Fig 2: ADAR1 deficiency decreases brain infarct volumes and improves neurobehavioral outcomes. (A) Mice were sham-operated or underwent MCAO for 60 min. 7 days later, mice were euthanized. Brain sections from WT and ADAR1 ± mice with MCAO were stained with Triphenyltetrazolium chloride (TTC). Shown are sections of anterior (top, 6 mm), middle (8 mm), and posterior (bottom, 10 mm) mouse brain tissues. Red-stained areas indicate normal healthy tissues, whereas the white areas indicate infarcted areas. (B) The brain infarction volumes at different distances (6 mm, 8 mm, or 10 mm) from the frontal pole were quantified as percentages of infarcted areas relative to the total brain areas. *P < 0.05, n = 12. ADAR1 ± decreased brain infarct volumes compared to WT mice. (C–F) Neurobehavioral tests were conducted 3, 5, 10, or 14 days after the MCAO for both WT and ADAR1 ± mice. The latency to fall in rotarod (C), time spent for right-biased bending in the elevated body swing (D), and grip strength (E) were all significantly improved in ADAR1 ± mice compared to WT mice. In addition, WT mice exhibited worse functional impairment in the sensorimotor asymmetry than ADAR1 ± mice in the corner test (F). Plots represent mean ± SD values. *P < 0.05 vs WT group, n = 9. ADAR1 ± attenuated neurobehavioral outcomes after stroke.

Fig 3: ADAR1 deficiency inhibits brain cell apoptosis and pre-inflammatory cytokine production in MCAO-operated mice. 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) TUNEL staining of brain tissue sections. (B) TUNEL + cells relative to the total brain cells. *P < 0.01 vs. WT group with Sham; #P < 0.01 vs. WT group with MCAO; n = 8. (C) Western blot analyses of cleaved (CL)-Caspase 3 and cleaved (CL)-PARP in ischemic brains. (D) Cleaved-Caspase 3 and Cleaved-PARP protein levels were normalized to GAPDH, respectively. *P < 0.01 vs WT group with Sham; #P < 0.01 vs WT group with MCAO; n = 6. (E) Western blot analyses of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in ischemic brains. (F) Protein levels shown in E were quantified by normalizing to GAPDH, respectively. *P < 0.01 vs. WT group with Sham; #P < 0.01 vs WT group with MCAO; 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: 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.