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. 2023 Dec;45(6):3307-3331.
doi: 10.1007/s11357-023-00927-x. Epub 2023 Oct 2.

Vascular senescence and leak are features of the early breakdown of the blood-brain barrier in Alzheimer's disease models

Ka Ka Ting  1   2 , Paul Coleman  3   4 , Hani Jieun Kim  5 , Yang Zhao  6 , Jocelyne Mulangala  3 , Ngan Ching Cheng  3 , Wan Li  7 , Dilini Gunatilake  3 , Daniel M Johnstone  4   8 , Lipin Loo  9 , G Gregory Neely  9 , Pengyi Yang  5 , Jürgen Götz  10 , Mathew A Vadas  3   11 , Jennifer R Gamble  12   13
Affiliations

Vascular senescence and leak are features of the early breakdown of the blood-brain barrier in Alzheimer's disease models

Ka Ka Ting et al. Geroscience. 2023 Dec.

Abstract

Alzheimer's disease (AD) is an age-related disease, with loss of integrity of the blood-brain barrier (BBB) being an early feature. Cellular senescence is one of the reported nine hallmarks of aging. Here, we show for the first time the presence of senescent cells in the vasculature in AD patients and mouse models of AD. Senescent endothelial cells and pericytes are present in APP/PS1 transgenic mice but not in wild-type littermates at the time of amyloid deposition. In vitro, senescent endothelial cells display altered VE-cadherin expression and loss of cell junction formation and increased permeability. Consistent with this, senescent endothelial cells in APP/PS1 mice are present at areas of vascular leak that have decreased claudin-5 and VE-cadherin expression confirming BBB breakdown. Furthermore, single cell sequencing of endothelial cells from APP/PS1 transgenic mice confirms that adhesion molecule pathways are among the most highly altered pathways in these cells. At the pre-plaque stage, the vasculature shows significant signs of breakdown, with a general loss of VE-cadherin, leakage within the microcirculation, and obvious pericyte perturbation. Although senescent vascular cells were not directly observed at sites of vascular leak, senescent cells were close to the leak area. Thus, we would suggest in AD that there is a progressive induction of senescence in constituents of the neurovascular unit contributing to an increasing loss of vascular integrity. Targeting the vasculature early in AD, either with senolytics or with drugs that improve the integrity of the BBB may be valid therapeutic strategies.

