Mitochondria-Endoplasmic Reticulum Contacts in Reactive Astrocytes Promote Vascular Remodeling

Abstract

Astrocytes have emerged for playing important roles in brain tissue repair; however, the underlying mechanisms remain poorly understood. We show that acute injury and blood-brain barrier disruption trigger the formation of a prominent mitochondrial-enriched compartment in astrocytic endfeet, which enables vascular remodeling. Integrated imaging approaches revealed that this mitochondrial clustering is part of an adaptive response regulated by fusion dynamics. Astrocyte-specific conditional deletion of Mitofusin 2 (Mfn2) suppressed perivascular mitochondrial clustering and disrupted mitochondria-endoplasmic reticulum (ER) contact sites. Functionally, two-photon imaging experiments showed that these structural changes were mirrored by impaired mitochondrial Ca²⁺ uptake leading to abnormal cytosolic transients within endfeet in vivo. At the tissue level, a compromised vascular complexity in the lesioned area was restored by boosting mitochondrial-ER perivascular tethering in MFN2-deficient astrocytes. These data unmask a crucial role for mitochondrial dynamics in coordinating astrocytic local domains and have important implications for repairing the injured brain.

Keywords: Mitofusin 2; angiogenesis; brain injury; brain repair; calcium imaging; contact sites; metabolism; mitochondrial dynamics; perivascular endfeet; proteomics; synthetic linker.

Graphical abstract

Figure 1

Astrocytic Endfeet Are Enriched in Mitochondria-ER Contact Sites (A) Experimental design. (B) Example of an astrocyte co-transduced with ER-GFP and mitoRFP viruses. Yellow arrowheads: endfeet. Scale bar, 10 μm. (C) Magnifications of the astrocyte shown in (B). Yellow arrowheads point to bundles of elongated mitochondria. Scale bar, 5 μm. (D and E) Example of astrocytes transduced with ER-GFP or mitoYFP (in gray) wrapping around dextran-labeled vessels (in red). Insets show zooms of the perivascular endfoot. Side panels: 3D rendering of the same astrocytes. Scale bars, 10 and 25 μm. (F) EM picture of a vessel cross-section showing the astrocytic endfoot (segmented black line) and its organelles (mitochondria, yellow; ER, red; contact sites, blue). Inset: mitochondria-ER contact sites lining the basal lamina. Scale bars, 2 and 1 μm. (G) Perisynaptic astrocytic processes and their organelles. Scale bar, 2 μm. (H) Quantification of mitochondrial parameters in branches (n = 21 cross-sections from 3 mice) and endfeet (n = 32 cross-sections from 3 mice; nonparametric Mann-Whitney t test). ^∗∗∗p < 0.001. PC, pericyte; EC, endothelial cell; BL, basal lamina. See also Figure S1.

Figure 2

Dynamic Remodeling of Astrocyte Mitochondrial and ER Networks following Injury (A) Experimental design. (B) Example of an hGFAP::CreER x R26^LSL-mitoYFP mouse at 7 days after cortical SW injury. Inset: extravasating CD45⁺ leukocytes in the lesion core. Scale bar, 150 μm. (C) Surface rendering of mitoYFP+ control (uninjured animals) or reactive astrocytes proximal to the lesion track. Arrowheads point to the soma. Zooms depict the network morphology in branches. Scale bar, 15 μm. (D) Density plots depicting the mitochondrial heterogeneity in resting (Ctrl, uninjured animals) or reactive astrocytes (SW 7 days). Threshold values for mitochondrial sphericity (0.8) and length (1 μm) are shown. (E) Time course of mitochondrial fragmentation quantified as in (D) (n ≥ 3 mice/time point, 8–15 astrocytes/mouse; one-way ANOVA followed by Dunnett’s post hoc test). (F) Volume reconstruction of mitoYFP+ reactive astrocytes (arrowheads) surrounding dextran-labeled vessels. Scale bar, 25 μm. (G) Vessel cross-sections showing astrocytic mitoYFP in control (uninjured animals) and injured conditions. Scale bar, 10 μm. (H) Quantification of perivascular mitoYFP (n ≥ 30 vessels/time point; nonparametric Kruskal-Wallis test). (I) Experimental design for analyzing the astrocytic ER. (J) 3D example of an astrocyte expressing ER-GFP (signal density shown in pseudocolors). Scale bars, 10 and 5 μm. (K) Quantification of the ER-GFP perivascular g-ratio at the indicated time points (n ≥ 35 vessels/time point; nonparametric Kruskal-Wallis test). ^∗∗p < 0.01, ^∗∗∗p < 0.001. See also Figure S2.

Figure 3

Mfn2 Deletion Affects Astrocytic Mitochondria-ER Tethering Domains (A) Proposed model. (B) Validation of Mfn2 knockout in astrocytes by MACS enrichment and proteomic analysis (n = 4 Mfn2^cKO mice and 3 Mfn2^WT mice). (C) EM pictures of astrocytic endfeet at 4 weeks post-tamoxifen treatment. Organelles are highlighted in different colors. Right panels: zooms of mitochondrial cristae. EC, endothelial cell; BL, basal lamina. Scale bars, 1 μm and 200 nm. (D) Perivascular distribution of ER tubules and their contact sites with mitochondria. Scale bars, 250 nm. (E) Quantification of the indicated parameters in Mfn2^WT (n = 85 cross-sections from 4 mice) and Mfn2^cKO perivascular endfeet (n = 145 cross-sections from 3 mice; nonparametric Mann-Whitney t test). ^∗∗∗p < 0.001. See also Figure S3.

