Fig 4. Increased astrocytic MFG-E8 signaling at the onset of synapse loss in the NL-F KI hippocampus.

A) Representative image of RNAScope probing for Mfge8 (green), Aldh1l1⁺ astrocytes (magenta) and Cx3cr1⁺ myeloid cells (yellow) in the hippocampus. Scale bar = 25 μm. Inset shows exemplary ROI. B) Quantification of the number of Mfge8 mRNA spots on Aldh1l1⁺ astrocytes, Cx3cr1⁺ myeloid cells or Aldh1l1⁻Cx3cr1⁻ cells in the hippocampus using Imaris. Data shown as a percentage of the total number of Mfge8 spots in a large area of interest. Each point = 1 mouse (n=5 female mice). One-way ANOVA (F (2, 12) = 378.9, p<0.0001) followed by Bonferroni’s multiple comparisons post-hoc test on mouse average. C) RT-qPCR probing for Mfge8 expression in FACS sorted ACSA2⁺CD45⁻ astrocytes and CX3CR1⁺CD45⁺CD11b⁺ microglia. Gene expression is normalized to the geomean of 3 house-keeping genes (Actb, Gapdh and Rpl32) by the Delta CT method. Each point = 1 mouse (n=3–4 female mice). Unpaired two-tailed student’s t-test with a Welch’s correction on mouse average. D) Representative images of MFG-E8 (magenta) and GFAP (green) immunoreactivity in the post-mortem human frontal cortex. Scale bar = 10 μm. E) RNAScope probing for Mfge8 (green) on Slc1a2⁺ (magenta) astrocytes in the 6-mo WT and NL-F KI hippocampus. Scale bar = 15 μm. F) Quantification of the total number of Mfge8 spots in a large region of interest using Imaris. Each point = 1 mouse (n=4–5 mixed male and female mice per genotype). Unpaired two-tailed student’s t-test on mouse average. G) Quantification of the number of Slc1a2⁺ astrocytes in a large region of interest using Imaris. Each point = 1 mouse (n=4–5 mixed male and female mice per genotype). Unpaired two-tailed student’s t-test on mouse average. H) Quantification of the number of Mfge8 mRNA spots in the hippocampus shown as percent distribution on Slc1a2⁺ astrocytic nuclei. Each point = mouse average (n=4–5 mixed male and female mice per genotype). Two-way ANOVA (Interaction: F (2, 21) = 6.188, p<0.01) followed by Bonferroni’s multiple comparisons post-hoc test on Y=log(Y) transformed mouse average. I) Representative images of MFG-E8 (white) immunoreactivity in the 6-mo hippocampal CA1 SR in WT and NL-F KI mice. Scale bar = 30 μm. J) Quantification of MFG-E8 immunoreactivity in large region using ImageJ. Each point = mouse average (n=7 male mice per genotype). Unpaired two-tailed student’s t-test on mouse average. K) Western blot probing for MFG-E8 (55–75 kDa) with respect to GAPDH loading control (39 kDa) in 6-mo WT and NL-F KI hippocampal homogenates. 1 lane is 1 mouse. L) Quantification of MFG-E8/GAPDH using ImageJ densitometry analysis. Each point = mouse average (n=5 male mice per genotype). Unpaired two-tailed student’s t-test on mouse average. M) Representative images of RNAScope followed by immunostaining probing for either Itga_v (cyan) and Itgb₅ (magenta) integrin mRNA subunits (left) or Itga_v (cyan) and Itgb₃ (magenta) integrin mRNA subunits (right) with microglial P2Y12 (green) in the hippocampus. Scale bar = 20 (left) and 15 (right) μm. Inset highlight ROIs. N) Flow cytometry probing for CD61 integrin on CD45⁺CD11b⁺CX3CR1⁺ hippocampal microglia from 6-mo WT and NL-F KI mice. O) Quantification of the levels of CD61 on CD45⁺CD11b⁺CX3CR1⁺ microglia. Each point = mouse average (n=5 female mice per genotype). Unpaired two-tailed student’s t-test followed by a Welch’s correction on mouse average. All data shown as mean ± SEM. p-values shown ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.