Fig 2: Trisomy of Hsa21 does not alter the localisation of cystatin B (CSTB) to the lysosome. a–c Total cellular proteins from disomic and trisomy 21 human primary fibroblasts were separated into cytosolic and nuclear fraction and the abundance of CSTB quantified by western blot, LSD1 was used as a marker of the nuclear fraction. b Relative CSTB abundance was increased by trisomy 21 (Mann–Whitney U test p = 0.0286) but the nuclear/cytosolic CSTB ratio was not altered (Mann–Whitney U test p = 0.8857). d, e Colocalisation of CSTB (cyan) with LAMP1 (magenta) did not differ between disomic (Pearson’s Correlation R = 0.555) and trisomy 21 cells (Pearson’s Correlation R = 0.582) (Mann–Whitney U test p = 0.8571). For d and e, individual data points are technical means of 3 technical replicates for n = 4 disomic and n = 3 trisomy 21, independent cell lines, error bars SEM. *p < 0.05

Fig 3: Increased abundance of CSTB protein in fibroblasts isolated from individuals who have trisomy of Hsa21 is not sufficient to decrease total or lysosomal cathepsin activity compared to disomic matched cells. Cathepsin B activity and CSTB protein abundance were measured in human primary fibroblasts. a, b Trisomy of chromosome 21 significantly increases CSTB protein abundance as measured by western blot (Mann–Whitney U test p = 0.0286). c Trisomy of chromosome 21 does not alter total cathepsin B activity as measured by biochemical assay (rate of cleavage of Ac-RR-AFC), (Mann–Whitney U test p = 0.8857) or d by BMV109 fluorescence (red) quantified by in gel assay based upon enzyme identification by molecular weight (28 kDa) normalized to total protein measured by Coomassie blue (Mann–Whitney U test p = 0.6857). e Trisomy of Hsa21 does not alter cathepsin activity within lysosomes as measured by lysotracker (cyan) colocalised BMV109 fluorescence (magenta) (Manders’ coefficient (disomic) = 0.302; (Trisomy 21) = 0.339) (Mann–Whitney U test p = 0.2000). Individual data points are group means for n = 4 disomic and n = 4 trisomy 21, independent cells lines (three technical replicates for western blots and 15–20 cells per lines for immunofluorescence), error bars SEM, p < 0.05

Fig 4: Cystatin B (CSTB) abundance is increased and cathepsin B activity is reduced in the brains of individuals with AD-DS, independently of changed cathepsin B protein level or cathepsin D activity. Cystatin B (CSTB) (a–d) and cathepsin B (a, e, f Ab-3 or b, g ab92955), protein abundance measured by western blot in human temporal cortex from people who had AD-DS, EOAD and healthy ageing. c, d Type of case altered the abundance of CSTB protein (ANOVA ß-actin F (2,18) = 9.087, p = 0.002; GAPDH F(2,21) = 7.500, p = 0.003). CSTB abundance was higher in individuals who had AD-DS than those with EOAD (pairwise comparisons with Bonferroni correction ß-actin p = 0.008, GAPDH p = 0.020) and healthy controls from the general population (pairwise comparisons with Bonferroni correction, ß-actin p = 0.024; GAPDH p = 0.013), with no difference in abundance between individuals with EOAD and control individuals (pairwise comparisons ß-actin and GAPDH p = 1.000). e–g The abundance of mature cathepsin B protein, e, f Ab-3 or g ab92955, was not altered by the type of case (Ab-3 ANOVA ß-actin F(2,16) = 3.051, p = 0.075; GAPDH F (2,21) = 2.141, p = 0.143) (ab92955 ANOVA F(2,18) = 0.970, p = 0.398). h Type of case affected cathepsin B activity (ANOVA F(2,22) = 9.027, p < 0.001); activity was significantly higher in individuals who had EOAD than in controls (pairwise comparisons with Bonferroni correction p = 0.004), but lower in individuals with AD-DS than those with EOAD (pairwise comparisons with Bonferroni correction, p = 0.012), with no difference between individuals with AD-DS and control individuals (pairwise comparisons p = 1.000). (i) Within case types, no significant correlations between age (in years) at time of death were observed (simple linear regression, Healthy ageing r2 = 0.1962 p = 0.199, EOAD r2 = 0.0989 p = 0.376 and AD-DS r2 = 0.0079 p = 0.807). j Type of case significantly affected cathepsin D activity (ANOVA F(2,16) = 6.360, p = 0.009); activity was significantly lower in individuals who had EOAD than in healthy controls (pairwise comparisons with Bonferroni correction p = 0.022), no significant difference in activity was observed between AD-DS and healthy ageing controls (pairwise comparisons with Bonferroni correction, p = 0.180), with no difference between in individuals with AD-DS and those with EOAD (pairwise comparisons p = 1.000). Individual data points are technical means for independent biological samples, error bars SEM. *p < 0.05 and **p < 0.01