Fig 2: Inpp5a Knockdown leads to Purkinje cell degeneration, whereas Inpp5a overexpression reduces Purkinje cell degeneration.a Schematic diagram of AAV-Inpp5a gRNA and AAV-Inpp5a vectors. b Western blotting analysis of INPP5A and calbindin in AAV-control gRNA or AAV-Inpp5a gRNA-injected 3-month-old EIIa-Cas9 transgenic mice cerebellum (left panel). The densitometric ratios of INPP5A to β-actin and calbindin to β-actin were normalized to control and analyzed with Student’s t test (right panel). Calbindin, t = 3.095, *P = 0.0213; Inpp5a, t = 5.79, **P = 0.0012, n = 4 mice per group. c Representative immunofluorescence images of AAV-gRNA-injected Cas9 mouse cerebellum without mutant TBP. d Western blotting showing the expression of AAV-Inpp5a or AAV-GFP in the injected KI mouse cerebellum. e Representative immunofluorescence images of AAV-Inpp5a or AAV-GFP-injected 5-month-old KI mouse cerebellum. f Western blotting showing increased calbindin in the cerebellum of SCA17 mice after injection of AAV-Inpp5a. AAV-GFP injection served as control. g Quantification of Purkinje cells (left panel) in e was analyzed with Student’s t test, t = 3.483, *P = 0.0011, n = 4 mice per group, three images were used to count from each mouse. The densitometric ratios of calbindin to vinculin in f were normalized to WT and analyzed with one-way ANOVA followed with Tukey’s multiple comparisons test. F = 12.32, *P < 0.05, **P < 0.005. n = 5 mice per group. h IP3 ELISA assay of cerebellar lysates from 2-month-old KI mice and age-matched WT mice (left) and from AAV-Inpp5a or AAV-GFP-injected 3-month-old KI mice (right). IP3 levels (fold of WT or fold of GFP) were analyzed with Student’s t test, t = 3.77, *P = 0.013 (left); t = 2.681, *P = 0.0365 (right), n = 3–4 mice per group. i A proposed model for selective neurodegeneration in SCA17. Mutant TBP downregulates INPP5A by inhibiting SP1 from activating Inpp5a transcription. The reduction of INPP5A dysregulates the IP3/Ca2+ pathway, leading to Purkinje cell degeneration. Scale bar = 100 μm. Data are represented as mean ± SEM. Source data and full blots are provided as a Source Data file.

Fig 3: Cerebellum-enriched INPP5A is downregulated in SCA17 knock-in mice.a Heatmaps of dysregulated genes hierarchically clustered from the cerebellum (CB), striatum (STR), and prefrontal cortex (PFC) of 3-month-old SCA17 KI and age-matched WT mice (n = 3–4 mice per group). Data were presented as log2 fold change with adjusted P value < 0.05. b Summary of the numbers of differentially expressed genes in the CB, STR, and PFC in KI mice. c Venn diagrams indicating the numbers of downregulated or upregulated genes in the CB, STR, and PFC. d Western blotting of INPP5A levels in different tissues from 3-month-old WT mice. Vinculin was used as a loading control. e Real-time PCR assay of Inpp5a mRNA levels in the CB, STR, and PFC from 3-month-old KI mice. The relative mRNA levels of Inpp5a were obtained by normalizing values to an internal control, GAPDH, and analyzed with Student’s t test, t = 5.493, **P = 0.0054, n = 3 mice per group. f, g Western blotting (f) and immunofluorescent staining (g) of INPP5A in the cerebellum from 3-month-old WT and KI mice. Scale bar = 100 µm. In f, densitometric ratios of NeuN to ß-actin were normalized to WT and analyzed with Student’s t test, t = 6.903, **P = 0.0023, n = 3 mice per group. Data are represented as mean ± SEM. Source data and full blots are provided as a Source Data file.

Fig 4: PolyQ expansion promotes TBP to preferentially form aggregates in the cerebellum.a A schematic diagram of AAV plasmids expressing human TBP with different polyQ repeat lengths. b Western blot analysis of HEK293 cells transfected with AAV-TBP plasmids confirming the expression of TBP with different polyQ repeats (13Q, 44Q, 68Q, and 105Q). Vinculin was used as a loading control. c A diagram of stereotaxic injection of AAV-TBP into the cerebellum, striatum, and prefrontal cortex in 3-month-old wild-type mice. d–f TBP immunofluorescent staining of the cerebellum (d), striatum (e), and prefrontal cortex (f) from AAV-TBP-injected mouse brains. Anti-1TBP18 antibody was used. Enlarged images in the boxed areas were also presented. Red arrows indicate aggregates. Scale bar = 20 μm. g Quantification of the accumulated TBP aggregates in the cerebellum, striatum, and prefrontal cortex. One-way ANOVA followed with Tukey’s multiple comparisons test was performed; cerebellum, F = 1491; striatum, F = 311.6; prefrontal cortex, F = 354.6, ****P < 0.0001. n = 4 mice per group, three images per brain region from each mouse were used to count. Data are represented as mean ± SEM. Source data and full blots are provided as a Source Data file.

Fig 5: Selective neuronal loss is also caused by endogenously expressed mutant TBP.a Western blotting analysis of TBP expression in the cerebellum, striatum, and prefrontal cortex from 3-month-old SCA17 knock-in (KI) and wild-type (WT) mice. Vinculin was used as a loading control. b Immunofluorescent staining of Purkinje neurons with anti-calbindin antibody in the cerebellum of 5-month-old WT and KI mice. Scale bar = 100 µm. c Western blotting analysis of calbindin and NeuN levels in the cerebellum of 5-month-old WT and KI mice. The densitometric ratios of calbindin to vinculin and NeuN to vinculin were normalized to WT and analyzed with Student’s t test, t = 6.305, **P = 0.0032, n = 3 mice per group. d Western blotting of DARPP32 levels in the striatum of 5-month-old WT and KI mice. ß-Actin was used as a loading control. The densitometric ratios of DARPP32 to ß-actin were normalized to WT and analyzed with Student’s t test, t = 5.872, **P = 0.0011, n = 4 mice per group. e Immunofluorescent staining with anti-DARPP32 antibody in the striatum of 5-month-old WT and KI mice. Scale bar = 50 µm. Quantitative assessment of the number of medium spiny neurons labeled by DARPP32 per image field are also presented, t = 2.301, *P = 0.0352, n = 3 mice per group, three images from each mouse were used to count. f Western blotting of NeuN levels in the prefrontal cortex of 5-month-old WT and KI mice. The densitometric ratios of NeuN to vinculin were normalized to WT and analyzed with Student’s t test. n = 3 mice per group. Data are represented as mean ± SEM. Source data and full blots are provided as a Source Data file.