Fig. 4

Differential regulation of synaptic proteins and inflammatory cytokines in cuprizone treated MyD88 deficient mice. a Pairwise sample correlation (left panel) and phylogenetic distance clustering (right panel) of samples based on protein expression in the cerebellar cortex of wild type (WT, gray and pink) and MyD88^−/− (white and red) mice with (Cup) or without (Ctrl) 5 week cuprizone feeding (n = 3 independent samples per group). Note that cuprizone treatment had a stronger effect than genotype in sample correlation and clustering. b Heatmap representation and Gene Set Z-score (GSZ) of proteome analysis revealing differential expression of proteins related to innate immune system response (reactome, left panel) and glutamatergic synapse (gene ontology, right panel). c Self organizing maps (SOM) and sample portraits for the different experimental conditions depicting overexpression spots (A-G) and their specific expression and function in different groups (table). Normalized Purkinje cell EPSC amplitudes at 50 Hz stimulation frequency of parallel fibers presented as a stimulus train (d) or 2nd EPSC (e) show a reduction in facilitation in WT animals after cuprizone treatment (wt CUP5W, pink) as compared to naive WT animals (wt naive, grey). Each point represents mean normalized EPSC value ± SEM. No differences in facilitation are observed in naive (MyD88^−/− naive, white) or cuprizone fed (MyD88^−/− CUP5W, red). Each point represents an individual cell. Bars’ heights represent mean ± SEM. f Quantification of relative mRNA expression (qPCR) of selected immune modulatory mediators in the cerebellar nuclei (CN) showing a significant increase in several genes (CX3CR1, CCL3, TNFα, and TGFβ) in WT animals after cuprizone treatment. A similar cytokine upregulation of CX3CR1 and TGFβ was observed in MyD88^−/− mice who also showed an increase in IL-1β and a trend towards a decrease in TNFα and CCL3 when compared to WT CUP5W mice. Each point represents mean relative expression of two technical replicates for a single animal. Bar height represents mean relative expression ± SEM. P values were obtained after two-way ANOVA followed by Tukey’s multiple comparisons test (e) and one-way ANOVA followed by Tukey’s multiple comparisons test (f). Asterisks represent significant p-values (*p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001)