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
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