Belgian study populations

The PD cohort consisted of 617 unrelated patients (mean age at onset (AAO) 60.0 ± 11.5 years, 31.8% women, 18.6% with a positive familial history) and the DLB cohort consisted of 226 unrelated patients (mean AAO 70.8 ± 9.8 years; 32.7% female, 23.0% with a positive familial history) recruited in the framework of the Belgian Neurology (BELNEU) Consortium, a multicenter collaboration of neurology expertise centers in Belgium [20, 41]. Index patients were evaluated with a detailed clinical history of patients and family, clinical neurological examination, and neuroimaging. All PD patients fulfilled the National Institute of Neurological Disorders and Stroke (NINDS) diagnostic criteria for PD [40], whereas DLB patients were diagnosed in accordance with the established criteria for possible, probable or pathological DLB [71]. In the DLB cohort, 70 patients received a neuropathological diagnosis of DLB and 113 patients received a clinical diagnosis of probable DLB. Patients with a positive familial history have at least one first-degree relative presenting with PD or dementia. EOPD, was defined as PD with age at onset ≤ 50 years, and was observed in 120 patients of the PD cohort (mean AAO 43.4 ± 5.9 years, 24.2% women, 18.3% with a positive familial history). Of the EOPD patients, we collected detailed familial information and bio-sampled available family members. The complete PD cohort was genetically profiled for the 5 major PD genes (SNCA, LRRK2, PARK2, PINK1 and PARK7) by means of Sanger sequencing for simple mutations, and multiplex amplicon quantification (MAQ, Agilent, Multiplicom, Niel, Belgium), quantitative real-time PCR or multiplex ligation-dependent probe amplification (MLPA) [50] for copy number variants [79]. Additionally, VPS13C was screened using a custom-designed gene panel (MASTR technology, Agilent, Multiplicom, Niel, Belgium) [42] followed by massive parallel sequencing on a MiSeq sequencing platform (Illumina, San Diego, CA, USA). We selected 52 unrelated EOPD patients (mean AAO 41.7 ± 6.7, 28.8% women, 23.1% with a positive familial history) with documented family history of disease and no disease-causing PD mutations for WES to search for novel PD genes.

A geographically-matched cohort, using self-reported ethnicity data, consisted of 598 unrelated control individuals with a mean age at inclusion (AAI) of 70.4 ± 7.9 years (67.7% female). Some control individuals were partners of the patients visiting a clinical neurology department, and who were screened for neurological or psychiatric antecedents and neurological complaints. The majority of the control individuals were community-dwelling volunteers, who tested with a score > 25 on a Montreal Cognitive Assessment (MoCA) [76] and without personal or familial history of neurodegenerative or psychiatric diseases.

