Recombinant pro-CTSD (cathepsin D) enhances SNCA/α-Synuclein degradation in α-Synucleinopathy models.

Cell culture and animal model HEK 293-EBNA culture

For production of recombinant human proCTSD (rHsCTSD), human embryonic kidney (HEK) 293 cells stably expressing the Epstein-Barr virus nuclear antigen 1 under the control of the CMV promoter (HEK 293-EBNA) were utilized (Invitrogen, R620-07). Cells were maintained in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, 41965) containing 4 mM L-glutamine and 4.5 g/L glucose and supplemented with 10% fetal calf serum (FCS), 1% PenStrep (Sigma, P0781) and 0.25 mg/mL G-418 (Life Technologies, 11811-023) in a humidified 5% CO 2 atmosphere at 37 °C. HEK 293-EBNA cells were transfected with pCEP-Pu containing proCTSD as described previously [ 47 ] using polyethyleneimine (PEI; Polysciences, 24765) in a 1:3 DNA to PEI ratio. Expressing cells were selected 48 h after transfection with 0.25 mg/mL G-418 and 1 µg/µL puromycin for 3 weeks. A high expressing clone was further selected by serial dilution. rHsCTSD production and purification Human recombinant proCTSD was produced as described previously [ 47 ]. Briefly, for one round of protein expression, 4 × 10 6 HEK 293-EBNA cells stably overexpressing rHsCTSD, were seeded in five 175 cm 2 flasks with 35 mL of DMEM containing 10% FCS, 1% PenStrep, 0.25 mg/mL G418 (Thermo Fisher Scientific, 10131035) and 1 µg/µL puromycin per flask. Once cells reached 80% confluence, the medium was discarded and replaced by 100 mL DMEM containing 2.5% FCS and 1% PenStrep per flask. After one week, the medium was harvested, vacuum-filtered (0.22 µm; Fisher Scientific, FB12566510) and concentrated to a final volume of 50 ml utilizing an Amicon system and ultracentrifugation disk (10MWCO; Millipore, PLGC07610). The recombinant enzyme was purified by binding of its N-terminal His-tag to a HisTrap 1-mL column (GE Healthcare, 29–0510-21) on an ÄKTA Purifier (GE Healthcare) and subsequent elution with 250 mM imidazole (Millipore, 104716) in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 0.8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , pH 7.4). An additional purification step was performed by size-exclusion chromatography via a Superdex 75 column (GE Healthcare, GE17-5174-01). Finally, monomeric rHsCTSD was concentrated using a Vivaspin 20 (10MWCO; Sartorious, VS2002) and purity checked by SDS-PAGE.

H4 cell culture

Inducible human H4 neuroglioma cells expressing WT SNCA under the control of a tetracycline inducible promoter (tet-off) have been previously described [ 57 ]. Cells were maintained in Optimem medium (Thermo Fisher Scientific, 31985070) containing 5% FCS (tet-free; PAN-Biotech, P30-3602), 200 µg/ml geneticin (Thermo Fisher Scientific, 10131035) and hygromycin (Thermo Fisher Scientific, 10687010), 1% Pen/Strep. If not indicated differently, SNCA expression was turned off by the addition of 2 µg/mL doxycycline (Dox; Sigma, D3447) for 72 h. H4 cells were seeded into 6-well plates for western blot at 2 × 10 5 or 1.2 × 10 5 (on 12 mm cover glasses) per well for immunostaining. On the following day, 20 µg/mL of rHsCTSD was added to the media. Cells were then incubated for 24, 48 and 72 h before harvesting. The cellular uptake of rHsCTSD and its effects on SNCA were then evaluated by western blot and immunostaining. To test for potential cytotoxicity, rHsCTSD of different concentration (20, 40, 60, 80, 100 µg/mL) were given to cells for 72 h and cell death was measured by LDH (lactate dehydrogenase) assay, ToxiLight assay (LONZA, LT27-066) and cleaved CASP3. Leupeptin (LeuP; Thermo Fisher Scientific, 78435) was given to the cells to inhibit cysteine-cathepsins (e.g., CTSB and CTSL) and was used at 5 µM for 72 h.

