Constructs and generation of stable cell lines
Constructs were purchased from IDT (gBlocks Gene Fragments) and subcloned into pcDNA5/frt/to (Thermo Fisher Scientific, # V652020). Site-directed mutagenesis was performed according to the manufacturer’s protocol (Agilent, QuickChange II XL), and base pair exchange was confirmed by Sanger sequencing. Flp-In™ 293 T-Rex cells (Thermo Fisher Scientific, # R78007) were grown in media composed of Dulbecco’s modified Eagle’s medium (DMEM, Sigma–Aldrich), 10% fetal bovine serum (Gibco), 1% GlutaMax (Gibco) supplemented with 100 mg/ml Zeocin (InvivoGen) and 15 mg/ml blasticidin (InvivoGen). Inducible Flp-In T-Rex 293 cells were generated according to the manufacturer’s protocol (Thermo Fisher Scientific). Selection was performed with DMEM supplemented with 15 mg/ml blasticidin and 100 mg/ml hygromycin B Gold (InvivoGen) 48 h after transfection and continued until the expression of the gene of interest was induced by treating the cells with 50 ng/ml doxycycline hyclate (Sigma–Aldrich) for 48 h. List of primer sequencing is provided in Supplementary Table 2.
Cells were detached using Accutase and washed 2x with ice-cold phosphate-buffered saline (PBS, Sigma–Aldrich). Cells were lysed in 1xTBS (pH7.4) containing 0.5% NP40 and protease/phosphatase inhibitor cocktail (Pierce, #A32959). The washed Protein G Agarose beads (Merck, #16-266) were preincubated with the respective antibody for endogenous PD while rotating for 2 h. Lysates were immunoprecipitated with M2 anti-flag agarose resin (Sigma–Aldrich, #A2220), antibody-decorated beads against the respective protein (for endogenous CoIP), or control IgG for 2 h at 4°C while rotating. For Flag coimmunoprecipitation, Flag was eluted with Flag Peptide (Sigma–Aldrich, #F3290) by moving on a wheel and spun for 1 min at 1000 × g. Control immunoprecipitation using only Protein G agarose was performed. The supernatant was collected and subjected to TMT labeling or western blot analysis. For endogenous coimmunoprecipitation, Protein G agarose beads (Merck, #16-266) were washed and preincubated with the antibody against the respective protein overnight. Next, the beads were washed three times with binding buffers containing 150 mM NaCl and 50 mM Tris-HCl. Elution was performed by boiling the beads twice with 2x Laemmli buffer. Then, the beads were spun for 1 min at 10,000 × g, and the supernatant was collected and subjected to western blot analysis. The antibodies are listed in Supplementary Table 3.
TMT-based quantitative proteomics
Lysates were pretreated with Benzonase ® Nuclease (Sigma) before further processing for LC-MS analysis. Samples were incubated with dithiothreitol at 56°C for 30 min (10 mM in 50 mM HEPES, pH 8.5) to reduce cysteine. This step was followed by sample alkylation with 2-chloroacetamide (20 mM in 50 mM HEPES, pH 8.5) while protected from light at RT for 30 min. The SP3 protocol was used as published by Hughes et al. for processing the samples 43. The proteins were digested on beads using Trypsin (sequencing grade, Promega) overnight at 37°C. The ratio between trypsin and proteins was 1:50. TMT11plex Isobaric Label Reagent (Thermo Fisher) was used as described in the manufacturer’s protocol. For sample clean up, an OASIS® HLB µElution Plate (Waters) was used. Six fractions were the outcome of offline high pH reversed-phase fractionation with an Agilent 1200 Infinity high-performance liquid chromatography system equipped with a Gemini C18 column (3 μm, 110 Å, 100 x 1.0 mm, Phenomenex). A trapping cartridge (µ-Precolumn C18 PepMap 100, 5 µm, 300 µm i.d. x 5 mm, 100 Å) and an analytical column (nanoEase™ M/Z HSS T3 column 75 µm x 250 mm C18, 1.8 µm, 100 Å, Waters) were attached to the UltiMate 3000 RSLC nano LC system (Dionex). For 6 min, continuous flow of trapping solution (0.05% trifluoroacetic acid in water) onto the trapping column at 30 µL/min allowed peptide trapping. Elution of trapped peptides was achieved by running solvent A (0.1% formic acid in water, 3% DMSO) with a constant flow of 0.3 µL/min through the analytical column, with an increasing percentage of solvent B (0.1% formic acid in acetonitrile, 3% DMSO). The Nanospray Flex™ ion source in positive ion mode allowed direct attachment of the valve of the analytical column to an Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer (Thermo Fisher Scientific). A Pico-Tip Emitter 360 µm OD x 20 µm ID; 10 µm tip (New Objective) was used to introduce the peptide into the Fusion Lumos using an applied spray voltage of 2.2 kV. The capillary temperature was 275°C. The resolution of the orbitrap used in profile mode was 120,000, and the mass range was set to 375-1500 m/z during the full mass scan. The maximum filling time was 50 ms. For data-dependent acquisition (DDA), the resolution of the Orbitrap was set to 30,000, and the fill time was set to 94 ms. The ion number was limited to 1x105 ions. A normalized collision energy of 36 was applied. Profile mode was used to acquire MS2 data. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD032155.