Whole exome sequencing

WES was performed on high molecular weight genomic DNAs of the selected 52 EOPD patients. The SeqCap® EZ Human Exome Probes v3.0 (Roche) was used for exome enrichment for 23 patients followed by library sequencing on a NextSeq 500 platform (Illumina; ≥ 20 × coverage of RefSeq [80] coding region: 90.3 ± 1.88%). The SureSelect All Human Exon V5 + UTR enrichment (Agilent) was applied for 10 patients (≥ 20 × coverage of RefSeq [80] coding region: 87.3 ± 0.80%), the Nextera Rapid Capture Exome (Illumina) for 1 patient (≥ 20 × coverage of RefSeq [80] coding region: 61.18%) and the Nextera Rapid Capture Expanded Exome (Illumina), for 2 patient (≥ 20 × coverage of RefSeq [80] coding region: 78.13 ± 0.78%) followed by sequencing on a HiSeq 2000 platform (Illumina). The Burrows-Wheeler Aligner (BWA) [62] was used to perform the sequence alignment to the reference genome GRCh37 (hg19, UCSC Genome Browser). Variant calling was done with Genome Analysis Toolkit (GATK) Unified Genotyper [27, 72]. For 16 patients, exome enrichment was performed using the TargetSeq Exome Enrichment kit (Thermo Fisher Scientific, https://www.thermofisher.com) followed by sequencing on the SOLiD™ 5500xl Wildfire system (Thermo Fisher Scientific; ≥ 20 × coverage of RefSeq [80] coding region: 64.1 ± 6.75%). Here, read alignment and variant calling ware performed using the LifeScope™ Genomic Analysis Software (Thermo Fisher Scientific). GenomeComb, an in house developed package designed for analysis of massive parallel sequencing data, was used to annotate and select variants [91]. Because recessive PD genes are associated with an early onset age, we focused on homozygous and putative compound heterozygous variants (≥ two variants in one gene) with the following criteria: No occurrence or a minor allele frequency (MAF) ≤ 5% in Genome Aggregation Database (gnomAD) and < 25% in an in-house next generation sequencing (NGS) database of probands with distinct neurodegenerative brain disorders; variants with impact on protein level (non-synonymous missense, nonsense, frameshift, and splice site variants); high quality variants (coverage ≥ 15, genomic location outside repeat regions marked as simple repeats or micro satellites by RepeatMasker v3.0 [103]. Genes with autosomal recessive variants in 3–4 EOPD patients received the highest priority as well as genes with functional annotations related to known PD pathways.

Whole genome sequencing

Whole genome sequencing (WGS) of EOPD patient DR621 and unaffected parents, subsequent read alignment to the human reference genome (NCBI build 37) and base and variant calling were performed by Complete Genomics™ Inc [31]. A minimum coverage of 20 × was obtained for 93.0 ± 2.2% of the bases. GenomeComb was used for variant annotation and selection [91]. Low coverage (< 20 ×) variants in addition to variants located in tandem repeats or high variability clusters were excluded from further analyses. We focused on exonic non-synonymous variants with a MAF less than 5% in the 1000 Genomes Project database or below 25% in an in- NGS database of probands with distinct neurodegenerative brain disorders. Variants were selected in line with the following inheritance patterns: X-linked, de novo, autosomal compound heterozygous recessive (in trans) or autosomal homozygous recessive. Validation of the selected variants in DNA samples of DR621 as well as variant genotyping in unrelated control individuals was performed using iPLEX Gold chemistry on the Sequenom MassARRAY platform followed by MALDI-TOF mass spectrometry, or by direct Sanger sequencing on an automated ABI3730xl DNA analyzer (Applied Biosystems). Primers were designed with either MassARRAY Assay Design software v.3.0.2.0 (Sequenom Inc) or Primer3. Genotypes were automatically called using MassARRAY Typer software v4.0 (Sequenom Inc) or the NovoSNP software package and visually checked according to GLP principles [116]. Variation combinations were assigned a lower priority for follow-up when present in aged control individuals. In case of X-linked variations, only genotypes of male individuals were accounted for. Homozygosity mapping of the WGS data using PLINK was performed as described elsewhere [55]. Homozygosity mapping did not reveal homozygous regions larger than 1 Mb in DR621, indicating that homozygous variants are unlikely to be pathogenic in the proband. Structural variants (SV) were called by Complete Genomics™ Inc [31] and a SV detection tool integrated in GenomeComb [91]. In silico disease-network analyses were performed using four algorithms: SUSPECTS, ToppGene, Endeavour and Biograph [4, 5, 63, 99]. These prediction programs rely on distinct combinations of features and metrics to link a predefined disorder with a set of candidate genes. The known genes for recessively inherited early-onset PD (PARK2, PINK1, and PARK7) and recessively inherited juvenile- or early-onset atypical parkinsonian syndromes (FBXO7, ATP13A2 and PLA2G6) were used as training parameters. Candidate gene prioritization programs nominated ATP10B as the most likely disease gene in this family. Sanger sequencing was used to sequence the coding region and splice site junctions of all eight candidate genes in 120 EOPD patients.

