Cytoplasmic Escape of Mitochondrial DNA Mediated by Mfn2 Downregulation Promotes Microglial Activation via cGas-Sting Axis in Spinal Cord Injury

Abstract

Neuroinflammation is associated with poor outcomes in patients with spinal cord injury (SCI). Recent studies have demonstrated that stimulator of interferon genes (Sting) plays a key role in inflammatory diseases. However, the role of Sting in SCI remains unclear. In the present study, it is found that increased Sting expression is mainly derived from activated microglia after SCI. Interestingly, knockout of Sting in microglia can improve the recovery of neurological function after SCI. Microglial Sting knockout restrains the polarization of microglia toward the M1 phenotype and alleviates neuronal death. Furthermore, it is found that the downregulation of mitofusin 2 (Mfn2) expression in microglial cells leads to an imbalance in mitochondrial fusion and division, inducing the release of mitochondrial DNA (mtDNA), which mediates the activation of the cGas-Sting signaling pathway and aggravates inflammatory response damage after SCI. A biomimetic microglial nanoparticle strategy to deliver MASM7 (named MSNs-MASM7@MI) is established. In vitro, MSNs-MASM7@MI showed no biological toxicity and effectively delivered MASM7. In vivo, MSNs-MASM7@MI improves nerve function after SCI. The study provides evidence that cGas-Sting signaling senses Mfn2-dependent mtDNA release and that its activation may play a key role in SCI. These findings provide new perspectives and potential therapeutic targets for SCI treatment.

Keywords: biomimetic nanoparticles; mitofusin 2; mtDNA; neuroinflammation; spinal cord injury; stimulator of interferon genes (Sting).

Figure 1

The cGas‐Sting pathway is activated in SCI mice. a) Volcano plot of RNA‐seq data from spine tissue from 8‐week‐old C57BL/6 uninjured and SCI mice. Red and blue dots represent genes with a log2 FC (fold change) of >0.5 and < −0.5, respectively. All other genes are colored gray. Selected genes such as Irf, Cxcl, and Ccl are labeled. b) The GO enrichment analysis revealed hallmark pathways associated with the DEGs, which are upregulated in the control samples compared to the SCI samples. c) KEGG enrichment analysis revealed hallmark pathways associated with the DEGs, which are upregulated in control samples compared to SCI samples. d) IPA prediction of Sting (Sting1, Tmem173) as an upstream regulator of upregulated DEGs identified using an activation z score of >1 and a p‐value overlap of <0.05. e) Dot plot of normalized cell‐type expression of Sting (Sting1, Tmem173) in snRNA‐seq samples (n = 1); OPCs, oligodendrocyte progenitor cells; ODC, oligodendrocytes. f) The GO enrichment analysis revealed hallmark pathways associated with spinal cord microglia clusters (subtypes). g) UMAP plots showing temporal changes of spinal cord microglia clusters (subtypes) and indicating temporal changes of Sting in microglia. h) Dot plot indicating temporal expression changes of Sting (Sting1, Tmem173) in spinal cord microglia clusters (subtypes). i,k) Single‐cell trajectory and pseudo‐time analysis of microglia clusters defined the proliferation advantage cluster and the metabolism advantage one. l) IPA prediction of Sting (Sting1, Tmem173) as an upstream regulator of upregulated DEGs identified using an activation z score of >1 and a p‐value overlap of < 0.05.

Figure 2

Activation of cGas‐Sting signal played a key role in the secondary SCI. a) Western blots analysis of cGas and Sting levels in the perilesional tissues at 1, 7, and 14 d after SCI. b) Sting /Iba1 double immunostaining in the perilesional tissues 7 d after SCI (Scale bar: 50 µm). c) Western blot analysis of cGas, Sting, Irf3, p‐Irf3, P65, and p‐P65 protein expression in the perilesional tissues 7 d after SCI. d) Diagram for construction of microglia‐specific Sting knockout mice. e) Effects of Sting knockout on neurological function scores at 1 d, 7 d, and 14 d after SCI. f) Representative confocal images of M1 state (iNOS⁺/Iba1⁺) were obtained from the perilesional tissues 7 d after SCI. g) Nissl staining in the perilesional tissues 7 d after SCI (Scale bar = 20 µm). h) Levels of pro‐inflammatory cytokines, including IL‐1β, IFN‐β, and TNF‐α in the perilesional tissues 7 d after SCI. n = 6 for each group. Error bars denote mean ± SEM, ns, no significance, ^*** p < 0.001 versus sham group in each strain of mice, # p < 0.05 versus Cx3cr1‐Cre ERT2; Stingfl/fl group.