MS data analysis
Data were processed with IsobarQuant (https://github.com/protcode/isob/archive/1.0.0.zip) and Mascot v2.2.07 (http://www.matrixscience.com/server.html; RRID:SCR_014322). The UniProt Homo sapiens database (UP000005640, https://www.uniprot.org/proteomes/UP000005640) was used as the proteome database. The search criteria were determined as follows: carbamidomethyl (C) and TMT11 (K) (fixed modification), acetyl (N-term), oxidation (M) and TMT11 (N-term) (variable modifications). For the full scan (MS1), a mass error tolerance of 10 ppm was set, and a spectrum of 0.02 Da was used for MS/MS (MS2). Two skipped cleavages by trypsin were the maximum permitted. For protein identification, the following criteria had to be fulfilled: a) at least two distinctive peptides were recognized per protein and b) the peptide length was required to be at least seven amino acids long. The false discovery rate (FDR) at the peptide and protein levels was set to 0.01. To filter out nonspecific proteins, a limma-based differential analysis was performed comparing FLAG and IgG control samples. Hit and candidate proteins were defined based on the following criteria. Candidate proteins were defined with an FDR ≤ 0.2 and a FC ≥ 1.5, and hit proteins were based on an FDR < 0.05 and a FC > 2. Statistically enriched hit and candidate proteins were analyzed using Panther pathway analysis software version 14.0 (FDR <5%) (http://www.pantherdb.org; RRID:SCR_004869). Mitochondrial proteins were identified according to the Human Mitocarta 3.0 database (https://www.broadinstitute.org/files/shared/metabolism/mitocarta/human.mitocarta3.0.html; RRID:SCR_004869). The subcellular localization of the top 100 candidate proteins was assigned according to the GeneCards database version 5.6 (https://www.genecards.org; RRID:SCR_002773) by using a confidence level ³ 4. The visualization of the protein subcellular localization was generated with Cytoscape version 3.9.0 (https://cytoscape.org; RRID:SCR_003032). The WT-GCase interactor list was used as the source node, fold-changes as the source attribute, and the subcellular localization as the target node. The distance and distribution between the nodes were randomly assigned.
iMTS-ls profile generation
Internal N-terminal matrix-targeting-like signal (iMTS-ls) analysis was performed as described in Boos et al. 18. Multiple truncated GCase protein sequences were generated by sequentially removing N-terminal amino acids. GCase suffix sequences were then subjected to TargetP prediction (TargetP 2.0) in standard FastA format. The mTP scores obtained were plotted against the corresponding amino acid position.
Split-GFP mitochondrial localization
For mitochondrial targeting of GFP1-10 (MTS-GFP1-10), the N-terminal matrix-targeting signal (MTS) of mitochondrial subunit VIII of cytochrome c oxidase (Cox8A) was added at the N-terminus of the GFP1-10 β-strand sequence, as described in Calì et al. 44. The MTS-GFP1-10 construct was obtained from Integrated DNA Technologies IDT and cloned into pcDNA™5/FRT (Thermo Fisher). The stable T-Rex HEK cell line was generated as described above. For GCase split-GFP, a 27-bp linker followed by the GFP S11 sequence 44 was added at the C-terminus of the GCase cDNA sequence. For MT-GCase split-GFP, we cloned a construct in which we inserted the mitochondrial targeting sequence of Cox8A along with a 9-amino-acid (aa)-long linker at the N-terminus and the 11th β-strand sequence of GFP at the C-terminus of GCase. Constructs were obtained from VectorBuilder. E326K or L444P mutation was inserted into the WT construct by site-directed mutagenesis using the QuickChange XL kit (Agilent) according to the manufacturer’s protocol. For mtGFP1-10 induction, 2x10^4 mtGFP1-10 T-Rex HEK cells were seeded on Geltrex (Thermo Fisher)-coated chambered cell culture slides (Ibidi) for live-cell imaging or immunostaining and treated with 50 to 200 ng/ml doxycycline, respectively. GCase split-GFP constructs were transfected with Viafect (Promega) according to the manufacturer’s protocol.
