Extended Data Fig. 7: In vitro and in vivo analysis of microglia challenged by myelin and amyloid.
From: Myelin dysfunction drives amyloid-β deposition in models of Alzheimer’s disease
(a) Scheme illustrating the experimental setup to study amyloid phagocytosis after myelin digestion. Bone marrow-derived macrophages (BMDMs) were isolated and treated with myelin debris for 12h (1) prior to incubation with HiLyte488-labelled Aβ42 for 4h (2). The internalisation of Aβ42 was assessed by microscopy. (b) Representative immunofluorescence images of BMDMs treated with HiLyte488-labelled Aβ42 with and without (control) prior myelin treatment. Quantification of amyloid internalisation normalised to Lectin+ area is given on the right. Bars represent means; dots represent replicates/coverslips/n. (n = 3 per group, with 2-3 quantified images per coverslips). Statistical analysis: two-sided, unpaired Student’s t-test (p-value is given in graph). (c) Representative images of microglia immunostainings (Iba1 and DAPI) in 6-months old Cnp−/−5xFAD mice and respective controls (WT, Cnp−/− and 5xFAD). Dotted line highlights the border between cortical (CTX) and callosal tissue (CC). (d) Quantification of microgliosis (number of microglia, Iba1+ area) in 6-months old Cnp−/−5xFAD mice and respective controls (WT, Cnp−/− and 5xFAD) in the corpus callosum (CC) and cortex (CTX). Bars represent means; dots represent biological replicates/mice (n = 5 for WT, n = 6 for 5xFAD, n = 3 for Cnp−/−, n = 5 for Cnp−/−5xFAD) Statistical analysis: ordinary one-way ANOVA with Tukey’s posthoc test for multiple comparisons (p-values for the posthoc test for all p < 0.05 are given in the graphs). (e) Representative images of microglia immunostainings (Iba1 and DAPI) in 6-months old Plp−/y5xFAD mice and respective controls (WT, Plp−/y and 5xFAD). Dotted line highlights the border between cortical (CTX) and callosal tissue (CC). (f) Quantification of microgliosis (number of microglia, Iba1+ area) in 6-months old Plp−/y5xFAD mice and respective controls (WT, Plp−/y and 5xFAD) in the corpus callosum (CC) and cortex (CTX). Bars represent means; dots represent biological replicates/mice (n = 4 for each experimental group). Statistical analysis: ordinary one-way ANOVA with Tukey’s posthoc test for multiple comparisons (p-values for the posthoc test for all p < 0.1 are given in the graphs). (g) Microscopic analysis of microglial corralling by amyloid (6E10), microglia (Iba1) and nucleus (DAPI) co-labelling in 6-month-old Cnp−/−5xFAD and 5xFAD control mice. Upper panel: overview images of the Iba1 staining. Lower panel: closeup of a cortical region. Quantification of microglia corralling (number of microglia per plaque) in the cortex is given on the right. Violin plots show the distributions for single biological replicates/mice/n (n = 3 per genotype, 50 plaques per replicate). Black lines in violin plots indicate medians. Bar graph on the right shows the comparison of means (n = 3 per genotype, 50 plaques per replicate). Statistical analysis: Two-sided, unpaired Student’s t-test (p-value is given above graph). Right, lower panel: closeup of a representative microglia-plaque interaction in each genotype. (h) Microscopic analysis of microglial corralling by amyloid (6E10), microglia (Iba1) and nucleus (DAPI) co-labelling in 6-month-old Cnp−/−AppNLGF and Cnp+/−AppNLGF control mice. Upper panel: overview images of the Iba1 staining. Lower panel: closeup of a cortical region. Quantification of microglia corralling (number of microglia per plaque) in the cortex of Cnp−/−AppNLGF and Cnp+/−AppNLGF. Violin plots show the distributions for single biological replicates/mice/n (n = 3 per genotype, 50 plaques per replicate). Black lines in violin plots indicate medians. Bar graph on the right shows the comparison of means (n = 3 per genotype, 50 plaques per replicate). Line indicates means. Statistical analysis: Two-sided, unpaired Student’s t-test (p-value is given above graph). Right, lower panel: closeup of a representative microglia-plaque interaction per genotype. (i) Microscopic analysis of microglial corralling by amyloid (6E10), microglia (Iba1) and nucleus (DAPI) co-labelling in 6-month-old Plp−/y5xFAD and 5xFAD control mice. Upper panel: overview images of the Iba1 staining. Lower panel: closeup of a cortical region. (j) Quantification of microglia corralling (number of microglia per plaque) in the cortex of 6-month old Plp−/y5xFAD and 5xFAD. Violin plots show the distributions for single biological replicates/mice/n (n = 3 per genotype, 50 plaques per replicate). Black line in violin plots indicate medians. Bar graph on the right shows the comparison of means (n = 3 per genotype, 50 plaques per replicate). Line indicates mean. Statistical analysis: Two-sided, unpaired Student’s t-test (p-value is given above graph). (k) Quantifications of microglia corralling (microglia coverage per plaque) in the cortex of 6-month-old Plp−/y5xFAD and 5xFAD. Violin plots show the distributions for single biological replicates/mice/n (n = 3 per genotype; 2010 plaques for 5xFAD and 2529 plaques for Plp−/y5xFAD genotype in total). Black line in violin plots indicate medians. Bar graph on the right shows the comparison of medians (n = 3 per genotype). Line indicates mean. Statistical analysis: Two-sided, unpaired Student’s t-test (p-value is given above graph). (l) Microscopic analysis of microglial corralling in Cuprizone-treated 5xFAD animals by amyloid (6E10), microglia (Iba1) and nucleus (DAPI) co-labelling. Note residual white matter gliosis in Cuprizone 5xFAD mice and small amyloid deposits highlighted by white arrowheads. The same observations were made in different independent samples/mice per group (n = 4 for 5xFAD and n = 5 for Cuprizone 5xFAD). (m) Microscopic analysis of microglial corralling in EAE 5xFAD animals by amyloid (6E10), microglia (Iba1) and nucleus (DAPI) co-labelling. A comparison of a non-lesion site versus a lesion site is shown. Note the massive microglia/macrophage infiltration at the lesion site that made corralling analysis difficult. The same observations were made in different independent samples/mice per group (n = 5 for 5xFAD and n = 5 for Cuprizone 5xFAD).
