Regulation of blood-brain barrier integrity by Dmp1-expressing astrocytes through mitochondrial transfer

Abstract

The blood-brain barrier (BBB) acts as the crucial physical filtration structure in the central nervous system. Here, we investigate the role of a specific subset of astrocytes in the regulation of BBB integrity. We showed that Dmp1-expressing astrocytes transfer mitochondria to endothelial cells via their endfeet for maintaining BBB integrity. Deletion of the Mitofusin 2 (Mfn2) gene in Dmp1-expressing astrocytes inhibited the mitochondrial transfer and caused BBB leakage. In addition, the decrease of MFN2 in astrocytes contributes to the age-associated reduction of mitochondrial transfer efficiency and thus compromises the integrity of BBB. Together, we describe a mechanism in which astrocytes regulate BBB integrity through mitochondrial transfer. Our findings provide innnovative insights into the cellular framework that underpins the progressive breakdown of BBB associated with aging and disease.

Fig. 1.. Astrocytes transfer mitochondria and alleviate oxidative stress of endothelial cells.

(A) Flowchart of 2D coculture of primary astrocytes^Mito-Dendra2 and bEnd.3 endothelial cells. (B) Representative confocal images and intensity wave showing Dendra2-labeled mitochondria translocated from primary astrocytes^Mito-Dendra2 (AQP4) to endothelial cells (CD31) in 2D coculture system. Scale bar, 50 μm. (C) Time lapse confocal images show astrocytes^Mito-Dendra2 mitochondria (Dendra2) moving dynamically toward the adjacent MitoTracker Red-labeled endothelial cell. Scale bar, 10 μm. (D) Representative TEM image shows the perivascular mitochondria (yellow) of astrocytes outside the endothelial plasma membrane and the mitochondria (yellow) within capillary. Scale bar, 10 μm. (E) Schematic of primary astrocytes^Mito-Dendra2 extending distal dendrites through the pores of a Transwell membrane (TM) and forming endfeet-like structures on the underside of the well. (F) The 3D lateral confocal images of TM show the distribution of mitochondria in the soma and distal dendrites of primary astrocytes^Mito-Dendra2 plated on the TM. Scale bars, 20 μm. (G) Flowchart of 3D coculture of primary astrocytes^Mito-Dendra2 and bEnd.3 endothelial cells. (H) Confocal images of the upper side (soma) and under side (endfeet) of TM showing the enrichment of mitochondria in astrocytes soma and distal end of processes (AQP4). Scale bars, 20 μm. (I) Confocal images of CD31-labeled bEnd.3 on coverslip show the mitochondria transferred from primary astrocytes^Mito-Dendra2 on TM. Scale bars, 20 μm. (J) Representative scatter plot and quantitative results of the percentage of bEnd. Three endothelial cells containing Mito-Dendra2 fluorescence in all endothelial cells pretreated with or without 2 μM A/R after coculturing with primary astrocytes^Mito-Dendra2 (n = 3). Two-tailed Student’s unpaired t test. Data are presented as means ± SEM. (K) Representative histogram overlays and quantitative results of MitoSOX intensity in healthy bEnd.3 cells, 2 μM A/R-damaged bEnd.3 cells, and 2 μM A/R-damaged bEnd.3 cells cocultured with primary astrocytes^Mito-Dendra2 cells (n = 3). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Data are presented as means ± SEM.

Fig. 2.. A subset of astrocytes that express Dmp1 specifically adhere to blood vessels.

(A) tSNE plot of reclustered astrocytes (n = 4531 cells) reveals cellular heterogeneity of brain astrocytes. (B) Heatmap of the top three marker genes for subcluster identification of all astrocytes. (C) Bubble plot of Aqp4 expression in different astrocytes subclusters. (D) Bubble plot of expression distribution for Dmp1 in all astrocytes subclusters. (E) Bubble plot of Dmp1 expression in different brain cells. (F) Feature plots of expression distribution for Dmp1 in all captured single-cell events of scRNA-seq. Expression levels are color-coded as in tSNE plot. (G) Pie diagram of the cell types proportion among all Dmp1⁺ cells from the pool of 1M, 6M, and 20M mouse brains. (H) Representative confocal images and fluorescence intensity analysis of CD31-labeled endothelial cells, S100β-labeled astrocytes and tdTomato-marked Dmp1⁺ cells in sagittal brain sections of 1M Dmp1^Cre-Ai9 male mice thalamus. Scale bar, 20 μm. (I) Quantitative analysis of the percentage of S100β⁺/Dmp1⁺ cell number or S100β⁺/Dmp1⁻ cell number among all S100β⁺ cells (n = 10 images from three mice). Two-tailed Student’s paired t test. Data are presented as means ± SEM. (J) Quantitative analysis of the percentage of cell number that are associated or nonassociated with CD31-labeled endothelial cells among all S100β⁺/Dmp1⁺ cells (left) or S100β⁺/Dmp1⁻ cells (right) (n = 10 images from three mice per group). Two-tailed Student’s paired t test. Data are presented as means ± SEM. (K) Gene expression level of Aqp4 in Dmp1⁺ and Dmp1⁻ astrocytes at 1M. Wilcoxon rank sum test. Data are presented as median. (L) Significantly up-regulated GO cellular component terms in Dmp1⁺ astrocytes compared to Dmp1⁻ astrocytes at 1M. (M) Representative confocal images and quantitation of AQP4 expression among S100β⁺/Dmp1⁺ astrocytes and S100β⁺/Dmp1⁻ astrocytes in sagittal brain sections of 1M Dmp1^Cre-Ai9 male mice thalamus (n = 12 images from three mice). Scale bar, 20 μm. Two-tailed Student’s paired t test. Data are presented as means ± SEM.

