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. 2013 Jul 25;4(2):385-401.
doi: 10.1016/j.celrep.2013.06.018. Epub 2013 Jul 11.

A Neurodegeneration-Specific Gene-Expression Signature of Acutely Isolated Microglia From an Amyotrophic Lateral Sclerosis Mouse Model

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Free PMC article

A Neurodegeneration-Specific Gene-Expression Signature of Acutely Isolated Microglia From an Amyotrophic Lateral Sclerosis Mouse Model

Isaac M Chiu et al. Cell Rep. .
Free PMC article

Abstract

Microglia are resident immune cells of the CNS that are activated by infection, neuronal injury, and inflammation. Here, we utilize flow cytometry and deep RNA sequencing of acutely isolated spinal cord microglia to define their activation in vivo. Analysis of resting microglia identified 29 genes that distinguish microglia from other CNS cells and peripheral macrophages/monocytes. We then analyzed molecular changes in microglia during neurodegenerative disease activation using the SOD1(G93A) mouse model of amyotrophic lateral sclerosis (ALS). We found that SOD1(G93A) microglia are not derived from infiltrating monocytes, and that both potentially neuroprotective and toxic factors, including Alzheimer's disease genes, are concurrently upregulated. Mutant microglia differed from SOD1(WT), lipopolysaccharide-activated microglia, and M1/M2 macrophages, defining an ALS-specific phenotype. Concurrent messenger RNA/fluorescence-activated cell sorting analysis revealed posttranscriptional regulation of microglia surface receptors and T cell-associated changes in the transcriptome. These results provide insights into microglia biology and establish a resource for future studies of neuroinflammation.