Keywords: Alzheimer’s disease; Blood–brain barrier; Pericytes; Senescence; VE-cadherin; Vascular leak.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Validation of senescence green probe (SGP) as a marker of senescence. Ai-vi shows a representative gating strategy used to identify senescent cells populations in H2O2-treated cells. Focused cells were gated into single cell population (green box). DAPI+ cells (magenta box) was analyzed for p16 expression followed by SGP expression against cell area/size. Small p16+ cells (yellow box) shows a population with high SGP expression (intermediate senescent cells) and another population negative for SGP expression (non-senescent cells). A majority of the large cells with high expression of p16 (orange box) also had high expression of SGP (committed senescent cells). Untreated, H2O2-treated, and unstained HUVECs were analyzed by Amnis ImageStream and gated in the same fashion. B Representative images of unstained HUVECs, non-senescent HUVECs, and HUVECs at different stages of senescence according to their cell size in brightfield (BF) and side scatter (SSC) as well as expression of senescent markers SGP (green) and p16 (red). Committed senescent cell in the representative image shown here has two DAPI (purple) positive nucleus. C Quantification of the percentage of SGP+ and p16+ large senescent cells in untreated and H2O2-treated HUVECs. D Representative images of HUVECs at different stages of senescence according to cell size and senescent markers SGP (green) and p21 (red). E Quantification of the percentage of SGP+ and p21+ large senescent cells in untreated and H2O2-treated HUVECs. In vitro data presented for p16- or p21-independent experiments are from 2 different HUVEC lines and are presented as mean ± standard deviation, scale bar = 20 µm. F Representative images of traditional SA-β-gal (blue) and SGP (green) stain in the brain taken from APP/PS1 mice after amyloid plaque development. Amyloid beta (gray) staining shows that SGP (green) staining on CD31+ vasculature (red) is localized to vascular cells, not plaques deposited on the blood vessel. Scale bar = 50 µm
Fig. 2
Fig. 2
SGP expression in mouse models of AD. Representative images of SGP expression in APP/PS1 (A) and APP23 (B) compared to wildtype (WT) mice over different stages of plaque development. Ai and Bi White arrows show the presence of vascular cells positive for SGP (green) in 2-month-old APP/PS1 and 3-month-old APP23 mice that have no detectable plaques. Aii and Bii Representative images show that more SGP-positive vascular cells (white arrows) can be detected in APP/PS1 and APP23 mice after plaque formation compared to controls. Arrowheads in Aii and Bi show non-vascular cells that have SGP expression. C and D Quantification of the number of SGP-positive cells on the vasculature relative to brain cortical area in APP/PS1 and APP23 mice, respectively. Data in C is from 3 independent experiments and represented as mean ± standard deviation. The Mann–Whitney t-test was used to determine difference in SGP expression between WT and APP/PS1 transgenic mice. *p-value ≤ 0.05, **p-value ≤ 0.01, WT = 5 per age group APP/PS1 = 8–10 per age group. Data in D is represented as mean ± standard deviation, WT = 2–3 per age group and APP23 = 2–3 per age group. Ei Localization of SGP (green) in pericytes stained with PDGFRβ (red) in large and small vessel. Scale bar = 20 µm. Eii Localization of SGP (green) found in the endothelium stained with CD31 (red). Scale bar = 15 µm
Fig. 3
Fig. 3
Association of albumin leak and endothelial junctions with SGP + vascular cells after plaque formation. A Representative image of SGP+ EC (green) localized to a capillary leak of endogenous albumin is indicated by dashed yellow box. Amyloid plaques were observed in 8-month-old APP/PS1 mice (white dashed lines). Claudin-5 (cyan) and VE-cadherin (red) expression appear to be reduced and disorganized, respectively. B Representative image of SGP+ perivascular cell (green) localized to large vessel leak of endogenous albumin. No leak was observed in similar vessel in WT mice. C Albumin leak was found more in large and small blood vessels (BV) in older APP/PS1 mice (red) compared to wildtype (WT) mice (blue). D Global Claudin-5 expression was significantly reduced in APP/PS1 mice compared to WT mice. E VE-cadherin was not significantly altered between 8-month-old WT and APP/PS1 mice. F Positive correlation of number of SGP+ cells within 50 microns to leak area in 8-month-old APP/PS1 mice. Gi Staining of blood vessels positive for CD31 (red) and senescence marker p21 (green) and nuclei (DAPI, blue) in post-mortem human mid-temporal cortical sections. Senescence of endothelial cells and perivascular cells were detected in human Alzheimer’s disease (AD) brain sections (white arrows). Gii Human brain sections had decreased expression of VE-cadherin (green) in blood vessels positive for CD31 (red). Giii Overall expression of VE-cadherin in human AD mid-temporal region. Mouse data is from 3 independent experiments; human data is from 3 non-demented controls and 3 AD age and sex-matched cases. All data are represented as mean ± standard deviation. Unpaired t-test was used to determine leak number per section, VE-cadherin, and claudin-5 expression between WT and APP/PS1 mice. *p-value ≤ 0.05, WT = 4–6, and APP/PS1 = 5–7 mice. Scale bar in Fig. A = 10 µm, Fig. Gi = 10 µm and Gii = 20 µm
