Extended Data Fig. 6: cGAS translocates and is indispensable for LSD pathogenesis.
From: Innate immune sensing of lysosomal dysfunction drives multiple lysosomal storage disorders
(a) Nucleoplasmic translocation of cGAS was detected in primary neurons with lysosome dysfunction induced by BafA1. (b) Nucleoplasmic translocation of cGAS was observed in primary neurons following HSV-1 infection. (c) The effect of BafA1-induced lysosomal dysfunction on cGAS nucleoplasmic translocation was detected in astrocytes. (d) BafA1-induced activation of STING signalling was abolished by Cgas deletion. (e) qRT-PCR assays measured the BafA1-induced expression of ISGs, which was blocked by cGAS deletion. n = 3 independent samples, two-way ANOVA with Bonferroni correction, mean ± SEM. (f, g) Depletion of cGAS reduced the constant expression of ISGs and chemokines in N2a cells that lacked Ctsd (f) or Gba (g). n = 3 independent samples in graphs f-g. (h, i) The autoactivation of STING-TBK1-IRF3 signalling in Ctsd knockout (h) or Gba knockout (i) N2a cells was attenuated by cGAS depletion. (j) Quantification of pSTING intensity in mice brainstem neurons. n = 15 slices pooled from 4 mice in each group. (k, l) qRT-PCR assays and immunoblotting showed that the constitutive activation of STING signalling in Npc1 KO N2a cells was abolished by cGAS deletion, as indicated by attenuated ISG expression (k, n = 3 independent samples), diminished phosphorylation levels of TBK1, IRF3, and STAT1, and decreased RIG-I protein levels (l). (m) The autoactivation of STING-TBK1-IRF3 signalling in Hexb KO N2a cells was attenuated by depleting cGAS but not RIG-I or SREBP2. (n) qRT-PCR assays revealed the high knock-down efficiency of siRNAs targeting SREBP2. (o–q) qRT-PCR assays revealed that depletion of cGAS, but not SREBP2, decreased ISG expression in Hexb KO (o), Npc1 KO (p), and Gba KO (q) N2a cells. n = 3 independent samples in graphs o-q. Unless otherwise specified, error bars in graphs e-q represent mean ± SEM and P values were calculated by one-way ANOVA with Bonferroni correction. NS, not significant. Results in a–q represent three independent experiments.