For live-cell imaging, cells were washed once with OptiMEM (Gibco) and then incubated for 30 min at 37°C with 100 nM MitoTracker red CM-H2Xros (Thermo Fisher Scientific) in OptiMEM. Cells were washed once with OptiMEM, in which the cells were kept for imaging. Images were acquired using a Leica TCS SP8 confocal microscope (Leica, Germany) equipped with a 100× /1.4 numerical aperture oil-immersion objective. Images were analyzed using Diffraction PSF 3D and DeconvolutionLab2 plugins in Fiji-ImageJ version 2.3.0/1.53q (https://fiji.sc; RRID:SCR_002285). For A-SYN PFF experiments, iPSC-derived neurons were treated with 0.25 mM Alexa Fluor 594-labelled PFFs (594-PFF) with or without cotreatment with 1 mM CDDO-Me (Cayman Chemical). After 24 h, the cells were incubated with 100 nM MitoTracker Green (Invitrogen, MA, USA) in neuronal medium for 30 min at 37 °C. A CellBrite™ Steady 488 Membrane Staining Kit (Biotium) was used to visualize cell membranes according to the manufacturer’s instructions. Images were acquired using a Leica TCS SP8 confocal microscope (Leica, Germany) with a 63 × /1.4 numerical aperture oil-immersion objective. Z-stacks were acquired for calculation of the PFF particle area and fluorescence intensity. For each condition, 5-8 images were acquired from at least four independent experiments. Data were obtained from n > 20 cells per experiment per condition. For the quantification of colocalization and image processing, images were analyzed using the “Analyze particles” and “EzColocalization” plugins in Fiji-ImageJ.
Generation of induced pluripotent stem cells and gene correction
Skin fibroblasts from all patients were obtained with informed consent approved by the Ethics Committee of the Medical Faculty and the University Hospital Tübingen. Skin fibroblasts were reprogrammed by nucleofection with pCXLE-hOct3/4 (RRID:Addgene_27076), pCXLE-hSK (RRID:Addgene_27078) and pCXLE-hUL (RRID:Addgene_27080) plasmids 45 using the Amaxa nucleofection kit for human dermal fibroblasts (Lonza, VPD-100) and program P-022 of the Nucleofector 2b (Lonza). Nucleofected fibroblasts were plated in 6-well plates coated with Matrigel (Corning) in DMEM supplemented with 10% FBS (Gibco) and 1% GlutaMAX Supplement (Gibco). The following day, the medium was changed to DMEM +/+ (DMEM with 10% FBS and 1% GlutaMAX Supplement and 1% Pen/Strep (Millipore)) supplemented with 2 ng/ml recombinant basic human fibroblast growth factor (FGF2, Peprotech). On day 3 or 4 postnucleofection, the medium was changed to E8 medium composed of DMEM F12 with HEPES (Gibco), 128 ng/ml ascorbic acid (Sigma–Aldrich), 1x insulin-transferrin-selenium (ThermoFisher Scientific), 10 ng/mL FGF2 (Peprotech), 500 ng/ml heparin (Sigma–Aldrich), and 2 ng/ml TGFβ1 (Peprotech). E8 medium was supplemented with 100 µm sodium butyrate and 0.1% Pen/Strep. Colonies started to appear from day 14 onward. Induced pluripotent stem cells (iPSCs) were cultured on Vitronectin XF (StemCell Technologies) in E8 medium. The gene correction for the L444P mutation was performed as previously described 46. The crRNA for the gene correction of the E326K mutation was designed with an online CRISPR design tool according to Ran et al 47. The guides and ssODN sequences are listed in Supplementary Table 2. One hour before nucleofection, 10 µM Rock inhibitor was added to the iPSC medium. 240 nM crRNA (IDT): Atto550-labeled tracrRNA (IDT) duplex was complexed with 124 µM Cas9 to form the ribonucleoprotein complex (RNP complex). iPSCs (1.6x10^6) were nucleofected with the RNP complex and 16 µg of ssODN using 100 µl of Ingenio nucleofection solution (Mirus) with program B-016 of Nucleofector 2b. Following nucleofection, the cells were FACS sorted for Atto550-positive cells using a FACS Aria II with a 100-µm nozzle. After sorting, 1x10^4 cells were plated per 10-cm dish. Colonies were picked and sequenced by Sanger sequencing. The top 5 possible exonic off-target effects predicted by https://cctop.cos.uni-heidelberg.de:8043/ (RRID:SCR_016963) were checked by Sanger sequencing.
