Fig. 1

Disrupted trafficking and activity of cathepsins in H4 cells overexpressing αSyn. a Representative Western blot analysis of αSyn in H4 cells overexpressing αSyn under an inducible tetracycline (tet)-off promoter. The addition of doxycycline (DOX) for 24 h, 48 h, and 72 h results in down-regulation of αSyn protein levels compared to control samples (cells treated with DMSO for 72 h). For loading control β-actin staining was performed (n = 3). b Representative Western blot analyses of the heavy chain (hc) representing the mature form of cathepsin D (CTSD), cathepsin B (CTSB) and cathepsin L (CTSL) in H4 cells overexpressing αSyn under an inducible tetracycline (tet)-off promoter. αSyn expression was downregulated by the addition of DOX for 24 h, 48 h and 72 h. Control cells were treated with the same amount of the DOX dissolvent DMSO (high αSyn) for 72 h. As a loading control β-actin was stained to ensure equal protein load. c Corresponding quantification of Western blot analysis for CTSD (left), CTSB (middle) and CTSL (right). Cells were treated with DMSO for 72 h or with DOX for 24, 48, or 72 h and were normalized to β-actin and expressed as fold change, compared to high αSyn levels (DMSO) (n = 3). d Schematic illustration of the maturation process of CTSD, CTSB, and CTSL. The inactive pro-forms of all three enzymes are generated in the endoplasmatic reticulum (ER). The pro-forms are transported into endosomes, where they are cleaved, resulting in the formation of single chains (sc) of the enzymes. The final maturation step takes places in the lysosomes, where a further cleavage of the sc leads to the production of a heavy and a light chain (hc and lc, respectively). e Western blot analysis of hc of CTSD, CTSB and CTSL in lysosome-enriched fractions of DMSO- and DOX (for 72 h)-treated H4 cells. All three investigated cathepsins show increased levels upon αSyn expression downregulation (cell treated with DOX) (n = 2–6). f Lysosomal CTSD, CTSL, and CTSB activity in H4 cells high in αSyn (DMSO) compared to low αSyn (treated with DOX for 72 h). Lysosomal CTSD activity was assessed by measuring activity within fractions enriched for lysosomes. Lysosomal CTSB and CTSL were determined by live-cell activity assay (n = 6–7). g Left: Representative immunofluorescence images of CTSD (red) co-stained with lysosomal protein LAMP2 (green) in H4 cells high in αSyn (DMSO) and low in αSyn (DOX). The nucleus is shown in blue and stained with DAPI. Scale bar: 10 µm. Right: Quantification of CTSD:LAMP2 co-staining determined by Pearson correlation coefficient (n = 11 individual cells per group). h Left: Representative immunofluorescence images of CTSB (red) co-stained with lysosomal protein LAMP2 (green) in H4 cells high in αSyn (DMSO) and low in αSyn (DOX). The nucleus is shown in blue and stained with DAPI. Scale bar: 10 µm. Right: Quantification of CTSB:LAMP2 co-staining determined by Pearson correlation coefficient (n = 23–24 individual cells per group). Statistical analyses were performed by using one-way ANOVA together with Dunnett’s multiple comparison test for a, c, and e, and two-tailed unpaired Student’s t-tests for f–h. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05