Figures

Fig. 1
Fig. 1
Transcriptional profiling of purified spinal cord microglia and parallel flow cytometry analysis. (A) Percoll gradient isolated leukocytes from SOD1G93A, non-Tg littermate, SOD1WT, and LPS injected mice were subjected to FACS analysis (T cells, microglia) and CD11b+ microglia purification for RNAseq. (B) Kaplan Meier curves of disease progression in SOD1G93A mice (n=35), showing onset of hindlimb weakness, peak weight, and death. Spinal cords were dissected at day 65, day 100, and day 130/end stage for microglia analysis. (C) Plots of relative transcript levels of purified microglia compared to whole spinal cord, examining oligodendrocyte (Sox10, Mag, Mog, Mobp, Cldn11), astrocyte (Gfap, Slc1a2, S100b, Aldh1l1), motor neuron (Mnx1, Isl1, Isl2, Chat), and microglia (Cd11b, Cd45, Cx3cr1, Iba1, Cd68) markers. Values are fold-change ± s.e.m. (*, p<0.05; **, p<0.01; ***, p<0.001 by t-test). See also Figure S1.
Fig. 2
Fig. 2
Microglia marker identification by transcriptome comparisons with other CNS and myeloid immune cell-types. (A) Heat map of top 50 genes enriched in microglia compared to CNS cell-types by RNAseq, ranked by fold-change (q<0.05, >5-fold enrichment). (B) Heat map of top 50 genes enriched in microglia compared to neutrophil, macrophage, and monocyte subsets by microarray, ranked by fold-change (q<0.05, >5-fold enrichment). (C) Venn diagrams show that 29 genes distinguish microglia from other CNS and peripheral myeloid cells. (D) Plots of transcript levels across neutrophil, macrophage, monocyte subtypes, and microglia (n=3–5 arrays/group; mean±sem). Olfml3, SiglecH, and Tmem119 are highly enriched markers for microglia. By contrast, Iba1 (Aif1), Cd68, and Cx3cr1 are expressed by other myeloid cell-types. (E) FACS of Siglec H expression on CD11b+CD45+ myeloid cells isolated from spleen, lymph node, liver, spinal cord, and brain of the same animals. Siglec H expression was restricted to CNS microglia (red histograms; grey, unstained cells). Error bars, mean±s.e.m. See also Figure S2.
Fig. 3
Fig. 3
SOD1G93A spinal cord immune activation is characterized by resident microglia (CD11b+Olfml3+) and not peripheral monocytes (CD11b+Ly6c+). (A) FACS analysis and cell quantification show increases in CD11b+ myeloid cells in SOD1G93A spinal cords over time. (**, p<0.01). (B) Transcript level comparisons of whole spinal cord RNA show increases in the microglia markers Olfml3, Tmem119, and Siglec H in SOD1G93A vs. SOD1WT mice. (C) Immunostaining shows Olfml3 expression in resting microglia (CD11b+) in non-Tg spinal cord. At end-stage, activated SOD1G93A microglia in ventral horns express Olfml3, but nerve root activated macrophages do not. Scale bars, 100 µm. (D) Microglia signature genes (red) show progressive increases on volcano plots of SOD1G93A vs.SOD1WT spinal cord RNAseq data (fold-change vs. p-value). Percentages denote proportion of microglia genes expressed >1.5-fold (dotted line) in SOD1G93A spinal cord. (E) CD45+ spinal cord leukocytes were analyzed for lymphocyte, monocyte, and microglia populations by FACS. At end-stage, SOD1G93A spinal cords showed greater proportions of CD11b–CD45hi lymphocytes compared to non-Tg controls (2.38% vs. 0.221%). Within CD11b+ myeloid cells, the majority are Ly6C- microglia (99.9% vs. 99.5%), and not Ly6C+ monocytes (0.37% vs. 0.0977%). (F) Quantification of FACS analysis (n=14–16 per group). (G) By qPCR, monocyte markers Ccr2 and Ly6c1 are not upregulated in purified SOD1G93A microglia at end-stage. Relative expression±SEM (**, p<0.01). See also Figure S3.
Fig. 4
Fig. 4
SOD1G93A microglia progressively upregulate potentially neuroprotective and neurotoxic factors. (A) Hierarchical clustering of RNAseq data from SOD1G93A, SOD1WT, and non-Tg microglia at different timepoints shows distinct segregation of mutant from control transcriptomes. (B) Differentially upregulated or downregulated genes (2-fold difference, p<0.05) in mutant microglia compared to control microglia at each timepoint, displayed as Venn diagrams. (C) Heat map displaying the 40 most differentially expressed genes across all datasets (ranked by coefficient of variance), shown by genotype and timepoint. (D) Microglia significantly upregulate potentially neuroprotective (IGF-1, Progranulin, DAP 12) and neurotoxic factors (MMP-12, Optineurin, Nox2) during disease progression (*, p<0.05; **, p<0.01; ***, p<0.001 by t-test). (E) Immunostaining for IGF-1, MMP-12, and microglia (Iba1) in SOD1G93A (end-stage) and non-Tg spinal cords (day 130). Top panels, ventral horn; lower panels, high magnification images of microglia. Scale bars, 100 µm. Error bars, mean±s.e.m. See also Figure S4 and S5.
Fig. 5
Fig. 5
ALS spinal cord microglia do not resemble LPS activated microglia or M1 and M2 macrophages. (A) SOD1G93A end-stage microglia profiles were compared to microglia activated by LPS injection. Lines show 2-fold change boundaries, specific enriched genes are highlighted. (B) SOD1G93A or LPS activation induced genes (1.5-fold, p<0.05 compared to naïve mice) show differential KEGG pathway enrichment (heat map, -log p-value). (C) Volcano plots identify M1 activation or M2 activation induced genes (2-fold, p<0.01). Venn diagram shows hallmark genes for each activation modality. (D) M1 or M2 macrophage genes were overlaid onto volcano plots of SOD1G93A vs. control microglia (p-value vs. fold-change). The number of M1 or M2 genes downregulated (left) or upregulated (right) in SOD1G93A microglia at each timepoint is displayed. (E) Fold-change induction for prototypic M2 macrophage genes (Arginase 1, YM1), for M1 genes (TNF-α, IL-1β), and for IGF-1 or Axl, during macrophage or SOD1G93A microglia activation. Error bars, mean±s.e.m. See also Figure S6.
Fig. 6
Fig. 6
Pathway analysis shows dysregulation of Alzheimer’s disease genes in mutant SOD1 microglia. (A) iPAGE analysis finds significant enrichment for ribosomal, lysosome, RNA splicing, and Alzheimer’s disease KEGG pathways in SOD1G93A microglia compared to control microglia (clusters C3, C4). (B) Transcriptional motif enrichment analysis finds 3 positively enriched transcriptional motifs linked to KEGG pathway changes. (C) SOD1G93A microglia show significant dysregulation in Alzheimer’s disease (AD) genes, as shown in this pathway diagram (adapted from KEGG). (D) AD pathway genes were grouped by K-means clustering and plotted over time by fold-difference between SOD1G93A vs. control microglia. Genes whose mutations directly link to AD and components of ATP synthase, NADH dehydrogenase, cytochrome C oxidase, and other AD pathway genes are differentially increased in SOD1G93A microglia (E) Presenilin 2 (Psen2) and Apolipoprotein E (Apoe) levels are upregulated in SOD1G93A microglia by RNAseq and qPCR analysis (*, p<0.05; **, p<0.01; ***, p<0.001 by t-test). (F) Immunostaining of spinal cord sections shows significant upregulation of ApoE throughout the ventral horns of SOD1G93A compared to non-Tg spinal cords. Higher magnification images show microglia (Iba1+) colocalization with ApoE. Scale bars, 50 µm. Error bars, mean±s.e.m.
Fig. 7
Fig. 7
Post-transcriptional regulation of microglia surface markers and T cell associated molecular pathways. (A) Representative histograms of CD11c and CD86 surface levels on microglia from SOD1G93A and control spinal cords. (B) Surface MFI and transcript levels (RPKM) for CD11c (Itgax) and CD86. (**, p<0.01; ***, p<0.001 by t-test). (C) FACS-RNAseq correlation shows that surface CD11c is directly regulated by transcription while surface CD11b and CD86 are regulated post-transcriptionally. (D–E) Infiltrating T cells quantified by FACS and cell count of spinal cord samples. Intra-sample comparative analysis with microglia transcriptome data shows specific microglia GO categories correlated to levels of CD4+ or CD8+ T cells (Spearman rank). Error bars, mean±s.e.m.

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