A shared disease-associated oligodendrocyte signature among multiple CNS pathologies

Abstract

Alzheimer's disease (AD) is a complex neurodegenerative disease, perturbing neuronal and non-neuronal cell populations. In this study, using single-cell transcriptomics, we mapped all non-immune, non-neuronal cell populations in wild-type and AD model (5xFAD) mouse brains. We identified an oligodendrocyte state that increased in association with brain pathology, which we termed disease-associated oligodendrocytes (DOLs). In a murine model of amyloidosis, DOLs appear long after plaque accumulation, and amyloid-beta (Aβ) alone was not sufficient to induce the DOL signature in vitro. DOLs could be identified in a mouse model of tauopathy and in other murine neurodegenerative and autoimmune inflammatory conditions, suggesting a common response to severe pathological conditions. Using quantitative spatial analysis of mouse and postmortem human brain tissues, we found that oligodendrocytes expressing a key DOL marker (SERPINA3N/SERPINA3 accordingly) are present in the cortex in areas of brain damage and are enriched near Aβ plaques. In postmortem human brain tissue, the expression level of this marker correlated with cognitive decline. Altogether, this study uncovers a shared signature of oligodendrocytes in central nervous system pathologies.

Extended Data Fig. 1. Quality control of CD45-libraries described in figure 1

Extended data Figure 1 associated to Figure 1. (a) Distribution of total cellular unique molecular identifiers (UMI). Dashed line marks the threshold for analysis. (b) Distribution of total gene UMIs. Dashed line marks the threshold for analysis. (c) Distribution of the proportion of mitochondrial genes. Dashed line marks the threshold for analysis. (d) Comparison of the total cellular UMI distribution in cells from 5xFAD and WT mice (left panel) and the proportion of mitochondrial genes in cells from 5xAD and WT mice (right panel). P-values were computed using a Kruskal-Wallis test. The box bounds the IQR. Line, median. Whiskers extend to a maximum of 1.5*IQR beyond the box. n=18 independent mice (9 5xFAD, 9 WT) (e) Violin plot of known marker genes expression across the different cell clusters. (f) Comparison of the main cell-type proportions between 5xAD and WT mice.

Extended Data Fig. 2. Quality control of GalC+ libraries described in figure 2

Extended data Figure 2 associated to Figure 2. (a) Gating strategy used to enrich for oligodendrocytes. (b) Proportion of oligodendrocytes isolated in each sequenced plate. Large bars correspond to the median and small bars to IQR. n=48 384-well plates (c) Number of oligodendrocytes sequenced for each 5xAD and WT mice (left panel) and across ages (right panel). Large bars correspond to the median and small bars to IQR. n=33 independent mice (17 5xFAD, 16 WT) (d) Proportion of cluster 14 in WT and 5xFAD mice across ages. Large bars correspond to the median and small bars to IQR. n=33 independent mice (across ages: 6-8m; n=4 5xFAD, 4 WT, 10-11m; n=6 5xFAD, 5 WT, 15m; n=3 5xFAD, 3 WT, 24m; n=4 5xFAD, 4 WT) (e) Volcano plot corresponding to the differential expression analysis between DOL-like and the rest of oligodendrocytes as identified by Zhou et al.11. DOL genes are colored in orange. (f) Proportion of DOL-like among oligodendrocytes between 5xFAD and WT mice in the data by Zhou et al.11. P-value was computed using the Kruskal-Wallis test.

Extended Data Fig. 3. Cell type annotation of dataset described in figure 3 and culture quality control

Extended data Figure 3 associated to Figure 3. (a) Spearman correlation between the mean transcriptomic profiles of the cell clusters identified in the dataset from Lee et al. (b) Violin plot of known marker genes across the different cell clusters. (c) Violin plot of known DAM marker gene expression across the different microglia clusters after refined clustering. (d) Representative bright-field microscopy image of the primary oligodendrocyte culture; scale bar corresponds to 50μm. Representative results from 12 independent experiments.

Extended Data Fig. 4. GSEA and quality control of datasets described in figure 4

Extended data Figure 4 associated to Figure 4. (a) GSEA analysis plot corresponding to the acute EAE dataset. (b) GSEA analysis plot corresponding to the multiphasic EAE dataset. (c) GSEA analysis plot corresponding to the aging SVZ dataset. (d) score of topic number 8, corresponding to DOL-like signature, in LPS-stimulated and control samples. n=6 independent samples (3 LPS-stimulated, 3 control). Thick line corresponds to the median, the bottom and upper limits of the box to the first and third quartile, respectively. The lower and upper whiskers correspond to the lowest and highest values respectively within the range of the first (third) quartile minus (plus) 1.5 times the Interquartile range. (e) GSEA analysis plot of the DOL signature in topic 8. (f) Serpina3n expression (transcripts per thousand) in LPS-treated and control mice. n=6 independent samples (3 LPS-stimulated, 3 control). p-value was computed by performing a Gene Set Enrichment Analysis as described by Subramanian et al. Thick line corresponds to the median, the bottom and upper limits of the box to the first and third quartile, respectively. The lower and upper whiskers correspond to the lowest and highest values respectively within the range of the first (third) quartile minus (plus) 1.5 times the Interquartile range. (g) Intensity of the oligodendrocyte signature across the sections of the three LPS treated mice.

