Circular RNA circ-FoxO3 attenuates blood-brain barrier damage by inducing autophagy during ischemia/reperfusion

Abstract

Blood-brain barrier (BBB) damage can be a result of central nervous system (CNS) diseases and may be a cause of CNS deterioration. However, there are still many unknowns regarding effective and targeted therapies for maintaining BBB integrity during ischemia/reperfusion (I/R) injury. In this study, we demonstrate that the circular RNA of FoxO3 (circ-FoxO3) promotes autophagy via mTORC1 inhibition to attenuate BBB collapse under I/R. Upregulation of circ-FoxO3 and autophagic flux were detected in brain microvessel endothelial cells in patients with hemorrhagic transformation and in mice models with middle cerebral artery occlusion/reperfusion. In vivo and in vitro studies indicated that circ-FoxO3 alleviated BBB damage principally by autophagy activation. Mechanistically, we found that circ-FoxO3 inhibited mTORC1 activity mainly by sequestering mTOR and E2F1, thus promoting autophagy to clear cytotoxic aggregates for improving BBB integrity. These results demonstrate that circ-FoxO3 plays a novel role in protecting against BBB damage, and that circ-FoxO3 may be a promising therapeutic target for neurological disorders associated with BBB damage.

Keywords: E2F1; autophagy; blood-brain barrier; circular RNA FoxO3; hemorrhagic transfromation; ischemic stroke; mTORC1 complex; proteins aggregates.

Graphical abstract

Figure 1

circ-FoxO3 is upregulated and autophagy is activated in the BBB after cerebral I/R injury (A and B) Images of circ-FoxO3 and CD31 and their quantification (integrated optical density) in brain tissues at the site of intracerebral hemorrhage from patients with HT and the peri-infarct area of MCAO/R mice. Ctrl group: brain tissues from non-stroke patients (six females and six males). HT group: patients with hemorrhagic transformation, six females and eight males. Sham group: male mice without ligation of the arteries and insertion of nylon monofilaments, n = 6. MCAO/R group: male mice under MCAO and reperfusion for 2 h, respectively, n = 6. White arrowheads indicate the co-localization of circ-FoxO3 and CD31. Scale bars, 50 μm. (C) The ultra-structures of the BBB in human. The cross-section of the BBB from patients with glioma and patients with HT were imaged in I and II, respectively. The autophagosome-like vacuoles were enlarged in III to VI. Scale bar, 500 nm. (D) Quantitative number of autophagosome-like vacuoles in the BMECs from patients with HT. n (Ctrl group): three females and three males. n (HT group): three females and four males. (E) Ultra-structure of BBB in mice. The cross-section of BBB from sham and MCAO/R mice was presented in I and II, respectively. Cell-cell junctions marked # in I and II was enlarged in III and IV, respectively. White dotted line: cell-cell junctions. Arrowheads indicate desmosomes. Scale bar, 500 nm. The autophagosome-like vacuoles enlarged into V to VIII. Scale bar, 500 nm. (F) Quantitative number of autophagosome-like vacuoles in the BMECs from male MCAO/R mice. n = 6. ECs, endothelial cells. Black arrowheads, autophagosome-like vacuoles; L, lumen; #, cell-cell junction; P, pericyte; Asp, astrocyte processes; MG, microglia; MS, medullary sheath; M, mitochondria; N, nucleus; AP, autophagosome; AL, autolysosome; MCAO/R, middle cerebral artery occlusion for 2 h followed by reperfusion for 2 h. p values indicate a two-tailed unpaired Student's t test. Data are provided as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.

Figure 2

circ-FoxO3 inhibits OGD/R-induced endothelial barrier permeability The siRNA targeting of circ-FoxO3 or LV-circ-FoxO3 was transfected into the BMECs (bEnd.3 and HBMEC). (A) The relative level of circ-FoxO3 or FoxO3 mRNA was measured by qRT-PCR in BMECs. n = 3. (B) The level of circ-FoxO3 and FoxO3 mRNA was compared in BMECs with circ-FoxO3 overexpression. n = 4. (C) The infiltration of FITC-dextran (10 kDa) across the monolayer of BMECs was measured to determine the role of circ-FoxO3 on the properties of the endothelial cell barrier. n = 6. (D) Representative photomicrographs of tube formation in BMECs with circ-FoxO3 knockdown or overexpression. n = 6. Scale bar, 200 μm. (E) Quantification of the number of tubes in (D). The p values indicate one-way ANOVA with Dunnett's multiple comparisons test. OGD/R, oxygen-glucose deprivation/reoxygenation; O3/R3, oxygen-glucose deprivation for 3 h and reoxygenation for 3 h (O6/R6 and O9/R9 indicate the same for 6 and 9 h, respectively). Data are provided as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.