Differentiation of iPSCs into neuronal precursor cells and dopaminergic neurons
The generation of neuronal precursor cells (NPCs) from patient and isogenic control iPSCs as well as the differentiation from NPCs to dopaminergic neurons was performed according to Reinhardt et al. 48. NPCs were cultured in N2/B27 medium, consisting of DMEM-Ham’s F12 medium and neurobasal medium (1:1), 0.5% N2 (Gibco), 1% B27 (Gibco), 1% P/S and 1% GlutaMAX supplemented with 3 µm CHIR 99021 (Axon Medchem), 0.5 µM puromycin (Merck Millipore), and 150 µM ascorbic acid (AA, Sigma–Aldrich). NPCs were split in a Matrigel-coated well every 5-6 days at a ratio of 1:10. To start differentiation, 1.25x10^6 cells were split into a 6-well and the following day the medium was changed to differentiation medium [N2/B27 medium supplemented with 100 ng/ml FGF8 (Peprotech), 1 µM phorbol 12-myristate 13-acetate (PMA) and 200 µM AA]. On day 8, the medium was changed to maturation medium [N2/B27 medium supplemented with 10 ng/ml brain-derived neurotrophic factor (BDNF) (Peprotech), 10 ng/ml glial cell-derived neurotrophic factor (GDNF) (Peprotech), 1 ng/ml TGF-β3 (Peprotech), 0.5 mM dibutyryl cAMP (dbcAMP) (Applichem), and 200 µM AA] supplemented with 1 µM PMA for 2 days. From day 10 onward, the cells were cultured in maturation medium without PMA. Maturation was reached after 14 days in maturation medium.
Western blot analysis
Cells were collected and washed once with PBS. The pellets were lysed, and the protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein (20-30 µg) was mixed with 6x Laemmli containing 12.5% β-mercaptoethanol (Roth). Protein samples, 20-30 µl of eluate of the Flag precipitate or the eluate of the endogenous PD were loaded on self-casted 7.5-15% acrylamide gels (Applichem) or on precast NuPage 4-12% Bis-Tris Protein Gels (Thermo Fisher Scientific). Proteins were transferred to PVDF membranes. The PVDF membranes were blocked in TBS 0.1% Tween containing 5% milk (nonfat dried milk powder, PanReac AppliChem) or 5% bovine serum albumin (Serva). The primary antibody was diluted in 5% Roche Block solution (Roche) or 5% BSA in TBS-T supplemented with 0.04% sodium azide. Secondary antibodies were diluted in blocking solution. The antibodies are listed in Supplementary Table 3.
Blue native electrophoresis and in-gel activity assay
Mitochondrial pellets were isolated from NPCs and iPSC-derived neurons and processed for blue native electrophoresis (BN-PAGE) as previously described 49 with minor modifications. In brief, samples were solubilized with digitonin at a detergent-to-protein ratio of 4:1, and 40 mg of mitochondrial protein was loaded per lane in precast NativePAGETM 3-12% Bis-Tris gels (InvitrogenTM). After electrophoresis, proteins were transferred to PVDF membranes at 30 V overnight and probed with the appropriate antibody.
