Fig 2: ADAR1 is induced in astrocytes in ischemic mouse brains with intraluminal middle cerebral artery occlusion (MCAO). Mice were sham-operated or underwent MCAO for 60 min. 7 days later, mice were sacrificed, and ischemic penumbra areas were collected and frozen sectioned. (A) ADAR1 expression in glial scar and ischemic area was detected by co-immunostaining with GFAP. (B) Quantification of ADAR1+GFAP + cells relative to the total cell numbers in the brain sections. Data are shown as mean ± SD. *P < 0.05 vs Sham group, n = 8. (C–D) Mice were sham-operated or underwent MCAO for 60 min. The mice were then euthanized 1, 3, 5, 7, or 14 days later. The ischemic penumbra areas were collected, and RNAs and proteins were extracted followed by RT-qPCR and Western blot analyses to detect ADAR1 mRNA (C) and protein expression (D-E), respectively. ADAR1 protein levels were quantified by normalizing to α-Tubulin. *P < 0.05, n = 3 in each group. Both p150 and p110 isoforms were significantly induced by MCAO.

Fig 3: Astrocyte ADAR1 promotes neuron apoptosis by secretion of pro-inflammatory cytokines IL-1, IL-6 and TNF-α. Astrocytes were primary cultured from wild type (WT) and ADAR ±mice. (A–B) Primary neurons were treated with astrocytes-conditioned medium with or without (Ctrl) oxygen-glucose-deprivation (OGD). Inflammation cocktail containing IL-1 (10 ng/ml), IL-6 (10 ng/ml) and TNF-α (10 ng/ml) was used as a positive control. TUNEL staining of cultured neuron (A), and TUNEL + cells were quantified relative to the total neuron cells (B). &P < 0.01 vs. neuron cells treated with WT astrocyte-conditioned medium without OGD (WT, Ctrl); **P < 0.01 vs. WT astrocyte-conditioned medium with OGD (WT, OGD); n = 8. (C-D) Western blot analyses of IL-1, IL-6, and TNF-α production in primary astrocytes cultured with or without Oxygen-Glucose-Deprivation (OGD) for 24 h (C). The protein levels in C were quantified by normalizing to α-Tubulin (D). *P < 0.01 vs. WT astrocytes without OGD (WT, Ctrl); #P < 0.05 vs. WT astrocytes with OGD (WT, OGD); n = 6.

Fig 4: ADAR1 deficiency inhibits post-stroke proliferation of astrocytes. (A–C) Wild type (WT) or ADAR1 ± mice were sham-operated or underwent MCAO for 60 min. 7 days later, mice were euthanized, and brain tissues collected. (A) Astrocyte proliferation in glial scar tissues was detected by co-immunostaining of Ki-67 with GFAP. (B) Western blot analyses of PCNA, Ki-67, phospho-ERK, ERK, phospho-Akt, Akt expression in the ischemic brains. (C) The protein expression levels in A were quantified by normalizing to α-Tubulin. *P < 0.01 vs. WT group with sham, #P < 0.01 vs. WT group with MCAO; n = 6. (D, E) Primary astrocytes were isolated from mouse brain, cultured, transduced with control (Ad-GFP), ADAR1 cDNA (Ad-ADAR1), or ADAR1 shRNA (Ad-shRNA)-expressing adenovirus, and then treated with vehicle (−), PI3/Akt (LY294002), or MEK1/MEK2 (U0126) inhibitor. ADAR1, phospho-Akt (p-Akt), Akt, phospho-ERK (p-ERK), ERK, PCNA, Ki67 expression was detected by Western blot (D) and quantified by normalizing to α-tubulin and relative to the level with Ad-GFP treatment for each protein. &P < 0.05 vs. Ad-GFP group; σP<0.05 vs. Ad-ADAR1 alone group; NS: non-significant; n = 6.

Fig 5: ADAR1 promotes astrocyte proliferation and migration in vitro. Primary cultured astrocytes were isolated from wild type (WT) and ADAR ±mouse brains. (A) Ki67 expression in astrocytes treated with vehicle or TGF-β was detected by immunostaining, and Ki67+ cells were quantified relative the total cell numbers. *P < 0.01 vs WT group with vehicle treatment; #p < 0.01 vs WT group with TGF-β treatment, n = 5. (B) Confluent monolayers of primary astrocytes from wild type (WT) or ADAR1 ± mice were scratched. The wound healing was observed by an inverted phase-contrast microscope at 0 and 24 h. Shown are representative Images of three independent experiments. Migration rates were calculated as wound closure percentage. &P < 0.01 vs. WT group, n = 5. (C) Transwell assay showed that ADAR ±significantly inhibited astrocyte migration. Migrated astrocytes were stained with crystal violet. &P < 0.05 vs. WT group, n = 5.