Fig. 3.. Astrocytes^Dmp1 transfer mitochondria to CECs in vivo.

(A) Representative FACS plots for Dendra2-containing cells isolated from 1M male Dmp1^Cre-Cox8^Dendra2 mouse brains (n = 2). Single cells isolated from age-matched male Cox8^Dendra2 mouse brains were used as negative control (n = 2). (B) tSNE plot of reclustered Dmp1-negative cells from all Dendra2-containing cells (n = 14,074 cells) shows the cells that containing extrinsic mitochondria. (C) Pie diagram of the percentages of different Dendra2-containing Dmp1-negative cell types indicates the acquisition of extrinsic mitochondria in different cell types. (D) Representative scatter plot and pie diagram of the average percentage of Dendra2⁺/CD31⁺ endothelial cells among all CD31⁺ endothelial cells in 1M male Dmp1^Cre-Cox8^Dendra2 mouse brains (n = 4 mice). (E) Confocal images with orthogonal views and fluorescence intensity analysis of Dendra2 (refers to mitochondria of astrocytes^Dmp1) and astrocyte endfeet marker AQP4 in sagittal brain sections of 1M Dmp1^Cre-Cox8^Dendra2 male mouse thalamus show Dendra2-labeled mitochondria colocalized with (insert I, white line) astrocyte endfeet or surrounded by (insert II, yellow line) astrocyte endfeet. Scale bar, 20 μm. (F) Confocal images with orthogonal views and fluorescence intensity analysis of Dendra2 (refers to mitochondria of astrocytes^Dmp1) and endothelial cell marker CD31 in 1M Dmp1^Cre-Cox8^Dendra2 male mouse thalamus show Dendra2-labeled mitochondria aggregating around (insert I, white line) endothelial cells or colocalized with (insert II, yellow line) endothelial cells. Scale bar, 20 μm.

Fig. 4.. MFN2-mediated mitochondrial transfer from astrocytes^Dmp1 to endothelial cells decreases with age.

(A) 3D confocal images of the Mito-Dendra2 and CD31-labeled endothelial cells within the thalamus of 1M young and 20M aged Dmp1^Cre-Cox8^Dendra2 male mice. Scale bar, 10 μm. (B) Quantification of total CD31 intensity, area of Dendra2 colocalized with CD31, total Dendra2 intensity, percentage of CD31-colocalized Dendra2 intensity/total Dendra2 intensity from images in (A) (n = 3 mice per group). Two-tailed Student’s paired t test. Data are presented as means ± SEM. (C) Images and quantification of immunoblotted MFN2 signal in thalamus tissues from 1M young and 20M aged WT mouse brains (n = 3 mice per group). Two-tailed Student’s unpaired t test. Data are presented as means ± SEM. (D) Representative histogram overlays and quantification of Dendra2 intensity in healthy bEnd.3 cells cocultured with astrocytes^Mito-Dendra2, 2 μM A/R-damaged bEnd.3 cells cocultured with astrocytes^Mito-Dendra2 or Mfn2^het-astrocytes^Mito-Dendra2 (n = 3). One-way ANOVA followed by Tukey’s post hoc test. Data are presented as means ± SEM. (E) Representative histogram overlays and quantification of MitoSOX intensity in healthy bEnd.3 cells, 2 μM A/R-damaged bEnd.3 cells, 2 μM A/R-damaged bEnd.3 cells cocultured with astrocytes^Mito-Dendra2 or Mfn2^het-astrocytes^Mito-Dendra2 (n = 3). One-way ANOVA followed by Tukey’s post hoc test. Data are presented as means ± SEM. (F) Representative TEM images from the thalamus of 6w Mfn2^fl/fl and Mfn2-cKO male mice and quantifications of the mitochondria (yellow) number in perivascular astrocyte endfeet and distance from mitochondrial outer membrane to capillary lumens (red) (n = 6 images from three mice per group). Scale bar, 2 μm. Two-tailed Student’s unpaired t test. Data are presented as means ± SEM. (G) 3D confocal images of Dendra2-labeled mitochondria and CD31-labeled endothelial cells from the thalamus of 1M Dmp1^Cre-Cox8^Dendra2 and Dmp1^Cre-Cox8^Dendra2-Mfn2^fl/fl male mouse brains. Scale bar, 10 μm. (H) Quantification of total CD31 intensity, area of Dendra2 colocalized with CD31, total Dendra2 intensity, percentage of CD31-colocalized Dendra2 intensity/total Dendra2 intensity from images in (G) (n = 3 mice per group). Two-tailed Student’s paired t test. Data are presented as means ± SEM.