Midbrain organoid generation
Midbrain-like organoids were generated by a protocol adapted from Jo et al. 23 (dx.doi.org/10.17504/protocols.io.5qpvobr8xl4o/v1). At day 0, iPSC colonies were dissociated into single cells using Accutase. In each well of a 96-well U-Bottom low-attachment plate, 10^4 iPSCs were seeded in a 1:1 mix of DMEM F12 and neurobasal medium supplemented with 0,5% N-2 supplement, 1% B27 supplement without vitamin A, 1% NEAA, 0.01% β-mercaptoethanol, 1 µg/ml heparin, 10 µM SB431542, 200 ng/ml LDN, 0.7 µM CHIR99021 and 50 µM Rock inhibitor. On day 4, the medium was changed to DMEMF12/neurobasal (1:1) supplemented with 0,5% N2 supplement, 1% B27 supplement, 1% NEAA, 0.1% β-mercaptoethanol, 1 µg/ml heparin, 10 µM SB431542, 200 ng/ml LDN, 100 ng/ml SHH-C25II, 1 µM purmorphamine, 7.5 µM CHIR99021 and 100 ng/ml FGF8. On day 7, embryoid bodies (EBs) were embedded in Matrigel using a protocol adapted from Qian et al. 50. Matrigel was diluted in a 3:2 ratio with medium and used as an embedding mixture. EBs were washed in fresh medium and embedded in Matrigel in a six-well ultra-low-attachment plate using the Matrigel-medium mixture. The Matrigel-EB mixture was incubated for 30 min at 37°C, and on day 7, medium containing the following was added: neurobasal medium supplemented with 0,5% N2 supplement, 1% diluted B27 supplement, 1% GlutaMax, 1% NEAA, 0.1% β-mercaptoethanol, 2.5 µg/ml insulin, 200 ng/ml laminin, 7.5 µM CHIR99021, 100 ng/ml SHH-C25II, 1 µM purmorphamine and 100 ng/ml FGF8. On day 8, the medium was changed to neurobasal medium supplemented with 0,5% N2, 1% B27, 1% GlutaMax, 1% NEAA, 7.5 µM CHIR99021, 0.1% β-mercaptoethanol, 10 ng/ml BDNF, 10 ng/ml GDNF, 100 µM ascorbic acid and 125 µM dbcAMP. On day 10, the medium was refreshed, and the concentration of CHIR99021 was reduced to 3 µM. On day 13, the concentration of CHIR99021 was further reduced to 0.7 µM until day 15. On day 20, the Matrigel was dissociated from the organoids.
10X genomics single-cell RNA sequencing
Midbrain organoids were dissociated into single-cell suspensions using a Worthington Papain Dissociation System kit (Worthington Biochemical), as previously described 51. Organoids were minced and incubated with 2,5 mL papain/DNAase solution was incubated on an orbital shaker for 30 min at 37°C. Organoid suspensions were triturated using 1 ml low-attachment pipette tips and then incubated on an orbital shaker for 20 min at 37°C. After enzyme inactivation, the cell suspensions were filtered with a 30-μm cell strainer and washed three times with HBSS. The final cell suspension was centrifuged at 300 × g for 7 min, and the cell pellet was resuspended in PBS-0.5% BSA. The cell concentration and viability were determined by microscopy using trypan blue staining (0.2%). scRNA-seq libraries were immediately generated using the 10X Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 (Cat. 1000128) according to the manufacturer’s instructions and paired-end sequenced on an Illumina NovaSeq 6000 (SP Flow Cell) with a sequencing depth of 300 million reads per library. GEO accession number: GSE198033.
Single-cell sequencing data analysis
The 10X Genomics pipeline cellranger count was run to generate filtered gene-barcode matrices that were used as input for downstream analysis using the R package Seurat version 4.1.0 (RRID:SCR_007322) (Butler et al. 2018, Stuart et al. 2019). Low-quality cells were filtered out (detected genes > 200, counts > 500, and mitochondrial ratio < 0.2), and genes with zero counts and genes that were expressed in less than 10 cells were also removed. For clustering analysis, the dataset was SCtransformed and normalized, and variation due to the cell cycle and mitochondrial expression was regressed out. Data from different 10x runs were integrated in Seurat using individual 10X runs as grouping variables. Principle components (PCs) were determined, and using the first 40 PCs, cells were clustered using a K-nearest neighbor (KNN) graph with a clustering resolution of 0.6, resulting in 21 clusters. Cell clusters were visualized using UMAP. Conserved markers were determined across all samples for each cluster (FindConservedMarkersfunction) using default settings (min.pct=0.25, logfc.threshold=0.25) and used for assigning cell type to cell clusters. The corresponding annotation file was downloaded from https://raw.githubusercontent.com/hbctraining/scRNA-seq/master/data/annotation.csv. Pseudo-time analysis was performed using Monocle 3 (version 1.0.0). After data normalization and integration, Monocle3 object was generated using as.cell_data_set function, and cells were clustered using cluster_cells function with a resolution of 1e-3. Clusters were manually assigned using La Manno et al as a reference 25. When ordering the cells along the trajectory using “order_cells” function, SOX1 expressing cells were specified as the starting state.