Extended Data Fig. 5. Quality control of image analysis described in figure 5

Extended data Figure 5 associated to Figure 5. (a) Separated channels corresponding to the 5xFAD brain sample in fig. 5a; scale bar corresponds to 20μm. (b) Distribution of cell size across mice samples. The box bounds the IQR. Line, median. Whiskers extend to a maximum of 1.5*IQR beyond the box. n=6 independent samples (4 5xFAD, 2 WT). (c) Distribution of OLIG2 intensity across mouse samples. The box bounds the IQR. Line, median. Whiskers extend to a maximum of 1.5*IQR beyond the box. n=6 independent samples (4 5xFAD, 2 WT). (d) Distribution of OLIG2 intensity and estimated threshold (vertical red line). (e) Proportion of OLIG2+ cells across mouse samples. (f) Number of plaques in 5xFAD (n=4) and WT (n=2) mice. (g) Distribution of SERPINA3N intensity and the estimated threshold (vertical red line). (h) The 15 most contributing genes to the macrophage signature. (i) The 15 most contributing genes to the inflammatory signature. (j) Separated channels corresponding to the postmortem AD brain sample in fig. 5i; scale bar corresponds to 50μm. (k) Distribution of cell size across human samples. The box bounds the IQR. Line, median. Whiskers extend to a maximum of 1.5*IQR beyond the box. n=16 independent samples (8 AD, 8 NDC). (l) Distribution of SERPINA3 intensity and the estimated threshold (vertical red line) for the two different batches of samples.

Figure 1:. Oligodendrocytes display major transcriptomic alteration in the 5xFAD mouse model.

(a) Single-cell expression heatmap of non-immune (CD45⁻) cells from 5xFAD (n=9) and WT (n=9) mice. (b) Two-dimensional Uniform Manifold Approximation and Projection (UMAP) embedding of the scRNA-seq data. Dots are colored based on the scRNA-seq clustering. (c) Number of differentially expressed genes between 5xFAD and WT mice across the major cell types. (d) Specificity of the differentially expressed genes in oligodendrocytes. (e) Power analysis- number of differentially expressed genes based on the number of cells used for the differential expression analysis. Red- down-sampling of oligodendrocytes, orange- astrocytes, green- endothelial cells. Red dots correspond to the mean number of DEG while the bar represents the standard deviation.

Figure 2:. Identification of a disease-associated oligodendrocyte state.

(a) Experimental strategy to study oligodendrocytes. Illustration was created with Biorender.com. (b) Expression heatmap of oligodendrocytes from 5xFAD and WT mice at various ages, GalC⁺ sorted and pooled together with the previously sequenced oligodendrocytes. n=33 independent mice (across ages: 6-8m; n=4 5xFAD, 4 WT, 10-11m; n=6 5xFAD, 5 WT, 15m; n=3 5xFAD, 3 WT, 24m; n=4 5xFAD, 4 WT). (c) Two-dimensional UMAP embedding of the scRNA-seq oligodendrocytes data. Dots are colored based on the scRNA-seq clustering. Top- cells from WT mice. bottom- cells from 5xFAD mice. (d) Z-score of the enrichment test comparing cluster frequency between 5xFAD and WT mice. P-values were computed by fitting a Poisson regression and corrected using multiple-testing (Methods). (e) Proportion of DOLs among oligodendrocytes between 5xFAD and WT mice (left panel) and across ages (right panel). P-value was computed using the Kruskal-Wallis test. Large bars correspond to the median and small bars to IQR. n=33 independent mice (across ages: 6-8m; n=4 5xFAD, 4 WT, 10-11m; n=6 5xFAD, 5 WT, 15m; n=3 5xFAD, 3 WT, 24m; n=4 5xFAD, 4 WT) (f) Volcano plot corresponding to the differential expression analysis between DOLs and the rest of the oligodendrocytes. (g) Results of the promoter analysis by iRegulon.

Figure 3:. DOLs are independent of dementia’s etiology.

