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Innate immune sensing of lysosomal dysfunction drives multiple lysosomal storage disorders

Nature Cell Biology volume 26, pages 219–234 (2024)Cite this article

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Abstract

Lysosomal storage disorders (LSDs), which are characterized by genetic and metabolic lysosomal dysfunctions, constitute over 60 degenerative diseases with considerable health and economic burdens. However, the mechanisms driving the progressive death of functional cells due to lysosomal defects remain incompletely understood, and broad-spectrum therapeutics against LSDs are lacking. Here, we found that various gene abnormalities that cause LSDs, including Hexb, Gla, Npc1, Ctsd and Gba, all shared mutual properties to robustly autoactivate neuron-intrinsic cGAS–STING signalling, driving neuronal death and disease progression. This signalling was triggered by excessive cytoplasmic congregation of the dsDNA and DNA sensor cGAS in neurons. Genetic ablation of cGAS or STING, digestion of neuronal cytosolic dsDNA by DNase, and repair of neuronal lysosomal dysfunction alleviated symptoms of Sandhoff disease, Fabry disease and Niemann–Pick disease, with substantially reduced neuronal loss. We therefore identify a ubiquitous mechanism mediating the pathogenesis of a variety of LSDs, unveil an inherent connection between lysosomal defects and innate immunity, and suggest a uniform strategy for curing LSDs.

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Fig. 1: STING is constitutively active in neurons of LSD mice.
Fig. 2: Sting1 ablation alleviates motor dysfunction in LSD mice.
Fig. 3: STING signalling drives Sandhoff disease through neuronal death.
Fig. 4: Various lysosome storages mutually induce STING signalling.
Fig. 5: Cytosolic dsDNA accumulation induces cGAS–STING signalling.
Fig. 6: cGAS translocates and is indispensable for LSD pathogenesis.
Fig. 7: Digestion of cytosolic dsDNA in neurons by a DNase cures LSDs.

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Data availability

There are no restrictions on data availability in this article, and all data supporting the findings of this study are provided. All original immunoblots and statistical data are included as source data. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

This research was sponsored by the NSFC Projects (31830052, 32321002 and 31725017 to P.X., and 82230038 to C.S.) and the National Key Research and Development Program of China (2021YFA1301401 to P.X.). We thank the staff at the Life Sciences Institute core facilities of Zhejiang University for technical assistance.

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Author notes

  1. These authors contributed equally: Ailian Wang, Chen Chen.

Authors and Affiliations

  1. MOE Laboratory of Biosystems Homeostasis and Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China

    Ailian Wang, Chen Chen, Chen Mei, Shengduo Liu, Cong Xiang, Wen Fang, Fei Zhang, Yifan Xu, Cunqi Ye, Xin-Hua Feng, Xinran Li & Pinglong Xu

  2. Institute of Intelligent Medicine, ZJU-Hangzhou Global Scientific and Technological Innovation Center, Hangzhou, China

    Ailian Wang, Chen Mei, Shengduo Liu, Xinran Li & Pinglong Xu

  3. Department of Hepatobiliary and Pancreatic Surgery and Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, University School of Medicine, Zhejiang University, Hangzhou, China

    Ailian Wang, Chen Mei, Fei Zhang, Qi Zhang, Xueli Bai, Tingbo Liang & Pinglong Xu

  4. Zhejiang Provincial Key Laboratory for Water Environment and Marine Biological Resources Protection, College of Life and Environmental Science, Wenzhou University, Wenzhou, China

    Shasha Chen

  5. MOE Laboratory of Biosystem Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou, China

    Aifu Lin

  6. Department of Cell Biology, Zhejiang University School of Medicine, Hangzhou, China

    Dante Neculai

  7. Department of Thoracic Cancer, Affiliated Hangzhou Cancer Hospital, School of Medicine, Zhejiang University, Hangzhou, China

