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. 2024 Mar 22;15:2553. doi: 10.1038/s41467-024-46705-x

Fig. 5. SNX8 overexpression rescues LSD phenotypes in Hexb−/− primary mouse fibroblasts and animals.

Fig. 5

A WT or Hexb−/− primary fibroblasts isolated from P1 neonatal mice were transfected with GFP or GFP-SNX8 as indicated for 24 h and then stained with lysotracker. Representative images (upper) with red asterisks indicating cell nuclei and quantifications (lower) are shown (n = 75 for all groups, p = 0.19 for WT + GFP vs. Hexb−/−+GFP-SNX8, and =0 for other two comparisons). Scale bar = 10 μm. B Sample groups same as in (A) were stained with filipin to visualize free cholesterol. Representative images (upper) with red asterisks indicating cell nuclei and quantifications (lower) are shown (n = 30, 30, 28, respectively, for each group, p = 0.022 for WT + GFP vs. Hexb−/−+GFP-SNX8, and <0.00001 for other two comparisons). Scale bar = 10 μm. C Sample groups same as in (A) were transfected with LAMP1-mCherry then imaged for LAMP1. LAMP1-positive tubules were counted. Representative images (upper) showing the whole cell (left panels) and enlarged insets (right panels, ROI are marked with red boxes) and quantifications (lower) are shown (n = 29, 25, 26, respectively, for each group, p < 0.00001 for WT + GFP vs. Hexb−/−+GFP-SNX8, and =0.0049 for Hexb−/−+GFP vs. Hexb−/−+GFP-SNX8). The fluorescent intensity was differentially boosted to clarify tubules’ visualization (pointed with red arrows). Scale bar = 20 μm for left panels and 3 μm for insets. D, E Representative images (left) and quantifications (right) of immunohistochemistry of GM2 ganglioside in the cerebellum regions (D), or NeuN in the brainstem regions (E) of 3-months-old WT and Hexb−/− mice injected with AAV-GFP or AAV-SNX8 (n = 7 for all groups, for (D), p = 0.93 (WT + GFP vs. WT + SNX8-GFP), and <0.00001 for the other two comparisons. For (E), p = 0.55 (WT + GFP vs. WT + SNX8-GFP), 0.00008 (WT + GFP vs. Hexb−/−+GFP), 0.011 (Hexb−/−+GFP vs. Hexb−/−+SNX8-GFP)). Scale bar = 50 μm for (D) and 100 μm for (E). F Quantifications of the grip strength of forelimbs of 3-months-old WT and Hexb−/− mice injected with AAV-GFP or AAV-SNX8 (n = 7 for all groups, p = 0.53 (WT + GFP vs. WT + SNX8-GFP), 0.00033 (WT + GFP vs. Hexb−/−+GFP), 0.87 (WT + GFP vs. Hexb−/−+SNX8-GFP), 0.0034 (Hexb−/−+GFP vs. Hexb−/−+SNX8-GFP)). G Same mice as in (F) were put onto rotating rotarods, and the duration they can stay on rotarods was recorded and quantified (n = 7 for all groups, p = 0.31 (WT + GFP vs. WT + SNX8-GFP), <0.00001 (WT + GFP vs. Hexb−/−+GFP), 0.018 (Hexb−/−+GFP vs. Hexb−/−+SNX8-GFP)). For graphs, error bars are s.e.m, statistical comparison was done using one-way ANOVA, Tukey test (two sided, no adjustments). Source data are provided as a Source Data file.