Fig. 9
Segmentation and differential ganglioside expression in the brain using MALDI-MS. A Unsupervised statistical analysis employing bi-sectioning k-means in SCiLS Lab reveals distinct regions in mouse brains, with segmentation analysis identifying green and orange regions of interest (ROIs) characterized by unique lipid profiles between wildtype Npc2+/+ and untreated Npc2−/−. High responder AAV-BR1-NPC2-treated Npc2−/− mice show lipid profiles comparable to Npc2+/+, while low responders are comparable to the untreated Npc2−/− mice. B Average spectra from the green and orange segmented regions, normalized for RMS intensity, highlight distinct lipid profiles characteristic of each segment. C Spatial distribution maps of gangliosides GM3(d20:1/18:0) at 1207.76 m/z (a), GM2(d18:1/18:0) at 1381.81 m/z (b), and GM2(d20:1/18:0) at 1410.84 m/z (c) illustrate their presence in the cortex, hippocampus, and cerebellum across different genotypes and treatment groups. Representative images are shown for Npc2+/+ (n = 4), Npc2−/− (n = 5), and AAV-BR1-NPC2-treated Npc2−/− high responders (n = 3/4), low responders (n = 1) mice. D Quantitative analysis of average ganglioside intensities: a GM3 (1207.76 m/z) in the cortex, b GM2 (1381.81 m/z) in the hippocampus, c GM2 (1410.84 m/z) in the cerebellum. Significant differences (*p < 0.05) are observed between Npc2+/+ and Npc2−/− mice. No significant differences are observed between Npc2+/+ and AAV-BR1-NPC2-treated Npc2−/− mice, indicating a therapeutic impact of AAV-BR1 treatment and that the variation in ganglioside accumulation is influenced by genotype and treatment efficiency. All data were analyzed with one-way ANOVA (FGM3 (20) (1207.76 m/z)[2,11] = 4.394, p < 0.0396), FGM2 (18) (1381.81 m/z)[2,11] = 4.735, p < 0.0328, F GM2(18) (1410.84 m/z)[2,11] = 5.067, p < 0.0276, with Tukey's multiple comparisons test. Data are presented as mean ± SD (n = 4–5). High responders (n = 4) are indicated with green/black triangles, while low responders (n = 1) are indicated with green triangles