Figure 3

cGas‐Sting signaling pathway was activated in LPS‐induced microglia. a) Western blot and quantitative analysis of cGas and Sting in LPS‐induced microglia of 0, 1, 5, and 10 µg mL⁻¹ after 24 h. b) Western blot analysis of cGas, Sting, Irf3, p‐Irf3, P65, and p‐P65 protein expression in LPS (5 µg mL⁻¹)‐induced microglia. c) Representative of immunofluorescence staining of Sting in microglia (Scale bar: 20 µm). d) Western blot and quantitative analysis of cGas, Sting, Irf3, p‐Irf3, P65, and p‐P65 in microglia of Control, LPS, LPS + vehicle, and LPS + C‐176. e) Representative immunostained images of M1 state (iNOS) microglia. f) Levels of pro‐inflammatory cytokines, including IL‐1β, IFN‐β, and TNF‐α in the microglia medium. g) Flow cytometric analysis on the expression levels of M1 microglia ratio (F4/80/ CD86+). n = 6 for each group. Error bars denote mean ± SEM, ns, no significance, *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4

Sting signaling activation in LPS‐induced microglia enhanced neuron death. a) Western blot analysis of cleaved‐Parp expression in the perilesional tissues at 7 d after SCI. b) NeuN/TUNEL double immunostaining in the perilesional tissues 7 d after SCI (Scale bar: 50 µm). c) The protocol of in vitro experiments for detecting neurons (CATH.a) death regulated by Sting activation in primary microglia (by Figdraw). Primary microglia treated with PBS or LPS (5 µg mL⁻¹) for 24 h. After removal of the supernatant, cells were cultured with DMEM culture medium for 24 h, and then the supernatant was collected as neurons conditioned medium for 24 h. d) Western blot analysis of cleaved‐Parp expression in neurons in each group. e) The neuron death rate was measured by Annexin V and PI staining. n = 6 for each group. Error bars denote mean ± SEM, ns, no significance, ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

Figure 5

Mitochondria swelling and expression of mitofusin decreased after SCI. a) Representative ultrastructure of microglia in the perilesional tissues at 7 d after SCI (Scale bar: 1 µm (a1,a2) and 0.5 µm (a3,a4)). b) Western blot analysis of the Drp1, Mfn1, and Mfn2 levels in microglia of Control and LPS. c) Western blot analysis of cGas, Sting, Drp1, and Mfn2 levels in microglia of Control and LPS, LPS+MASM7, and LPS+Mdivi‐1. d) Representative of immunofluorescence staining of M1 state (iNOS) in microglia (Scale bar: 20 µm). e) Levels of pro‐inflammatory cytokines, including IL‐1β, IFN‐β, and TNF‐α in the medium of microglia. f) Flow cytometric analysis on the expression levels of M1 ratio (F4/80/ CD86+). g) The neuron death rate measured by Annexin V and PI staining. n = 6 for each group. h) Mfn2 /Iba1 double immunostaining in the perilesional tissues 7 d after SCI (Scale bar: 50 µm). Error bars denote mean ± SEM, ns, no significance, ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

Figure 6

Mitochondrial fission‐induced mtDNA release mediates activation of Sting signaling in LPS‐induced microglial. a) Representative of immunofluorescence staining of Mfn2 in microglia in different groups (Scale bar = 20 µm). b) Western blot and quantitative analysis of Mfn2, cGas, Sting, Irf3, p‐Irf3, P65 and p‐P65 in microglia of Control, LPS, LPS + vehicle, and LPS + MASM7. c) dsDNA and HSP60 double immunostaining microglia in different groups (Scale bar: 20 µm). d) Representative MitoTracker fluorescence images illustrating mitochondrial morphology in microglia (Scale bar: 20 µm). e) Representative fluorescence staining of JC‐1 aggregates (red)/JC‐1 monomers (green) illustrating the MMP (Scale bar: 20 µm). f) Representative MitoSOX fluorescence images of mitochondria‐derived ROS (Scale bar: 20 µm). Error bars denote mean ± SEM, ns, no significance, ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

Scheme 1

Schematic process of the activation of cGas‐Sting signaling mediated by Mfn2‐dependent release of mtDNA in microglia in SCI.

Figure 7

Nanomaterials carrying MASM7 promoted neuroprotection and functional recovery after SCI. a) The diameter distribution of MSNs, MSNs‐MASM7@AS, MSNs‐MASM7@NE, or MSNs‐MASM7@MI. b) The zeta potential distribution of MSNs, MSNs‐MASM7@AS, MSNs‐MASM7@NE, or MSNs‐MASM7@MI. c) Flow cytometric analysis of nanomaterials (coated by microglia membranes, neuron, and astrocyte membranes) uptake by primary microglial. d) TEM images of the MSNs and MSNs‐MASM7@MI (Scale bar = 50 nm). e) Fluorescence images of cellular morphology (F‐actin) in primary microglial, neuron, and astrocyte cells after 24 h (Scale bar = 20 µm). f) Fluorescence images of MSNs‐MASM7@MI and mitochondria (HSP60) in primary microglial after 24 h (Scale bar = 20 µm). g) Effects of different treatments on neurological function scores at 1, 7, and 14 d after SCI. h) Nissl staining in the perilesional tissues 7 d after SCI (Scale bar = 20 µm).