Figure 4

Mfn2 Deletion Prevents Astrocytic Perivascular Clustering of Still Functional Mitochondria (A) Example of cortical SW injury in Mfn2^cKO mice at 7 days. Inset: CD45⁺ leukocytes in the lesion core. Scale bar, 100 μm. (B) Time-course analysis of mitochondrial fragmentation in Mfn2^cKO and Mfn1^cKO astrocytes (n ≥ 3 mice/time point, 8–15 astrocytes/mouse; two-way ANOVA followed by Tukey’s post hoc test). (C) Mitochondrial morphologies in astrocytes (arrowheads point to soma) proximal to the lesion site at 28 days post-SW. Zooms depict peripheral branches. Scale bar, 20 μm. (D) Top view projections (100 μm deep) of whole-mount injured cortices (7 days) following tissue clearing. The injury site is indicated by a dashed yellow line. Middle panels: zooms of astrocytes proximal to the lesion track. Right panels: vessel cross-sections. Scale bars, 50, 10, and 10 μm. (E) Quantification of mitoYFP perivascular density (n = 3 mice/condition, at least 80 vessel sections quantified; the contralateral sides were utilized as controls; one-way ANOVA followed by Holm-Sidak’s post hoc test). (F) Experimental approach. (G) Heatmaps of TCA cycle and associated enzymes in reactive Mfn2^cKO and Mfn1^cKO astrocytes. Asterisks indicate significant changes (−log₁₀ of the p ≥ 1.3) (n = 4 Mfn2^cKO mice, 4 Mfn1^cKO mice and 3 Ctrl mice). (H) Atom transition model of ¹³C labeling after ¹³C₆-glucose supplementation. (I and J) Graphs depicting the mass isotopomer enrichment analysis for the indicated metabolites (n = 5 Mfn2^cKO mice, 6 Mfn1^cKO mice, and 5 Ctrl mice; two-way ANOVA followed by Dunnett’s test). (K) Heatmaps of proteins regulating Ca2+ transport. Asterisks indicate significant changes (−log₁₀ of the p ≥ 1.3) (n = 4 Mfn2^cKO mice, 4 Mfn1^cKO mice, and 3 Ctrl mice). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. See also Figures S3 and S4.

Figure 5

Compromised Mitochondrial Ca²⁺ Uptake and Abnormal Cytosolic Activity in Mfn2^cKO Astrocytic Endfeet (A) Experimental design. (B) MitoGCaMP6-expressing astrocyte following AstroSparks processing and ROI detection. Inset displays cytosolic mCherry. Scale bar, 10 μm. Right panels: individual ROI traces and corresponding raster plot. (C and D) Quantification of mitochondrial Ca²⁺ transient parameters, including active fraction, frequency, duration, and amplitude, in Mfn2^WT astrocytes (n = 41–53 cells from 3 mice). (E) Example of an Mfn2^cKO astrocyte. Scale bar, 10 μm. (F) Quantification of active mitochondria in Mfn2^WT (n = 40–56 cells, 3 mice/condition) and Mfn2^cKO (n = 36–73 cells, 2–3 mice/condition) astrocytic endfeet. (G) Ca²⁺ transient parameters of the astrocytes in (F). (H) Mitochondrial and cytosolic Ca²⁺ traces. (I) Experimental in vivo setting. (J) Example of GCaMP3-expressing astrocyte following AstroSparks processing. Scale bar, 20 μm. (K) ROI traces and corresponding raster plots of Mfn2^WT and Mfn2^cKO astrocytes. (L and M) Average frequency (endfeet) (L) and area of Ca²⁺ transients (M) in Mfn2^WT (n = 35–111 cells, 2–3 mice/condition) and Mfn2^cKO astrocytes (n = 51–73 cells, 2–3 mice/condition). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 (nonparametric Mann-Whitney t test). See also Figure S5.

Figure 6

Impaired Angiogenesis and Vascular Remodeling in Mfn2^cKO Mice (A) Experimental approach. (B) Top views of reconstructed vascular networks. Arrowheads point to the lesion track. Insets: zooms of the lesioned core region (circled in white). Scale bar, 200 μm. (C) Pipeline used for vasculature quantification. Scale bar, 30 μm. (D) Vascular network analysis (n = 3 mice/condition; two-way ANOVA followed by Tukey’s post hoc test). (E) Experimental protocol for EdU labeling. Lower pictures: views of injured cortices. Scale bar, 200 μm. (F) Magnification showing the presence of proliferating endothelial cells (CD31⁺/ERG+). (G) Quantification of proliferating ERG+ cells at 7 days post-SW (n = 4–5 mice/condition; nonparametric Mann-Whitney t test). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. See also Figures S6 and S7.

Figure 7

Forced Astrocytic Mitochondria-ER Tethering Rescues Vascular Remodeling in Injured Mfn2^cKO Mice (A) Experimental approach. (B) Example of an AAV-linker-expressing Mfn2^cKO astrocyte (arrowhead points to the soma). Zooms depict the vessel cross-section. Scale bar, 10 μm. (C) Examples of vessel cross-sections following AAV expression in Mfn2^cKO astrocytes. Scale bar, 5 μm. (D) Quantification of perivascular mitochondrial density (n ≥ 21 vessel sections; one-way ANOVA followed by Tukey’s post hoc test). (E) Quantification of mitochondrial Ca²⁺ uptake in Mfn2^cKO astrocytes (n ≥ 30 cells; nonparametric Mann-Whitney t test). (F) Vasculature density in Mfn2^WT and Mfn2^cKO sections (dashed line: lesion track). Scale bar, 80 μm. (G) Vasculature density in AAV-transduced Mfn2^cKO cortices. Scale bar, 80 μm. (H) Quantification of CD31 area fraction (n ≥ 3 mice/condition; one-way ANOVA followed by Tukey’s post hoc test). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. See also Figure S7.