Cells were washed once with PBS and fixed for 10 min with 4% paraformaldehyde (PFA, Sigma–Aldrich) at RT. After fixation, the cells were washed twice with PBS. The cells were blocked and permeabilized in PBS containing 0.1% Triton-X (PBS-T) and 10% normal goat serum (NGS) for 1 h at RT. The primary antibody was diluted in PBS-T containing 5-10% NGS and incubated overnight at 4°C. Secondary antibody conjugated with Alexa 488 or 568 (Invitrogen) was diluted in 5% NGS in PBS-T and incubated for 1 h at RT. The nuclei of the cells were stained by incubation with 1 µM 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, Invitrogen), and coverslips were mounted on glass slides with mounting medium (DAKO). Images were acquired with a 63x 1.4NA plan-apochromat oil objective of a TCS SP8 confocal microscope (Leica Biosystems). Midbrain organoids were fixed in 4% (w/v) PFA for 1 h, and individual organoids were equilibrated in 30% sucrose in PBS overnight at 4°C. The next day, the organoids were embedded in blocks in optimal cutting temperature compound (OCT, Tissue-Tek). Slices with a thickness of 20 µm were cryosectioned and mounted on noncharged slides. Tissues were blocked in 10% (v/v) NGS in 0.5% Triton X-100 in PBS and incubated with primary antibodies overnight and secondary antibodies for 3 h. DAPI staining was performed with a concentration of 1 µM for 5 min, and after three PBS washes, tissues were mounted with DAKO mounting medium (DAKO) for image acquisition. The antibodies are listed in Supplementary Table 3.
Expansion microscopy (ExM) was performed as described 52 with some modifications. Cells were blocked with 10% (v/v) normal goat serum (NGS) in 0.1% (v/v) Triton X-100 in PBS and incubated with primary antibodies in blocking solution overnight. After a 3-h incubation with the corresponding secondary antibody (Alexa Fluor, Invitrogen), the samples were washed and treated with 0.1 mg/ml Acryloyl-X SE solution (Thermo Scientific) in PBS for 3 h at room temperature. The freshly prepared gelling solution consisted of Stock X solution (8.6% (w/v) sodium acrylate 33% (w/v), 2.5% (w/v) acrylamide 50% (w/v), 0.15% (w/v) N,N´-methylenebisacrylamide 2% (w/v), 11.7% (w/v) NaCl 5 M, and PBS 1X), water, 10% (w/v) TEMED and 10% (w/v) APS stock solution in a 47:1:1:1 ratio. Gel digestion was performed overnight in digestion buffer (0.5% (w/v) Triton X-100, 0.2% (v/v) EDTA 0.5 M, pH 8, 5% (v/v) Tris-Cl 1 M, pH 8, 4.67% (w/v) NaCl and 8 U/ml proteinase K). The gelling solution was added to each well and covered by a 15-mm coverslip to ensure the formation of a smooth, flat and thin gel. Coverslips were then incubated for 1 h at 37°C for complete polymerization. The gel was expanded in water for 1 h and mounted in 10 µg/mL poly-L-ornithine-coated coverslips to immobilize the gel for picture acquisition. Midbrain organoids were fixed, and immunofluorescent staining was performed as described above. Sections were treated with 0.1 mg/ml acryloyl-X SE solution in PBS at room temperature overnight. Gelation was performed in a 47:1:1:1 ratio of Stock X, 10% (w/v) TEMED, 10% (w/v) APS, and 0.5% (w/v) 4-hydroxy-TEMPO stock solutions. Gel digestion and expansion were performed as described above. Images were acquired using a Leica TCS SP8 confocal microscope (Leica, Germany) equipped with a 100× /1.4 numerical aperture oil-immersion objective. For each condition, 5 images were acquired from at least three independent experiments. Images were analyzed using Diffraction PSF 3D, DeconvolutionLab2, and EzColocalization plugins in Fiji-ImageJ. GraphPad Prism version 9.0.0 (RRID:SCR_002798) was used for calculating Spearman’s rank correlation value (ρ) to identify colocalization of fluorescence signals. The antibodies are listed in Supplementary Table 3.
