Reactive astrocytes function as phagocytes after brain ischemia via ABCA1-mediated pathway

Abstract

Astrocytes become reactive following various brain insults; however, the functions of reactive astrocytes are poorly understood. Here, we show that reactive astrocytes function as phagocytes after transient ischemic injury and appear in a limited spatiotemporal pattern. Following transient brain ischemia, phagocytic astrocytes are observed within the ischemic penumbra region during the later stage of ischemia. However, phagocytic microglia are mainly observed within the ischemic core region during the earlier stage of ischemia. Phagocytic astrocytes upregulate ABCA1 and its pathway molecules, MEGF10 and GULP1, which are required for phagocytosis, and upregulation of ABCA1 alone is sufficient for enhancement of phagocytosis in vitro. Disrupting ABCA1 in reactive astrocytes result in fewer phagocytic inclusions after ischemia. Together, these findings suggest that astrocytes are transformed into a phagocytic phenotype as a result of increase in ABCA1 and its pathway molecules and contribute to remodeling of damaged tissues and penumbra networks.Astrocytic phagocytosis has been shown to play a role in synaptic pruning during development, but whether adult astrocytes possess phagocytic ability is unclear. Here the authors show that following brain ischemia, reactive astrocytes become phagocytic and engulf debris via the ABCA1 pathway.

Fig. 1

Astrocytes become phagocytic after transient ischemic injury in vivo. a Representative images of the ischemic penumbra showing FJ-labeled and GFAP-immunostained brain sections at 7 days after MCAO (n = 10 mice). Arrowheads indicate FJ⁺-degenerating neuronal debris enwrapped by GFAP⁺ astrocytes. Fourteen images per z-stack image (0.49 µm step). b Representative image of GFAP⁺, Galectin-3⁺ astrocyte enwrapped NeuN⁺, FJ⁺ large neuronal debris. Arrowheads indicate FJ⁺ degenerating neuronal debris enwrapped by GFAP⁺ astrocytes. Thirty-four images per z-stack image (0.3 µm step). c Representative images of NeuN⁺ signals in LAMP2⁺ lysosomes in GFAP⁺ astrocytes (arrowheads). d Processes of Galectin-3 and GFAP double-positive astrocytes enwrapping numerous FJ⁺ small debris (n = 8 mice). The white squares in the left panel image indicate the region of high magnification of shown in the right panel. e Immunoelectron microscopic (iEM) images of GFAP⁺ astrocytes (gold particles: GP; blue). Left, an image of an astrocyte in the striatum of an intact mouse. Middle, an image of an astrocyte in the ipsilateral striatum at 3 days after MCAO. Arrowheads indicate phagocytic inclusions. Right, high-magnification image of the box shown in the middle panel. Arrow indicates myelin-like structure. N, nucleus; M, mitochondria; in, phagocytic inclusion. f Percentage of GFAP⁺ astrocytes with phagocytic inclusions in total in the intact (n = 461 cells, 4 mice) and MCAO-treated striatum (n = 315 cells, 4 mice, **P = 0.0056, unpaired t-test). Values represent means ± SEM

Fig. 2

Phagocytosis of cellular debris by reactive astrocytes in the ischemic penumbra. a–c Compared with astrocyte processes in the contralateral striatum (a, blue), those in the ischemic penumbra were characterized by prominent intermediate filament bundles (b, yellow, arrowheads) and abundant glycogen granules (b, asterisks), and often contained small pieces of cellular debris (b, red, arrow). The debris was completely included in the astrocytic cytoplasm (c-i–c-x, arrows). d–f Three-dimensional reconstruction shows several pieces of debris (e, f, pink) are scattered within single processes (e, f, white). g, h Astrocyte processes surround blood vessels (g, BV), and engulf large debris (g, arrows) and myelin debris (g, h, red, arrowheads). Bars: 2 μm a, b, g, h or 5 μm d–f. i Dot plots of debris density (number/volume (µm³)) in astrocytes in the contralateral and ipsilateral striatum (n = 5, eight cells, three mice. **P < 0.01 vs. control, Mann–Whitney U-test)

Fig. 3

Comparison of enwrapped neuronal debris by astrocytes and microglia. a Representative images showing immunohistochemical (IHC) staining for GFAP, and Iba1 with FJ staining in the ischemic penumbra at 7 days after MCAO. Iba1⁺ microglia (yellow arrowheads) and GFAP⁺ astrocytes (red arrowheads) enwrapped FJ⁺ large neuronal debris by their processes. b Representative images showing IHC staining for Galectin-3 and GFAP with FJ staining in the ischemic penumbra at 7 days after MCAO. Galectin-3 and GFAP double-positive processes enwrapped FJ⁺ large neuronal debris (yellow arrowheads). c Representative images showing quantification of the amount of FJ⁺ large debris (size over 10 µm²) enwrapped by Iba1⁺ microglia or Galectin-3⁺, GFAP⁺ astrocytes. d Quantification of FJ+ large debris enwrapment by Iba1⁺ microglia and Galectin-3⁺, GFAP⁺ astrocytes (n = 13 fields, four mice, ***P < 0.001 vs. astrocytes). All images are single plane images

