Age-impaired remyelination is associated with dysregulated microglial transitions

Abstract

Multiple sclerosis (MS) is a chronic, inflammatory condition characterized by neurodegeneration and lost myelin, or demyelination. This lost myelin may be regenerated in people with MS through a process called remyelination, that is prone to failure and is impaired with age. Remyelination is facilitated by microglia but our understanding of the microglial response during remyelination is incomplete. Here, we profile the microglial response during remyelination in the lysolecithin mouse model using single-cell RNA sequencing and find several distinct microglial states during the early stages of remyelination that coalesce into a resolved state defined by the presence of myelin transcripts, a state also present in MS brains. We also observe a delay in the appearance of several microglial states with age, in concordance with delayed remyelination. This multi-faceted microglial response during efficient remyelination provides the basis of multi-faceted microglia-specific targets for future MS therapies.

Fig. 1. Microglia adopt Igf1 ReAM, Ccl3 ReAM, proliferative microglia, or IRM signatures at 7 DPI and an MTEM signature at 21 DPI in young mice.

a scRNAseq experimental workflow. Created in BioRender. Ho, M. (2025) https://BioRender.com/sm1j9hf. b UMAP showing 26,424 microglia collected from (c) naïve (n_M = 5 pooled per control sample) and (d) sham (n_M = 5) controls and at (e) 7 (n_M = 10 pooled per LPC-injected sample), (f) 14 (n_M = 10) and (g) 21 DPI (n_M= 12) from young, 2-month-old mice. h Proportion and (i) total counts of each sample present in each microglial cluster identified in (b). j Differentially expressed genes used to define microglial states. Representative and 3D rendered images (scale bar = 10 μm) of spinal cord lesions from Tmem119^creERT2; Rosa26^tdTomato mice with tdTomato (magenta) labeling microglia, k mRNA probes for Igf1 (green) and Irf7 (gray) and m Plp1 (gray) l Quantification of Igf1, Irf7 (3 DPI, n_F = 0, n_M = 5; 7 DPI, n_F = 5, n_M = 0; 21 DPI, n_F = 3, n_M = 2) positivity in tdTomato⁺ microglia at 3, 7 and 21 DPI. Two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons post-hoc test. n Quantification of Plp1 (3 DPI, n_F = 0, n_M = 4; 7 DPI, n_F = 4, n_M = 0; 21 DPI, n_F = 2, n_M = 2) positivity in tdTomato⁺ microglia at 3, 7 and 21 DPI. One-way analysis of variance (ANOVA) with Sidak’s multiple comparisons post-hoc test. Each point represents one animal, and all data are presented as mean ± SEM. All library cell numbers are normalized to the library with the lowest cell count. Source data are provided as a source data file. n_F=  n_{Female Mice} and n_M = n_{Male Mice}.

Fig. 2. MTEM prune newly laid myelin while Igf1 ReAM are associated with lipid processing and Ccl3 ReAM with cytokine release.

a Heatmap showing gene regulatory networks active in Igf1 ReAM, Ccl3 ReAM, MTEM (1 and 2), and IRM where each network contains the active transcription factors, regulators, and the downstream target genes. The networks are denoted by ‘(+)’ following the transcription factor’s name. b Functional terms associated with the differentially expressed genes in Igf1 ReAM, Ccl3 ReAM, MTEM (1 and 2), and IRM as determined by the KEGG, REACTOME and Gene Ontology databases. c Dot plot describing the mean expression level of the cytokine, chemokine and growth factor genes expressed by microglial states. d Electron microscopy image of a microglia at 21 DPI from a Cx3Cr1^CreERT2+/−; ROSA26^iDTR- mouse showing the cell membrane (red outline), nuclear membrane (yellow outline), lysosomes (green), autophagolysosomes (yellow), endoplasmic reticulum (red), dilated endoplasmic reticulum (white asterisk), empty phagosome (black asterisk), myelin containing phagosome (blue) and lipid droplets (red arrows). Individual image taken. e Schematic describing tamoxifen injection followed by LPC injection into the corpus callosum of PDGFRα^CreERT2; Rosa26^mGFP mice, and tissue isolation at 28 DPI. Created in BioRender. Ho, M. (2025) https://BioRender.com/1yvoyb1. f 3D rendering of IBA1⁺ microglia (red), MBP (blue), GFP (yellow) and colocalization of MBP with GFP (teal) from the corpus callosum of PDGFRα^CreERT2; Rosa26^mGFP mice at 28 DPI.

