Fig. 6: Abnormal increase of ISGylated proteins in SCA1 Purkinje cells.

a ISG15 immunohistochemistry of Atxn1-KI and non-transgenic sibling mouse cerebellar cortexes during aging. Purkinje cells and neuropils of the molecular layer were stained with ISG15 from 4 to 56 weeks of age, while the increase of ISGylated proteins was highest at 4 weeks. b Quantitative analyses of ISG15 signals in three layers of the cerebellum. Twenty visual fields per mouse were observed in three C57BL6 and three Atxn1-KI mice. The two-sided Student’s t-test was used for statistical comparisons. The box plot shows the median and 25–75th percentile, and the whiskers represent data outside the 25–75th percentile range. c Western blot analysis of whole cerebellum tissue with anti-ISG15 antibody confirmed increased ISGylated proteins in Atxn1-KI mice. Anti-GAPDH served as loading control. Band intensities were obtained from four independent experiments. Tukey’s HSD test was used for multiple comparisons. The box plot shows the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. d Western blot analysis of whole cerebellar tissue with anti-IFNβ and anti-pTyr701-STAT1 antibodies. Anti-GAPDH served as loading control. Band intensities were obtained from four independent experiments. Tukey’s HSD test was used for multiple comparisons. The box plot shows the median and 25–75th percentile, and whiskers represent data outside the 25–75th percentile range. e The left scheme shows sequential activation of IFNβ, JAK-STAT, and ISGs. The right graph shows a summary of chronological changes of IFNβ, pSTAT, and ISG15 protein expression based on the ratios of their mean values between Atxn1-KI and non-transgenic sibling mice (N = 4), which was consistent with the expected scheme. f mRNA expression patterns of ISGylation-related genes during differentiation of human iPSC lines (N = 3), suggesting the significance of UbE2L6 in abnormal ISGylation associated with SCA1 pathology. The two-tailed Student’s t-test with the post-hoc Bonferroni correction was used for multiple comparisons. In RNA-seq, P = 0.036 in the right panel of UbE2L6 at the Purkinje cell stage, P = 0.005 (**) and P = 0.014 (#) in the left panel of ARIH1 at the EB stage, and P = 0.036 in the right panel of ARIH1 at the EB stage. In qPCR, P = 0.021 in the panel of UbE2L6 at the Purkinje cell stage, and P = 0.005 in the panel of ARIH1 at the EB stage. Corrected values mean cycle threshold (CT) values in qPCR that were normalized to GAPDH and referenced to iPSC stage in the calculations. Three independent RNA samples were used for analysis.