Transcriptomic and functional studies reveal undermined chemotactic and angiostimulatory properties of aged microglia during stroke recovery

Abstract

Age-dependent alterations in microglia behavior have been implicated in neurodegeneration and CNS injuries. Here, we compared the transcriptional profiles of young versus aged microglia during stroke recovery. CD45^intermediateCD11b⁺ microglia were FACS-isolated from the brains of young (10-week-old) and aged (18-month-old) male mice with sham operation or 14 days after distal middle cerebral artery occlusion and subjected to RNA-sequencing analysis. Functional groups enriched in young microglia are indicative of upregulation in cell movement, cell interactions, inflammatory responses and angiogenesis, while aged microglia exhibited a reduction or no change in these features. We confirmed reduced chemoattractive capacities of aged microglia toward ischemic brain tissue in organotypic slide co-cultures, and delayed accumulation of aged microglia around dead neurons injected into the striatum in vivo. In addition, aging is associated with an overall failure to increase the expression of microglial genes involved in cell-cell interactions, such as CXCL10. Finally, impaired upregulation of pro-angiogenic genes in aged microglia was associated with a decline in neovascularization in aged mice compared to young mice after distal middle cerebral artery occlusion. This study provides a new resource to understand the mechanisms underlying microglial alterations in the aged brain milieu and sheds light on new strategies to improve microglial functions in aged stroke victims.

Keywords: Microglia; aging; angiogenesis; cell movement; transcriptome.

Figure 1.

Isolation of microglia for RNAseq analysis. Young (10 weeks old) or aged (18 months old) mice were subjected to distal middle cerebral artery occlusion (dMCAO) or sham operation. Microglia were purified from sham brains and brains 14 days after dMCAO using FACS. (a) Schematic experimental design to compare gene profiles between young and aged microglia in ischemic brains. (b) Gene expression of cell-specific markers in purified microglia. Specific markers for microglia, neurons, astrocytes, oligodendrocytes (Oligo), endothelial cells (Endo) and peripheral immune cells (Immune) were compared. Data are expressed as log₂(FPKM + 1). (c–d) Volcano plot of gene expression profiles in microglia collected after dMCAO or after sham operation in young (c) and aged (d) group, showing distribution of significance [−log₁₀(adjusted Pvalue)] vs. fold change [log₂(fold change)] for all genes. The green dots indicate genes down regulated (fold change <−2, adjusted Pval <0.1), the red dots indicate genes upregulated (fold change >2, adjusted Pval <0.1), and the gray dots indicate genes with no significant change after dMCAO. Horizontal dashed lines indicate adjusted p-value = 0.1. Vertical dashed lines indicate expression fold change equal to −2 and 2, respectively.

Figure 2.

Gene ontology enrichment analysis of DEGs (dMCAO vs. sham) identifies key biological processes in young and aged stroke mice. (a–b) Heatmap plot of DEGs between dMCAO and sham microglia in young and aged groups (left). The bubble plots illustrate the highest differentially regulated biological processes, graphically displayed according to Z scores and significance [−log₁₀(adjusted Pvalue)]. Each circle indicates a term. Larger size indicates more DEGs involved in that term. Reduction of the bubble allowed better visualization (see methods). (c–d) The enrichment functional analysis was performed in IPA for young (c) and aged (d) groups. Top enriched functions were clustered into five groups: cell movement, cell to cell signaling and interaction, cardiovascular development and function, inflammatory response, and cell death/survival/function. The bars indicate z-score calculated in IPA, with a z-score >2 suggests activation and a z-score <−2 suggests inhibition. The black dots indicate statistical significance [–log₁₀(P value)] for each term.

Figure 3.

Microglia demonstrate age-dependent transcriptional profiles and functional alterations 14 days after stroke. (a) Scatterplot shows comparison of −log₁₀(adjusted Pval) for DEGs in young and aged microglia at 14 days after stroke. The DEG distributions within each subgroup (1–8) are listed in a table on the right. Quadrants 1, 3, 5, and 7 (with light grey background) indicate DEGs in both young and aged microglia. Quadrants 2, 4, 6, and 8 with light blue background indicate genes significantly changed in either young or aged group. (b) Metascape enrichment analysis for genes in second (a), fourth (b) and eighth (c) quadrants. Each node indicates a term. Clustering was made based on similarity (similarity > 0.3). (c) Function enrichment analysis performed by IPA identifies differences in intrinsic functions (left) and regulatory functions (right) between young and aged microglia. Functional terms were sorted according to z-score (high to low) in the young microglia.

