Fig. 5

Microglial clusters enriched ALS motor cortex express high levels of L-ferritin. Microglial single-cell average intensities of L-ferritin, HLA-DR, CD68, CD74, and Iba1 were used to identify and characterise microglial subpopulations that were changed in ALS relative to controls. A t-SNE plot was generated using random subset of 124,800 microglia (31,200 each from control motor cortex, ALS motor cortex, control hippocampus, and ALS hippocampus), and 27 clusters were identified using a k nearest neighbour and Louvain clustering approach (A). The total contribution of control versus ALS microglia to each cluster was visualised with a percentage bar to determine clusters most different between control and ALS: clusters 2, 4, 6, 11, and 19 (B₁-F₁). The percentage of total microglia in clusters 2, 4, 6, 11, and 19 were statistically compared between control and ALS motor cortex and hippocampus (B₂-F₂). Data presented as mean ± SD; control n = 10 and ALS n = 9–10. Cluster percentages were compared between case groups with multiple Mann–Whitney tests and multiple comparisons were controlled for using a False Discovery Rate of 0.01, as determined by the two-stage step-up method of Benjamini, Krieger, and Yekutieli. Significance of differences between case groups: *p ≤ 0.05. Microglial single-cell average intensities of L-ferritin, HLA-DR, CD68, CD74, and Iba1 were mapped on the merged t-SNE plot (all microglia from ALS and control motor cortex and hippocampus) using a binary high-low approach to visualise MOI^low and MOI^high microglia (G). To phenotype clusters based on MOI^high percentage, for each functional marker the percentage of MOI^high microglia in each cluster for a given case to the percentage of MOI^high microglia in that case overallThe percentage MOI^high for each functional marker was normalised to the total percentage of MOI^high in each case for clusters 2, 4, 6, 11, and 19 in all cases (H–L). Data presented as mean ± SD; n = 20. Presence of CD68^high L-ferritin^high ‘cluster 2’ microglia confirmed in stage 4 ALS case, MN15 (M). Representative image is a confocal Z-projection of Hoechst (blue; M₁), L-ferritin (green; M₂), CD68 (red; M₃), and Iba1 (cyan; M₄) co-labelling; scale bar = 20 µm