Microglial CMPK2 promotes neuroinflammation and brain injury after ischemic stroke

Abstract

Neuroinflammation plays a significant role in ischemic injury, which can be promoted by oxidized mitochondrial DNA (Ox-mtDNA). Cytidine/uridine monophosphate kinase 2 (CMPK2) regulates mtDNA replication, but its role in neuroinflammation and ischemic injury remains unknown. Here, we report that CMPK2 expression is upregulated in monocytes/macrophages and microglia post-stroke in humans and mice, respectively. Microglia/macrophage CMPK2 knockdown using the Cre recombination-dependent adeno-associated virus suppresses the inflammatory responses in the brain, reduces infarcts, and improves neurological outcomes in ischemic CX₃CR1^Cre/ERT2 mice. Mechanistically, CMPK2 knockdown limits newly synthesized mtDNA and Ox-mtDNA formation and subsequently blocks NLRP3 inflammasome activation in microglia/macrophages. Nordihydroguaiaretic acid (NDGA), as a CMPK2 inhibitor, is discovered to reduce neuroinflammation and ischemic injury in mice and prevent the inflammatory responses in primary human monocytes from ischemic patients. Thus, these findings identify CMPK2 as a promising therapeutic target for ischemic stroke and other brain disorders associated with neuroinflammation.

Keywords: CMPK2; NLRP3 inflammasome; Ox-mtDNA; human PBMC cells; microglia; stroke.

Graphical abstract

Figure 1

Peripheral blood expression of CMPK2 is elevated and positively correlative with infarct volume in human ischemic subjects (A) Quantification for CMPK2 gene expression (control = 21, stroke = 31). (B) Representative magnetic resonance imaging (MRI) images (diffusion-weighted imaging) of patients with stroke with CMPK2 gene expression (fold change < 10 and fold change > 10). (C) The correlation analysis was performed between CMPK2 transcript levels in blood and infarct volume in patients with stroke. n = 20. (D) The correlation analysis was performed between CMPK2 transcript levels in blood and NIHSS score in patients with stroke. n = 20. (E and F) Representative immunofluorescence images and quantification of 8-OHdG expression levels in the peripheral blood of ischemic patients. Scale bar: 20 μm. n = 6. (G and H) Representative immunofluorescence images and quantification of CMPK2 expression levels in the peripheral blood of patients with acute ischemic stroke. Scale bar: 20 μm. n = 6. Results are represented as means ± SD. ∗∗∗p < 0.001 vs. non-stroke control subjects.

Figure 2

CMPK2 expression is mainly upregulated in microglia/macrophages of the brain after ischemia onset (A and B) Western blotting images and quantification for CMPK2 protein expression in the peri-infarct region of sham-operated and ischemic mice at 3 h, 12 h, 1 day, 3 days, and 7 days following operation. n = 6. (C and D) Representative immunofluorescence images and quantitative analysis of CMPK2⁺ Iba1⁺ cells in the peri-infarct region of the ischemic mice. Scale bar: 50 μm. n = 6. (E and F) Western blotting images and quantification for CMPK2 protein expression in microglia/macrophages isolated from the peri-infarct region with CD11b⁺ magnetic beads at 24 h following operation. n = 6. (G and H) Western blotting images and quantification for CMPK2 expression in primary mouse microglia exposed to OGD conditions for 0, 2, 4, 6, 9, or 12 h. n = 4. (I and J) Western blotting images and quantification for CMPK2 expression in primary mouse microglia, astrocytes, or cortical neurons exposed to OGD conditions for 0 and 6 h. n = 6. Results are represented as means ± SD. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. sham or 0 h group.

Figure 3

Microglia/macrophage CMPK2 silencing reduces brain ischemic volume and improves short-term neurological function in the mouse tMCAO model (A) Schematic diagram about experimental strategy for CMPK2 knockdown. (B) Representative immunofluorescence images of mCherry⁺ cells stained with Iba1. Scale bar: 50 μm. n = 4. (C) Representative immunofluorescence images of mCherry⁺ cells stained with Iba1 and CMPK2. Scale bar: 20 μm. n = 6. (D) Western blotting images for CMPK2 protein expression in microglia/macrophages isolated from the brain of AAV-shCon and AAV-shCMPK2 CX₃CR1^Cre/ERT2 mice with CD11b⁺ magnetic beads. n = 6. (E and F) Regional cerebral blood flow. Results are expressed as means ± SD. n = 4. (G and H) AAV-shCMPK2 administration reduced cerebral infarct volume and promoted neurological functions in CX₃CR1^Cre/ERT2 male mice. n = 12. (I and J) AAV-shCMPK2 administration toned down cerebral infarct volume and improved neurological function in the CX₃CR1^Cre/ERT2 female mice. n = 8. Results are represented as means ± SD. ∗∗∗p < 0.001 vs. sham plus AAV-shCon group and ^###p < 0.001 vs. tMCAO plus AAV-shCon group.

