Fig. 5: Astrocytic ALKBH5 modulates glutamate levels through methylation modification of GLT-1.
From: Astrocytic ALKBH5 in stress response contributes to depressive-like behaviors in mice
a Targets to specifically alter m6A modification sites of GLT-1. b Dual-luciferase reporter constructs with the psiCHECK-2 vector contain Renilla luciferase and firefly luciferase driven by two different promoters. The effects of GLT-1 m6A were examined by transfection of a dual-luciferase reporter. (n = 4 biological replicates). c For single-base editing to construct and cotransfect dCas9-mCherry and sgRNA-puro-eGFP into mixed primary cultured astrocytes and representative images. Scale bars = 20 μm (right). (n = 6 replicates from three wells). d Western blotting analysis of ALKBH5 of sgRNA and Ctrl group. (n = 7 wells per group). e Glutamate uptake ability in the medium of astrocytes transfected with sgRNA and Ctrl plasmids. (n = 3 wells per group). f Western blotting analysis of ALKBH5 and GLT-1 in Alkbh5loxP/loxP astrocytes infected pLent-GFAP-Cre virus (cKO) and Ctrl virus. (n = 4 wells per group). g Glutamate uptake ability in the medium of cKO and Ctrl astrocytes. (n = 6 mice per group). h Western blotting analysis of GLT-1 in the mPFC of astrocyte cKO and Ctrl mice. (n = 8 mice per group). i, j Schematic illustrating fiber placement and representative images of AAV-Syn-iGluSnFR(A184S) expression. Scale bars = 500 μm (left), 20 μm (right). (n = 6 replicates from three mice). k Representative heatmaps of z-scores changes over all trials from single mice. Each row plots one trial, and a total of four trials are illustrated. l, m Time course of average iGluSnFR transient z-scores event locked to social interaction (l) and peak z-score (m) during social interaction. (Astrocyte cKO, n = 5; Ctrl, n = 7 mice). n, o Statistical analysis of GFAP△Alkbh5 mice injected with AAV-shRNA(GLT-1) or AAV-mCherry in FST (n n = 6 mice per group); SI test (o Ctrl, n = 6; GFAP△Alkbh5, n = 8; GFAP△Alkbh5-shGLT-1, n = 7 mice). All data were presented as the mean ± SEM. Two-sided unpaired t-test (d–h) or one-way ANOVA followed by Bonferroni’s test for multiple comparisons (b, n), or two-way ANOVA (m, o) with Bonferroni’s multiple comparisons test. *p < 0.05; ***p < 0.001; ***p < 0.001; n.s. no significance. Source data are provided as a Source Data file. See Supplementary Data 4 for statistical details.
