Fig. 2: Cellular senescence-associated transcriptional signatures predominate in the white matter endothelia, microglia, and astrocytes.
a Schematic representation of the consensus-based strategy used for senescent cell identification. Cell cycle arrest was evaluated by scoring cell cycle-related gene signatures using the senescence index tool (SIT), followed by protein validation of p16INK4a levels in MS tissues. Cell cycle arrest and SASP were assessed by scoring SenMayo gene signature. DNA damage response and nuclear reorganization were validated by identifying TBP53 and nuclear lamin B1 protein levels in MS tissues. Created in BioRender. Absinta, M. (2025) https://BioRender.com/d0hxq2g. b UMAP showing mapping SenMayo gene signature scores in the cortex and white matter from MS cases vs. controls. c Quantification (%, mean ± SEM) of SenMayo-informed senescent cells by location (white matter and cortex) in MS tissues vs. non-neurological controls (n = 43 biological samples, two-way ANOVA, p = 0.05, post-hoc multiple comparison analysis * p = 0.028). d Random Forest on the percentage of SenMayo-defined senescent cells highlighting the CNS cell type, pathological stage, and subject age as the most relevant factors. e Heatmap showing the percentage of SenMayo-defined senescent cells by CNS cell type and pathological stage (linear mixed model with fixed effects [sex, age, and pathological lesion stage] by each CNS cell type, * p < 0.05, **p < 0.01). f Correlation between SenMayo- and SIT-defined senescent-like cells (Pearson correlation coefficient r = 0.47, p < 0.001, equation y = 0.6278*x + 7.077). g A representative example of the CA lesion edge. Within the CA lesion edge, most cells (both microglia and endothelia) are positive for the senescence marker p16INK4a (arrows). Separate channels are shown to facilitate the visualization of different markers. Dotted lines indicate the lesion edge. h A representative example of the lesion core. Within the lesion core, the arrows indicate astrocytes positive for the senescence marker p16INK4a. i A representative example of p16INK4a+ ependymal cells. j Violin plot showing the quantification of p16INK4a+ cells (%) on human brain tissue by pathological condition from eighteen 5-μm paraffin-embedded tissue sections (ANOVA p < 0.0001; Tukey’s multiple comparisons * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). The violin plot shows the median (black line), quartiles (dotted lines), as well as the minima and maxima. snRNA-seq single-nucleus RNA sequencing, UMAP uniform manifold approximation and projection, MS multiple sclerosis, CA chronic active, CI chronic inactive, NAWM normal appearing white matter, OPC oligoprecursor cells, SASP senescence-associated secretory phenotype.
