Figure 3. Cell autonomous requirements for Ttbk2 in the cerebellum.

(A,B) Representative images of Control and Ttbk2^f/f;Pcp2Cre+ (Ttbk2^Pcp2) animals at age P30 (A) and P90 (B), immunostained for Calbindin to label PCs (red), VGLUT2 to label synapses (green), and nuclei (blue). VGLUT2 terminals are reduced in P90 Ttbk2^Pcp2 animals compared to P30 Ttbk2^Pcp2 animals. Scale bar = 20 μm. (C) Quantification of molecular layer thickness in P30 and P90 Ttbk2^Pcp2 and Control animals (each point represents one measurement, 75 measurements per genotype. n = 3 animals. No significant difference reported by one-way ANOVA with Tukey correction, error bars indicate SEM. (D) Quantification of VGLUT2+ puncta analysis in P30 and P90 Ttbk2^Pcp2 and Control animals. There are no differences in the number of puncta at P30; however these are significantly reduced by P90 (each point represents one field analyzed, five fields analyzed per animal, n = 3 animals. p=0.0098 by one-way ANOVA with Tukey correction, error bars indicate SEM). (E) Accelerating rotarod performance test of Ttbk2^Pcp2and littermate Controls from P30 to P90. Ttbk2^Pcp2 animals have a significantly shorter latency to fall time at P90 compared to P30 (a two-way ANOVA with Bonferroni’s multiple comparison test was used for calculating significance. p=0.1051 for P30 accelerating rotarod test, and p=0.0161 for P90 accelerating rotarod test). (F) Steady speed rotarod performance test of Ttbk2^Pcp2 and littermate Controls aging from P30 to P90. At P30 Ttbk2^Pcp2 animals do not have a shorter latency to fall time compared to Controls on the steady speed rotarod. However, by P90 there is a drastic reduction in latency to fall time for Ttbk2^Pcp2 animals compared to Controls, indicative of impaired motor ability with age (a two-way ANOVA with Bonferroni’s multiple comparison test was used for calculating significance. p=0.7819 for P30 steady speed rotarod test, and p=0.0023 for P90 steady speed rotarod test. n = 6 animals for Control, n = 4 animals for Ttbk2^Pcp2). (G) Representative images of Control and Ttbk2fl/fl;Slc1a3-CreER (Ttbk2^Slc1a3) animals at 4 months of age (3 months post TMX) treatment, immunostained for Calbindin to label PCs (red), VGLUT2 to label synapses (green) and nuclei (blue). Unlike Ttbk2^c.mut and Ttbk2^Pcp2 mice, there is no loss of VGLUT2 synapses throughout the PC dendrites of Ttbk2^Slc1a3 mice relative to Controls. (H) Quantification of molecular layer length in Ttbk2^Slc1a3 and Control animals (each point represents one measurement, 75 measurements per genotype. n = 3 animals. No significance reported by student’s unpaired t-test, error bars indicate SEM). (I) Quantification of VGLUT2+ puncta analysis in Ttbk2^Slc1a3 and Control animals. There is no difference in the numbers of puncta between these conditions (each point represents a field analyzed, five images analyzed per animal, n = 3 animals. No significance reported by student’s unpaired t-test, error bars indicate SEM).

Figure 3—figure supplement 1. Cilia are lost from PCs in Ttbk2^Pcp2 cerebellum.

(A) Images showing cilia localized to PCs soma in Control, and a centrosome lacking a primary cilium (arrowhead) in Ttbk2^Pcp2 PCs. PCs are stained for Calbindin (magenta), cilia are stained for ARL13B (red), and centrosomes are stained for γ-tubulin (green). Scale bar = 15 μm. (B) Quantification of cilia loss on PCs. Each point represents a 10 μm z-stack image of Purkinje cell layer (PCL )counted using a 63x objective. Control = 120 PCs counted, Ttbk2^Pcp2 = 84 PCs counted. n = 3 animals. p<0.0001 by unpaired student’s t-test, error bars represent SEM.