Journal: Nature

Article Title: Lymphoid gene expression supports neuroprotective microglia function

doi: 10.1038/s41586-025-09662-z

Figure Lengend Snippet: ( a, b ) PU.1 low plaque-associated microglia show lymphoid gene expression. Single-cell mRNA sequencing of forebrain microglia from 8-month-old wild-type (2♂) and 5xFAD mice (2♂) from Figs. a and , Supplementary Fig. , and Supplementary Table . ( a ) Top: Isolation of microglia by fluorescence-activated cell sorting (FACS) followed by single-cell mRNA sequencing. Bottom: The balloon plot shows differentially regulated gene ontologies (GOs, Fisher’s exact test) in the PU.1 low and PU.1 high cortical disease-associated microglia (DAM) subpopulations. ( b ) Scatter plot shows the correlation of upregulated genes in cortical PU.1 high (red) vs. PU.1 low microglia (blue) between two independent experiments (n = 1 mouse/group per experiment) using FindMarkers() analysis in Seurat—Wilcoxon rank sum test. 558 genes are significantly upregulated and 445 genes are significantly downregulated in PU.1 low microglia in two independent experiments, while 0 genes are regulated in opposing directions. ( c ) Plaque-associated, CSF1R-independent PU.1 low microglia show lymphoid gene expression. Single-nuclei sequencing of cortical microglia from 6-month-old control (♀) and two CSF1Ri-treated 5xFAD mice (2♂ pooled). Left: Isolation of lineage-traced microglial nuclei from the cortex by fluorescence-activated nuclear sorting (FANS) followed by single-nuclei sequencing. Right: The volcano plot shows the differentially up- (red) and down- (blue) regulated genes in cortical microglia control and CSF1Ri-treated mice from Fig. by FindMarkers() function in Seurat—Wilcoxon rank sum test. Please also see Supplementary Fig. and Supplementary Table . ( d ) PU.1 low microglia-expressed lymphoid-associated signaling molecules (ligands, surface receptors, and downstream signaling proteins) in T-cells are shown. Adapted from “T-cell and B-cell signaling” and “TCR downstream signaling” generated by BioRender.com. ( e ) Heatmap shows log2 (fold change) of selected differentially expressed genes in the PU.1 low DAM compared to the PU.1 high DAM, two independent experiments: (1) single-cell sequencing of forebrain microglia from wild-type and 5xFAD mice (Figs. , , Extended Data Fig. 3a) and (2) single-nuclei sequencing of cortical microglia from control diet- and CSF1Ri diet-fed 5xFAD mice (Fig. , Extended Data Fig. 3c). The color indicates log2 (fold change). ( f ) Top: Schematic shows multiplexed error-robust fluorescent in situ hybridization (MERFISH) within the brain of the 5xFAD model (8-month-old, ♂). Bottom: The representative image shows the identification of cells and plaques in the cortex. DAPI+ nuclei: red, 6E10+ amyloid β plaques: blue, Meg3+ neurons: brown, segmented plaques: blue dots. Scale bar: 1 mm. ( g ) Representative images show different cell types as well as a PU.1 low plaque-associated, and PU.1 high distal microglia. Macrophage-identifying gene Mrc1 : dark blue. Endothelial cell identifying gene Cldn5 : orange. T cell identifying genes Cd3e and Cd8a : green. Microglia homeostatic genes Cx3cr1, Tmem119, P2ry12, Csf1r, Hexb, Aif1, Tnf, Trem2, Itgam, C1qa, C1qb, C1qc, Sall1, Selplg : red. Neuron-identifying gene Meg3 : pink. Oligodendrocyte-identifying genes Mog, Sox10 , and Mag : brown. Lymphoid genes Pdcd1, Cd274, Cd28, Cd72, Tnfsf13b, Cd4: cyan. DAM identifying genes Itgax, Clec7a, Igf1, Cst7, Spp1 : yellow. Spi1 (PU.1): white. Amyloid β (6E10): blue. DAPI+ nuclei: gray. Marker genes are shown in Supplementary Table . Illustrations in a , c , d and f were created using BioRender ( https://biorender.com ).

Article Snippet: The primary antibodies used were Syk (Cell Signaling Technology; 2712; 1:500), phospho-Syk Y352 (Cell Signaling Technology; 2717; 1:500), PLCG2 (Cell Signaling Technology; 3852; 1:500), phospho-PLCG2 Y1217 (Cell Signaling Technology; 3871; 1:500), PU.1 (Cell Signaling Technology; 2266; 1:500), CD28 (Abcam; ab243228; 1:500), ACTB (Abcam; ab8227; 1:2,000), H3 (Abcam; ab1791; 1:5,000), IBA1 (1:500; Wako; 016-20001) and C1Qa (1:500; Proteintech; 11602-1-AP).