Fig. 4

Spatiotemporal differences in Galectin-3 between astrocytes and microglia. a Representative images showing IHC staining for GFAP, Iba1, and Galectin-3 in the ischemic penumbra at 3 and 7 days after MCAO. Arrowheads indicate Galectin-3 in astrocytes. Asterisks indicate the ischemic core (n = 5 mice). Thirteen images per z-stack image (0.5 µm step). b Left, quantification of spatiotemporal profile of Galectin-3⁺ astrocytic population (3PGDH⁺ cells) after MCAO (n = 4, 4, 5, 15, 15, 15, 4, 12, 12, 4, 12, 12 fields, 3–5 mice, *P < 0.05, ***P < 0.001 vs. contra (corresponding day), ^### P < 0.001 ipsi proximal vs. ipsi distal, ^§§§ P < 0.001 vs. ipsi proxymal D7, one-way ANOVA (P < 0.0001) with Tukey’s multiple comparison test). Right, quantification of spatiotemporal profile of Galectin-3 immunoreactivity mean intensity in 3PGDH⁺ astrocytes in the ipsilateral striatum after MCAO (n = 30, 51, 208, 76 cells, 3–5 mice, **P < 0.01, ***P < 0.001 vs. D1, ^### P < 0.001 vs. D7, one-way ANOVA (P < 0.0001) with Tukey’s multiple comparison test). c Left, quantification of mean intensity of Galectin-3 immunoreactivity in Iba1⁺ microglia after MCAO (n = 12, 12, 15, 15, 10, 14, 10, 15 fields, 4–5 mice, ***P < 0.001 vs. contra (corresponding day), ^## P < 0.01, ^### P < 0.001 vs. ipsi D3, ^§§§ P < 0.001 vs. ipsi D7, one-way ANOVA (P < 0.0001) with Tukey’s multiple comparison test). Right, quantification of Galectin-3⁺ microglia population (Iba1⁺) in the ipsilateral striatum after MCAO (n = 12, 15, 14, 15 fields, 4–5 mice, ***P < 0.001 vs. D3, ^# P < 0.05 vs. D7, one-way ANOVA (P < 0.0001) with Tukey’s multiple comparison test). Values represent means ± SEM

Fig. 5

Spatiotemporal differences in lysosomal protein between astrocytes and microglia. a Representative images of GFAP, 3PGDH, and LAMP2 immunoreactivity in the contralateral striatum (upper) and the ischemic penumbra of the ipsilateral striatum (lower) at 7 days after MCAO. Arrowheads indicate astrocytes (n = 10 mice). Forty images per z-stack image (0.47 µm step). b Quantification of LAMP2 immunoreactivity mean intensity in 3PGDH⁺ astrocytes after MCAO (n = 9, 9, 9, 16, 24, 9, 18, 26, 9, 16, 24 fields, 3, 4 mice, *P < 0.05, ***P < 0.001 vs. contra (corresponding day), ^# P < 0.05, ^## P < 0.01 ipsi (proximal) vs. ipsi (distal), ^§§§ P < 0.001 vs. ipsi proxymal D7, one-way ANOVA (P < 0.0001) with Tukey’s multiple comparison test). c Representative images of GFAP, CD68, and Iba1 immunoreactivity in the ischemic core, penumbra, and contralateral striatum at 3 and 14 days after MCAO (n = 4 mice). High-magnification images are shown in the right panel. Eighteen images per z-stack image (1.0 µm step). d Quantification of CD68 immunoreactivity mean intensity in Iba1⁺ microglia after MCAO (n = 8, 8, 10, 8, 4, 4, 12, 11, 10, 10, 8, 8, 8, 8 fields, 4–6 mice, ***P < 0.001 vs. contra (corresponding day), ^# P < 0.05, ^### P < 0.001 vs. ipsi core (corresponding day), ^§§§ P < 0.001 vs. respective ipsi core, one-way ANOVA (P < 0.0001) with Tukey’s multiple comparison test). Values represent means ± SEM

