TTBK2 and primary cilia are essential for the connectivity and survival of cerebellar Purkinje neurons
- Duke University, United States
Figures
Figure 1 with 4 supplements
Loss of Ttbk2 causes SCA-like phenotypes.
(A, B) Accelerating and steady speed rotarod performance test between Ttbk2c.mut and littermate Controls. Ttbk2c.mut animals have a shorter latency to fall time in both tests, indicative of impaired …
Figure 1—figure supplement 1
Ttbk2c.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 …
Figure 1—figure supplement 2
Dendritic trees of Ttbk2c.mut PCs do not exhibit gross morphological changes.
Representative images of Golgi stained PCs throughout the cerebellum of Control (A) and Ttbk2c.mut (B) mice. Dendritic trees are nearly indistinguishable between Controls and Ttbk2c.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 Ttbk2c.mut cerebella injected at P45 showing VGLUT2+ synapses in green. Scale bar = 30 μm. (B) Quantification of the loss of VGLUT2 puncta in …
Figure 1—video 1
Ttbk2c.mut mice have apparent motor coordination deficiencies.
Video showing the gait of age P45 Control animal versus the gait of a Ttbk2c.mut animal treated with TMX at P21.
Figure 2 with 1 supplement
ION and glial cells are affected by loss of Ttbk2.
(A) Representative images of neurons in the inferior olivary nucleus (ION) located in the medulla. Neural somata are immunostained with NeuN (green). Insets show how the area was measured. Scale bar …
Figure 2—figure supplement 1
Identification of ION neurons within the medulla.
(A) The location of the mouse inferior olivary nucleus (ION, dotted outline) within the medulla oblongata of the brain stem. Scale bar = 10 mm. (B) Neurons within the ION are stained for Calbindin …
Figure 3 with 1 supplement
Cell autonomous requirements for Ttbk2 in the cerebellum.
(A,B) Representative images of Control and Ttbk2f/f;Pcp2Cre+ (Ttbk2Pcp2) animals at age P30 (A) and P90 (B), immunostained for Calbindin to label PCs (red), VGLUT2 to label synapses (green), and …
Figure 3—figure supplement 1
Cilia are lost from PCs in Ttbk2Pcp2 cerebellum.
(A) Images showing cilia localized to PCs soma in Control, and a centrosome lacking a primary cilium (arrowhead) in Ttbk2Pcp2 PCs. PCs are stained for Calbindin (magenta), cilia are stained for …
Figure 4 with 2 supplements
Aged Ttbk2c.mut animals lose Purkinje cells.
(A) Representative images showing folia of 4-month-old Ttbk2c.mut (top) and 6-month-old Ttbk2c.mut animals (bottom) with respective littermate Controls. Cerebellum tissue is stained for Calbindin to …
Figure 4—figure supplement 1
PC gaps are present throughout most cerebellar folia in 6-month -old Ttbk2c.mut animals.
(A) Representative images of Calbindin staining (red) throughout the cerebellum of 6-month-old Control and Ttbk2c.mut animals. Arrowheads denote gaps in folia present in Ttbk2c.mut animals. Scale …
Figure 4—figure supplement 2
Neurons in the cortex of 6-month-old Ttbk2c.mut animals have some Tau accumulation.
(A) Representative images of neurons in the cortex of 6-month-old Control, 4-month-old Ttbk2c.mut, 6-month-old Ttbk2c.mut and 55-week-old JNPL3(P301L) (B) animals showing AT8 phosphorylated Tau in …
Figure 5 with 1 supplement
Ttbk2 is critical for primary cilia stability on neurons.
(A–C) Representative images of cilia on indicated cell types throughout parts of the cerebellum and medulla. Sections from a mouse expressing an ARL13B-mCherry transgene (green) (Bangs et al., 2015) …
Figure 5—figure supplement 1
Cilia are lost throughout the brain of Ttbk2c.mut animals.
(A,B) Sagittal sections of Ttbk2c.mut brains stained for AC3 to label cilia (red) and DAPI for nuclei. Neurons in the hippocampus (A) and the cortex (B) have lost cilia 3 months after tamoxifen …
Figure 6
Ift88c.mut have fewer, shorter cilia throughout the cerebellum and mislocalization of ciliary membrane markers.
(A) Representative images illustrating cilia loss in the cerebellum of Ift88c.mut animals, immunostained for ARL13B to label cilia (red), Pericentrin (PCNT) to label centrosomes (green) and nuclei …
Figure 7
Loss of IFT88 recapitulates neurodegenerative phenotypes of Ttbk2c.mut animals.
(A) Cerebellar tissue from Control and Ift88c.mut mice at 3 months after loss of Ift88, immunostained for Calbindin to label Purkinje cells (red) and VGLUT2 to show climbing fiber synapses (green). …
Figure 8
Loss of PCs occurs in Ift88c.mut mice by 6 months of age.
(A) Cerebellar folia from Control and Ift88c.mut at 6 months of age, 5 months after loss of Ift88, stained for Calbindin (red) and DAPI (blue). 6-month-old Ift88c.mut mice begin to show PC gaps …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | Ttbk2tm1a(EUCOMM)Hmgu | International Mouse Strain Resource | ||
| Genetic reagent (M. musculus) | Actb:FLPe | The Jackson Laboratory | #003800 | |
| Genetic reagent (M. musculus) | Ift88flox | The Jackson Laboratory | #022409 | |
| Genetic reagent (M. musculus) | Ubc-CreER | The Jackson Laboratory | #007001 | |
| Genetic reagent (M. musculus) | Slc1a3-CreER | The Jackson Laboratory | #012586 | |
| Genetic reagent (M. musculus) | Pcp2-Cre | The Jackson Laboratory | #010536 | |
| Chemical compound, drug | Tamoxifen | Sigma | T5648 | Working concentration: 20 mg/mL |
| Antibody | Mouse anti-ARL13B (monoclonal) | NeuroMabs | N295B/66 | 1:500 |
| Antibody | Rabbit anti-ARL13B (polyclonal) | Proteintech | 17711–1-AP | 1:500 |
| Antibody | Mouse anti-gamma-tubulin (monoclonal) | Sigma | T6557 | 1:1000 |
| Antibody | Rabbit anti- Calbindin D28K (monoclonal) | Cell signalling technologies | 13176S | 1:250 |
| Antibody | Guinea pig anti-VGLUT2 (polyclonal) | EMD Millipore | AB2251 | 1:2500 |
| Antibody | Rabbit anti-NeuN (monoclonal) | Abcam | Ab177487 | 1:1000 |
| Antibody | Rabbit anti-AC3 (polyclonal) | Santa Cruz | SC-588 | 1:10, discontinued |
| Antibody | Rabbit anti-AC3 (polyclonal) | Abeomics | 34–1003 | 1:100 |
| Antibody | Chicken anti-GFAP (polyclonal) | EMD Millipore | AB5541 | 1:500 |
| Antibody | Rabbit anti-IP3 (monoclonal) | Abcam | AB108517 | 1:200 |
| Antibody | Rabbit anti-FoxP2 (polyclonal) | Abcam | AB16046 | 1:400 |
| Antibody | Mouse anti-AT8 (monoclonal) | Thermo | MN1020 | 1:100 |
| Software | ImageJ | ImageJ: https://imagej.nih.gov/ij/ | ||
| Software | GraphPad Prism | GraphPad Prism: https://www.graphpad.com/scientific-software/prism/ | ||
| Commercial assay or kit | FD Rapid Golgistain Kit | FD Neurotechnologies | ||
| Commercial assay or kit | BSA Protein Assay Kit | Thermo | #23227 |
