Fig. 5

Increased SphK1 improves microglial phagocytosis. a Colocalization of microglia (Iba1, red) with Aβ (Aβ42, green) and quantification. Scale bars, 10 μm; 3D reconstruction from confocal image stacks scale bars, 10 μm (n = 7 per group). b Left, representative photomicrograph of live slice section incubated with fluorescent beads (green). Scale bar, 10 μm. White arrow point to phagocytotic microglia with fluorescent beads. Right, quantification of the number of microglial phagocytes normalized to the total number of microglia (n = 4–6 per group). c Up, immunofluorescence images of thio S (Aβ plaques, green) encapsulated within Lamp1⁺ structures (phagolysosomes, blue) in microglia (Iba1, red) present in brains of APP/PS1 and APP/PS1/SphK1 tg mice. Low-magnification scale bars, 50 μm; High-magnification scale bars, 10 μm; 3D reconstruction from confocal image stacks scale bars, 10 μm. Down, quantitation of microglial volume occupied by Lamp1⁺ phagolysosomes, percent of microglia containing Aβ-loaded phagolysosomes and Aβ encapsulated in phagolysosomes (n = 5 per group). d Morphology of microglia (Iba1, red) surrounding Aβ (ThioS, green) in cortex of APP/PS1 and APP/PS1/SphK1 tg mice. Up, high magnification (Scale bars, 10 μm) and Imaris-based three-dimensional images (Scale bars, 5 μm) of microglia surrounding Aβ. Down, Imaris-based automated quantification of microglial morphology (n = 7–8 per group). e Morphometric analysis of Aβ plaques in APP/PS1 and APP/PS1/SphK1 tg mice (n = 5–6 per group). Brain sections were labeled with thio S and plaques were counted and assigned to three mutually exclusive size categories based on maximum diameter: small <25 μm; medium 25–50 μm; or large >50 μm. All data analysis was performed on 9-month-old mice. a, c−e, Student’s t test. b One-way analysis of variance, Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. All error bars indicate s.e.m.