Article Figures & Data
Figures
- Figure 1
Single-cell landscape analysis based on single-cell sequencing in mouse middle cerebral artery occlusion/reperfusion (MCAO/R) samples. (A) Behavioural tests were conducted on days 1, 7 and 14 after MCAO/R, and mRNA transcripts from individual cells extracted from the brain were analysed. Single cells were extracted from the brain for analysis using the 10x Genomics technique. (B) t-SNE clustering analysis was performed on the annotated cell types after batch effect removal. (C) The distribution of different cell types in the sham group and samples on days 1, 7 and 14 after MCAO/R was compared. (D) Bar graphs illustrate the proportions of overall cell composition in the sham group and samples on days 1, 7 and 14 after MCAO/R, with cell types colour-coded. (E) t-SNE clustering plots demonstrate the proportions of individual cell types in the sham group and brain tissue samples on days 1, 7 and 14 after MCAO/R.
- Figure 2
Identification of cell-cell interactions in the cellular microenvironment of ischaemia-reperfusion brain tissue. (A) Statistical bar graphs showing the number of cell-cell communications and interaction strength between nine cell subtypes in the sham group and at 1, 7 and 14 days after mouse middle cerebral artery occlusion/reperfusion (MCAO/R). (B) Circos plots depicting the number of cell communications in the sham group and at 1, 7 and 14 days after MCAO/R, with line thickness representing the number of pathways. (C) Circos plots illustrate the number of cell communications between microglia and other cells in the sham group and at 1, 7 and 14 days after MCAO/R, with line thickness representing the number of pathways. (D) Heatmaps displaying significant correlations (L-R pairs) with microglia in the sham group and at 1, 7 and 14 days after MCAO/R.
- Figure 3
Expression of activating transcription factor 3 (Atf3) in ischaemia-reperfusion brain tissue and microglia. (A, B) Enrichment bubble and circos plots showing GO analysis of 86 intersecting genes. (C) t-SNE clustering plot illustrating the expression of Atf3 in cells from sham and mouse middle cerebral artery occlusion/reperfusion (MCAO/R) samples. (D) Violin plots displaying the expression of Atf3 in sham and samples on days 1, 7 and 14 after MCAO/R. (E) qRT-PCR analysis of Atf3 expression in brain tissue at different time points after arterial occlusion/reperfusion, with n=5 for each time point. (F) Western blot analysis (top) and quantification (bottom) of Atf3 expression in mouse brain homogenate at designated time points after MCAO/R injury, with n=5 for each time point. (G, H) Immunofluorescence staining showing the expression of Atf3 (red) in the adult mouse cerebral cortex, hippocampal CA1 region and striatum at 2 hours after MCAO surgery and 3 days of reperfusion, labelled with Iba-1 (green) to mark microglia and cell nuclei (4',6-diamidino-2-phenylindole (DAPI)), scale bar=50 µm. (I) qRT-PCR analysis of Atf3 mRNA expression levels in primary cultured microglia, astrocytes and neurons. (J) Western blot analysis (top) and quantification (bottom) of Atf3 expression in homogenates of primary cultured microglia subjected to oxygen-glucose deprivation (OGD) and reoxygenation, with n=3 experiments. (K) Immunofluorescence staining showing the expression of Atf3 (red) with DAPI (blue, nucleus) labelling, scale bar=25 µm. *p<0.05, cell experiments repeated at least three times.
- Figure 4
Neurotoxic effect of primary microglia-induced co-culture with neurons by activating transcription factor 3 (Atf3) induction. (A) Schematic diagram of primary microglia treated with LV-sh-NC or LV-sh-Atf3 and co-cultured with neurons via the Transwell system during OGD/R. (B) TUNEL staining shows neuronal apoptosis, scale bar=25 µm. (C) Quantification of cell death by measuring LDH release. (D) Assessment of neuronal viability using the CCK-8 assay. (E) Live-cell staining of ROS production in neurons using MitoSOX Red (superoxide) and MitoTracker Green (mitochondria). Merged images show the co-localisation of MitoTracker Green and MitoSOX Red. Scale bar=15 µm. Quantitative analysis was performed using ZEISS Zen lite software, calculating the ratio of average MitoSOX fluorescence intensity to average MitoTracker green fluorescence intensity in 100 cells per group. (F) Detection of cleaved caspase-3, cleaved caspase-9 and cleaved PARP levels in neurons by western blot. *p<0.05, cell experiments repeated at least three times.
