Fig. 4: 5xFAD XO4⁺ microglia contain less post-synaptic material than 5xFAD XO4⁻ microglia in the dentate gyrus.

a Representative 3D reconstructions of confocal z-stacks showing PSD95 internalized within WT, 5xFAD XO4⁻ or 5xFAD XO4⁺ microglia cells (scale bars = 15 μm). b PSD95 within microglia quantified as the average volume of phagocytosed PSD95 volume per microglia volume in each dentate gyrus section (n = 6 z-stacks per condition; *p = 0.0057, using one-way ANOVA and Tukey’s multiple comparison test). All data are from n = 3 WT and n = 6 5xFAD animals and is presented as mean ± SEM per individual section. c Functional analysis of ex vivo mouse microglia phagocytosis following 1 h incubation with (c_i) pHrodo-green-labelled E. coli, (c_ii) pHrodo-red-labelled synaptosomes or (c_iii) pHrodo-green-labelled fAβ by FACS. Each population is gated based on XO4⁺ signal and compared to controls not incubated with pHrodo particles. d_i Quantitation of the percentage of XO4⁺ and XO4⁻ microglia that phagocytose pHrodo-red-labelled synaptosomes or pHrodo-green-labelled E. coli (comparing XO4⁻ and XO4⁺ microglia from n = 4 animals), or d_ii pHrodo-green-labelled fAβ (comparing XO4⁻ and XO4⁺ microglia from n = 3 animals). Data in (d) are presented as mean ± SEM. ^*p = 0.0233, ^**p = 0.0027 and ^****p = 9.2 × 10⁻⁷ by paired 2-tailed t-test. e SCENIC regulon analysis showing that Hif1a and Elf3 are predicted to control the XO4⁺ gene regulatory network. The number of genes in each regulon is shown in parentheses. f, g BV2 cells were stably transduced with mCherry or mCherry.shHif1a lentivirus and treated with DMSO or AF488-labelled fAβ for 24 h, then blue-labelled synaptosomes for 1.5 h. mCherry⁺ cells were FACS sorted for AF488-fAβ. f Normalized heatmap of gene expression, measured by qPCR, of signature genes associated with XO4⁺ microglia in fAβ⁺ and non-treated (un) BV2 cells with or without shHif1a, including Hif1a regulon genes (Igf1, Spp1, Ctsa, Hif1a) and genes not part of the Hif1a regulon (Apoe, Trem2, P2ry12). Data are expressed as fold change relative to non-treated mCherry transduced cells, based on ΔCt values relative to Actb. The data are from 3 independent experiments. g The proportion of cells that are highly phagocytic for blue-bead-labelled synaptosomes. Data are expressed as fold change in % phagocytosis relative to non-treated mCherry transduced cells (mean ± SEM). The data are from 3 independent experiments performed in triplicate. n.s., p = 0.22, ^**p = 0.0026, ^****p = 6.0 × 10⁻⁶ by two-way ANOVA using Tukey’s multiple comparison test. h Histograms showing fluorescence intensity of HIF1A intracellular staining in AF488-fAβ⁺ and non-treated BV2 cells. Secondary antibody control cells are stained with Pacific-blue-labelled secondary antibodies alone. i The proportion of dox-treated (or not) and fAβ⁺ or non-treated (un) BV2 cells transduced with dox-inducible Hif1a expression constructs that are highly phagocytic for blue-bead-labelled synaptosomes. Data are expressed as fold change in % phagocytosis relative to non-treated mCherry transduced cells (mean ± SEM). The data are from 3 independent experiments performed in triplicate. ^*p = 0.0253 by one-way ANOVA using Holm-Sidak’s multiple comparison test.