Extended Data Fig. 4: Analysis of ganglioside storage in a temporospatial manner.

A) Bar graphs depicting dysregulated ganglioside deposition in brain homogenates of Hexb^−/− (n = 6) and Hexb^+/− mice (n = 3) at 120 days of age measured by untargeted lipidomics (LC-MS). B) Spatial MALDI mass spectrometry imaging (MALDI-MSI) on Hexb^−/− and Hexb^+/− brains at P0 (upper row), P7 (mid row), and P120 (bottom row). For each indicated ganglioside, ion images representative for three biological replicates are shown. Color scale represents a visual map of the intensities (in arbitrary units) of the ion images. C) Heatmap showing all differentially regulated and annotated lipids in the brain of P0, P7, and P120 Hexb^−/− (n = 3) and Hexb^+/+ (n = 3) control mice. Color scale indicates the z-score. D) MALDI MSI on the thalamus of Hexb^−/− and Hexb^+/− mice at P120. For each indicated ganglioside, ion images representative for three biological replicates are shown. Color scale represents a visual map of the intensities (in arbitrary units) of the ion images. E) Representative immunofluorescence images of GM2 storage in neurons and microglia in Hexb^−/− mice. Triangles point to GM2⁺IBA1⁺ microglia or GM2⁺NeuN⁺ neurons. F) Representative immunofluorescence images of GM2 storage (green) in lysosomes (LAMP1⁺, yellow) in microglia (IBA1⁺, red, top panel) or neurons (NeuN⁺, red, bottom panel), respectively. Orange color indicates Hexb^−/− mice and blue color Hexb^+/− mice. G) Quantification of lysosomal GM2, shown as the integrated fluorescence intensity of GM2 signal within LAMP1⁺ lysosomal areas, normalized to the cell area defined by IBA1⁺ (microglia) and NeuN⁺ (neurons) signals, respectively. Each symbol indicates one mouse. Data shown as mean ± s.e.m. Statistical analyses: Unpaired Student’s t-test comparisons were used for statistical testing (A); two-way ANOVA followed by Tukey’s test for correcting multiple comparisons was used for statistical testing (G).

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