Cell culture, transient transfections and treatments
Human SH-SY5Y neuroblastoma cells were maintained in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Waltham, MA) supplemented with 15% fetal bovine serum (Invitrogen) and 1% Penicillin-Streptomycin (Gibco, Waltham, MA) in a 37 °C incubator with 5% CO2. Transient plasmid transfections were performed with plasmid DNAs using Lipofectamine (Thermo Fischer Scientific, Waltham, MA), according to the manufacturer’s instructions. To induce mitochondrial depolarization, the SH-SY5Y cells were treated with 10 μM or 20 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Sigma-Aldrich, Saint Louis, MO), 1 μM oligomycin (Sigma-Aldrich), 1 μM antimycin A (Sigma-Aldrich), or 1 μM antimycin A and 1 μM oligomycin (AO), for the indicated times, prior to cell harvesting or fixation. DMSO treatment was used as a control.
Expression plasmids and antibodies
The plasmids used were pEGFP-Parkin  (a gift from Prof. Edward Fon (McGill University, Montreal, Quebec, Canada), Addgene plasmid #45875) and pEGFP-LC3 (a gift from Prof. Toren Finkel (University of Pittsburgh, Pittsburgh, PA), Addgene plasmid #24920) constructs . The primary antibodies used for immunoblotting were mouse anti-ATPIF1 (1:1000, Abcam, Cambridge, UK, ab110277), mouse anti-β-actin (1:5000, MP Biomedicals, Irvine, CA, 8691001), mouse anti-β-tubulin (1:5000, Sigma-Aldrich T4026), mouse anti-Parkin (1:500, Santa-Cruz Biotechnology, Dallas, TX; sc-32,282), rabbit anti-PINK1 (1:1000, Cell signaling, Danvers, MA, #6946), mouse anti-TOM20 (1:500, BD Biosciences, San Jose, CA; 612278) and goat anti-VPS35 (1:1000, Abcam, ab10099). The primary antibodies used for immunofluorescence (IF) were mouse anti-TOM20 (1:200, Santa-Cruz Biotechnology sc-17764) and rabbit anti-PINK1 (1:200, Abcam ab216144). Secondary antibodies for immunoblotting were HRP-conjugated goat anti-rabbit IgG (H + L) (1:10000, Bio-Rad, Hercules, CA), HRP-conjugated goat anti-mouse IgG (H + L) (1:10000, Bio-Rad) and HRP-conjugated donkey anti-goat IgG (H + L) (1:10000, Abcam). Secondary antibodies for IF were Cy3-conjugated donkey anti-mouse IgG (H + L) (1:250, Jackson ImmunoResearch, West Grove, PA) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H + L) (1:250, Jackson ImmunoResearch).
Generation of VPS35 D620N/wild-type (WT) SH-SY5Y cells
The D620N mutation in the VPS35 gene was obtained by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9–mediated genome editing in the SH-SY5Y neuroblastoma cell line, as previously described . Briefly, a 20-nt single guide RNA (sgRNA) sequence that targets exon 15 of the VPS35 gene and is predicted to cut approximately 9 base-pairs (bp) upstream of the GAT triplet encoding the aspartic acid residue on location 620 was cloned into the pSpCas9(BB)-2A-GFP (PX458) plasmid (a gift from Prof. Feng Zhang (Broad Institute, Cambridge, MA); Addgene plasmid #48138) using the BbsI restriction enzyme to form the targeting plasmid expressing Cas9-GFP. In addition, a single-stranded oligodeoxynucleotide sequence was designed to facilitate homology-directed repair of the endogenous locus and included the substitution of five nucleotides: a nucleotide substitution G > A that leads to the D620N mutation of VPS35 and four synonymous substitutions that create a novel EcoRI restriction site that also destroys the protospacer-adjacent motif sequence to avoid repetitive cutting of Cas9 by the repair template. Following validation, the PX458-sgRNA plasmid and the single-stranded oligonucleotides were transfected into the SH-SY5Y cells following the manufacturer’s protocol (Lonza, Basel, Switzerland). GFP-positive cells were single-cell sorted 48 h post-transfection using a SH800S cell sorter (Sony Biotechnology, San Jose, CA) and grown in separate cultures that were subsequently screened for the D620N mutation using the restriction enzyme EcoRI. In parallel, we mock-electroporated and sorted the same batch of cells, which were used as WT control in the following experiments. Finally, we sequenced the top three predicted off-target genomic regions within coding regions (obtained from http://crispr.mit.edu) of genes POU6F1, ZNF318 and KY, but found no off-target edits (not shown). Detailed primer and template sequences are provided in Table S1.