Fig. 4
Fig. 4
Pathways associated with adhesion and the blood-brain barrier are dysregulated in endothelial cells of Alzheimer’s disease mice. A Schematic of single-cell transcriptomic profiling of mouse brain (generated using BioRender). B tSNE representation of the transcriptomes of single cells. Cells are colored by their type in healthy (left) and diseased brain (right). OLG, oligodendrocytes; CPC, choroid plexus cells; ArtEC, CapECs, and VenECs, arterial, capillary, and venous endothelial cells. C Expression pattern of cell type marker genes. The size of circles denotes the average gene expression across cells and the color denotes the proportion of cells expression the gene. D Bar plot showing the number of significantly upregulated (red) and downregulated (down) genes in Alzheimer diseased versus healthy brain. E Bar plot of gene set over-representation of pathways associated with adhesion and the blood brain barrier among genes significantly downregulated in healthy (left) and diseased (right) mouse brain for each cell type. Y-axis denotes the degree of enrichment, and the X-axis denotes the pathways. F Volcano plot showing genes differentially regulated in capillary endothelial cells (CapECs). X-axis denotes the log2 fold-change in gene expression, and the Y-axis denotes the − log10 p-value (FDR-adjusted). G Visualization of the signature score denoting the transcriptional activity of 80 adhesion and blood-brain barrier (BBB)-associated genes on tSNE. Cells are colored by the signature where a stronger yellow color denotes higher activity, and an opaquer color denotes higher average gene expression. H The average normalized gene expression of the signature genes in healthy (WT, green) and Alzheimer’s disease (AD, red) brain cells is visualized as split violin and box plots for each cell type. The Student’s t-test was used to compare the difference in mean gene expression between healthy and diseased cells. ns, p-value > 0.05, *p-value ≤ 0.05; **p-value ≤ 0.01; ***p-value ≤ 0.001; ****p-value ≤ 0.0001
Fig. 5
Fig. 5
Association of vascular leak and endothelial junctions with SGP + vascular cells before plaque formation. Ai Representative image of normal capillary with endogenous albumin restricted to the microvessel in the 2-month-old WT mice. Aii Representative image of capillary leak of endogenous albumin (white) is indicated by the dashed yellow box is observed in 2-month-old APP/PS1 mice not associated with SGP+ cells. A lack of VE-cadherin (red) and claudin-5 (cyan) expression is observed at site of leak. Aiii, F In areas of no leak, VE-cadherin is also evidently decreased. B Albumin leak was localized to small blood vessels compared to large blood vessels (BV) in APP/PS1 mice (red) compared to wildtype (WT) mice (blue). C Image analysis of claudin-5 shows that there is no difference in expression between APP/PS1 and WT littermates. D Global VE-cadherin expression is decreased in APP/PS1 mice compared to WT mice. E Correlation analysis of leak size and number of SGP+ cells within 50 microns to the area of leak in APP/PS1 mice. F Capillary leak of biotin (white, dashed yellow box) is also observed, where there is a reduced expression of claudin-5. P21 expression (red) was identified in the DAPI + nuclei (blue) of endothelial cell (EC) and neurons (n) at areas of biotin leak. G Capillary leak of biotin (white, dashed yellow box) is also observed, where there is lack of VE-cadherin (red) expression. Data is from 3 independent experiments and represented as mean ± standard deviation. Unpaired t-test was used to determine leak number per section, VE-cadherin, and claudin-5 expression between WT and APP/PS1 mice. *p-value ≤ 0.05, WT = 4–6, and APP/PS1 = 5–7 mice. Scale bar in Fig. A = 10 µm, and scale bar in Fig. F and G = 20 µm
Fig. 6
Fig. 6
Pericyte morphology at sites of albumin leakage. A Representative images of PDGFRβ-positive pericytes (red) showing pericyte coverage in comparison to wildtype (WT) mice, even at sites of albumin leak (yellow dashed box) in the brain parenchyma of APP/PS1 mice before plaque development. Bi Pericytes can be detected migrating between the microvessel in the APP/PS1 mice (white arrows) before and after plaque formation. Migrating pericytes were characterized by the extension of pericyte cell body and processes towards another neighboring microvessel, which is outlined by vasculature containing albumin (white). Bii After plaque formation, pericyte coverage and migration were still detected on multiple sites of albumin leak (yellow dashed box). Scale bar = 20 µm
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