Angiopoietin-2 aggravates Alzheimer’s disease by promoting blood-brain barrier dysfunction and neuroinflammation

Figures

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  Figure 1.. Increased ANGPT2 is associated with AD progression in patients (A) Overview of the ROSMAP sample cohort (n = 436). (B) Uniform manifold approximation and projection (UMAP) visualization showing 14 distinct cell types identified from postmortem brain tissue in the ROSMAP sample cohort. (C) Average relative expression of marker genes across the 14 identified cell types. (D) Functional annotation of upregulated and downregulated DEGs in endothelial cells between patients with AD (diagnosis value = 4, n = 141) and patients with MCI (diagnosis value = 2, n = 110), showing significantly enriched biological processes, corrected for multiple comparisons using the false discovery rate (FDR). (E) Volcano plot showing DEGs in endothelial cells between patients with AD (diagnosis value = 4, n = 141) and patients with MCI (diagnosis value = 2, n = 110). DEGs involved in the regulation of the vascular permeability pathway (GO: 0043114) are labeled. (F) Violin plot showing relative ANGPT2 expression across the 14 identified subclusters. ANGPT2 was highly enriched in endothelial cells compared to other cell types. (G–I) Boxplot of log-normalized ANGPT2 expression levels stratified into high-…

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  Figure 2.. ANGPT2 exacerbates β-amyloid deposition in 5xFAD mice (A) Schematic diagram of the AAV-CAG-EGFP (control AAV) and AAV-CAG-ANGPT2-p2a-EGFP (ANGPT2 AAV) viruses, which are designed to target brain endothelial cells. (B) Experimental timeline for AAV treatment in 5xFAD mice to induce ANGPT2 overexpression. (C and D) Elevated endothelial ANGPT2 levels in the cortical region of 6-month-old 5xFAD mice treated with ANGPT2 AAV compared to control-AAV-treated 5xFAD mice (C), with the corresponding quantification of ANGPT2/PECAM-1 (%) (D). Scale bars: 100 μm. (E and F) Increased β-amyloid accumulation in whole-brain cross-sections of 6-month-old 5xFAD mice treated with ANGPT2 AAV compared to age-matched control-AAV-treated 5xFAD mice (E). Scale bars: 1 mm. Quantification of β-amyloid accumulation is shown as the percentage of the area in the cortex and hippocampus (F). Each dot in (D) and (F) represents an individual mouse. Data are presented as the mean ± SEM.

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  Figure 3.. ANGPT2 impairs BBB integrity in AD mice (A and B) Reduced vascular lectin perfusion in the brain cortex of 6-month-old 5xFAD mice treated with ANGPT2 AAV compared to control-AAV-treated mice (A), with the corresponding quantification (B). Scale bars: 100 μm. White arrows indicate regions lacking a lectin signal in blood vessels. (C and D) Increased fibrinogen extravasation in the brain cortex of ANGPT2-AAV-treated 5xFAD mice compared to controls (C), with quantification (D). Arrowheads indicate extravasated fibrin. Scale bars: 50 μm. (E and F) Increased cadaverine deposition in the brain cortex of ANGPT2-AAV-treated 5xFAD mice compared to controls (E), with the corresponding quantification (F). Scale bars: 50 μm. (G–J) Decreased claudin-5 and occludin levels in ANGPT2-AAV-treated 5xFAD mice compared with controls (G and I), with quantification (H and J). Arrowheads indicate vessels negative for claudin-5 or occludin. Scale bars: 50 μm. (K–N) Increased PLVAP and caveolin-1 expression in cortical vessels of ANGPT2-AAV-treated 5xFAD mice compared with controls (K and M), with quantification (L and N). Scale bars: 50 μm. (O and P) Decreased pTIE2 immunoreactivity in…

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  Figure 4.. ANGPT2 promotes neuroinflammation in AD (A and B) Increased CD74+ activated microglia co-labeled with Iba1 in the brain cortex of 6-month-old 5xFAD mice treated with ANGPT2 AAV compared to control-AAV-treated mice (A), with corresponding quantification (B). Arrowheads indicate CD74+ activated microglia. Scale bars: 50 μm. (C and D) Representative images (C) and quantification (D) showing increased cell body diameter and decreased dendritic length of Iba1+ microglia in ANGPT2-AAV-treated 5xFAD mice compared with controls. Asterisks mark the centers of Iba1+ microglial nuclei. Scale bars: 10 μm. (E and F) Elevated GFAP+ reactive astrocytes co-labeled with S100β in the cortex of ANGPT2-AAV-treated 5xFAD mice compared with controls (E), with corresponding quantification (F). Arrowheads indicate GFAP+ reactive astrocytes. Scale bars: 50 μm. (G and H) Morphological analysis of GFAP+ reactive astrocytes showing increased branching and area coverage in ANGPT2-AAV-treated 5xFAD mice compared to control-AAV-treated mice (G), with corresponding quantification (H). Scale bars: 50 μm (white) and 20 μm (yellow). (I) Cytokine profile analysis showing increased levels of pro-inflammatory…