- Figure 5
Impact of microglial activating transcription factor 3 (Atf3) knockdown on neuronal behavioural function and oxidative damage after mouse middle cerebral artery occlusion/reperfusion (MCAO/R) injury. (A) Experimental design for microglial-specific knockout of wild-type Atf3, with a stereotaxic injection of AAV2/6 vector encoding CMV-DIO-EGFP-U6-s-hAtf3 (or CMV-DIO-EGFP-U6-sh-NC) into the hippocampal CA1 region, cerebral cortex and striatum of Cx3cr1-Cre mice. (B) Schematic diagram of the experimental procedure. (C) 2,3,5-Triphenyl tetrazolium chloride (TTC) staining of mouse brain slices after MCAO/R, with quantification of infarct volume (d) .(D) Assessment of neurological deficit scores after MCAO/R. (E) Rotarod test and (F) ELISA detection of expression of oxidative damage-related markers ROS, 4-HNE, 8-OHDG, NT-3, PARP1 and 3-NT. n=5. *p<0.05.
- Figure 6
Transcriptional regulation of lon protease homolog 1 (Lonp1) by activating transcription factor 3 (Atf3) . (A) Average escape latency in the Morris water maze test from day 1 to day 6. (B) Latency to reach the hidden platform on day 6. (C) Time (in seconds) spent in the target quadrant during the probe trial on day 7. (D) Number of crossings over the target platform location during the probe trial on day 7. (E) Representative swim tracks on day 7. (F) Novel object recognition (NOR) test, exploration time during the familiarisation phase, and the percentage of time spent exploring the novel object during the test phase. n=5. *p<0.05. (G) Western blot analysis of Lonp1 expression.
- Figure 7
Effects of the activating transcription factor 3 (Atf3)/lon protease homolog 1 (Lonp1) axis on neuronal mitochondrial inner membrane abnormalities and mitochondrial hyperpolarisation. (A) Diagram illustrating the co-culture of primary microglia and neurons for OGD/R experiments. (B) Live-cell staining of neurons with MitoSOX Red (superoxide) and MitoTracker Green (mitochondria). The merged images show the colocalisation of MitoTracker Green and MitoSox Red. Scale bar: 15 µm. Quantitative analysis was performed using ZEISS Zen lite software to determine the ratio of average MitoSOX fluorescence intensity to average MitoTracker green fluorescence intensity in 100 cells per group. (C) Representative histogram of MitoSOX flow cytometry analysis and quantification of MitoSOX-positive cells. (D) Representative image of TMRE staining in neurons. Scale bar: 25 µm. (E) Flow cytometry analysis of mitochondrial membrane potential using JC-1 dye, the aggregated JC-1 was recorded at 590 nm emission in the PE-A channel, and the monomeric JC-1 was recorded at 529 nm emission in the FITC channel. (F) Transmission electron microscopy (TEM) images of mitochondria. The lower image is magnified more, with a scale bar of 1 µm/ 500 nm. Red arrows, compressedOM, compressed crista (CC); yellow arrows, vacuolar membrane structures. IM, inner membrane, crista; m, mitochondrial matrix; OM, outer membrane. Cell experiments were independently repeated three times. *p<0.05.
- Figure 8
Molecular mechanism of transcription factor activating transcription factor 3 (Atf3) -mediated transcriptional activation of lon protease homolog 1 (Lonp1) protein expression to alleviate cerebral ischaemia-reperfusion injury.
Supplementary Materials
Supplementary data
Supplementary data