Fig. 5.. Deletion of Mfn2 in astrocytes^Dmp1 leads to BBB disruption.

(A) The top 30 significantly down-regulated GO enrichment terms of biological process in Dmp1-negative endothelial cells of Mfn2-cKO mice compared with that of WT mice. (B) GSEA of Dmp1-negative endothelial cells for angiogenesis and blood vessel development in Mfn2-cKO mice compared with WT mice. (C) Gene expression level of Cldn5 and Col4a1 between WT and Mfn2-cKO mice Dmp1-negative endothelial cells. Wilcoxon rank sum test. Data are presented as median. (D and E) Comparisons between WT and Mfn2-cKO mice Dmp1-negative endothelial cells for ssGSEA on bicellular tight junction assembly and endothelial migration (D), and establishment of BBB, angiogenesis, and branching involved in blood vessel morphologies (E). Wilcoxon rank sum test. Data are presented as median. (F) Representative images and quantification of Evans Blue–filled blood vessel density and the number of vessel branches in the thalamus capillaries of 1M Mfn2^fl/fl and Mfn2-cKO male mice (n = 9 images from three mice for each group). Scale bar, 20 μm. Two-tailed Student’s paired t test. Data are presented as means ± SEM. (G) Representative images and quantification of the leaked Evans Blue dye fluorescence intensity in the thalamus of Mfn2^fl/fl and Mfn2-cKO 1M male mouse brains (n = 12 captured image areas from three mice for each group). Scale bar, 50 μm. Two-tailed Student’s unpaired t test. Data are presented as means ±SEM. (H) Representative images and quantification of the leaked Evans Blue dye fluorescence intensity in the thalamus of young (1M) and aged (20M to 22 M) WT mouse brains (n = 12 captured image areas from three mice for each group). Scale bar, 50 μm. Two-tailed Student’s unpaired t test. Data are presented as means ± SEM. (I) Representative images and the quantification of COL4A1 intensity in the thalamus of 1M Mfn2^fl/fl and Mfn2-cKO male mouse brains (n = 9 images from three mice for each group). Scale bar, 20 μm. Two-tailed Student’s paired t test. Data are presented as means ± SEM.

Fig. 6.. Astrocytes-derived mitochondria restore endothelial dysfunction.

(A) Work flow of 2/2 μM A/R treatment on bEnd.3 endothelial cells followed by mitochondrial transplantation and H₂DCFDA staining for flow cytometry analysis. (B) Representative histogram overlays and quantitative results of ROS (H₂DCFDA) intensity in healthy (vehicle), 2/2 μM A/R-damaged bEnd.3 endothelial cells, and 2/2 μM A/R-damaged bEnd.3 endothelial cells after transplanted with 21.25-, 42.5-, and 85-μg astrocytes-derived mitochondria (Mito-T) (n = 4). One-way ANOVA followed by Tukey’s post hoc test. Data are presented as means ± SEM. (C) Top 10 up-regulated GO enrichment terms of cellular components (CC) in bEnd.3 cells transplanted with primary WT astrocytes-derived mitochondria compared with normal control bEnd.3 cells. (D) Workflow for 4/4 μM A/R treatment on bEnd.3 endothelial cells followed by mitochondrial transplantation and cell scratch assay. (E) Representative images of scratched bEnd.3 endothelial cells without A/R treatment (vehicle), with 4/4 μM A/R treatment, with 4/4 μM A/R treatment and subsequent transplantation of approximately 170 μg of mitochondria isolated from primary astrocytes (up panel), followed by 24 hours of normal culture (below panel). Scale bar, 200 μm. (F) Quantification of wound healing rate after 24 hours of normal culture in bEnd.3 endothelial cells without 4/4 μM A/R treatment (vehicle), with 4/4 μM A/R treatment, with 4/4 μM A/R treatment and subsequent transplantation of approximately 170 μg of mitochondria (n = 4). One-way ANOVA followed by Tukey’s post hoc test. Data are presented as means ± SEM.