Fig. 4: Histological, TEM, and immunofluorescent analyses of the ΔLYST-B6 forebrain, cerebellum and sciatic nerve.

a Representative image of Calbindin-28k stained cerebellum of 18-month-old WT control and ΔLYST-B6. Purkinje cells are dark brown cells above the blue-grey granular layer. ΔLYST-B6 mice showed a large loss of the Purkinje cell layer. b Average linear density of Purkinje cells counted in the cerebellum per millimeter (mm) of 3-, 6-, 9-, 12-, 15-, 18-month-old mice. 3 mo: n = 10 ΔLYST-B6 (5 male, 5 female), n = 10 WT (5 male, 5 female); 6 mo: n = 9 ΔLYST-B6 (4 male, 5 female), n = 10 WT (5 male, 5 female); 9 mo: n = 9 ΔLYST-B6 (4 male, 5 female), n = 9 WT (4 male, 5 female), n = 1 Heterozygous (male); 12 mo: n = 8 ΔLYST-B6 (4 male, 4 female), n = 7 WT (5 male, 2 female), n = 1 Heterozygous (male); 15 mo: n = 6 ΔLYST-B6 (3 male, 3 female), n = 4 WT (female only); 18 mo: n = 10 ΔLYST-B6 (5 male, 5 female), n = 10 WT (5 male, 5 female). ANOVA analysis: *** = P < 0.001. c Immunofluorescent staining of LAMP1 (lysosomes), GFAP (glial cells), and PCP4 (Purkinje cells), and fluorescent staining using DAPI (nuclei) of WT and ΔLYST-B6 brains at 3-, 6-, and 12-months old. Perinuclear lysosomes (arrowheads) were visible in glial cells of ΔLYST-B6 mice. d Representative TEM images of murine sciatic nerve of WT and ΔLYST-B6 at 3 and 24 months of age. Myelin is dark grey surrounding neurofibrils. Apoptotic whirling (arrows) in myelin layers and accumulation of cellular debris was observed in ΔLYST-B6. Scale bar, 2 µm e. Average number of toluidine-blue stained sciatic nerves in 3- and 24-month WT (n = 2) and ΔLYST-B6 (n = 2) mice. Student’s two-sample t-test: *P < 0.05, **P < 0.01. WT, wild-type; ΔLYST-B6, Lyst homozygous knockout (C57BL/6J background).