Mitochondria were isolated from 10x10^6 cells or individual organoids using the Qproteome Mitochondrial isolation kit (Qiagen) with slight alterations to the manufacturer’s protocol. Cells or individual organoids were harvested, washed in 0.9% NaCl and incubated for 10 min at 4°C in lysis buffer. The homogenate was centrifuged at 1000 × g for 10 min at 4°C, and the supernatant was designated the cytosolic fraction. The pellet was resuspended in disruption buffer and mechanically passed through a 26-gauge needle 10 times. The enriched nuclear fraction was pelleted by centrifugation at 1000 × g for 10 min and homogenized in disruption buffer. Next, the supernatant was centrifuged at 6000 × g for 10 min at 4°C and resuspended in mitochondria storage buffer (enriched mitochondrial fraction). All buffers (except mitochondria storage buffer) were supplemented with 1:100 protease inhibitors (provided with the kit). Where indicated, cells were treated with 20 mg/ml digitonin (Sigma-Aldrich) or 0.01% Triton X-100 in PBS. For the proteinase K protection assay, freshly isolated mitochondria were resuspended in mitochondria storage buffer and treated with 2-20 mg/ml proteinase K (New England Biolabs GmbH) for 30 min on ice to digest surface-exposed proteins. The reaction was stopped by adding 2 mM PMSF Protease Inhibitor (Thermo Fisher).
Complex I activity assay
Mitochondria were isolated from HEK cells, iPSC-derived neurons, or midbrain organoids using the Qproteome Mitochondrial isolation kit as described above. Complex I (NADH oxidase/coenzyme Q reductase) was measured using the MitoCheck Complex I Activity Assay kit (Cayman Chemical, cat# 700930). The rate of NADH oxidation, which is proportional to CI activity, was determined by a decrease in absorbance at 340 nm over 15 min in the presence of ubiquinone and potassium cyanide to inhibit complex IV and prevent oxidation of ubiquinone.
The production and purification of recombinant human A-SYN was conducted according to Martinez et al. 53. A-SYN cDNAs were cloned into pET 21D (Novagen, Merck Millipore, #69743), and the plasmids were expressed in BL21DE3 E. coli (Novagen, Merck Millipore, #69450). Cultures of 750 ml were grown to midlog phase, and isopropyl-1-thio-3-d-galactopyranoside was added to 0.4 mM. After 2 h, the cells were pelleted, washed in PBS, and resuspended in 50 ml of 20 mM HEPES, 100 mM KCl, pH 7.2. The resuspended bacteria were heated to 90°C for 5 min. Aggregated protein was removed by centrifugation (40 min at 40,000 × g at 4°C). Contaminating nucleic acids and proteins were removed by ion exchange chromatography on Q-Sepharose Hi-Trap columns equilibrated with Solution A (50 mM Tris pH 7.4; Amersham Biosciences). Purified A-SYN was eluted by loading the soluble fraction and applying an increasing gradient of Solution B (50 mM Tris, pH 7.4, 1 M KCl). SYN-containing fractions were pooled and chromatographed on a Superose 12 column (Amersham Biosciences) in 20 mM HEPES, pH 7.4, 100 mm KCl. The monomer was aliquoted and frozen at −80°C. For preparation of PFFs, A-SYN monomer was shaken at 1,000 rpm for 7 days. PFFs were validated by electron microscopy, a sedimentation assay at 100,000 × g for 60 min, the thioflavin T assay, and on primary cortical neurons by pS129-syn immunostaining. Preformed fibrils were labeled using the Alexa Fluor 594 kit (Thermo Fisher, #A10239) according to the manufacturer’s instructions. For the treatment of neurons and organoids, A-SYN PFFs, which were generated at a concentration of 5 mg/mL, were vortexed and diluted with Dulbecco’s phosphate-buffered saline to 100 μg/mL and then sonicated (10-s pulses with 30% amplitude six times every 2 min) using a HTU SONI-130 sonicator (G. Heinemann, Germany). A-SYN PFFs were then diluted in neuronal or organoid media and added to cultures.