Figure 4

Microglia/macrophage CMPK2 silencing reduces brain infarction and improves long-term functional performances in the mouse tMCAO model (A) Schematic diagram about experimental procedures. (B and C) Representative T2-weighted MRI images of CX₃CR1^Cre/ERT2 mice on day 14 post-tMCAO. n = 4 or 5. (D) The long-term functional performances were investigated in CX₃CR1^Cre/ERT2 mice. n = 8. (E) The survival proportions. n = 12–15. Results are represented as means ± SD. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs. sham plus AAV-shCon group and ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 vs. tMCAO plus AAV-shCon group.

Figure 5

Microglia/macrophage CMPK2 silencing promotes microglial ramification and disrupts Ox-mtDNA-induced NLRP3 inflammasome activation in the brain of ischemic mice (A) Imaris-based 3D reconstruction images of microglia immunofluorescently stained with Iba1. Scale bar: 15 μm. (B) The process length and the number of branches of microglia stained with Iba1 in AAV-shCon and AAV-shCMPK2 CX₃CR1^Cre/ERT2 mice at 3 days after ischemia. n = 10. Results are represented as means ± SD. (C–F) Representative immunofluorescence images and quantification of microglia/macrophages stained with 8-OHdG and quantitative analysis of 8-OHdG⁺ Iba1⁺ cells in the peri-infarct region of CX₃CR1^Cre/ERT2 mice at 24 and 72 h after ischemia. Scale bar: 50 μm. n = 6. (G) Western blotting images and quantification for cleaved caspase-1, mature IL-1β, and N-GSDMD in the peri-infarct region of AAV-shCon and AAV-shCMPK2 CX₃CR1^Cre/ERT2 mice at 3 days following ischemic stroke. n = 6. Results are represented as means ± SD. ∗∗∗p < 0.001 vs. sham plus AAV-shCon group and ^###p < 0.001 vs. tMCAO plus AAV-shCon group.

Figure 6

The CMPK2-specific inhibitor NDGA suppresses Ox-mtDNA formation and NLRP3 inflammasome activation in microglia in vitro (A) The molecule structure of nordihydroguaiaretic acid (NDGA) and quantification of CMPK2 kinase assay with indicated concentrations of NDGA. n = 3. (B) The real-time binding kinetics of NDGA to immobilized CMPK2 protein were examined by SPR assay with the K_D value of 0.6 μM. (C) NDGA reduced the protein expression of cleaved caspase-1, N-GSDMD, and mature IL-1β in the cell supernatant of LPS-primed microglia cells followed by ATP stimulation. (D and E) NDGA prevented LDH release in microglia subjected to LPS plus ATP stimulation or OGD model. n = 4. Results are represented as means ± SD. ∗∗∗p < 0.001 vs. control (CN) group and ^##p < 0.01 and ^###p < 0.001 vs. LPS plus ATP or OGD group. (F) NDGA decreased the Cox-1, CYTB, and D-loop expression in primary mouse microglia cells. n = 3. Results are represented as means ± SD. ∗∗∗p < 0.001 vs. CN group and ^##p < 0.01 and ^###p < 0.001 vs. LPS group. (G) NDGA reduced the newly synthesized DNA, as visualized by EdU labeling in primary mouse microglia cells. Scale bar: 5 μm. n = 6. (H) NDGA reduced 8-OHdG expression in LPS-primed microglia followed by ATP stimulation. Scale bar: 20 μm. n = 4.

Figure 7

The CMPK2 inhibitor NDGA reduces brain ischemic volume and improves neurological performances in the mouse tMCAO model (A–C) NDGA reduced cerebral infarct volume and promoted short-term neurological functions in the male mice. n = 8. (D) NDGA improved long-term functional performances, including mNSS test, wire-hanging test, forelimb foot fault test, and rotarod test in the male mice. n = 8. (E) NDGA improved long-term survival proportions in the male mice. n = 12–15. Results are represented as means ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 vs. sham group and ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 vs. tMCAO plus vehicle group.