Targeted resequencing of ATP10B

PCR amplification of all 26 exons and flanking splice sites of ATP10B (NM_025153) was performed by a custom-designed amplicon-target PCR amplification assay (MASTR technology, Agilent, Multiplicom, Niel, Belgium) [42]. Amplicons were uniquely tagged; based on the Nextera XT shotgun library preparation protocol (Illumina, San Diego, CA, USA), containing sample-specific indices [59]. Libraries (n = 975) were pooled and sequenced in one run on the MiSeq platform using the MiSeq V3 chemistry, generating paired-end sequence reads of 300 nucleotides (Illumina, San Diego, CA, USA). After sample de-multiplexing, adapter clipping was performed with fastq-mcf [10] and sequence reads were mapped using the Burrows–Wheeler Aligner (BWA) [62] to a mini-genome, combining the target sequences extracted from the human genome reference sequence hg19. Primer sequences were clipped out using the sam_clipamplicons tool in Genomecomb [91]. The mean percentage > 20 × coverage of all target amplicons in 1441 samples (PD, DLB and control cohort) was 99.32 ± 1.92%. Sequence variants were called with GATKv2.4 UnifiedGenotyper and GATKv3.5 HaplotypeCaller [27, 72], and annotated using GenomeComb [91]. The option -dcov 1000 in GATK was used to downsample reads as of > 1000 reads per sample. We focused on homozygotes and compound heterozygotes (e.g. ≥ 2 variants per individual) with a frequency < 0.01%. Coding variants were numbered according to the GenBank Accession Number NM_025153 and amino acid changes according to the GenPept Accession Number NP_079429.

Variant genotyping

Candidate variants identified in the WES/WGS data were validated and genotyped in relatives whenever possible by PCR amplification of genomic DNA followed by Sanger sequencing using the BigDye® Terminator Cycle Sequencing kit v3.1 (Applied Biosystems) on an ABI3730 automated sequencer (Applied Biosystems). Primers were designed using the online Primer3 software [95]. Sanger sequencing was also used for the validation of rare (MAF < 5%) non-synonymous coding and splice site variants identified by targeted resequencing of ATP10B.

Haplotype sharing analysis

Haplotype sharing between the relatives was analyzed by genotyping 15 polymorphic short tandem repeat (STR) markers surrounding ATP10B at chromosome 5q34: GATA139B09, D5S2090, D5S2013, D5S673, D5S2007, D5S1507, D5S2049, D5S412, D5S2038, chr5:159987161–159987527, chr5:160120387–160120617, D5S529, D5S422, D5S2066 and D5S2040. The STR markers were PCR amplified using fluorescently-labeled primers and size-separated using GeneScan 600 LIZ Dye Size Standard (Applied Biosystems) on an ABI3730xl DNA Analyzer (Applied Biosystems). Fragment lengths were scored using the in-house developed Local Genotype Viewer genotyping software (https://www.neuromicssupportfacility.be/).

In silico splicing prediction

The splicing effect was evaluated in silico according to 4 splicing prediction programs (SpliceSiteFinder-like, MaxEntScan, NNSPLICE and GeneSplicer) integrated in Alamut Visual version 2.11.0 (Interactive Biosoftware, Rouen, France).