Figure 4.

Age-dependent changes in microglia migration after dMCAO. (a) Scatterplot shows comparison of log₂(fold change) for DEGs in young and aged microglia at 14 days after stroke. Green dots are DEGs that are selectively upregulated in young mice. Purple dots are DEGs upregulated distinctively in aged mice. Grey dots are DEGs downregulated in aged mice. Spp1 (blue dot) was upregulated in both young and aged mice. The horizontal and vertical dashed lines indicate expression fold change equal to 2. (b) Schematic illustration showing cellular distribution of some migration related DEGs in young microglia and their functions in cell migration. (c) Representative images showing migration of Iba1⁺GFP⁻ microglia out of young and aged brain slices cultured alone or together with CX3CR1-GFP ischemic brain at 11 DIV. Scale bar, 100 μm. (d) Quantification of cell migration distance for Iba1⁺GFP⁻ microglia. Totally 372 cells from 14 young brain slices and 178 cells from 10 aged brain slices were analyzed. Aged microglia migrated much shorter distances compared to young microglia. ***p < 0.001 Student’s t test. (e) The distribution of Iba1⁺GFP⁻ cells was quantified as the percentage of cells located within 400 µm, 400–800 µm, and beyond 800 µm from their original young or aged brain slices. ***p < 0.001 *p < 0.05, two-way ANOVA followed by Bonferroni post hoc. (f) Representative images showing migration of Iba1⁺ microglia (green) towards injected dead neurons in young and aged mouse brains. Red line indicates needle track (injection channel). White dashed lines indicate 50 µm and 100 µm distance marks from the injection site. Scale bar, 50 μm. (g) Quantification of Iba1 staining intensity within 50 µm, 50–100 µm, and 100–200 µm from the injection track. **p < 0.01 one-way ANOVA followed by Bonferroni post hoc. There are significant differences between dead neuron injection groups and PBS injection groups at both time points and in all areas.

Figure 5.

Aged microglia show reduced capacities for cell–cell interaction and regulation compared to young counterparts 14 days after stroke. (a) Circle plot shows eight cell-cell interaction related GO terms in young (left) and aged (middle) group. The outer circle shows a scatterplot for each term with log₂(fold change) of assigned genes. Red dots indicate upregulated genes, and blue dots indicate downregulated genes. The color of the inner circle displays the z-score, with the area of trapeziform indicating the p value of each term. The table shows the description of each term. (b) IPA analysis identified chemotaxis-related functions enriched in young microglia. The color of the outer circle nodes indicates the expression of assigned genes. The color of the inner circle nodes indicates the z-scores of enriched functions. The color of segments between gene nodes or function nodes implies gene activation or inhibition. Red indicates upregulation and green indicates downregulation. (c) Genes related to recruitment of T lymphocyte and monocytes were upregulated in young microglia 14 days after stroke. (d) Immunostaining of CXCL10 (red) and Iba1 (green). The left panels (bar = 20 µm) show the staining in young and aged sham. The middle and right panels (bar = 10 µm) show the staining in young and aged mice, respectively, 14 days after stroke. (e) Quantification of the number of CXCL10⁺Iba1⁺ microglia in young and aged mice 14 days after stroke. n = 5 for young and n = 3 for aged. *p < 0.05, Student’s t test. (f) Quantification of the mean intensity of CXCL10 signal in Iba1⁺ microglia. Violin plots are shown, with n = 71 cells for young and n = 47 cells for aged. ***p < 0.001. Mann-Whitney U test.

Figure 6.

Senescent microglia are associated with impaired angiogenesis in the aged brain after stroke. (a,b) DEGs related to angiogenesis were explored in both young (a) and aged (b) groups, respectively, in IPA. The genes were indicated by nodes and scatter plotted in different parts of cells. The color of the nodes indicates the expression levels of genes, and the dashed line indicates the predicated activation or inhibition of genes. (c) IPA analysis in young microglia showed that Vegf, Igf1 and Spp1 were predicted as upstream regulators of genes involved in angiogenesis. (d) BrdU (red) and CD31 (green) double staining was performed in both young and aged group 14 days after dMCAO. (e) BrdU⁺CD31⁺ cells were quantified in peri-infarct areas in both cortex and striatum.