Fig 2: Clec7a promotes microglia‐mediated synaptic phagocytosis after ischemic stroke. A) Schematic of the experimental design. B) PCR genotyping showed that Clec7ai∆MG (lanes 1, 3, 4, and 6) and WT littermate control (lane 7) mice were successfully generated by crossing Cx3cr1CreERT2 transgenic mice with Clec7afl/fl mice. Heterozygotes (Ht) that contained both Clec7a WT and Clec7aflox bands are shown. C) Representative photographs of TTC staining (left) after tMCAO and quantification of the relative infarct volume (right). n = 7 mice per group. D) T2 images at 24 h after the onset of ischemia in both groups. n = 8 mice per group. E) DW images of both groups at 24 h after ischemic stroke. n = 8 mice per group. F) CD68+ lysosome content (green) in Iba‐1+ microglia (red) in the ischemic penumbra from Clec7ai∆MG and Clec7afl/fl mice subjected to tMCAO. Scale bars, 5 µm. G) Quantification of the lysosomal area relative to the microglial area. H) Representative single‐plane images of Syn and PSD95 staining in the ischemic penumbra. Dotted circles highlight colocalized synapses. Scale bars, 5 µm. I) Quantification of excitatory synapses. J–M) Confocal images and quantification of synaptic puncta engulfed by microglia showing the colocalization of PSD95 (J, K) or Syn (L, M) with CD68 within microglia in the ischemic penumbra from sham and tMCAO mice. The white arrows indicate PSD95+CD68+ (J) or Syn+CD68+ (L) puncta within microglia. Scale bars, 10 µm. Statistics were derived from 18 slices, n = 6 mice per group. Mice aged 8–10 weeks were used for the experiments shown in this figure. Significance was calculated using either two‐tailed unpaired Student's t‐test (C–E, G, and I) or one‐way ANOVA, Tukey's multiple comparisons test (K and M). Data are presented as mean ± SD. ***p < 0.001 versus Sham; ## p < 0.01 and ### p < 0.001 versus tMCAO.

Fig 3: Microglial Clec7a knockout restores neuronal activity and synaptic transmission after ischemic stroke. A) Representative traces of the action potentials recorded from ischemic penumbra neurons in +180 pA current injection of Clec7afl/fl and Clec7ai∆MG mice subjected to tMCAO and sham operation. Scale bar, 20 mV, 50 ms. B) Statistics showed that as the injection current increased (from 0 to +360 pA), the number of spikes was significantly increased in tMCAO Clec7afl/fl mice compared to tMCAO Clec7ai∆MG and sham mice (Clec7afl/fl group, n = 16 neurons in 3 mice, Clec7ai∆MG group, n = 16 neurons in 3 mice; Clec7afl/fl + tMCAO group, n = 22 neurons in 5 mice, Clec7ai∆MG + tMCAO group, n = 24 neurons in 6 mice). C) Representative PPR traces of Clec7afl/fl and Clec7ai∆MG mice subjected to tMCAO and sham operation after two consecutive stimuli. Scale bar, 100pA, 10 ms. D) Statistical analysis showed that the ratios of tMCAO Clec7afl/fl mice was increased compared to tMCAO Clec7ai∆MG and sham mice (Clec7afl/fl group, n = 10 neurons in 3 mice, Clec7ai∆MG group, n = 12 neurons in 3 mice; Clec7afl/fl + tMCAO group, n = 12 neurons in 5 mice, Clec7ai∆MG + tMCAO group, n = 16 neurons in 6 mice). Statistical significance was determined by two‐way ANOVA (B) and one‐way ANOVA (D) followed by Tukey's multiple comparisons test. Data are presented as mean ± SD. *p < 0.05 and ***p < 0.001 versus Sham; ### p < 0.001 versus tMCAO.

Fig 4: Clec7a interacts with MD2 in the ischemic brain. A) Immunoprecipitates were subjected to SDS‐PAGE and silver staining. B) Annotated LC–MS/MS spectra of 150‐LGAHLLKIDNSKEFEFIESQTSSHR‐174 from Clec7a and 85‐IELPKRKEVLCHGHDDDYSFCRALK‐109 from MD2 in anti‐Clec7a immunoprecipitates from tMCAO mice. C) Normalized binding curve of Clec7a and MD2. The binding curve yielded a Kd of 2.96 ± 1.11 µm. The concentration of Clec7a was kept constant at 50 nm while the MD2 concentration was varied from 10 µM to 1.2 nM. n = 3 per group. D) Endogenous Clec7a was coimmunoprecipitated with MD2 after ischemic stroke. The experiments were repeated independently three times. E) The interaction between Clec7a (cyan) and MD2 (pink). The yellow dashed line indicates hydrogen bonding. F) Representative images and analysis of the ischemic penumbra showing the colocalization of MD2 with PSD95, as indicated by the white arrowheads; scale bars, 5 µm. G) Representative images and analysis of the ischemic penumbra showing the colocalization of MD2 with Syn as indicated by the white arrowheads; scale bars, 5 µm. H) Schematic illustrating the in vitro phagocytosis assay in which primary microglia isolated from Clec7ai∆MG or Clec7afl/fl mice phagocytosed MD2ko/ko or MD2wt/wt synaptosomes tagged with pHrodo. I) Representative images of cultured Clec7ai∆MG or Clec7afl/fl microglia engulfing pHrodo‐conjugated synaptosomes (top: engulfed pHrodo; bottom: merged image of Iba‐1 and pHrodo). Scale bars, 20 µm. Statistics were derived from 18 slices, n = 6 per group. Mice aged 8–10 weeks were used for the experiments shown in this figure. Significance was calculated using either two‐tailed unpaired Student's t‐test (F and G) or one‐way ANOVA, Tukey's multiple comparisons test (I). Data are presented as mean ± SD. ***p < 0.001.

Fig 5: Transcriptome sequencing reveals microglial responses to ischemic stroke. A) Schematic of RNA‐seq analysis of microglia isolated from the ischemic brain 7 days after tMCAO and from sham mice, n = 3 per group, each n consists of 30 mice. B) Principal component analysis (PCA) plot. The two groups of samples were separated when plotting the first (PC1) versus the second component (PC2). C) Scatterplot showing the comparison of log2CPM (counts per million) in sham microglia versus tMCAO microglia; red dots represent DAM genes. The orange dashed line indicates a fold change of 1. D) GO analysis of DEGs identified key biological processes in tMCAO microglia compared with sham microglia, which are graphically displayed in bubble plots according to Z scores and significance (log10[adjusted p value]). E–H) Heatmap showing genes related to neuronal death (E), immune response (F), homeostatic state (G) and DAM state (H) in tMCAO microglia versus sham microglia. I) qRT‒PCR analysis showing the expression of phagocytosis‐related genes in isolated microglia from the ischemic penumbra of tMCAO and sham mice, n = 6 per group, each n consists of 3 mice. J) Protein‒protein interaction (PPI) network for microglial phagocytosis‐related genes. K,L). Representative single‐plane images (K) and quantitative analyses (L) of Clec7a (red) in Iba‐1+ microglia (green) in the ischemic penumbra from sham and tMCAO mice. Scale bars, 100 µm. Mice aged 8–10 weeks were used for the experiments shown in this figure. Statistics were derived from 12 slices, n = 4 mice per group. In I and L significance was calculated using two‐tailed unpaired Student's t‐test. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001.