Figure 1. Loss of Ttbk2 causes SCA-like phenotypes.

(A, B) Accelerating and steady speed rotarod performance test between Ttbk2^c.mut and littermate Controls. Ttbk2^c.mut animals have a shorter latency to fall time in both tests, indicative of impaired motor ability (a two-way ANOVA with Bonferroni’s multiple comparison test was used for calculating significance. p<0.0001 for accelerating rotarod test, and p=0.0001 for steady speed. n = 9 animals for Control, n = 8 animals for Ttbk2^c.mut). (C) Cerebellar tissue from Control and Ttbk2^c.mut mice at 3 months after loss of Ttbk2, immunostained for Calbindin to label Purkinje cells (red) and VGLUT2 to show climbing fiber synapses (green). Ttbk2^c.mut animals show a reduction in VGLUT2 positive synapses throughout the cerebellum 3 months after loss of TTBK2. Scale bar = 50 μm. (D) Quantification of molecular layer length in Ttbk2^c.mut cerebellar tissue (each point represents one measurement, 75 measurements overall. n = 3 animals. p=0.0011 by student’s unpaired t-test, error bars indicate SEM). (E) Quantification of VGLUT2+ puncta throughout PC dendrites. Ttbk2^c.mut animals show a significant reduction in these VGLUT2+ synapse terminals (each point represents one measurement, 15 measurements per genotype, n = 3 animals. p<0.0001 by student’s unpaired t-test, error bars indicate SEM). (F) Golgi stain showing spines on proximal dendrites of PCs in Control and Ttbk2^c.mut animals. Scale bar = 2 μm. (G) Quantification of number of spines per micron of dendrite. Ttbk2^c.mut PCs do not lose spine density on proximal dendrites at 4 months of age. Each point represents a measurement taken from a singular dendrite, n = 3 animals. (H) Immunostaining for IP3R (red) and nuclei (blue). Loss of IP3R expression is seen as early as P45 in Ttbk2^c.mut cerebellum. By 3 months after TMX injection, IP3R expression is no longer localized to secondary dendrites throughout the dendritic tree of PCs in Ttbk2^c.mut animals. Scale bar = 50 μm.

Figure 1—figure supplement 1. Ttbk2^c.mut animals have phenotypes shared with other ciliopathy models.

(A–C) Quantification of cilia abundance (A), molecular layer thickness (B), and VGLUT2 puncta (C) across various Controls. There is no significant difference between Control genotypes compared to each other for any of these metrics using a one-way ANOVA with Tukey’s correction In (A), each point represents a field counted, 16 fields were counted in total. In (B), each point represents a singular measurement, three distinct primary fissures were included per group, 25 measurements were counted per animal. In (C), each point represents puncta quantified from a 10 μm z-stack on the caudal side of the primary fissure. n = 1 animal per genotype for (A–C), error bars indicate SEM. (D–D’) Representative images of Control (D) and Ttbk2^c.mut (D’) mice. Scale bar = 2.5 cm. (E) Quantification of weight gain in Ttbk2^c.mut mice compared to Controls. (n = 7 animals, p=0.0003). (F) H and E staining of kidneys from Control and Ttbk2^c.mut mice. Ttbk2^c.mut mice have polycystic kidneys. Scale bar = 100 μm. (G) Western blot analysis of cerebellum lysate from Ttbk2^c.mut animals showing no TTBK2 expressed 3 months after tamoxifen injection. (H) Representative images of Control and Ttbk2^c.mut brains 3 months after tamoxifen treatment. Scale bar = 1 mm.

Figure 1—figure supplement 2. Dendritic trees of Ttbk2^c.mut PCs do not exhibit gross morphological changes.

Representative images of Golgi stained PCs throughout the cerebellum of Control (A) and Ttbk2^c.mut (B) mice. Dendritic trees are nearly indistinguishable between Controls and Ttbk2^c.mut mice.

Figure 1—figure supplement 3. Loss of Ttbk2 starting at P45 results in loss of VGLUT2 synapses.

(A) Representative images of 4-month-old Control and Ttbk2^c.mut cerebella injected at P45 showing VGLUT2+ synapses in green. Scale bar = 30 μm. (B) Quantification of the loss of VGLUT2 puncta in P45-injected, 4-month-old Ttbk2^c.mut compared to Control. Each point represents one measurement. n = 3 animals, p<0.0001 by unpaired student’s t-test, error bars represent SEM.

Figure 1—video 1. Ttbk2^c.mut mice have apparent motor coordination deficiencies.

Download video file ^{(2.7MB, mp4)}

Video showing the gait of age P45 Control animal versus the gait of a Ttbk2^c.mut animal treated with TMX at P21.