Fig 2: rHsCTSD treatment decreases lysosomal SNCA and improves endo-lysosomal and autophagic system in SNCA overexpressing H4 cells. (A) Representative immunoblot of lysosomal fractions enriched from H4 cells overexpressing SNCA (tet-off). Cells were incubated with PBS, 20 µg/mL rHsCTSD or Dox for 72 h. Western blot shows LAMP2 signal and CTSD signal with pro and single chain forms (~52-kDa and ~48-kDa) as well as mature form (~34-kDa). (B) SNCA signals were detected and quantified using SNCA antibodies C-20 (top) and syn-1 (bottom). CBB-stained SDS-PAGE gel was used as protein loading control and the corresponding quantification is expressed as fold change (n =3). (C) Immunoblot of H4 SNCA cells incubated with PBS or 20 µg/ml rHsCTSD. Antibodies against lysosomal and autophagic marker LAMP1 and SQSTM1 were utilized. As loading controls GAPDH and CBB-stained SDS-PAGE gel were used. (D) Signal intensities of LAMP1 (top) and SQSTM1 (bottom) were normalized to GAPDH signals and expressed as fold change (n = 5). Representative western blot of PBS or Dox treated H4 SNCA cells is shown in Figure S3. (E) Quantification of lysosomal mass by Dextran-Cascade Blue staining, normalized to overall cell stain (Celltag700) and expressed as fold change (n = 4). (F) Lysosomal GBA activity was determined in H4 cells overexpressing SNCA after incubation with PBS, 20 µg/mL rHsCTSD or Dox for 72 h (n = 3). All data represent mean ± SEM. Statistical analyses were performed by using one-way ANOVA together with Dunnett’s multiple comparison test (B, D-F). Statistical differences are shown toward PBS treatment. **p < 0.01, *p < 0.05; n.s., not significant.

Fig 3: Intracranial injection of rHsCTSD decreases insoluble SNCA in the brain of ctsd deficient (KO) mice. (A) Representative immunoblot of Triton-soluble (T-sol) fraction of whole brain lysates from wildtype (WT) and ctsd KO (KO) mice injected with 10µL PBS or 100 µg rHsCTSD (KO rHsCTSD) each at day P1 into the left hemisphere and on day P19 into the right hemisphere (illustrative timeline of rHsCTSD treatment in mice is depicted in Figure S6A). Western blot stained for CTSD (pro/single chain ~52/~48-kDa and mature form ~34-kDa) as well as SNCA (C-20; immunoblots of syn-1 can be found in Figure S6B). TUBB3 and CBB-stained SDS-PAGE gels were used as control for equal protein load. (B) Quantification of pro-/single chain form and mature CTSD normalized to TUBB3 and normalized to WT (n = 3 mice per group). (C) Quantification of soluble SNCA (C-20; analysis for syn-1 can be found in Figure S6D), normalized to TUBB3 shown as fold change (n = 3 mice per group). (D) Immunoblot of Triton-insoluble (T-insol) fraction of whole brain lysates from WT, KO and rHsCTSD treated KO mice. Right, quantification of insoluble SNCA levels detected by C-20 representative for three individual experiments, normalized to TUBB3 expressed as fold change (n = 3 mice per group; immunoblot and quantification of syn-1 antibody can be found in Figure S6C-6D). (E) Representative dot blot from T-sol whole brain lysates of WT, ctsd KO and ctsd KO mice i.c. injected with rHsCTSD. Pathology-associated SNCA was detected by the conformation-specific SNCA antibody MJFR-14-6-4-2. ACTB was used as a loading control. Right, fold change quantification of SNCA-filament-MJFR signal normalized to ACTB (n = 3–4 mice per group). (F) Representative images of co-stained brain regions hypothalamus, fornix, basal forebrain/thalamus, hippocampus and midbrain of ctsd KO and ctsd KO +rHsCTSD. Pathology-associated SNCA was detected by the conformation-specific antibody MJFR-14-6-4-2 (red) and co-stained with the neuronal marker neurofilament (neurofil., green). Scale bar: 50 µm. For images of WT mice and in lower magnification refer to Figures S7A-7B. (G-I) Analyses of average SNCA aggregate size (µm) and SNCA intensity signal per aggregate from (G) hypothalamus, (H) fornix and (I) basal forebrain and thalamus. Dots represents SNCA aggregates found per image in the respective brain area (n = 3 mice per group). Quantification of SNCA intensity in (J) midbrain and (K) hippocampus (n = 3 mice per group). (L) Representative immunofluorescence pictures of primary neurons derived from wildtype (WT), heterozygous (Het) and ctsd-deficient (KO) mice stained for SNCA (syn1; red), CTSD (green) and nucleus (DAPI; blue). Primary neurons of KO animals were treated with 20 µg/ml rHsCTSD. Individual channels for CTSD (green) and SNCA (red) are shown in Figure S7F. Scale bar: 10 µm. Quantification of (M) SNCA and (N) CTSD signal intensity, normalized to WT and expressed as fold change (n = 6 individual neurons per group). Data represent mean ± SEM. Statistical analyses were performed by using one-way ANOVA together with a Tukey’s multiple comparison test and a two-tailed unpaired Student’s t-test in (G-I). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05; n.s., not significant.

Fig 4: Intraneuronal effect of rHsCTSD on SNCA and synaptical vesicles in primary neurons and in DA-iPSn. (A) Representative immunostaining of neuronal processes of primary neurons derived from WT, ctsd KO and ctsd KO +rHsCTSD animals. SNCA shown by antibody anti-syn-1 in red and SYN-1 (synapsin I) in green. Scale bar: 5 µm. (B) Quantification of SYN-1-positive vesicle cluster number per µm and (C) of the average SYN-1-positive vesicle size per punctum (µm) in primary neurons from WT, ctsd KO and ctsd KO +rHsCTSD (analysis of n = 11-13 individual neurons per group). (D) Representative immunostaining of SNCA (LB509; red) and SYN-1 (green) in neuronal processes of DA-iPSn of isogenic control (A53T corr) and mutant line (A53T) with and without rHsCTSD treatment. Scale bar: 5 µm. (E) Quantification of SYN-1-positive vesicle cluster numbers per µm and (F) analyses of average SYN-1-positive vesicle size per punctum (µm) in neuronal processes of DA-iPSn A53T corr. +PBS, A53T +PBS and A53T +rHsCTSD (n = 13-16 individual neurons per group). (G and H) Representative structured illumination microscopy (SIM) images providing detailed localization of SNCA respective to SYN-1 signal in (G) primary neurons derived from KO +PBS and KO +rHsCTSD animals and (H) DA-iPSn from A53T mutant +PBS and +rHsCTSD. Neuronal processes were immunostained with SNCA (Antibody: syn-1 for primary neurons and LB509 for DA-iPSn; magenta) and SYN-1 (green). Scale bar: overview 2 µm and inset 1 µm. Images highlighted here (G and H) correspond to experimental conditions in (A) and (D) and highlighted findings from respective experiment. Data represent mean ± SEM. All statistical analyses were performed by using one-way ANOVA followed by Tukey’s multiple comparison test. ****p < 0.0001, ***p < 0.001, *p < 0.05 n.s., not significant.