Generation of CTSD-deficient H4 cells

CTSD knockout cells were established using the CRISPR-Cas9 technique as described in Bunk et al. [ 91 ]. In brief, H4 cells were transfected by Neon Transfection System (10 μL-Kit; Invitrogen, MPK1025) with ribonucleoprotein (RNP), complexes of CTSD multi-RNA guides and Cas9 protein (Gene Knockout Kit v2; Synthego). The multi-RNA guides used in this study target the exon 2 of the CTSD gene. Successful editing efficiency was determined by western blot analysis and Sanger sequencing of CTSD exon 2. Single CTSD KO cells were grown and expanded for western blot and Sanger sequencing analysis. Clone 2 was used for further analyses in this study. Cells were maintained as described in the section “H4 cell culture”. iPSC culture and neuronal differentiation PD patient derived human induced pluripotent stem cells (iPSC) expressing SNCA A53T and the matched isogenic corrected iPSC line were generously provided by Dr. R. Jaenisch (Whitehead Institute MIT) and were previously characterized extensively [ 63 ]. iPSCs were maintained in mTeSR1 media (Stemcell, 85850) media or mTeSR plus (Stemcell, 100–0276) on Matrigel (Corning, 354234)-coated plates and differentiated into DA neurons using an established protocol [ 113 ]. Between day 25–30 after differentiation, neurons were seeded on coated plates with poly-D-lysine (Merck, P1149) and LAM/laminin (Merck, 11243217001) at a cell number of 3 × 10 5 per well (24-well plate) on 12-mm cover glasses for immunofluorescence or at 4 × 10 5 for western blot. Neurons were maintained in Neurobasal media (Thermo Fisher Scientific, 21103049) containing NeuroCult SM1 Neuronal Supplement (Stemcell, 05711) and 1% PenStrep until ~ day 90, when neurons were treated with 10 µg/mL rHsCTSD, replenished every 4 days with media replacement for 21–25 days. Detailed describtion for iPSn culturing during the microelectrode array (MEA) analysis is mentioned in the section ”Microelectrode array (MEA) analysis”.

Animals

CTSD knockout animals (ctsd KO; ctsd −/− ) mice were bred from heterozygous founders and genotyped as previously described [ 47 , 114 ]. All animals were housed in individually ventilated cages (IVC) and kept under a day/night rhythm of 12 h:12 h, with free access to water and food ad libitum. The room temperature was maintained at 19–22°C with a humidity of 45–60%. Animal handling and care were performed in agreement with the German animal welfare law according to the guidelines of the Christian-Albrechts University of Kiel. Animal experiments were approved by the Ministry of Energy, Agriculture, the Environment and Rural Areas Schleswig-Holstein under the reference number V242–40,536/2016 (81–6/16). Mice were genotyped at postnatal day 0 (P0). CTSD wildtype ( Ctsd WT; Ctsd +/+ ) or ctsd −/− were selected to be dosed intracranially (i.c.) at P1 and P19 with either PBS or 100 µg rHsCTSD (in 10 µl) using a micro syringe (30 G) connected to a spacing device with an injection depth of 1.15 mm. over a period of 3 min. P1 injections were done in the cauda putamen in the right hemisphere, while P19 injections were done in the left hemisphere. For P19 injections mice were anesthetized with 2% isoflurane in oxygenated air. All mice were sacrificed at P23 as chosen humane end point for untreated CTSD KO mice [ 47 ]. The brains were cut into 35 µm thick sections using a Leica SM 2000 R sliding microtome (Leica Microsystems) with dry-ice cooling and stored in PBS containing 0.02% (w:v) sodium azide.

Primary neuronal culture

Primary cortical neurons were cultured from the cortex of crossed heterozygous ( ctsd Het; ctsd +/− ) mice embryos (resulting in wild type ( ctsd +/+ ), het ( ctsd +/− ) and homozygous ( ctsd −/− )) at day 18.5 as previously described [ 114 ]. Briefly, the cortices of the embryo’s brains were aseptically dissected, freed of meninges and minced in Hanks’ Balanced Salt Solution (HBSS; Life Technologies, 14,175–095). After trypsin digestion at 37°C for 15 min and washing, disassociated cells were plated at 7,5 x 10 4 cells per 12-mm coverslip coated with a 3:1 COL1/collagen 1, rat tail (Thermo Fisher Scientific, A10483-01):poly-D-lysine solution and maintained in Neurobasal Medium supplemented with B27 Supplement (Thermo Fisher Scientific, 17504044), 0.5 mM GlutaMax (Thermo Fisher Scientific, 35050061), 25 µM glutamate (Sigma, G-6904), 5% PenStrep, 1 mM HEPES (Roth, 6761.1), pH 7.4, 10% heat-inactivated horse serum (Thermo Fisher Scientific, 26,050,070). The next day, the medium was replaced by Neurobasal Medium supplemented with B27, 0.5 mM GlutaMax, 5% PenStrep and 1 mM HEPES. Neurons were treated with 20 µg/mL rHsCTSD on day 7 and 10 after seeding and fixed on day 14 for immunofluorescence studies.

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