Cerebellum-enriched protein INPP5A contributes to selective neuropathology in mouse model of spinocerebellar ataxias type 17

Spinocerebellar ataxias 17 (SCA17) is caused by polyglutamine (polyQ) expansion in the TATA box-binding protein (TBP). The selective neurodegeneration in the cerebellum in SCA17 raises the question of why ubiquitously expressed polyQ proteins can cause neurodegeneration in distinct brain regions in different polyQ diseases. By expressing mutant TBP in different brain regions in adult wild-type mice via stereotaxic injection of adeno-associated virus, we found that adult cerebellar neurons are particularly vulnerable to mutant TBP. In SCA17 knock-in mice, mutant TBP inhibits SP1-mediated gene transcription to down-regulate INPP5A, a protein that is highly abundant in the cerebellum. CRISPR/Cas9-mediated deletion of Inpp5a in the cerebellum of wild-type mice leads to Purkinje cell degeneration, and Inpp5a overexpression decreases inositol 1,4,5-trisphosphate (IP3) levels and ameliorates Purkinje cell degeneration in SCA17 knock-in mice. Our findings demonstrate the important contribution of a tissue-specific protein to the polyQ protein-mediated selective neuropathology.


Supplementary Figure 1
Overexpression of mutant TBP in different brain regions. a Representative immunofluorescence images showing the transduction of AAV-TBP in the cerebellum, striatum and prefrontal cortex. Scale bar = 20 μm. b Immunofluorescence staining (Scale bar = 100 μm) and Nissl staining (Scale bar = 10 μm) indicated that TBP-105Q dramatically decreased the number of neuronal cells in the cerebellum. c The number of Nisslstained cells per image is presented as mean ± SEM. One-way ANNOVA followed with Tukey's multiple comparisons test was performed, F = 14.36, *** P < 0.0005, **** P < 0.0001. Data are represented as mean ± SEM. n = 3 mice per group, three images were used to count from each mouse. Source data are provided as a Source Data file.

Supplementary Figure 2
Overexpression of mutant TBP causes increased reactive astrogliosis in different brain regions. a-c Immunofluorescence staining showing increased reactive astrogliosis in a polyQ length dependent manner in the cerebellum (a), striatum (b) and prefrontal cortex (c). Scale bar = 50 μm. d Quantification of GFAP staining intensity. Data are presented as fold change compared to TBP-13Q (dashed line) in the cerebellum, striatum, and prefrontal cortex (mean ± SEM). One-way ANNOVA followed with Tukey's multiple comparisons test was performed, cerebellum, F = 78.13; striatum, F = 10.58; prefrontal cortex, F = 152; * P < 0.05, *** P < 0.0005, **** P < 0.0001. Data are represented as mean ± SEM. n = 3 mice per group, three images were used to count from each mouse. Source data are provided as a Source Data file.

Supplementary figure 3 (continued)
Supplementary Figure 3 Gene enrichment analyses of differentiated expressed genes in the cerebellum, striatum and prefrontal cortex in SCA17 knock-in mice. a-f Top 10 enriched pathways of differentiated expressed genes in the cerebellum (a-b), striatum (c-d), and prefrontal cortex (e-f). g Common 28 up-regulated genes in three brain regions was enriched in immune related pathways. h-m Top 10 enriched GO process of differentiated expressed genes in the cerebellum (h-i), striatum (j-k), and prefrontal cortex (l-m). Source data are provided as a Source Data file.

Supplementary Figure 4
Three isoforms of mouse Inpp5a gene. a Schematic map of three known isoforms of the mouse Inpp5a gene. The coding sequences of isoform A, B and C are 1239, 1269 and 1263 bp respectively. Sequence alignment reveals that isoform A and isoform B differ in their C terminus; isoform C has a distinct promoter and exon1 sequence. The location of the GC box (red rectangle), gRNA targeting sites (red triangle), antibody epitope (curly bracket) and PCR primers (arrow) are indicated. b Polymerase chain reaction (PCR) analysis using isoform-specific primers showed different expression levels of two Inpp5a isoforms in the cerebellum (CB) and prefrontal cortex (PFC). Common primers, Forward 1 (1F): 5'-GCA TCC TCA TGT CCC TGT CT-3' and reverse 1 (1R): 5'-TTA GGA GGA TGA GTT GGA TA-3', amplified 272 bp PCR products for isoform A or C and 221 bp for isoform B. Isoform C specific primers, 2F: 5'-GAA AGA CAT GGC CTG GAG AG-3' and 2R: 5'-GGA GGC CTC GTA GTT TTT CC-3', amplified a 146 bp PCR product. Since there were no 221 bp PCR products, isoform B may not be expressed or may be expressed at an undetectable level in the cerebellum and prefrontal cortex. A positive control (upper band: 300 bp, lower band: 250 bp) demonstrated that the resolution of 2% agarose gel is sufficient to distinguish small bands. c Quantification of PCR products. GAPDH is used as the internal control. Densitometric ratios of Inpp5a to GAPDH were normalized to CB, and were analyzed with Student's t test. t = 2.519, # P = 0.0655, t = 8.059, ** P = 0.0013, n = 3 mice per group. d RNA sequencing identified that Inpp5a, but not other Inpp5 paralogs, was significantly downregulated in the cerebellum of SCA17 knock-in (KI) mice, compared to wild type (WT) mice. The data were analyzed with Student's t test, t = 8.474, ** P = 0.0011. n = 3 mice per group. Data are represented as mean ± SEM. Source data are provided as a Source Data file. Figure 5 Mutant TBP affects SP1 transcriptional activity. a Western blotting shows the expression of SP1 with TBP-13Q, -44Q, -68Q, or -105Q in transfected HEK293 cells. b Luciferase activity analysis of HEK293 cells transfected with SP1 and TBP-13Q, -44Q, -68Q or -105Q, suggesting that mutant TBP affects SP1's transcriptional activity on Inpp5a independent of polyQ repeat length. Luminescence intensity was analyzed with One-way ANNOVA followed with Tukey's multiple comparisons test. F = 22.87, ** P < 0.005, *** P < 0.0005, **** P < 0.0001. Data are represented as mean ± SEM. Source data and full blots are provided as a Source Data file. Figure 6 Verification of targeting the mouse Inpp5a by its gRNA and CRISPR/Cas9. a T7 Endonuclease I (T7E1) assay using genomic DNA from Neuro2a cells transfected with Cas9 plus control gRNA or Inpp5a gRNA verified the targeting of the mouse Inpp5a gene by its gRNA. b Representative immunofluorescence images of AAV-control gRNA-or AAV-Inpp5a gRNA-injected cerebellum in Cas9 transgenic mice without mutant TBP. RFP (red) represents viral transduction and gRNA expression. Arrow indicates AAV-Inpp5a gRNA-injected region that shows loss of calbindin-labeled Purkinje cells (green) as compared with the region without AAV transduction or with AAV-control gRNA injection.

Supplementary
Scale bar = 100 μm. c Representative Nissl staining images (left panel) of AAV-GFP or AAV-Inpp5a injected cerebellum in SCA17 mice. Scale bar = 50 μm. Quantification of Purkinje cells (right panel) was analyzed with Student's t test, t = 3.581, ** P = 0.0025. Data are represented as mean ± SEM. n = 3 mice per group, three images were used to count from each mouse. Source data are provided as a Source Data file.