Generation of stable COX8-EGFP-mCherry reporter SH-SY5Y cells
The COX8-EGFP-mCherry sequence was obtained from the pCLBW COX8-EGFP-mCherry construct  (a gift from Prof. David Chan (Caltech, Pasadena, CA), Addgene plasmid #78520) through restriction enzyme digestion with ApaI and EcoRI, and was ligated into the mammalian expression vector pcDNA 3.1(+). Subsequently, the vector was transfected into WT and VPS35D620N SH-SY5Y cells using Lipofectamine 3000 (Thermo Fischer Scientific), following the manufacturer’s protocol. Forty-eight hours after transfection, the growth medium was replaced with selection medium containing 800 ng/μl G-418 (Sigma-Aldrich). The selection medium was refreshed every other day for 10 days until only cells with the plasmid remained. Stable cell lines were cultured for three passages before performing the experiments.
Protein extraction and immunoblotting
SH-SY5Y cells were harvested in 2% sodium dodecyl sulfate (SDS)/phosphate-buffered saline (PBS) buffer containing a proteinase inhibitor cocktail (Roche, Basel, Switzerland) and sonicated. Crude mitochondrial fractions were isolated as previously described . Briefly, SH-SY5Y cells were collected and homogenized using a Dounce homogenizer in ice-cold isolation buffer containing 320 mM sucrose and a proteinase inhibitor cocktail. The homogenized samples were differentially centrifuged at 1500 g for 15 min and 17,000 g for 30 min to obtain nuclei and crude mitochondria, respectively. The cytosolic fraction was obtained from the final supernatant. Protein concentrations were quantified using the Pierce™ BCA protein assay kit (Thermo Fischer Scientific), and samples were mixed with loading buffer containing 10% β-mercaptoethanol before being boiled at 95 °C for 5 min. Subsequently, equal amounts of total protein extracts were subjected to SDS-PAGE, transferred to nitrocellulose membranes, blocked for 1 h in skimmed milk, incubated overnight with primary antibody at 4 °C and then with the corresponding secondary antibody for 1 h at room temperature (RT). The blots were imaged on a Chemidoc™ MP Imaging System (Bio-Rad). Protein levels were quantified by densitometry using the ImageJ software (NIH, Bethesda, MD).
WT and VPS35D620N SH-SY5Y cells that were seeded on glass coverslips in 24-well plates were fixed in 4% paraformaldehyde in PBS for 10 min at RT. Cells were then permeabilized in 0.1% Triton X-100 in PBS for 10 min and blocked with 5% donkey serum (Abcam) in PBS for 1 h. The coverslips were then incubated overnight at 4 °C with the primary antibodies diluted in blocking buffer and for 1 h at RT for secondary antibody incubation. Coverslips were finally mounted onto glass slides in 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting medium (Vector Laboratories, Burlingame, CA). The slides were analyzed using either structured illumination microscopy (SIM) or confocal microscopy. SIM images were acquired with an AxioObserver Z1 compound microscope (Carl Zeiss, Oberkochen, Germany) equipped with an Apotome, 63x oil objective and an AxioCam MRm3 CCD camera (Carl Zeiss). Confocal images were acquired with a TCS SP8 high-resolution confocal laser scan microscope (Leica Microsystems, Wetzlar, Germany) and an HC PL APO CS2 63x/1.4 oil objective. For quantitative analysis, maximum intensity projections were generated from all Z-stacks, which were captured for each condition with identical exposure times or laser settings.
All image analyses were performed using the ImageJ software (NIH). Colocalization analyses of PINK1 and TOM20 were performed using ImageJ plugin Coloc 2 (https://imagej.net/Coloc_2). Regions of interest (ROIs) were created per cell in the TOM20 channel (n = ~ 80–100 cells per condition in each experiment). Pearson’s correlation coefficients were subsequently determined per ROI using the Costes method for threshold regression .
Mitochondrial morphology was quantified as previously described . Briefly, images of single cells were pre-processed and binarized, followed by particle analysis and computation of several metrics. The number of mitochondria was determined as the number of individual particles. The aspect ratio was determined by dividing the major axis by the minor axis of each particle. A total of 80–100 cells were quantified per condition in each experiment.
For the EGFP-Parkin translocation experiment, a blinded observer scored each cell for either diffuse EGFP-Parkin or mitochondria-localized EGFP-Parkin (n = ~ 50 cells per condition in each experiment), as previously described .
Mitophagy in COX8-EGFP-mCherry stable cell lines was quantified by determining the ratio of the number of particles obtained from the mCherry channel (mitophagolysosomes) to the number of particles obtained from the EGFP channel (mitochondria) per cell (n = ~ 50–70 cells per condition in each experiment). Particles were analyzed in a similar fashion to the mitochondrial morphology quantification.