Techniques: Gene Expression, Sequencing, Isolation, Fluorescence, FACS, Control, Generated, In Situ Hybridization, Marker

Journal: Nature

Article Title: Lymphoid gene expression supports neuroprotective microglia function

doi: 10.1038/s41586-025-09662-z

Figure Lengend Snippet: ( a ) 5xFAD-PU.1-low mice show similar numbers of distal microglia. The bar graphs show the density of distal and total microglia in the cortex of 6-month-old 5xFAD (n = 6 mice, 3♀, 3♂) and 5xFAD-PU.1-low mice (n = 5 mice, 3♀, 2♂)—unpaired two-tailed t-test. ( b ) CD28 protein expression. Western blot shows CD28 levels in ex vivo microglia acutely isolated from the forebrain of 12-month-old control (n = 3 pooled mice, 1♀, 2♂) and 5xFAD; PU.1-low mice (n = 3 pooled mice,1♀, 2♂). Normalized CD28 quantification is shown below the blot. ( c-e ) Microglia in 5xFAD-PU.1-low mice show reduced neutral lipid droplet load and complement C1Q expression. ( c ) Western blot (left) with quantification (right). The bar graph shows the C1QA level normalized to TUBB3 per sample in the hippocampi of 6-month-old control (n = 6 mice, 6♂), 5xFAD (n = 4 mice, 4♂), and 5xFAD-PU.1-low mice (n = 3 mice, 3♂)—ordinary one-way ANOVA with multiple comparisons was used. ( d-e ) Representative immunofluorescence images (left) with quantification (right) from the cortex of 6-month-old mice—unpaired two-tailed t-test. Scale bar: 10 μm. ( d ) CLEC7A+ disease-associated microglia: green, TMEM119+ homeostatic microglia: cyan, C1QA: red. The bar graph shows the percent C1QA in the cortex of 5xFAD (n = 8 mice, 5♀, 3♂) and 5xFAD-PU.1-low mice (n = 6 mice, 4♀, 2♂). ( e ) CD11B+ microglia: red, BODIPY 493/503: green, ThioS+ dense-core plaques: blue. The bar graph shows the BODIPY+ volume within microglia normalized to microglia volume in 5xFAD (n = 3 mice, 2♀, 1♂) and 5xFAD-PU.1-low mice (n = 4 mice, 2♀, 2♂). ( f ) 5xFAD-PU.1-low mice show a reduced soluble amyloid load. The bar graphs show the percent area covered by 6E10+ soluble Aβ or ThioS+ dense-core plaques in the subiculum from 6-month-old 5xFAD (n = 7 mice, 2♀, 5♂) and 5xFAD; PU.1-low mice (n = 5 mice, 2♀, 3♂)—unpaired two-tailed t-test. Representative images are shown in Fig. . ( g ) 5xFAD-PU.1-low mice show a reduced spread of p-Tau following intracerebral injection of human pathogenic Tau isolated from the brain (schematic, top). Bar graphs (bottom) show the percent AT8+ phosphorylated Tau (p-Tau) area and HJ3.4+ amyloid β in the indicated brain regions of 10-month-old 5xFAD (n = 12 mice, 7♀, 5♂) and 5xFAD; PU.1-low mice (n = 9 mice, 4♀, 5♂) ( h ) LTP: The line graph shows the time course of long-term synaptic plasticity (LTP) quantified as a percentage change in excitatory postsynaptic potential (EPSP) slope from baseline (10-min period of a pre-and 60-min period of a post-100 Hz stimulation) in CA1 neurons of the hippocampus in 9-10-month-old control (n = 12 slices from 5 mice, 3♀, 2♂), 5xFAD (n = 13 slices from 6 mice, 4♀, 2♂), and 5xFAD; PU.1-low mice (n = 11 slices from 5 mice, 1♀, 4♂)—two-way ANOVA with repeated measures, P time = 0.0183, P genotype = 0.1031. ( i ) Open field analysis: Experimental design (left) and quantification (right) of the open field experiment. The area identified as “center” is marked by a red square. The bar graph shows the total distance traveled (m), time spent in the center (seconds), and the periphery (seconds) in a 10-min-long open field assay from 9–10 month-old control (n = 15 mice, 9♀, 6♂), 5xFAD (n = 12 mice, 6♀, 6♂), and 5xFAD; PU.1-low (n = 13 mice, 7♀, 6♂)—ordinary one-way ANOVA with multiple comparisons. ( j ) Novel object recognition: The schematic (top) and quantification of the novel object recognition experiment in a 3-room box. The bar graphs show the distance traveled during 20-min habituation, 10-min training, and 10-min testing; the total time spent sniffing any object during training and testing; and the “recognition score” calculated as the percent time spent sniffing the constant object to the total time spent sniffing either object during training from 9-10-month-old control (n = 10 mice, 5♀, 5♂), 5xFAD (n = 11 mice, 6♀, 5♂), and 5xFAD; PU.1-low mice (n = 15 mice, 7♀, 8♂)—ordinary one-way ANOVA with multiple comparisons. No sex differences are observed in behavioral analyses, and trends between genotypes were preserved in sex-specific analyses. Bar graphs with individual data points show mean ± SEM. Illustrations in g , i and j were created using BioRender ( https://biorender.com ).

Article Snippet: The primary antibodies used were Syk (Cell Signaling Technology; 2712; 1:500), phospho-Syk Y352 (Cell Signaling Technology; 2717; 1:500), PLCG2 (Cell Signaling Technology; 3852; 1:500), phospho-PLCG2 Y1217 (Cell Signaling Technology; 3871; 1:500), PU.1 (Cell Signaling Technology; 2266; 1:500), CD28 (Abcam; ab243228; 1:500), ACTB (Abcam; ab8227; 1:2,000), H3 (Abcam; ab1791; 1:5,000), IBA1 (1:500; Wako; 016-20001) and C1Qa (1:500; Proteintech; 11602-1-AP).

Techniques: Two Tailed Test, Expressing, Western Blot, Ex Vivo, Isolation, Control, Immunofluorescence, Injection