Fig. 3. Microglial transitions result in three terminal states: Igf1 ReAM, MTEM 2 and TM3.

a Microglial trajectories mapped using RNA velocity. Microglial trajectories terminate in (b) Igf1 ReAM, MTEM 2 and TM3 states as identified by a stability index > = 0.96. c Circular projection plot of cell fates towards the terminal states (vertices), where the cells closer to the terminal states are committed to the trajectory while the cells present in the centre of the triangle have multi-lineage potential. Random walk analyses, where the orange circles represent the supervised origin point and the yellow circles represent the unsupervised termination point, showing that (d) proliferative microglia terminate in Igf1 ReAM, e IRM terminate in MTEM 2 and f schematic describing microglial transitions. Pseudotime-determined trajectory showing the driver genes of the Igf1 ReAM state, g Igf1 and h Gpnmb increase towards the termination of the Igf1 ReAM trajectory, but not the MTEM 2 or TM3 trajectories. i Density of IRM lineage (YFP⁺IBA1⁺) microglia from Mx1^cre; Rosa26^YFP mice at 3 DPI (n_F = 1, n_M = 4), 7 DPI (n_F =  3, n_M = 2) and 21 DPI (n_F = 2, n_M = 2). One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons post-hoc test. j, l Representative images (scale bar = 10 μm) of spinal cord lesions from Mx1^cre; Rosa26^YFP mice with YFP (green) labeling Interferon responsive lineage cells, IBA1 (magenta) labeling microglia and probes for (j) Irf7 (top gray) and Igf1 (bottom gray) showing interferon lineage derived IRM and Igf1 ReAM respectively at 7 DPI or (l) Plp1 probe (gray) showing IRM lineage derived MTEM at 21 DPI. k Quantification of Igf1, Irf7 positivity in YFP⁺IBA1⁺ microglia shown in (j). Two-tailed unpaired t-test. m Quantification of Plp1 positivity in YFP⁺Iba1⁺ microglia shown in (l) at 3 DPI (n_F = 1, n_M = 40), 7 DPI (n_F = 3, n_M = 2) and 21 DPI (n_F = 2, n_M = 2). One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons post-hoc test. Each point represents one animal, and all data are presented as mean ± SEM. Source data are provided as a source data file. n_F= n_{Female Mice} and n_M = n_{Male Mice}.

Fig. 4. Absence of IRM in IFNAR1KO mice reduces OPC proliferation, late stages of oligodendrocyte differentiation and remyelination.

Representative images and quantification of (a) IBA1 (green), Irf7 (gray) density at 7 DPI from WT (n_F= 3, n_M = 3) and IFNAR1KO (n_F = 3, n_M = 2) mice. Arrowheads highlight Irf7⁺IBA1⁺ cells. Representative microglia for each group (gray square) are shown in the inset. b IBA1 (green), MAC2 (red) density at 7 (WT, n_F = 4, n_M = 3; IFNAR1KO, n_F = 4, n_M = 4) and 21 DPI (WT, n_F = 3, n_M = 3; IFNAR1KO, n_F = 4, n_M = 2). Inset shows MAC2 in greyscale. c IBA1 (green), IL1β (gray) density at 7 (WT, n_F = 4, n_M = 3; IFNAR1KO, n_F = 4, n_M = 4) and 21 DPI (WT, n_F = 3, n_M = 3; IFNAR1KO, n_F = 4, n_M = 2). Inset shows IL1β in greyscale. d OLIG2 (gray), PDGFRα (red), KI67 (green) density at 7 DPI (WT, n_F = 4, n_M = 2; IFNAR1KO, n_F = 3, n_M = 2). Arrowheads highlight OLIG2⁺PDGFRα⁺KI67⁺ cells. e MYRF (gray) density at 7 (WT, n_F = 4, n_M = 3; IFNAR1KO, n_F = 4, n_M = 2) and 21 DPI (WT, n_F = 3, n_M = 3; IFNAR1KO, n_F = 4, n_M = 2). f CNP (green), OLIG2 (red) density at 21 DPI (WT, n_F = 3, n_M = 3; IFNAR1KO, n_F = 4, n_M = 2). g CAII (gray) density at 21 DPI (WT, n_F = 3, n_M = 3; IFNAR1KO, n_F = 4, n_M = 2). Arrowheads highlight CAII⁺ cells. h MBP (gray) density at 21 DPI (WT, n_F = 3, n_M = 3; IFNAR1KO, n_F = 4, n_M = 2). i IBA1 (green), Plp1 (gray) density at 21 DPI (WT, n_F = 3, n_M = 3; IFNAR1KO, n_F = 4, n_M = 2). a, d, h, f, i: two-tailed Student’s t-test; a: Irf7⁺IBA1⁺ density: Mann–Whitney test; b, c, e: two-way ANOVA with Sidak’s post-hoc. Dotted lines outline lesions. Each point represents one animal, and data are presented as mean ± SEM. Source data are provided as a source data file. n_F= n_{Female Mice} and n_M= n_{Male Mice}.