    Bing Xia

  8. Eye Center of the Second Affiliated Hospital, Institutes of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China

    Jian Zou

  9. Cancer Center, Zhejiang University, Hangzhou, China

    Tingbo Liang, Xin-Hua Feng & Pinglong Xu

  10. Department of Neurobiology of The First Affiliated Hospital, Institute of Translational Medicine, MOE Frontier Science Center for Brain Research and Brain-Machine Integration, School of Medicine, Zhejiang University, Hangzhou, China

    Chengyong Shen

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Contributions

A.W. and C.C. conducted most of the experiments, C.M., S.L., C.X., W.F., F.Z., Y.X. and S.C. contributed to several experiments, and Q.Z., X.B., A.L., D.N., B.X., C.Y., J.Z., T.L., X.-H.F., X.L. and C.S. helped with data analyses and discussions. P.X. and A.W. conceived the study and designed the experiments. P.X. and A.W. wrote the manuscript.

Corresponding authors

Correspondence to Xinran Li, Chengyong Shen or Pinglong Xu.

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Extended data

Extended Data Fig. 1 STING is constitutively active in neurons of LSD mice.

(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.

Source data

Extended Data Fig. 2 Sting1 ablation alleviates motor dysfunction in LSD mice.

Sex-separate statistics of motor function behaviours (a–d) showed that Hexb-/- female mice were more stable in the phenotype of Sandhoff disease by STING knockout. Open field test (a), n = 6 male mice per group, 3 female mice per group. Rotarod (b) and hindlimb clasping (d), n = 5 male mice in Hexb-/- group and n = 7 male mice in other groups; n = 4 female mice in WT group, n = 5 female mice in Hexb-/- group and n = 6 female mice in Hexb-/-; Sting1-/- group. Grip strength (c), n = 4 male mice in Hexb-/- group and n = 5 male mice in other groups; n = 4 female mice in the WT group and n = 5 female mice in other groups. Error bars in a-d represent mean ± SEM, and P values were calculated by one-way ANOVA with Bonferroni correction.

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Extended Data Fig. 3 STING signalling drives Sandhoff disease via neuronal death.

(a, b) Immunofluorescence imaging detected STING aggregation in primary neurons upon 2-hour treatment of STING agonist DMXAA (a) and their quantification (b). Scale bars, 50 μm (left), 10 μm (right). n = 20 cells per group. (c) Immunoblotting revealed that cGAS-STING signalling was activated in primary mixed glial cells upon HSV-I infection. (d, e) Both immunoblotting (d) and qRT-PCR (e) showed that cGAS-STING signalling was activated in primary astrocytes upon DMXAA treatment (n = 3 independent samples). (f, g) TUNEL staining indicated neuronal death (FITC-positive) upon DMXAA treatment for 12-hour and 24-hour (f) and their quantification (g) Scale bars, 50 μm. n = 8 images per group. (h, i) Levels of c-PARP protein showed that prolonged DMXAA treatment was more likely to induce death of primary neurons (h) than glial cells (i). (j) A schematic model of the generation of transgenic mice carrying caSTING in excitatory neurons was shown. (k) Conditional induction of caSTING (STING R281Q) expression in excitatory neurons profoundly activated STING signalling as indicated by IRF3 phosphorylation and accompanied by neuronal loss. (l, m) Nissl staining of mice brains revealed that expression of caSTING in cre-positive mice resulted in neuronal death in the CA1 regions of the hippocampus (l), and their quantification of the neuron density was shown (m). Scale bars, 20 μm. n = 4 mice per group. Error bars in graphs b-m represent mean ± SEM, and P values were calculated by one-way ANOVA with Bonferroni correction. Results in a–m represent three independent experiments.

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Extended Data Fig. 4 Various lysosome storages mutually induce STING signalling.

