Liver-specific expression of ANGPTL8 promotes Alzheimer’s disease progression through activating microglial pyroptosis

Figures

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  Fig. 1. ANGPTL8 KO improved the cognitive function of 5xFAD mice. A Representative images of the tracks of 9-month-old WT and ANGPTL8 KO mice in the Morris water maze (MWM) test. B-C Statistical results of the escape latency (B) and number of platform crossings (C) of 9-month-old mice (n = 8) on d 7. D Schematics of the experimental procedures for the four groups of mice (WT, ANGPTL8 KO, 5 × FAD, and ANGPTL8 KO:5xFAD; every group included half male and half female mice). E Representative plot of the MWM test results of 3-month-old mice in the four groups (n = 8–10). F The time needed to reach the hidden platform (escape latency) was plotted across training days. Statistical analysis revealed a difference in 3-month-old ANGPTL8 KO:5 × FAD mice compared with WT, ANGPTL8 KO and 5 × FAD mice on d 7 (n = 8–10). G‒I. Escape latency, number of platform crossings and swimming speed (mm/s) were analysed during the MWM test (d 7) in 3-month-old mice. J‒K The escape latency and number of platform crossings were analysed during the MWM test (d 7) in 6-month-old mice (n = 6‒8). L‒M Recognition indices of the four groups of 3- and 6-month-old mice were detected via a novel object recognition…

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  Fig. 2. ANGPTL8 promotes AD progression by inducing microgliosis and microglial activation. A Changes in cortical cells in the brains of 6-month-old 5xFAD: ANGPTL8-KO and Con-5xFAD mice were analysed via single-nucleus RNA sequencing (snRNA-seq). Joint uniform manifold approximation and projection (UMAP), colored by total major cell type (17 main clusters) in mice. B-C. Global breakdown, region composition, enrichment and number of nuclei for 17 main cluster subtypes in the Con-5xFAD mouse group (B) and 5xFAD:ANGPTL8-KO mouse group (C). D-F. Subpopulations of microglia. UMAP embeddings of single nuclei profiled via snRNA-seq, colored according to clusters, and annotated for each subpopulation, indicating the proportion of each subpopulation in the Con-5xFAD mouse group (E) and the 5xFAD:ANGPTL8-KO mouse group (F). G. Immunostaining for Iba-1 and DAPI in the CA1 area of the hippocampus. Microglia were labelled with an Iba-1 antibody. H. Colocalization of Aβ and Iba-1 in the hippocampus of the mice in the indicated groups. Aβ plaques and microglia were labelled with anti-Aβ (red) and anti-Iba-1 antibodies (green), respectively

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  Fig. 3. Liver-derived ANGPTL8 activated microglia. A-D. ANGPTL8 mRNA was detected by qPCR in liver of 1, 3, 6 and 9 months old WT and 5xFAD mice. E. The effect of Aβ stimulation on ANGPTL8 expression was analyzed by qPCR in LO2 cells. F-I. Serum ANGPTL8 levels were detected by ELISA in 1-, 3-, 6-, and 9-month-old WT and 5xFAD mice (n ≥ 3). J-K. ANGPTL8 protein levels in the hippocampal and cortical homogenates of 6-month-old 5xFAD and WT mice were determined via ELISA. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant. L. Schematic of the establishment of a model for long-term complementation of the liver expression of ANGPTL8 in ANGPTL8 KO:5xFAD mice via hydrodynamic tail vein injection of AAV8. M. Representative images of the tracks of 6-month-old ANGPTL8 KO:5xFAD-AAV8-con and ANGPTL8 KO:5xFAD-AAV8-ANGPTL8 mice in the Morris water maze (MWM) test. N‒O. Statistical analysis of the number of platform crossings (N) and escape latency (O) of 6-month-old ANGPTL8 KO:5xFAD-AAV8-con and ANGPTL8 KO:5xFAD-AAV8-ANGPTL8 mice (n = 8) on d 7. P‒R. IHC staining (P) and statistical analysis (Q‒R) of Aβ plaques per pixel area in the hippocampus and cortex of two groups of mice (ANGPTL8…

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  Fig. 4. ANGPTL8 reduces microglial phagocytosis to inhibit the clearance of Aβ. A Morphological changes (resting ramified microglia and activated amoeboid microglia) in BV2 cells treated with rANGPTL8 (600 ng/ml) for 6 h under a bright field. Scale bar: 50 μm. B-E. Flow cytometry analysis of the phagocytic ability of fluorescence microspheres in BV2 cells or primary microglia after treatment with Aβ1-42 (400 ng/ml) and/or ANGPTL8 (600 ng/ml) for 6 h. Representative scatter plots showing the frequencies of the cells containing fluorescent microspheres (B, C) and summary bar graphs (D, E). F‒I. Microscopic analysis of the phagocytosis of fluorescent microspheres by BV2 cells or primary microglia after treatment with Aβ1‒42 (400 ng/ml) and/or ANGPTL8 (600 ng/ml) for 6 h. Representative images of fluorescent microspheres (green) engulfed by BV2 cells (F) or primary microglia (G) (marked with anti-Iba1 (red) and DAPI for the nucleus (blue)) and a summary bar graph (H‒I). Scale bar: 50 μm. *P < 0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001