(a) Experimental strategy in the scRNA-seq dataset from Lee et al. (b) Two-dimensional UMAP embedding of the oligodendrocytes in each mouse model. Cluster 11 cells are in orange . (c) Comparison of the mean transcriptional profile of oligodendrocytes from cluster 11 with the rest of the oligodendrocytes. DOL genes are colored in orange. (d) Result of GSEA analysis performed on the log2FC list between oligodendrocytes from cluster 11 and the rest of the oligodendrocytes using the DOL genes as a query gene set. p-value was computed by performing a Gene Set Enrichment Analysis as described by Subramanian et al. (e) Proportion of DOLs among oligodendrocytes across mouse strains (n=3 for each strain). Large bars correspond to the median and small bars to IQR. (f) Proportion of DAM among microglia across mouse strains. (n=3 for each strain). Large bars correspond to the median and small bars to IQR. (g) Log2FC of DOL genes in cultured oligodendrocytes after treatment with Aβ at different aggregation stages.

Figure 4:. DOL signature in non-AD pathologies.

(a) Analytical approach used to identify DOL signature in additional CNS pathologies. (b) Volcano plot corresponding to the differential expression analysis between oligodendrocytes from control (CFA) and EAE spinal cord, Falcão et al.. DOL genes are colored in orange. (c) Volcano plot corresponding to the differential expression analysis between oligodendrocytes from control (CFA) and peak EAE spinal cord, Wheeler et al.. DOL genes are colored in orange. (d) Volcano plot corresponding to the differential expression analysis between oligodendrocytes from control young and old mice, Dulken et al.. DOL genes are colored in orange. (e) Kinetics of the mean expression level of key DOL genes across various EAE stages. p-values were computed using a binomial regression with complementary log-log link function (Methods). (f) Intensity of the DOL-like signature across the sections of the three LPS treated mice (left). H&E staining of the corresponding slides (right).

Figure 5:. Spatial analysis of DOLs in mouse and human brain sections.

Immunohistochemistry of DOL-like cells and analysis of spatial association with areas enriched with plaques in 5xFAD mice (a-f). (a) Representative image of DOLs (OLIG2+ (red) SERPINA3N+ (cyan)) from cortex of 16 months-old 5xFAD and WT mice. in 5xFAD, DOLs are in proximity to Aβ-plaques (yellow); magnification 63x, scale bar corresponds to 20μm. Arrowheads point to OLIG2+SERPINA3N+ cells. (b) Description of the experimental and computational approach used to study DOLs location in mouse and human brains. Illustration was created with Biorender.com. (c) Proportion of SERPINA3N+ cells in 5xFAD (n=4) and WT (n=2) mice. Large bars correspond to the median and small bars to IQR. (d) Proportion of SERPINA3N+ cells among OLIG2+ cells in 5xFAD and WT mice. Large bars correspond to the median and small bars to IQR. (e) Normalized L-functions of the SERPINA3N+/OLIG2+ cells in 5xFAD sections. Each curve corresponds to a unique sample. (f) Estimated interaction functions within SERPINA3N+/OLIG2+ cells (blue curves) or between them and plaques (red curves) in 5xFAD mice. Each curve corresponds to a unique sample. Analysis of Visium data from postmortem human AD brains (g-h). (g) Intensity of the macrophage (left) and inflammatory (right) topics across the four samples. n=4 biologically independent samples (2 AD, 2 NDC). Thick line corresponds to the median, the bottom and upper limits of the box to the first and third quartile, respectively. The lower and upper whiskers correspond to the lowest and highest values respectively within the range of the first (third) quartile minus (plus) 1.5 times the Interquartile range. (h) H&E staining of the AD2 sample (left panel), spatial pattern of the macrophage (middle panel) and inflammatory (right panel) topics. Immunohistochemistry of postmortem human AD brains (i-k). (i) Representative image of postmortem AD patient (right) and NDC (left) temporal cortex stained for oligodendrocytes marker (CC1, red), SERPINA3 (cyan), Aβ (Thioflavin S, yellow), and nuclei (Hoechst, blue); Magnification 20x, scale bar corresponds to 50μm. Arrowheads point to CC1+SERPINA3+ cells. Inset marks position of right image in 63x magnification, scale bar corresponds to 20μm. (j) Proportion of SERPINA3N+ cells in AD (n=8) and NDC (n=8) samples. Large bars correspond to the median and small bars to IQR. p-value was computed using a Wilcoxon rank test. (k) Association between MMSE score and proportion of SERPINA3+ cells. Dashed line corresponds to an ordinary least square linear. p-value was computed by testing the significance of the contribution of the MMSE score to predict the proportion of SERPINA3+ cells using a likelihood ratio test.