Cx3cr1-deficient microglia exhibit a premature aging transcriptome

Microglia were isolated from 2-mo-old Cx3cr1^+/+ (WT) or Cx3cr1^eGFP/eGFP (KO) mice, pooling brains from three gender-matched mice to obtain samples for chromatin immunoprecipitation followed by sequencing. (A) ChIP-seq for Rbp2, a mark of actively transcribed genes, indicates few significant differences between the genotypes. Only genes with at least four sequencing reads in at least one of the genotypes are shown, with genes reaching statistical significance highlighted in blue. (B) There is minimal correlation between mRNA expression (fold change determined by RNA-seq) and active transcription (Rbp2 binding determined by ChIP-seq). Genes with fold change > |1.5| are highlighted in coral. (C) ChIP-seq for H3K27Ac, a mark of open and active chromatin, indicates few significant differences between the genotypes. Only genes with at least four sequencing reads in at least one of the genotypes are shown, with genes reaching statistical significance highlighted in blue and coral. (D) The UCSC browser snapshots of Rbp2 binding for selected genes. Exons are represented by boxes, and direction of transcription is indicated by arrows in introns.

2-mo-old Cx3cr1^+/+ (WT, n = 7), Cx3cr1^+/eGFP (Het, n = 6), or Cx3cr1^eGFP/eGFP (KO, n = 6) mice were injected with 1 mg/kg LPS i.p. (or saline control, four WT and three KO animals only). Twenty-four hours later, peritoneal cells were collected, and microglia were sorted from isolated brains. Both cell populations were used for RNA-seq. (A) PCA of all samples. Each dot within a cell type represents an individual animal, but there are matched microglia and peritoneal cells from the same animal. Samples, coded based on the cell type (open or solid fill), genotype (color), and treatment (symbol), separate by cell type and treatment. (B) The PCA of microglia only shows separation by treatment, but not genotype in LPS-injected mice. Samples are coded by gender (open or solid fill), genotype (color), and treatment (symbol). (C) Expression of selected genes that are modulated by Cx3cr1 genotype after LPS injection.

Microglia were isolated from 2-mo-, 1-yr-, or 2-yr-old Cx3cr1^+/+ (WT), Cx3cr1^+/eGFP (Het), or Cx3cr1^eGFP/eGFP (KO) mice. (A) Fewer microglia were recovered from 2 yr mice, but the recovery was independent of genotype. (B, C) RNA-seq was performed on all samples together. Each symbol represents microglia from an independent animal. (B) The PCA for all samples suggests separation by age as the primary factor driving variability, with genotype second and gender third (B). (C) For microglia from 2-yr-old mice (C), the samples separate by genotype and by gender within each genotype. (D) Overlap of DEGs between 2-yr and 2-mo microglia in WT and in KO mice. There is a large amount of shared DEGs between the genotypes.

Microglia were isolated from 2-mo-, 1-yr-, or 2-yr-old Cx3cr1^+/+ (WT), Cx3cr1^+/eGFP (Het), or Cx3cr1^eGFP/eGFP (KO) mice and used for RNA-seq analysis, followed by quantification and unsupervised hierarchical clustering for the top 100 DEGs (KO versus WT, by P-value) at each time point, resulting in a combined list of 254 genes. In 2-mo-old mice, the samples separate by genotype, whereas in older mice, they first separate by age and then genotype. Overall, 2-mo-old Cx3cr1-Het and KO microglia resemble aged mice of any genotype. Some genes increase with both age and Cx3cr1 deletion and show the highest expression in aged Cx3cr1-KO microglia.

Quantification and unsupervised hierarchical clustering for microglia from 2-yr-old Cx3cr1^+/+ (WT, n = 9), Cx3cr1^+/eGFP (Het, n = 8), or Cx3cr1^eGFP/eGFP (KO, n = 11) mice highlights a strong effect of gender on gene expression in old mice. The samples primarily separate by gender and not Cx3cr1 genotype. Selected genes and associated pathways (manual annotation) are represented in different colors.

(A, B) Representative images of brain sections from the cortex (A) or cerebellum (B) of young (2 mo) or old (18–24 mo) Cx3cr1^+/+ or Cx3cr1^eGFP/eGFP male or female mice immunoreacted with anti-Iba1 (purple) and anti-Pu.1 (brown). Note the overall different microglial morphology in the cerebellum in both young and old mice compared with the cortex. Many microglial clusters were detectable in the cerebellum of old animals of either genotype. Scale bar: large image, 200 μm; inset, 50 μm. (C, D) Quantification of microglial cell numbers and morphology in the cortex (C) and the cerebellum (D). Microglial cell numbers were quantified as the number of Pu.1-positive nuclei per unit area. Microglial morphology was assessed as the total Iba1 immunoreactive area per Pu.1⁺ nucleus, dendritic process area per Pu.1⁺ nucleus, and dendritic process perimeter per Pu.1⁺ nucleus. Only processes attached to nuclei were included in the dendritic process calculations. Statistics: two-way ANOVA (for genotype and age) and Tukey’s post hoc test. Only select significant intergroup comparisons are shown. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Ctx cell number: genotype: F(1,32) = 4.892, P = 0.0342; age: F(1,32) = 0.0001061, P = 0.9918; interaction: F(1,32) = 0.61, P = 0.4405. Ctx Iba1 area: genotype: F(1,32) = 8.461, P = 0.0065; age: F(1,32) = 3.225, P = 0.0820; interaction: F(1,32) = 2.182, P = 0.1494. Ctx process area: genotype: F(1,32) = 9.908, P = 0.0035; age: F(1,32) = 5.665, P = 0.0234; interaction: F(1,32) = 3.165, P = 0.0847. Ctx process perimeter: genotype: F(1,32) = 11.31, P = 0.0020; age: F(1,32) = 2.868, P = 0.1000; interaction: F(1,32) = 2.777, P = 0.1054. Cbm cell number: genotype: F(1,32) = 4.982, P = 0.0327; age: F(1,32) = 31.08, P < 0.0001; interaction: F(1,32) = 1.119, P = 0.2817. Cbm Iba1 area: genotype: F(1,32) = 2.602, P = 0.1165; age: F(1,32) = 6.502, P = 0.0158; interaction: F(1,32) = 0.6807, P = 0.4154. Cbm process area: genotype: F(1,32) = 0.5592, P = 0.4601; age: F(1,32) = 113, P < 0.0001; interaction: F(1,32) = 2.322, P = 0.1374. Cbm process perimeter: genotype: F(1,32) = 0.5271, P = 0.4731; age: F(1,32) = 118.5, P < 0.001; interaction: F(1,32) = 2.745, P = 0.1073.