The verified Mission lentiviral plasmids encoding nontargeting shRNA (#SHC016) and HSPA8/HSC70 shRNA (#TRCN0000017279, #TRCN0000017281) in the pLKO.1-puro vector backbone were purchased from Sigma–Aldrich. The sequences of shRNA-TIM23 used by Goemans et al. 54 were cloned into pLV(shRNA)-Puro-U6 (purchased from VectorBuilder); pLV[shRNA]-Puro-U6>Scramble-shRNA (purchased from VectorBuilder) was used as a nontargeting shRNA. For lentiviral production, HEK cells were transfected with the plasmids expressing the shRNAs together with the lentiviral packaging plasmid psPAX2 (RRID:Addgene_12260) and the envelope plasmid pMD2.G (RRID:Addgene_12259) using TransIT-X2 (Mirus). In short, HEK 293T cells were seeded in a 10-cm dish to reach 80% confluency the day after transfection. The next day, the medium was changed. On days 4 and 5 after transfection, the medium was collected and filtered through a 0.45-µm PVDF membrane. To concentrate the virus, the filtered supernatant was centrifuged in a Vivaspin column (Sartorius Stedim Biotech) at 3000 RCF at 4 °C until the volume reached 500-1000 µl. The virus-containing supernatant was collected, and the concentration of p24 particles was determined with Lenti-X GoStix Plus (Takara). Cells were infected with equal concentrations of p24 particles for scramble and respective shRNA.
GCase activity assay
Cells or midbrain organoids were lysed by sonication in H2O containing 0.01% Triton, and the protein concentration was determined by BCA. For enzymatic assay measurement, 10-20 µg of protein was incubated for 30 min at room temperature with 25 µl of McIlvaine buffer 4X (0.4 M citric acid, 0.8 M Na2HPO4), pH 5.2, AMP-DNM (N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin) at a final concentration of 5 nM, and H2O to a final volume of 100 µl. 4-Methylumbelliferyl-β-D-glucopyranoside (MUB-Glc; Glycosynth, Warrington, UK) was dissolved in 200 mM sodium citrate phosphate buffer by heating to 60°C. At the end of incubation, 25 µl of 4-MU was added at a final concentration of 6 mM and incubated for 2 h at 37°C. As a control, 1 mM condurbitol B epoxide (CBE, Calbiochem) was used. The fluorescence was recorded after transferring 20 μl of the reaction mixture to a microplate and adding 180 μl of 0.25 M glycine, pH 10.2. Data were calculated as picomoles of converted substrate per milligram of cell protein per hour.
The levels of total A-SYN were measured in midbrain organoids homogenates using the hSYN total ELISA kit (847-0108000103, Roboscreen Diagnostic, Liepzig, Germany), according to the manufacturer’s instructions. The optical density was read at 450 nm on a microplate reader (Bio-Rad). Data were normalized on protein content.
Mitochondrial function was assessed in live cells using an XFp Extracellular Flux Analyzer (Agilent Technologies). A total of 1x10^4 T-Rex HEK or 1.5x10^5 iPSC-derived neurons were seeded in XFpSeahorse microplates and allowed to adhere overnight (for HEK cells) or for seven days (iPSC-derived neurons). Measurements of the oxygen consumption rate (OCR) were performed in freshly prepared assay medium, pH 7.4 (Seahorse XF DMEM Medium), according to the manufacturer’s protocol. The OCR was measured before and after the serial addition of 20 µM or 1 μΜ oligomycin, 1 µM or 5 μΜ carbonyl cyanide p-trifluoromethoxyphenylhydrazone (CCCP), and 2 µM or 1 μΜ antimycin A and 2 µM or 1 µΜ rotenone to T-Rex HEK cells or iPSC-derived neurons, respectively (all from Sigma–Aldrich). Following each injection, three measurements for a total period of 15 min were recorded. The data were analyzed using Wave 2.6 software (RRID:SCR_014526), and OCR parameters (basal respiration, maximal respiratory capacity, respiratory reserve, and ATP-linked respiration) were calculated. At least three technical replicates per condition were used, and the experimental values were normalized to the protein content per well via a BCA assay.