Endo-lysosomal genes are dysregulated in the SCA6 cerebellum.

(a) Schematic showing the endo-lysosomal system. (b) Heat map showing relative expression of endo-lysosomal associated transcripts in the cerebellum of 5 WT and 5 SCA6 mice (1 column per mouse). Genes are separated into groups based on their gene ontology categorization (GO term Endosome and Lysosome; the group “Endolysosome” denotes genes that belong to both GO term categories), only genes with an adjusted P value < 0.05 are shown.

List of DEGs in Endosome and Lysosome pathways

Early endosomes are enlarged and accumulate BDNF and TrkB in SCA6 Purkinje cells.

(a) Schematic showing BDNF and TrkB endocytosis and trafficking to early endosomes labeled with EEA1. (b) The early endosome marker EEA1 stains early endosomes in lobule 3 of the cerebellar vermis in WT and SCA6 mice, calbindin labels Purkinje cells. Scale bar, 100µm. (c) Closeup images of the early endosome marker EEA1 in lobule 3 of cerebellum, calbindin labels Purkinje cells (outlined). Scale bar, 10µm. (d) The area covered by EEA1 staining in Purkinje cells is increased in SCA6 mice compared to WT (P < 0.001; N = 6 WT mice, 173 cells; 5 SCA6 mice, 171 cells). (e) Representative images of the early endosome marker EEA1 and BDNF within Purkinje cells (outlined) in the anterior vermis. Scale bar, 10µm. (f) Relative staining level of BDNF within early endosome compartment is higher in the Purkinje cells of SCA6 mice compared to WT (P < 0.001; N = 6 WT mice, 173 cells; 5 SCA6 mice, 171 cells). (g) Representative images of the early endosome marker EEA1 and TrkB within Purkinje cells (outlined) in the anterior vermis. Arrowheads denote locations of significant TrkB accumulations. Scale bar, 10µm. (h) Relative staining level of TrkB within early endosome compartment is higher in the Purkinje cells of SCA6 mice compared to WT (P = 0.013; N = 4 WT mice, 79 cells; 4 SCA6 mice, 58 cells). Mann Whitney U test used for all statistical comparisons, * P < 0.05, *** P < 0.001.

Recycling of TrkB in recycling endosomes is impaired in SCA6 Purkinje cells.

(a) Schematic showing the return of TrkB to the cell membrane via recycling endosomes labeled with Stx13. (b) Representative images of the recycling endosome marker Stx13 and TrkB within Purkinje cells (outlined) of the anterior vermis. Scale bar, 10µm. (c) The area covered by Stx13 staining in Purkinje cells is unchanged between WT and SCA6 mice (P = 0.96; N = 4 WT mice, 67 cells; 4 SCA6 mice, 66 cells). (d) The relative level of TrkB within recycling endosomes was significantly decreased in SCA6 Purkinje cells (P = 0.0034; N = 4 WT mice, 67 cells; 4 SCA6 mice, 66 cells). Mann Whitney U test used for all statistical comparisons, ** P < 0.01, n.s. P > 0.05.

Late endosomes are reduced in SCA6 Purkinje cells and carry less BDNF.

(a) Schematic showing the transport of BDNF in late endosomes expressing Rab7. (b) Representative images of the late endosome marker Rab7 and BDNF within Purkinje cells (outlined) of the anterior vermis. Scale bar, 10µm. (c) The area covered by Rab7 staining in Purkinje cells is decreased in SCA6 mice compared to WT (P = 0.0019; N = 4 WT mice, 93 cells; 4 SCA6 mice, 81 cells). (d) The relative level of BDNF within late endosomes was significantly decreased in SCA6 Purkinje cells (P < 0.001; N = 4 WT mice, 93 cells; 4 SCA6 mice, 81 cells). Mann Whitney U test used for all statistical comparisons, ** P < 0.01, *** P < 0.001.

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.

Trafficking of an exogenous peptide to the lysosome shows impairment in lysosomal action on endocytosed cargo in SCA6.

(a) Schematic showing the use of Pepstatin A BODIPY FL construct to visualize endosome trafficking and Cathepsin D activity. (b) Representative images of Pepstatin A staining in the anterior vermis. Top row shows colocalization with lysosome marker Lamp1, Calbindin labels Purkinje cells (outlined). Scale bars, 10µm (c) The area covered by Pepstatin A staining in Purkinje cells was significantly decreased in SCA6 mice (P = 0.00076; N = 3 WT mice, 78 cells; 3 SCA6 mice, 66 cells). (d) The intensity of Pepstatin A staining within Purkinje cells was significantly decreased in SCA6 mice (P < 0.0001; N = 3 WT mice, 78 cells; 3 SCA6 mice, 66 cells). Mann Whitney U test used for all statistical comparisons, *** P < 0.001.

TrkB activation with 7,8-DHF rescues early endosomes deficits in SCA6 Purkinje cells.

(a) Schematic showing 7,8-DHF administration to SCA6 mice and the previously described therapeutic benefits of 7,8-DHF in SCA6 mice. (b) Representative images of the early endosome marker EEA1 and BDNF within Purkinje cells (outlined) of the anterior vermis. Scale bar, 10µm. (c) The area covered by EEA1 staining in Purkinje cells is decreased in SCA6 mice given DHF compared to those that received control water (P < 0.001; Student’s t test; N = 3 SCA6 mice, 50 cells; 3 SCA6 DHF mice, 63 cells). (d) Relative staining level of BDNF within early endosome compartment is decreased in the Purkinje cells of SCA6 mice that received DHF compared to controls (P < 0.001; Mann Whitney U test; N = 3 SCA6 mice, 50 cells; 3 SCA6 DHF mice, 63 cells). *** P < 0.001.

Multiple deficits in the endo-lysosomal system lead to abnormal localization of BDNF and TrkB in the Purkinje cells of SCA6 mice.

List of antibodies