EGFP-LC3 puncta on mitochondria were quantified as follows: a mask was created from the TOM20 mitochondrial staining and used as overlay over the EGFP-LC3 image. The puncta were subsequently counted for each cell (n = ~ 40–50 cells per experiment).
Mitochondrial membrane potential quantification
Mitochondrial membrane potential was measured using fluorescence-activated cell sorting (FACS). WT and VPS35D620N SH-SY5Y cells were incubated for 30 min with 100 nM tetramethylrhodamine methyl ester (TMRM) dye (Thermo Fischer Scientific) and 100 nM MitoTracker Green FM dye (Thermo Fischer Scientific) diluted in culture medium. Cells were rinsed, dissociated with 0.05% Trypsin-EDTA (Thermo Fischer Scientific) and aliquoted in multiple FACS tubes. FACS measurements were performed with a FACSCalibur flow cytometer (BD Biosciences) or a Novocyte Quanteon flow cytometer (Agilent, Santa Clara, CA ). For the timeline measurements, baseline measurements were taken, after which CCCP was added to a final concentration of 10 μM, upon which measurements were taken at each time point. For the dose-response measurements, CCCP was added to the indicated final concentrations, and 1 min later measurements were made. Slopes were determined using a simple linear regression method. For 24-h treatments, cells were treated with 10 μM CCCP or 1 μM antimycin A and 1 μM oligomycin for 24 h, then the cells were dissociated and TMRM was measured as indicated above. Three independent sorts measuring at least 10,000 cells were performed per clone per data point for all experiments. Data analysis was performed using the Kaluza Analysis software (Beckman Coulter, Brea, CA). Mitotracker Green median fluorescence intensity was used to correct for mitochondrial mass fluctuations.
For conventional transmission electron microscopy (TEM), WT and VPS35D620N SH-SY5Y cells were treated with DMSO or 10 μM CCCP for 6 h. Then an equal volume as the culture media of double-strength fixative (4% paraformaldehyde, 5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4) was then added to the cells and incubated for 20 min at RT, followed by further fixing the cells with the same volume of single-strength fixative (2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4) for 2 h at RT. After five washes with 0.1 M sodium cacodylate buffer (pH 7.4), the cells were scraped and embedded as previously described . Subsequently, 70-nm ultrathin sections were cut using a Leica EM UC7 ultra microtome (Leica Microsystems) and stained with uranyl acetate and lead citrate as previously described . The cell sections were analyzed using an 80 kV transmission electron microscope CM100bio TEM (FEI, Eindhoven, The Netherlands).
The analysis of the different mitochondrial profiles per cell type was performed by random screening of sections derived from at least three different grids per sample. The mitochondrial profiles were categorized as follows: classical mitochondria with well-defined cristae (category I), dark mitochondria with well-defined cristae often swelling (category II), mitochondria with undefined cristae (category III), dark mitochondria with undefined contours and cristae (category IV), and large mitochondria with very light content and few remnant cristae (category V). The number of each mitochondrial type per condition was determined by counting 665, 579 and 521 mitochondria profiles from the DMSO-treated WT and VPS35D620N cell (clones 1 and 2) sections, respectively, as well as 727, 1028 and 914 mitochondria profiles from the CCCP-treated WT and VPS35D620N cell (clones 1 and 2) sections, respectively.
Cell viability assay
Cell viability upon treatment with CCCP was determined using a 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay (Abcam). The SH-SY5Y cells were plated in 96-well plates 12 h prior to incubation with CCCP for 24 h. MTT assay compounds were added following the manufacturer’s protocol and absorbance was measured using a Synergy HT optical plate reader (Biotek, Winooski, VT).
Data of Western blot densitometry measurements, mitochondrial membrane potential measurements, TEM and mitochondrial morphology were analyzed using a linear model by one-way or two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Count data, such as the EGFP-LC3 and mitochondrial particle quantification data were modelled using a generalized linear model followed by one-way or two-way ANOVA and Tukey’s post-hoc test. PINK1-TOM20 colocalization data were analyzed using the Kruskal-Wallis test followed by pairwise Mann-Whitney U-test with Benjamini-Hochberg multiple testing correction. Proportional data of COX8-EGFP-mCherry mitophagy and EGFP-Parkin localization were analyzed using beta regression analysis. Data are means ± SEM from at least three independent experiments, unless otherwise specified. P < 0.05 was considered as statistically significant. Statistical analyses were performed in the statistical computing environment R (version 1.3.959; The R Foundation for Statistical Computing; Vienna, Austria).