Quantitative real time PCR

Total RNA of five substantia nigra and six medulla oblongata samples from six idiopathic PD patients without ATP10B mutations and four substantia nigra and medulla oblongata samples from four age- and gender-matched control individuals without neurologic pathology (provided by the Antwerp Biobank at the institute Born-Bunge) was extracted using the Ribopure kit (Ambion) and DNAse treated (Turbo DNAse kit, Ambion). Total RNA originating from a variety of human tissues (Life Technologies, AM6000) and brain regions, including substantia nigra (Clontech), cerebellum (Agilent Technologies), frontal cortex (Agilent Technologies & Stratagene), temporal cortex (Clontech & Biochain), parietal cortex (Aligent Technologies & Ambion), occipital cortex (Clontech), medulla oblongata (Biochain), striatum (Agilent Technologies), hippocampus (Clontech & Biochain), and basal ganglia were purchased. RNA concentrations and integrity were evaluated on a 2100 Bioanalyzer (Agilent Technologies). cDNA synthesis (Superscript III First-Strand synthesis, Thermofisher) was performed with both random hexamer and oligo (dT) primers. ATP10B expression was examined by SYBR green-based quantitative real time PCR (qRT-PCR, ATP10B ex10 FW: TCATCCTCATGTGCCTTATTGG & Rev: TGTTCTTCAAA GGTCCCATTCC; ATP10B ex17 FW: TCATGGAAACTGCACAGCATCT & Rev: CTGCAGCCGGTCTTCGAT). At least two reference genes were included in the experimental setup, based on their stability as calculated by Qbase+ (Biogazelle, HPRT1 FW: TGACACTGGCAAAACAATGCA & Rev: GGTCCTTTTCACCAGCAAGCT; GAPDH FW: ACGGGAAGCTTGTCATCAATG & Rev: GCATCGCCCCACTTGATTT; YWHAZ FW: CACAAGCAGAG AGCAAAGTCTTCTAT & Rev: AGCTTCTTGGTATGCTTGTTGTGA). All samples were run in triplicates. Normalization of ATP10B values and calculation of the relative mRNA expression levels was performed using Qbase+ software. Knockdown was verified with quantitative RT-PCR on RNA isolated from transduced and selected cells with the following primers: FW CTTCTACATGTTCCTCACAATGATCA, Rev GCTCAATGGAGACATACAAAGAGATG (human); FW CTT CTATATGTTCCTCACAATGATCA, Rev GCTCAATGGACACATACAAGGAGATC (mouse).

Tissue culture maintenance

HeLa, Hek293T (Dharmacon, HCL4517), Im95m (JCRB cell bank, JCRB1075.1) and WM115 cell lines (Sigma, 91061232) were cultured in Dulbecco's Modified Eagle Medium (DMEM) culture media containing 1% l-glutamine and penicillin/streptomycin (Sigma-Aldrich; G7513 and P0781, respectively) as well as 10% fetal bovine serum (FBS; Life Technologies, 10270106). Im95m cells were cultured with 10 µg/ml insulin in the medium (Sigma-Aldrich; I9278). Cells were cultured for a maximum of 20 passages.

Viral transductions

For viral transductions, 100,000 of either ATP10B negative HeLa, or endogenously expressing WM-115 or Im95m cells or 200,000 isolated cortical neurons were plated per well in 24-well plates. For overexpression, HeLa cells were transduced with lentiviral vectors coding for Cell Cycle Control Protein 50A (CDC50A) and ATP10B (encoding for the wild type (WT), catalytic dead mutants p.E210A and p.D433N, disease mutants p.R153*, p.G671R/p.N865K, p.V748L, p.E993A, p.I1038T, p.T161N, p.G393W, p.G6487R, p.I1222T and the polymorphism p.C217R or an enhanced green fluorescent protein (eGFP)-tagged WT and p.D433N variant). After lentiviral transduction, cells were selected with hygromycin (CDC50A, 200 µg/ml; Invitrogen, Ant-hg-1) or puromycin (ATP10B variants, 2 μg/mL; Invitrogen, Ant-pr-5) before confirmation by immunoblotting. For double transduction cells were first transduced with CDC50A vector, selected with hygromycin, and then transduced with the different ATP10B viral vectors. For knockdown, microRNA (mir) based short-hairpin lentiviral vectors were generated as described [82]. Viral vectors against 5 (human) to 7 (mouse) different target sequences were produced and validated for functionality. The most potent mir against each target were further used in this study (human mir2: ATGATTCAAGCTGCTGATATTG, human mir3: ACTTTGCCATCACCCGCTTTAA, human mir5: ACCTTAAGCTAGTACCTATATA, mouse mir5: TCCTGGTGATTCTGAACTGGAT, mouse mir7: CCCTAAGACAGTGCCTATACAT). A mir against firefly luciferase (mirFluc, ACGCTGAGTACTTCGAAATGTC) was used as control [82]. Transduced knockdown cell lines were selected with blasticidin (10 µg/ml, Invivogen).

