Fig 1: Activity of cathepsin B in cortical lysates of 3- and 6- month-old mice.Total cortical proteins were used to determine enzyme activity, as the gain in fluorescence during the linear portion of the reaction curve relative to the wildtype mean. (A) Cortical relative cathepsin B activity at 3-months of age, graphed as group means ± SEM (n = 18 per genotype; (22 females and 14 males in total), revealed no statistically significant effects of dupCstb or sex (univariate ANOVA). (B) Cortical relative cathepsin B activity at 6-months of age, graphed as group means ± SEM, (dupCstb n = 7, wildtype n = 9; 7 females and 9 males in total), revealed no statistically significant effects of dupCstb or sex (univariate ANOVA). Recombinant human cathepsin B (R&D Systems, Cat. No. 953-CY-010) was used as a positive control. (C) Representative western blot probed with an anti-cathepsin B antibody that recognises pro-cathepsin B and cathepsin B heavy chain and an anti-β-actin antibody. (D) Protein band densities of pro-cathepsin B and cathepsin B heavy chain were quantified using ImageJ, normalised to β-actin, and are shown as cathepsin B/pro-cathepsin B ratio in the dupCstb group relative to the ratio in wildtype, graphed as group mean ± SEM (dupCstb n = 7, wildtype n = 9; 7 females and 9 males in total).
Fig 2: Restoring lysosomal acidification promoted the clearance of protein aggregates and the combined treatment of LPPs facilitated the removal of protein aggregates. (A) GFP intensity was measured per field after GFP-HTT Q74 exon 1 overexpression and treatment with various pH LPPs (100 µg/ml) for 72 h in U87MG cells (n = 3 biological replicates). Scale bars: 275 μm (overview), 55 μm (magnified). (B) Aβ oligomers were stained and measured per field after treatment with 10 µM Aβ oligomers with various pH LPPs (100 µg/ml) for 72 h in SK-N-SH cells (n = 5 biological replicates). Scale bars: 20 μm (overview), 4 μm (magnified). (C, D) Western blot analysis of protein expression of HTT Q74 exon 1 and Aβ oligomers after LPPs treatment. (E, F) Western blot analysis of protein expression of HTT and Aβ after treatment of pH 4 LPPs in a dose-dependent manner (50–200 µg/ml). (G) Western blot analysis of protein expression of Aβ oligomers and Cathepsin B activity after treatment with CTSB-LPPs (50–200 µg/ml) for 24 and 6 h, respectively (n = 6 biological replicates). (H) Western blot analysis of Aβ oligomers and Cathepsin B activity after combined treatment with pH 4 LPPs (100 µg/mL) and CTSB-LPPs (100 µg/mL) for 24 and 6 h, respectively (n = 6 biological replicates). Band intensities were analyzed using ImageJ and normalized to actin as a loading control. Relative intensity values are shown below each band. Data are presented as bar graphs showing mean ± S.E. The p-values were calculated using one-way ANOVA with Bonferroni correction (*, p < 0.05, **, p < 0.01; ***, p < 0.001)
Fig 3: Acidification of lysosomes through LPPs restored lysosomal dysfunction caused by protein aggregates. (A) LysoTracker staining intensity in U87MG cells after 24-hour transfection with HTT Q74 exon 1 (1 µg) and 6-hour treatment with 100 µg/ml LPPs of different pH was quantified per field (n = 5 biological replicates). Scale bar represents 275 μm. (B) CTSB activity was analyzed under identical conditions of (A) (n = 4 biological replicates). (C) CTSB activity was analyzed at various concentrations of pH 4 LPPs (50–200 µg/ml) (n = 6 biological replicates) for 6 h. (D) Cell viability analysis under HTT Q74 exon 1 transfection with 100 µg/ml LPPs of different pH (n = 10 biological replicates). (E) LysoTracker activity in SK-N-SH cells after 24-hour treatment with 10 µM Aβ oligomers followed by 6-hour treatment with 100 µg/ml LPPs of different pH was quantified per field (n = 5 biological replicates). Scale bar represents 125 μm. (F, G) CTSB activity was analyzed under identical conditions of (E) (n = 4 biological replicates) and different concentration of pH 4 LPPs (50–200 µg/ml) (n = 6 biological replicates) for 6 h. (H) Cell viability analysis under Aβ oligomers treatment with 100 µg/ml LPPs of different pH (n = 10 biological replicates). (I) SK-N-SH cells were treated with 10 µM of Aβ oligomers for 24 h and visualized using LAMP2A antibody, and quantified per cell (n = 5 biological replicates). Scale bars: 10 μm (overview), 3 μm (magnified). Data are presented as bar graphs showing mean ± S.E. The p-values were calculated using one-way ANOVA with Bonferroni correction (*, p < 0.05, **, p < 0.01; ***, p < 0.001)
Fig 4: Acidic LPPs restored lysosomal acidification and CTSB activity. (A) Analysis of LysoTracker staining intensity in U87MG cells after treatment with 200 nM BafA1 and 100 µg/mL LPPs for 6 h. LysoTracker fluorescence intensities were quantified per field and normalized to the control (n = 6 biological replicates). Scale bar represents 125 μm. (B) The autophagy flux was measured after mCherry-GFP-LC3 transfection in U87MG cells under treatment with 100 µg/mL LPPs of different pH. The ratio of red to yellow puncta was analyzed per cell using ImageJ-based analysis (n = 3 biological replicates). Scale bar represents 5 μm. (C) Western blot analysis of autophagy flux markers after treatment with 200 nM BafA1 and 100 µg/mL LPPs for 6 h. Protein expression levels of p62 and LC3 were assessed by Western blot. Band intensities were analyzed using ImageJ and normalized to actin as a loading control. Relative intensity values are shown below each band. (D, E) Cathepsin B activity in U87MG treated with BafA1 (n = 4 biological replicates) and different pH LPPs or different concentrations of pH 4 LPPs (n = 6 biological replicates) for 6 h. (F) Cell viability analysis under 200 nM BafA1 treatment with 100 µg/ml LPPs of different pH (n = 10 biological replicates). Data are presented as bar graphs showing mean ± S.E. The p-values were calculated using one-way ANOVA with Bonferroni correction (*, p < 0.05, **, p < 0.01; ***, p < 0.001)
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