Lysosomes in SCA6 Purkinje cells are morphologically normal but there may be a deficit in late endosome maturation.
(a) Schematic showing the endo-lysosomal system as a series of compartments of increasingly acidic pH with lysosomes, expressing Lamp1, being most acidic. (b) Representative images of Purkinje cells in the anterior vermis stained for the lysosome membrane marker Lamp1 and LysoTracker as a marker of acidic compartments. Calbindin labels Purkinje cells (outlined). Scale bar, 10µm. The area covered by (c) Lamp1 staining and (d) LysoTracker staining in Purkinje cells is unchanged between WT and SCA6 mice (P = 0.26 for Lamp1; P = 0.20 for LysoTracker; N = 3 WT mice, 78 cells; 3 SCA6 mice, 76 cells). (e) The area of colocalization between Lamp1 and Lysotracker is unchanged between WT and SCA6 mice (P = 0.34; N = 3 WT mice, 78 cells; 3 SCA6 mice, 76 cells). (f) The number of lysosomes that were both Lamp1 positive and LysoTracker positive was not significantly different between genotypes (P = 0.72; N = 3 WT mice, 78 cells; 3 SCA6 mice, 76 cells). (g) The number of lysosomes that were Lamp1 positive, but LysoTracker negative was unchanged between genotypes (P = 0.83; N = 3 WT mice, 78 cells; 3 SCA6 mice, 76 cells). (h) The number of putative late endosomes undergoing maturation that were Lamp1 negative, but LysoTracker positive was significantly decreased in the Purkinje cells of SCA6 mice (P = 0.034; N = 3 WT mice, 78 cells; 3 SCA6 mice, 76 cells). Mann Whitney U test used for all statistical comparisons except number of Lamp1+ puncta (f) which was normally distributed and so a Student’s t test was used, * P < 0.05, n.s. P > 0.05.