Figure 3

circ-FoxO3 attenuates BBB damage in MCAO/R mice (A) The flowchart of the experimental procedures in mice. (B) The schematic diagram of LV injection (top panel) and the localization of LV in brain tissues of mice (lower panel). White arrowhead indicates that the LV-EGFP-shcirc-FoxO3 or LV-EGFP-circ-FoxO3 localized in vessels. Scale bar, 50 μm. (C) The expression of circ-FoxO3 in the peri-infarct area of mice, n = 4. (D) Representative images of Evans blue extravasation into entire brains of mice with circ-FoxO3 knockdown or overexpression. The white dotted lines in the top panels highlight the injection site of LV. n = 5. Scale bar, 2 mm. (E) Vascular permeability was detected by measurement of Evans blue extravasation in brain tissue using spectrophotometry at 610 nm. n = 5. (F) The cell-cell junctions were imaged by TEM analysis in brain tissue after circ-FoxO3 knockdown or overexpression. n = 5. Scale bar, 250 nm. L, lumen; MCAO/R, middle cerebral artery occlusion for 2 h and followed by reperfusion for 2 h. p values indicate one-way ANOVA with Dunnett's multiple comparisons test. All data are presented as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.

Figure 4

The role of circ-FoxO3 in alleviation of BBB damage depends on autophagy (A and B) Evans blue extravasation was detected in male mice with circ-FoxO3 knockdown or upregulation that were pretreated with Rapa or CQ for 1 week before MCAO/R. CQ, chloroquine, 5 mg/kg/daily. Rapa, rapamycin, 0.75 mg/kg/daily. Vehicle control, saline containing 5% polyethylene glycol 400 and 5% Tween 80. White arrowheads indicate cerebral hemorrhage. Four of five mice displayed cerebral hemorrhage. Scale bar, 2 mm. n = 5. (C) The level of BBB-related proteins such as ZO-1, Mfsd2a, and Cldn5 in BMECs (bEnd.3 and HBMEC) with circ-FoxO3 knockdown or overexpression was analyzed by immunoblotting when cells were pretreated with CQ (or Rapa) before OGD/R. Rapa, 50 nmol/L; CQ, 30 μmol/L. n = 4. p values indicate one-way ANOVA with Dunnett's multiple comparisons test. MCAO/R, middle cerebral artery occlusion for 2 h followed by reperfusion for 2 h; OGD/R, oxygen-glucose deprivation for 3 h followed by reoxygenation for 3 h. All data are presented as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.

Figure 5

circ-FoxO3 promotes autophagy in OGD/R-treated BMECs Autophagy was detected in the monolayer of endothelial cells (bEnd3 and HBMEC) with circ-FoxO3 knockdown or overexpression after OGD/R. (A) Representative images show LAMP1 and LC3B staining. (B) The quantitative integrated optical density of LAMP1 and LC3B in (A). n = 6. Scale bar, 10 μm. (C) Immunoblotting analysis of LC3B II/I and SQSTM1 levels. OGD/R, oxygen-glucose deprivation for 3 h followed by reoxygenation for 3 h. (D) Quantitative results of the bands in (C). n = 4. p values indicate one-way ANOVA with Dunnett's multiple comparisons test. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.

Figure 6

circ-FoxO3 interacts with mTOR and inhibits mTORC1 activity (A) The biotin-labeled circ-FoxO3 probe was incubated in lysates of BMECs with circ-FoxO3 overexpression, and mTOR was identified using an immunoblotting assay. (B) circ-FoxO3 was immunoprecipitated with anti-mTOR-coated magnetic beads, and then detected by qRT-PCR. n = 4. (C) Representative images showed co-localization of circ-FoxO3 and mTOR in BMECs (bEnd.3 and HBMEC). Scale bar, 10 μm. n = 6. White arrowheads, co-localization of circ-FoxO3 and mTOR. (D) The activity of the mTORC1 complex was determined by immunoblotting assay to measure the phosphorylation of S6k1. n = 4. p values indicate one-way ANOVA with Dunnett's multiple comparisons test. OGD/R, oxygen-glucose deprivation for 3 h followed by reoxygenation for 3 h. Data are provided as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.

Figure 7

circ-FoxO3 sequesters E2F1 to inhibit mTOCR1 activity and promotes autophagy (A) circ-FoxO3 was immunoprecipitated with anti-E2F1-coated magnetic beads, and then detected using qRT-PCR. n = 3. p values indicate one-way ANOVA with Dunnett's multiple comparisons test. (B) The localization of circ-FoxO3 and E2F1 in BMECs (bEnd.3 and HBMEC) was determined by FISH and IF. n = 5. Scale bar, 10 μm. The dotted line is the boundary of the nucleus. White arrowheads show the co-localization of circ-FoxO3 and E2F1 in cytosol. Yellow arrowheads indicate the E2F1 in the nucleus. (C) Quantitative integrated optical density of E2F1 in nucleus in (B). p values indicate a two-tailed unpaired Student's t test. (D) The levels of E2F1, PFKFB3, p-S6K1, S6K1, SQSTM1, and LC3B were measured by western blotting. n = 3. (E) Quantitative results of the bands in (D). n = 3. p values indicate one-way ANOVA with Dunnett's multiple comparisons test. (F) The schematic diagram of circ-FoxO3 in the attenuation of BBB damage during I/R. OGD/R, oxygen-glucose deprivation for 3 h followed by reoxygenation for 3 h. I/R, ischemia/reperfusion. Data are provided as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01.