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  Fig. 5. ANGPTL8 induced inflammatory factor expression and microglial polarization. A. Volcano plot visualizing the differential expression of the genes of the heart transcriptome (n = 3 in each group). B. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of the identified differentially expressed genes on the basis of the RNA-seq dataset from the hippocampi of 6-week-old 5 × FAD (WT) and ANGPTL8-KO:5 × FAD mice (n = 3 in each group). C. Representative immunoblots probed with antibodies against NLRP3, p-NF-κBp65, NF-κBp65, and β-tubulin and the statistical results of Western blotting. D-F. UMAP gene expression patterns of NF-κB pathway markers in 17 labelled clusters (D). Gene expression patterns of NF-κB pathway markers in UMAPs of microglial subpopulations (E). Distinct gene expression patterns of NF-κB pathway markers in microglial subpopulations in 5 × FAD (WT) and ANGPTL8-KO:5 × FAD mice. The dot color shows the mean expression in expressing cells (column scale); the dot size shows the percentage of cells expressing the gene (F). G. Representative images of CD86 and CD206 expression detected by flow cytometry in the indicated groups. H. Quantification of CD86/CD206…

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  Fig. 6. ANGPTL8-induced microglial pyroptosis. A. Representative morphological images of pyroptosis in BV2 cells treated with Aβ1-42 (400 ng/ml) and/or rANGPTL8 (600 ng/ml) for 36 h. B. Statistical analysis of pyroptotic cell numbers/mm.2 in each group. C. Representative morphological images of the indicated groups observed via transmission electron microscopy. LPS (500 ng/ml) + PMA (500 ng/ml) was used as a positive control. D. Quantification of the levels of GSDMD-N normalized to those of β‐tubulin. Experiments were performed in the indicated groups (n = 3 mice/group) E. Representative western blot bands of GSDMD-FL, GSDMD-N, and β-tubulin in 3-month-old mice in each group. F-I. IHC staining (F&H) and statistical analysis (G&I) of NLRP3 and ASC in the hippocampus and cortex of four groups of mice (WT, ANGPTL8 KO, 5xFAD, and ANGPTL8 KO:5xFAD). N = 7 to 11 slices from three mice per group, *P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant. J‒K. Colocalization of ASC and Iba-1 (J) or NRLP3 and Iba-1 (K) in the hippocampus of the mice in the indicated groups. L. Representative immunoblots probed with antibodies against GSDMD-FL, GSDMD-N, cleaved caspase-1, ASC and β-tubulin in…

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  Fig. 7. ANGPTL8 promoted microglial pyroptosis via the PirB/NLRP3 pathway. A. To analyse the role of NLRP3 in BV2 cell pyroptosis after treatment with ANGPLT8 and/or Aβ1-42, a siRNA strategy was used to manipulate the expression of NLRP3 in BV2 cells. Immunoblots of NLRP3, GSDMD-FL, GSDMD-N, and β-tubulin (loading control) in BV2 cells and NLRP3-knockdown cells treated with Aβ1-42 (400 ng/ml) and/or rANGPTL8 (600 ng/ml) for 6 h. LPS (500 ng/ml) + PMA (500 ng/ml) was used as a positive control. B-C. The levels of NLRP3 and GSDMD-N were quantified and normalized to those of β-tubulin. D. To analyse the role of PirB in BV2 cell pyroptosis after treatment with ANGPLT8 and/or Aβ1-42 by western blotting, a siRNA strategy was used to manipulate the expression of PirB. E–F. Quantification of NLRP3 and GSDMD-N in BV2 and PirB-knockdown BV2 cells treated with Aβ1-42 with or without rANGPTL8. G-H. Representative images of NLRP3 and ASC expression detected by immunofluorescence in the indicated groups. I‒J. Flow cytometry analysis of the phagocytic ability of fluorescent microspheres in BV2 and PirB-knockdown BV2 cells after treatment with ANGPTL8 (600 ng/ml) for 6 h. Representative scatter plots…

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  Fig. 8. Metformin improved cognitive dysfunction in 5xFAD mice by inhibiting ANGPTL8. A. Schematics of the experimental procedures for the eight groups of mice. B. A representative locus plot of the MWM test results of 4-month-old WT, ANGPTL8KO, 5 × FAD, ANGPTL8KO: 5 × FAD mice that received without or with Met (300 mg/kg, i.g.) for 4 weeks is shown. C‒E. The swimming speed, number of platform crossings, and escape latency of the mice in the indicated groups were analysed during the Morris water maze test. F. The recognition index was detected by a novel object recognition experiment in 4-month-old mice in the indicated groups. G. Serum ANGPTL8 levels were detected by ELISA in 4-month-old 5 × FAD mice without or with Met (300 mg/kg, i.g.) treatment for 4 weeks. H. Amyloid plaques were detected by thioflavin-S staining (scale bars, 100 μm) in the cortex and hippocampus of the brains of 4-month-old mice from the indicated groups. I‒J. Quantitative analysis of amyloid plaques in the cortex (I) and hippocampus (J) in each group (the average value of 8‒10 slices from each mouse, n = 3 mice/group), *P < 0.05, ** P < 0.01, ns = not significant. K. Schematic representation of the proposed…