(a, b) 24-hour treatment of BafA1 in HeLa cells led to clustering and co-localizing STING with the ERGIC, GM130 (Glogi marker), and LAMP1 (Lysosome marker) (a) and quantification (b). Scale bar, 20 μm. n = 22 cells per group, two-way ANOVA with Bonferroni correction, mean ± SEM. (c) Quantitative real-time RT-PCR indicated that BafA1-induced lysosomal storage promotes IFN-I and ISG mRNA expression, downstream effectors of STING signalling. n = 4 independent samples. (d, e) Immunoblottings and qRT-PCR assays indicated a profound increase of cGAS-STING signalling and ISGs expression in Npc1 KO N2a cells upon treating STING agonist DMXAA. n = 3 independent samples, two-way ANOVA with Bonferroni correction, mean ± SEM. (f) Immunoblottings revealed an enhanced STING signalling upon diABZI treatment without Gla. (g) Increased clustering STING and phosphorylated STING in Gla-/- DRG neurons were detected and quantificated. n = 10 images per group. Unless otherwise specified, error bars in graphs c and g represent mean ± SEM, and P values were calculated by one-way ANOVA with Bonferroni correction. Results in a–g represent three independent experiments.

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Extended Data Fig. 5 Cytosolic dsDNA accumulation induces cGAS-STING signalling.

(a, b) An increase in the accumulation of dsDNA was detected (a, as dsDNA puncta, indicated by yellow arrows) and quantified (b, n = 40 dsDNA puncta pooled from 4 mice in each group) in Gla-/- mice DRGs by immunostaining. Scale bars, 100 μm (left), 50 μm (right). Scale bars, 100 μm. Error bars in graph b represent mean ± SEM, and P values were calculated by one-way ANOVA with Bonferroni correction.

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Extended Data Fig. 6 cGAS translocates and is indispensable for LSD pathogenesis.

(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.

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Extended Data Fig. 7 Digesting cytosolic dsDNA in neurons by DNase cures LSDs.

(a, b) Immunohistochemistry staining of NeuN (a) and its quantification (b) indicated that mTrex1 therapy prevented the neuronal death of 12-week-old mice with Hexb deletion. Scale bars, 1 mm (left), 100 μm (right). n = 20 slices pooled from 4 mice in each group. (c, d) Immunofluorescence imaging displayed that the number of surviving Purkinje cells was significantly increased in Npc1-/- mice with AAV-mTrex1-3x Flag administration, compared to AAV-mCherry administration (c). n = 20 slices pooled from 4 mice in WT + AAV-mCherry and WT + AAV-mTrex1 groups; n = 25 slices pooled from 5 mice in Npc1-/- + AAV-mCherry and Npc1-/- + AAV-mTrex1 groups (d). Scale bars, 100 μm. (e, f) mTrex1 therapy partially rescued symptoms of Niemann-Pick disease, including impairment of motor coordination and balance (e) and hindlimb clasping (f). n = 6 mice in WT + AAV-mCherry and WT + AAV-mTrex1 groups, and n = 5 mice in Npc1-/- + AAV-mCherry and Npc1-/- + AAV-mTrex1 groups. (g) Diagram indicating the experiment schedule of AAV administration, DRGs, and the analyses of footpads skin nerve fibres in WT and Gla-/- mice. (h, i) Immunofluorescence imaging revealed a diminished STING signalling in DRG neurons of Gla-/- mice in response to Trex1 therapy (h), as quantified by relative STING signal strength in individual mCherry+ or mTrex1-3xFlag+ DRG neurons in 6-month-old WT and Gla/- mice (i, n = 40 neurons). Scale bars, 20 μm. (j, k) Immunofluorescence imaging showed that mTrex1 therapy partially rescued the reduction in the number of small fibres from Gla-/- mice footpads (j) and quantified (k), n = 4 mice in WT + AAV-mCherry group, n = 5 mice in other groups. Scale bars, 100 μm. Error bars in graphs b-k represent mean ± SEM and P values were calculated by one-way ANOVA with Bonferroni correction.

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Supplementary information

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Wang, A., Chen, C., Mei, C. et al. Innate immune sensing of lysosomal dysfunction drives multiple lysosomal storage disorders. Nat Cell Biol 26, 219–234 (2024). https://doi.org/10.1038/s41556-023-01339-x

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