Extended Data Fig. 1: STING is constitutively active in neurons of LSD mice.
From: Innate immune sensing of lysosomal dysfunction drives multiple lysosomal storage disorders
(a) Quantifying GM2-positive cells in WT and Hexb-/- mice brain regions. Purkinje cell layer, n = 16 slices pooled from 4 WT mice, and n = 14 slices pooled from 4 Hexb-/- mice. Cerebellar deep nuclei, n = 7 slices pooled from 4 mice in each group. Brainstem, n = 16 slices pooled from 4 mice in each group. (b, c) Genotyping of mice with genetic deletion of Hexb and Sting1, which were both generated by CRISPR strategy. (d) Quantifying relative STING signal in WT and Hexb-/- mice neurons. n = 80 Purkinje cells pooled from 4 mice in each group (left); n = 4 slices captured from cerebellar deep nuclei of 4 mice per group (middle); n = 16 slices captured from brainstem of 4 mice per group (right). (e) Quantification of pSTAT1 and RIG-I protein levels showed an increased IFN-I signalling in the cerebellum of Hexb-/- mice at 12 weeks old. n = 3 mice per group. (f) Immunoblottings of phosphorylated proteins revealed that STING signalling was activated in the primary neurons of Hexb-/- mice. (g) Sequencing verified Hexb knockout clones generated by CRISPR. (h) Immunoblotting revealed that RIG-I as an ISG was highly induced in Hexb KO cells and increased by DMXAA treatment in WT and Hexb KO N2a cell lines. (i) qRT-PCR indicated that siRNA-mediated depletion of RIG-I in HEXB KO N2a cells failed to prevent ISG expression. n = 3 independent samples. (j) Quantification of pSTAT1 and RIG-I protein levels showed AAV-SNX8 infection reduced IFN-I signalling in Hexb-/- mouse brains at 3 months old. n = 3 mice per group. Graphs in a–j represent mean ± SEM, and P values were calculated by one-way ANOVA with Bonferroni correction. NS, not significant. Results in f and h represent three independent experiments.
