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. 2020 Mar 3;48(7):3678–3691. doi: 10.1093/nar/gkaa140

Figure 4.

Figure 4.

Itpr1 expression is reduced the most in the cerebella of PolbNes-CreAtm−/− and Xrcc1Nes-CreAtm−/− animals that show severe ataxia. (A) Hierarchical cluster analysis of cerebellar RNAseq data. The Venn diagram indicates the numbers of genes significantly and uniquely up- or downregulated in each mutant cerebellum. Hierarchical clustering of the top 14 genes among 44 upregulated and 23 downregulated genes unique to the PolbNes-CreAtm−/− cerebellum is shown. The color scale of the normalized expression values ranges from 0 (blue) to 12 (yellow) to 18 (red). The gene list also includes related human genetic disease information obtained from the public database (http://omim.org and http://rarediseases.info.nih.gov). The most differentially expressed gene in the PolbNes-CreAtm−/− cerebellum was Itpr1, compared with that in the control, Atm−/− and PolbNes-Cre cerebella at 3 weeks of age. ITPR1 mutations or deletions are responsible for Spinocerebellar ataxia 15/29 or Gillespie syndrome. (B) RNAseq profiling of Itpr1 in the mouse cerebellum at 3 weeks of age; the Itpr1 gene is located on chromosome 6 (Chr6) 108,213,083–108,551,116. The vertical lines in the gene structure indicate exons, and the zigzag lines indicate introns. The black peaks matched with exon locations represent the normalized RNAseq reading (y-axis scale, 0–1900), and these peaks are negligible in the PolbNes-CreAtm−/− cerebellum, indicating the reduction of Itpr1 expression in this particular genetic background. (C) Immunostainings of ITPR1 and CAR8, which are highly and exclusively expressed in the Purkinje cell layer of the cerebellum (coronal plane). ITPR1 immunopositivity is almost absent in the PolbNes-CreAtm−/− cerebellum. Also reduced CAR8 staining is visible in the double knockout cerebellum. Mo, Molecular layer; Pur, Purkinje cell layer; Gr, Granule cell layer. (D and E) Western blots (D) and quantification (E) analyses for ITPR1, ITPR2, ITPR3 and CAR8 in the cerebral cortices and cerebella at 3 weeks of age. A dramatic reduction of ITPR1 is evident only in the PolbNes-CreAtm−/− and Xrcc1Nes-CreAtm−/− cerebella. Both PolbNes-CreAtm−/− and Xrcc1Nes-CreAtm−/− animals displayed severe ataxia. The expressions of ITPR1 and CAR8 in the cerebral cortex were barely detected. The protein levels of indicated proteins were measured using ImageJ (densitometry) and were normalized to those of tubulin (E). N = 3. All bars indicate mean ± SEM; **, P < 0.01.