Extended Data Fig. 3: Myelin status in AD mouse models and myelin mutant crossbreedings and their behavioural analysis.

(a) Representative EM images of regions of the cingulate cortex and medial corpus callosum in App^NLGF and 5xFAD mice at 6 months of age. (b) Quantification of myelinated axon counts per field of view (FOV) of EM images as shown in (a). Dots represent single micrographs analysed. 5 images per animal/biological replicat/n were analysed (n = 5 for WT and App^NLGF, n = 6 for 5xFAD). Contoured dots represent the mean for each biological replicate. Statistical analysis: ordinary one-way ANOVA (p-value given in graphs) with Tukey’s multiple comparisons (no significant differences were found). (c) Analysis of myelin thickness by g-ratio measurements of single axons in the medial corpus callosum of WT, 5xFAD and App^NLGF mice at 6 months of age. Dots represent single axons analysed (WT = 477, 5xFAD = 512, App^NLGF = 533 from 3 animal/biological replicat/n per group); lines represent linear regression, dotted line represent 95% confidence bands. In the bar graph, bars represent means and dots represent biological replicates/mice/n (n = 3). Statistical analysis: ordinary one-way ANOVA (p-value given in graphs) with Tukey’s multiple comparisons. (d) Western blot analysis of myelin proteins MBP and CNP in 5xFAD and App^NLGF mice at 6 months of age in different brain regions (hemisphere, cortex, hippocampus). Molecular weight marker (in kDa) is indicated on the left. (e) Quantification of western blot analysis shown in (d). Relative protein abundance was determined by normalisation to total protein staining and wildtype levels. Bars represent means; dots represent biological replicates/mice/n (n = 4 per group). Statistical analysis: ordinary one-way ANOVA per group (antibody, brain region). p-values are given above bar graphs. (f) Closeups of myelin (anti-MBP) labelling in 6-months old WT, 5xFAD and App^NLGF mice in (DG), cornu ammonis (CA1), corpus callosum (CC), and cortex (CTX). (g) Quantification of myelin profiles (MBP⁺ area) in DG, CA1 and CTX and intensity in CC. Bars represent means; dots represent biological replicates/mice/n (n = 6 for WT, n = 6 for 5xFAD, n = 5 for App^NLGF). Statistical analysis: ordinary one-way ANOVA with Tukey’s multiple comparison test was performed. No significant (p < 0.05) differences were found. (h) Immunofluorescence closeups of myelin (MBP labelled) in plaque proximity (labelled with 6E10) in WT, 5xFAD and App^NLGF cortex. The same observations were made in different independent samples/mice per group (n = 6 for WT, n = 6 for 5xFAD, n = 5 for App^NLGF). (i) Closeups of myelin (anti-PLP) labelling in Cnp^−/−5xFAD mice, corresponding single mutants (Cnp^−/− and 5xFAD) and wildtype (WT) controls in the CA1, CTX and CC at 6 months of age. (j) Closeups of myelin (anti-CNP labelling in Plp^−/y5xFAD mice, corresponding single mutants (Plp^−/y and 5xFAD) and wildtype (WT) controls in the CA1, CTX and CC region at 6 month of age. (k) Quantification of myelin profiles (PLP+ area) in CA1 and CTX in Cnp^−/−5xFAD brains shown in (i). Bars represent means; dots represent biological replicates/mice/n (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 multiple comparison tests. P-values for p < 0.05 are shown in the bar graph. Non-significant p-values are not shown. (l) Quantification of myelin profiles (CNP+ area) in CA1 and CTX in Plp^−/y5xFAD brains shown in (j). Bars represent means; dots represent biological replicates/mice/n (n = 4 per group). Statistical analysis: ordinary one-way ANOVA with Tukey’s multiple comparison tests. P-values for p < 0.05 are shown in the bar graph. Non-significant p-values are not shown. (m) Representative tracks and images for the behaviour of Cnp^−/−5xFAD female mice in the elevated plus maze (EPM), Y maze (YM) and the clasping test paradigms. (n) Quantification of the behaviour of Cnp^−/−5xFAD female mice in the paradigms shown in (m). Bars represent means; dots represent biological replicates/mice/n (n = 10 for WT, n = 8 for 5xFAD, n = 9 for Cnp^−/−, n = 10 for Cnp^−/−5xFAD). (o) Representative tracks and images for the behaviour of Plp^−/y5xFAD male mice in the elevated plus maze (EPM), Y maze (YM) and the clasping test paradigms. (p) Quantification of the behaviour of Plp^−/−5xFAD female mice in the paradigms shown in (m). Bars represent means; dots represent biological replicates/mice/n (n = 10 for WT, n = 8 for 5xFAD, n = 8 for Plp^−/y, n = 10 for Plp^−/y5xFAD). Statistical analysis for (n) and (p): two-way type III ANOVAs probing the main effects for the 5xFAD genotype (p_AD) and the respective myelin-mutant genotype (p_MY) as well as their interaction (p_INT) followed by Tukey’s multiple comparisons posthoc test (p-value is given in the bar graphs).

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