Fig. 5. The appearance of Ccl3 and Igf1 ReAM is delayed in middle-aged mice, but that of IRM and proliferative microglia is not.

a UMAP showing 38,592 microglia collected from young mice from naïve (n_M= 10, pooled per sample), 7 DPI (n_M= 20) and 21 DPI (n_M  = 22) and middle-aged mice from naïve (n_M= 5), 7 DPI (n_M= 10) and 21 DPI (n_M= 10) conditions, resulting in (b) 13 clusters. c Proportion of each condition present in each microglial cluster identified in (b). d Differentially expressed genes used to define microglial states. e Representative images (scale bar = 10 μm) of lesions from Tmem119^creERT2; Rosa26^tdTomato mice with tdTomato (magenta) labeling microglia, mRNA probes for Igf1 (green) and Irf7 (gray). Quantification of (f) Igf1 positivity in tdTomato⁺ microglia and (g) Irf7 positivity in tdTomato⁺ microglia in young (3 DPI, n_F = 0, n_M = 5; 7 DPI, n_F = 5, n_M = 0; 21 DPI, n_F = 3, n_M = 2) and middle-aged (3 DPI, n_F = 2, n_M = 3; 7 DPI, n_F = 2, n_M = 3; 21 DPI, n_F = 2, n_M = 3) mice. h Quantification of Plp1 positivity in tdTomato⁺ microglia in young (3 DPI, n_F = 0, n_M = 4; 7 DPI, n_F = 4, n_M = 0; 21 DPI, n_F = 2, n_M = 2) and middle-aged (3 DPI, n_F = 2, n_M = 3; 7 DPI, n_F = 2, n_M = 3; 21 DPI, n_F = 1, n_M = 4) mice. Two-way analysis of variance (ANOVA). i Representative images (scale bar = 100 μm) of lesions from middle-aged, C57BL/6 mice with MBP (gray) labeling myelin. j Rank order of MBP inside lesions in middle-aged (21 DPI, n_M = 5; 45 DPI, n_M = 5; 60 DPI, n_M = 5) mice. One-way analysis of variance (ANOVA). k Quantification of Plp1 positivity in IBA1⁺ microglia present in middle-aged (21 DPI, n_M = 5; 45 DPI, n_M = 5; 60 DPI, n_M = 5) mice. One-way analysis of variance (ANOVA). All ANOVA were performed  with Tukey’s multiple comparisons post-hoc test. Each point represents one animal, and data are presented as mean ± SEM. Source data are provided as a source data file. n_F= n_{Female Mice} and n_M= n_{Male Mice}.

Fig. 6. Protein-level validation of microglial states during remyelination using high-dimensional flow cytometry.

a Representative scatterplot showing Ly6g⁺ neutrophils, Ly6c⁺ monocytes and Ly6c^-Ly6g^- microglia from Cd45⁺Mrc1^-Cd11b⁺ cells. b Unsupervised clustering of 47,871 microglia and the (c) proportional contribution of each cluster isolated from d–f young, 2-month-old and g–i middle-aged, 10-month-old male mice from naïve (n_young= 10, n_middle-aged = 10), 7 DPI (n_young= 8, n_middle-aged = 12) and 21 DPI (n_young= 10, n_middle-aged = 12) conditions, respectively. j UMAP depicting the expression of Idh2—a protein involved in the oxidative phosphorylation pathway. k Proportions of each sample (naïve (n_young= 10, n_middle-aged = 10), 7 DPI (n_young= 8, n_middle-aged = 12) and 21 DPI (n_young= 10, n_middle-aged = 12)) present in neutrophil and monocyte populations. Two-way analysis of variance (ANOVA) with Sidak’s post-hoc test. Each point represents two animals combined, and all data are presented as mean ± SEM. Metabolic pathway activity across (l) microglial states identified in (Fig. 1), throughout remyelination in (m) young mice and (n) young combined with middle-aged mice as a function of the genes found in the KEGG metabolic pathways. Source data are provided as a source data file.

