey´s test, * p<0.05, ** p<0.01). (f) Probe trial day 9. Q1=quadrant where platform was located day 1–8. Time spent in all other (o.a.) quadrants was averaged for all of the above mice (mean ±SEM; one-way ANOVA, Tukey´s test, * p<0.05, *** p<0.001). Representative runs (right panels). (g) LTP was induced by TBS 20 min after baseline recordings in hippocampal slices from mice. LTP is expressed as % potentiation ~50 min post TBS (mean of WT n=16, APP/PS1 n=23, APP/PS1/NLRP3^−/− n=16; hippocampal slices measured ±SEM from n=6–9 animals per group; ANOVA, Tukey´s test, *** p<0.001). (h) Open field test, age=16 months. Vertical locomotor activity (distance travelled) decreased over three consecutive days in WT (n=16) and NLRP3^−/− (n=12) mice. Habituation was not observed in APP/PS1 mice displaying a hyperdynamic behavioral phenotype. APP/PS1/NLRP3^−/− (n=15) were indistinguishable from NLRP3^−/− (n=12) (mean±SEM, ANOVA, Tukey´s test, * p<0.05).

Fig. 2. NLRP3 gene deficiency leads to decreased Aβ levels and deposition

(a) Aβ plaque deposition was quantified in the hippocampus (HC), frontal cortex (FC) and motor cortex (MC) using thioflavin S. (b) Quantification of number and surface area of Aβ plaques was performed in 5 consecutive sections per animal and is given as count per area or area fraction (%) (n=7–8, mean±SEM, Students t-test, * p<0.05, ** p<0.001). (c) WB analysis of RIPA and FA brain extracts of 16 month old APP/PS1 (n=3) and APP/PS1/NLRP3^−/−(n=3) mice. Densitometrical quantification of APP, FA-soluble Aβ and CTFs as ratios (n=5, mean ± SEM, Student's t-test, ** p<0.01). (d) ELISA of RIPA and SDS fractions for Aβ1–40 and 1–42 from 16 month-old mice (n=5, mean ± SEM, Student's t-test, * p<0.05, ** p<0.01, *** p<0.001).

Fig. 3. NLRP3 or caspase-1 deficiency increases microglial Aβ phagocytosis

(a) Quantification of Aβ phagocytosis by flow cytometry of microglia isolated from adult mice 3h after intraperitoneal injection of methoxy-X04 (n=5, mean±SEM, ANOVA, Tukey´s test, ** p<0.01) (b) Same as A with APP/PS1 and APP/PS1/Casp-1^−/− mice (n=5, mean±SEM, ANOVA, Tukey´s test, * p<0.05) (c) Immunohistochemistry staining of ASC specks in CD11b-positive microglia. H3342 is a nuclear stain. (d) Immunocytochemistry of mAb IC16 (anti-Aβ), methoxy-X04 labelled Aβ within Lamp2+ intracellular structures in CD11b+ microglia from 16 month old APP/PS1 mice. (e) Quantification of CD11b+, Aβ+ microglia in the hippocampus (HC) and frontal cortex (FC) of 16 month old mice (n=5, mean±SEM, Student's t-test, ** p<0.01) (f) Representative micrographs from methoxy-X04-treated APP/PS1 and APP/PS1/NLRP3^−/− mice stained for Iba-1 and Aβ. (g) Average IC16-positive Aβ plaque size, determined by co labelling with methoxy-XO4, was markedly reduced in APP/PS1/NLRP3^−/− mice (n=150 plaques were assessed from each group of four mice, mean±SEM, Student's t-test, *** p<0.001). A scatter blot of all plaques that was analyzed by linear regression is shown at the right (150 plaques/group; lines: linear regression analysis, dashed lines: 95% confidence intervals, R²=0.5588 for APP/PS1 and R²= 0.4431 for APP/PS1/NLRP3^−/− mice).

Fig. 4. NLRP3 gene deficiency conveys a M2 microglial phenotype, decreases NOS2 expression and strongly reduces 3NTyr-Aβ formation

(a) WB detection of insulin degrading enzyme (IDE) in cerebral lysates of mice at 16 months of age. Quantification by densitometry is to the right of each WB (n=5, mean±SEM, ANOVA, Tukey´s test, ** p<0.01, *** p<0.01). (b) Corresponding analysis of IDE gene transcription in caspase-1 deficient mice (n=5/group, mean ± SEM, Student´s t-test, *** p<0.001). (c) Transcription of Arginase-1 (Arg-1), (d) Found in inflammatory zone-1 (FIZZ1,) (e) interleukin-4 (lL-4) and (f) nitric oxide synthase 2 (NOS2) at 16 months of age. (n=5, mean±SEM, ANOVA, Tukey´s post hoc test, * p<0.05, ** p<0.01, *** p<0.01) (g) WB detection and quantification of NOS2 in cerebral lysates from 16 month old mice (n=5, mean±SEM, ANOVA, Tukey´s post hoc test, * p<0.05). (h) Representative brain sections were analyzed by immunohistochemistry for nitrated Aβ (3NTyr-Aβ). (i) ELISA detection of 3-NTyr-Aβ in RIPA; SDS and FA extracts showed a robust reduction of 3NTyr-Aβ in APP/PS1/NLRP3^−/− mice at 16 month (n=4–5, Student's t-test, * p<0.05). (j) Cortical sections from 16-month-old mice were probed for 3NTyr-Aβ and Aβ using mAb IC16. NLRP3 gene deficiency reduced both IC16-positive Aβ and 3NTyr-Aβ plaque size (n = 85 plaques were assessed from each group of four mice, mean±SEM, Student's t-test, * p<0.05, *** p <0.001). Scatter blot of all plaques analyzed by linear regression (n = 4 mice, 85 plaques/group, lines: linear regression analysis, dashed lines: 95% confidence intervals, R²= 0.4920 for APP/PS1 and R²= 0.3884 APP/PS1/NLRP3^−/− mice).