Fig. 6

Transient ischemic injury induces ABCA1 upregulation exclusively in reactive astrocytes. a In situ hybridization (ISH) analysis of Abca1 mRNA at 7 days after MCAO. Abca1 ISH signals (purple) are strongly upregulated in the ischemic penumbra. b Representative images show that increased Abca1 ISH signals colocalize with GFAP immunoreactivity (DAB: brown). c Increased ABCA1 ISH signals colocalize with GFAP and 3PGDH, but not with NeuN and Iba1 in the ischemic penumbra at 7 days after MCAO. Low-magnification images from the contralateral side are shown in Supplementary Fig. 6b. d Real-time PCR analysis of Abca1 mRNA in total RNA extracted from the ipsilateral and contralateral striatum after MCAO. Values represent the relative ratio of Abca1 mRNA (normalized to GAPDH mRNA levels) to the corresponding contralateral striatum (D1: n = 4; D2: n = 3; D3: n = 8; D6: n = 9; D14: n = 6; *P < 0.05, **P < 0.01, ***P < 0.001 vs. contra (corresponding day)). e Immunostaining for ABCA1 and GS in the contralateral and ipsilateral striatum at 7 days after MCAO. Fifteen images per z-stack image (1.14 µm step). f Increased ABCA1 IHC signals colocalize with GFAP, but not with NeuN, CD11b, or CD31 in the ischemic penumbra at 7 days after MCAO. g Quantification of ABCA1 immunoreactivity mean intensity in GS⁺ astrocytes after MCAO (n = 9, 8, 12, 12, 21, 11, 12, 20, 12, 11, 21 fields, 3–4 mice, *P < 0.05, **P < 0.01, ***P < 0.001 vs. contra (corresponding day), ^## P < 0.01 ipsi proximal D1, one-way ANOVA (P < 0.0001) with Tukey’s multiple comparison test). Asterisks indicate the ischemic core. Values represent means ± SEM

Fig. 7

ABCA1 mediates astrocytic phagocytosis in vitro. a Confocal images show astrocytes (GFAP, cyan), ABCA1 (green), and 4-µm fluorospheres (red). Astrocytes were incubated with 4-μm fluorospheres (30 min after incubation). Arrowheads indicate ABCA1 accumulation and points of attachment between captured beads and astrocytes. Forty-eight images per z-stack images (0.38 µm step). b Phagocytic activities in the presence of ABCA1 inhibitors. Astrocytes were pretreated Glyburide or PSC833 for 15 min prior to the addition of 4-μm fluorospheres, and uptake was assessed by FACS after 1 h (n = 15, 6, 6, 15, 6, 6, *P < 0.05, ***P < 0.001 vs. control, unpaired t-test). c Phagocytic activities of astrocytes transfected with control siRNA or Abca1 siRNAs (n = 11, 14, 10, 11, ***P < 0.001 vs. control siRNA, unpaired t-test). d Phagocytic activities of astrocytes from wildtype (WT), Abca1^+/−, or Abca1^−/− mice (n = 3, 13, 7, 14, ***P < 0.001 vs. littermate control (Litter), one-way ANOVA (P < 0.0001) with Tukey’s multiple comparison test). e Phagocytic activities of astrocytes from WT or Abca1^−/− mice with or without T0901317 pretreatment (100 nM, 48 h) (n = 11, 13, 7, 8, *P < 0.05 vs. WT naïve, one-way ANOVA (P < 0.0001) with Tukey’s multiple comparison test). Values represent means ± SEM for b–e

Fig. 8

ABCA1 mediates astrocytic phagocytosis in vivo. a, b Serial electron microscopic images were acquired from the ischemic penumbra of control and ABCA1-cKO (cKO) mice, and three-dimensionally reconstructed (7 days after MCAO). Ischemic penumbra of control contains extracellular debris (a, green, arrows), and astrocyte processes (a, b, left, blue) with glycogen granules (a, asterisks) include cellular debris (a, b, red, arrowheads). By contrast, ischemic penumbra of cKO contains numerous extracellular debris (a, green, arrows), but the astrocyte processes (a, b, yellow) rarely contains debris. Bars: 1 μm a or 2 μm b. c, d Dot plots c and scatter plots d of debris densities (number/volume (µm³)) in astrocytes in control (blue) and cKO (red) mice (n = 12, 11 cells, three, four mice, respectively. ***P < 0.001 vs. control, Mann–Whitney U-test). e Dot plots show extracellular debris densities (%: volume/volume). The % volume occupied by extracellular debris tended to be higher in cKO, although the difference was not significant because of a large variance (n = 19, 22 regions, three, four mice, respectively. Mann–Whitney U-test). f Small debris densities in microglia are shown in a dot plot (n = 7 cells, three, four mice, respectively. Mann–Whitney U-test). These data excluded that microglia engulfed large cellular debris (maximum Feret diameter > 4 µm; control = 3, cKO = 3 cells). g Dot plots replotted from c and f show small debris densities in astrocytes and microglia in control mice (n = 12, 7 cells, three mice each. Mann–Whitney U-test)