Fig. 7. Lipid peroxidation is discordant with ROS production in middle-aged mice.

a Representative images (scale bar = 50 μm) of spinal cord lesions from Cx3cr1^creERT2; Rosa26^tdTomato mice with MDA (gray) showing lipid peroxidation in young (top) and middle-aged (bottom) mice at 21 DPI. b Quantification of the MDA present in lesions from Cx3cr1^creERT2; Rosa26^tdTomato (3 DPI, n_M = 4; 7 DPI, n_F = 1, n_M = 4; 21 DPI, n_F = 3, n_M = 2) mice. Two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons post-hoc test. c Representative flow cytometry plots of Cd11b⁺ microglia and CellROX intensity. Cells from 7 DPI (n_young= 5, n_middle-aged = 5) and 21 DPI (n_young= 6, n_middle-aged = 5) conditions were previously gated to include single cells based on forward scatter area and height then live cells based on live dead blue staining. Geometric mean intensity of CellROX in (d) microglia (Ly6c⁻Ly6g⁻Cd11b⁺), e Igf1 ReAM (Ly6c^-Ly6g^-Cd11b⁺Cd11c⁺), f OPC (Cd140a⁺), g astrocytes (Aqp4⁺) and MitoSOX in (h) microglia (Ly6c^-Ly6g^-Cd11b⁺), i Igf1 ReAM (Ly6c^-Ly6g^-Cd11b⁺Cd11c⁺), j OPC (Cd140a⁺), k astrocytes (Aqp4⁺) in young mice at 7 DPI (Dark blue) and 21 DPI (Dark Orange) and middle-aged mice at 7 DPI (Light blue) and 21 DPI (Light Orange). Two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons post-hoc test. l Split violin plots showing the differences between the Naïve, 7 DPI and 21 DPI conditions isolated from young and middle-aged mice in the ROS producer score and ROS scavenger score. m UMAPs depicting the gene-weighted density estimation of ROS producer score and ROS scavenger score showing increased expression in Igf1 ReAM 1 and 2. n Heatmap of genes for ROS producer enzymes and ROS scavenger enzymes across HM, IRM, Ccl3 ReAM, Igf1 ReAM (1–4), MTEM and proliferative microglia shown in (o) describing increased expression in Igf1 ReAM 1 and 2. o UMAP describing microglia from young and middle-aged mice across naïve, 7 and 21 DPI as shown in Fig. 5a. Each point represents one animal, and all data are presented as mean ± SEM. Source data are provided as a source data file. n_F= n_{Female Mice} and n_M = n_{Male Mice}.

Fig. 8. MTEM is present in shadow plaques and NAWM of MS brains.

a Unsupervised clustering of 1815 microglia isolated from chronic active and chronic inactive MS lesions along with healthy controls in a published dataset. b Unsupervised classification of HM, IRM, MTEM, Igf1 ReAM and Ccl3 ReAM identified from young mice during remyelination. A classifier was trained using SVMradial and avNNet machine learning models on the principal components used to cluster the mouse dataset and identified the HM, IRM, MTEM, Igf1 ReAM and Ccl3 ReAM states in the human MS dataset. c UMAP showing the expression level of PLP1 restricted to a portion of cells classified as MTEM in (b). d Proportion of classified microglial states present in each lesion type in (a). e Unsupervised clustering of 4137 microglia isolated from chronic active and chronic inactive MS lesion edges, lesion core, periplaque white matter, and control white matter in a published dataset. f Unsupervised classification of HM, IRM, MTEM, Igf1 ReAM and Ccl3 ReAM states as described in (b). g UMAP showing the expression level of PLP1 restricted to a portion of cells classified as MTEM in (f). h Proportion of classified microglial states present in each lesion type in (e). Representative images showing identification of MS lesions using (i) Luxol fast blue and PLP1 staining, where intense staining indicates NAWM (n = 5), lack of staining indicates a lesion and intermediate staining indicates a shadow plaque (n = 3). Further classification of shadow plaque as regions of intermediate, but distinct, BCAS1 staining (arrowheads) and classification of the identified lesion in (i) and (j) as inactive (n = 4) by lack of LN3 expression, or mixed (n = 1) by LN3 expression at the peripheries of the lesion. Lesions were collectively isolated from six brain regions isolated from five patients. Representative images of MS shadow plaque with PLP1 mRNA (white) and IBA1 (red) to identify IBA1⁺ microglia expressing PLP1 mRNA. j Quantification of PLP1 positivity in IBA1⁺ microglia. Each point represents one lesion, and all data are presented as mean ± SEM. Source data are provided as a source data file.