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  Figure 5.. Endothelial ANGPT2 deletion attenuates vascular leakage and neuroinflammation in AD (A) Experimental timeline for endothelial-specific ANGPT2 conditional knockout in 5xFAD mice (5xFAD;ANGPT2iECKO). Tamoxifen was administered at 2 months, and brains were harvested at 12 months of age. (B) No detectable ANGPT2 immunoreactivity in the blood vessels of the cortex in 12-month-old 5xFAD;ANGPT2iECKO mice compared to 5xFAD;ANGPT2WT mice. Scale bars: 100 μm. (C and D) Comparison of β-amyloid deposition in whole-brain cross-sections of 5xFAD;ANGPT2iECKO and age-matched control 5xFAD mice (C) and its quantifications in the hippocampus and cortex (D). The yellow line outlines the brain boundary, and the yellow dotted line indicates the hippocampal regions. Scale bars: 1 mm. (E and F) Decreased fibrin leakage into the hippocampus and cortex of 5xFAD;ANGPT2iECKO mice compared to controls (E), with corresponding quantification (F). Scale bars: 100 μm. (G and H) Enhanced endothelial pTIE2 levels in the hippocampus and cortex of 5xFAD;ANGPT2iECKO mice compared to controls (G), with quantifications (H). Arrowheads indicate pTie2-expressing regions. Scale bars: 50 μm. (I and J) Decreased microglial…

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  Figure 6.. Transcriptomic analysis in brains following ANGPT2 overexpression in 5xFAD mice (A) Graphical abstract depicting the experimental procedure for snRNA-seq. Brains were harvested, trimmed in the frontal cortex, and dissected to include the upper right cortical region and hippocampus. Nuclei were isolated and subjected to snRNA-seq followed by transcriptomic analysis. Control AAV: n = 3 (2 males and 1 female); ANGPT2 AAV: n = 4 (2 males and 2 females). (B) UMAP visualization showing 12 distinct cell types identified from 5xFAD mice injected with control AAV or ANGPT2 AAV. Clusters were annotated based on marker gene expression profiles. (C) Proportional representation of each cell type in 5xFAD mice injected with control AAV or ANGPT2 AAV. (D–F) Functional annotation of upregulated and downregulated DEGs showing significantly enriched biological processes, corrected for multiple comparisons by FDR. Bar plots show the top enriched GO terms for biological processes associated with upregulated (left, purple) and downregulated (right, blue) genes in specific cell types: microglia (D), astrocytes (E), and excitatory neurons (F) in the cortex. The x axis represents −log10 (FDR) values,…

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  Figure 7.. ANGPT2 impairs memory function in AD mice (A) Sequence of behavioral tests for ANGPT2 gain-of-function and loss-of-function studies in 5xFAD mice. The testing timeline included a 2-day radial arm water maze (RAWM), a 3-day fear conditioning test, a 2-day visible platform test, a 2-day open-field test, and a 1-day sensory threshold assessment (STA). (B) Experimental timeline for ANGPT2 gain-of-function studies. Control or ANGPT2 AAV was administered at 4 months of age, and mice were tested at 6 months. (C) ANGPT2 overexpression impaired RAWM performance in 5xFAD compared to controls. Two-way ANOVA for repeated measures between groups: F(1,22) = 6.565, p = 0.018. Control AAV: n = 12 (6 males and 6 females); ANGPT2 AAV: n = 12 (7 males and 5 females). (D) ANGPT2 overexpression did not affect associative memory function in 5xFAD mice, as assessed by fear conditioning. Two-way ANOVA for repeated measures: contextual: F(1,17) = 0.095, p = 0.762 and cued: F(1,17) = 0.079, p = 0.783. Control AAV: n = 9 (3 males and 6 females); ANGPT2 AAV: n = 10 (4 males and 6 females). (E) Experimental timeline for ANGPT2 loss-of-function studies. After administering tamoxifen at 2 months of age to…