Transient transfection

For the subcellular localization of ATP10B, stable eGFP-ATP10B WT or eGFP-ATP10B p.D433N cell lines were transiently transfected with either an endo-/lysosomal marker (Rab5-mCherry, Rab7-mCherry, LAMP1-mCherry) or an endoplasmic reticulum (ER) marker (Serca-mCherry) using Lipofectamine 2000 (Thermo Fischer Scientific, 11668-027, at a ratio of 3:1) for 8 h in FBS free media prior to overnight incubation in full culture media. The cells were washed with PBS (Life Technologies, 14190169) and fixed (30 min, 37 °C) with 4% paraformaldehyde (PFA, Affymetrix, 199431LT).

Neuron Isolation and experimental culture

Primary cortical neurons were derived from E16 FVB/N mice embryos. Pregnant mice were sacrificed on gestation day 16 by cervical dislocation. E16 mice pup brains were collected and placed in a dish containing calcium- and magnesium-free Hanks’ Balanced Salt Solution (HBSS, Life Technologies, 14180-046) on ice. Both cerebral hemispheres were separated from the cerebellum. Meninges were removed from the cerebral hemispheres and brain cortices were dissected. Brain cortices were collected, washed twice and digested with 0.05% trypsin (Life Technologies, 25300-054, 10 min at 37 °C). Trypsin reaction was stopped by adding 7 ml of HBSS and 1 ml of horse serum. Cells were separated by pipetting and filtration through a 40 μm cell strainer (Falcon, 352340). Cells were centrifuged for 5 min at 1000 rpm (4 °C), the supernatant removed and the pellet suspended in 5 ml Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich, D6546) + GlutaMAX (Life Technologies, 31966-021) containing 5% horse serum (Life Technologies, 26050-088) and 20 mM glucose (Sigma-Aldrich, 8270). Primary cortical neurons were plated in the relevant well plates, coated with poly-d-lysine (Sigma-Aldrich, P6407). After an overnight incubation, cell medium was exchanged for Neurobasal medium (Life Technologies, 21103-049) supplemented with 2% B27 (Life Technologies, 17504-044) and 2 mM l-glutamine (Life Technologies, 25030-24). At 3 days in vitro (DIV) the neurons were transduced with two independent ATP10B targeting mir’s, utilizing mirFluc as a control. For recovery of ATP10B expression within experimental knockdown conditions, lentiviral based overexpression of ATP10B or p.D433N was performed on DIV 4. Fluc was used as a control. At DIV 6, neurons were treated and experiments were terminated at DIV 7.

Treatments and reagents

Cell lines and isolated neurons were treated with indicated doses of rotenone (dimethyl sulfoxide (DMSO), 0–10 µM; Sigma-Aldrich, R8875), zinc (ZnCl 2 , 0–10 µM, Sigma-Aldrich, Z0152), the proteasome inhibitor bortezomib (phosphate buffered saline (PBS), Bort, 100 nM; Bio-Connect BV, 354938) or manganese (PBS, MnCl 2 0–1 mM; CASP, 25,605) for 48 h. Apoptosis was blocked by a 1 h pre-incubation with the caspase inhibitor Zvad-fmk (DMSO, Zvad, 50 µM; Bachem, N1560-0005). Lysosomal functionality was blocked by a 1 h pre-treatment with bafilomycin (BAF A1, 50 nM or 1 µM; Sigma-Aldrich, B1793-10UG). In non-treated conditions, vehicle was used as a control. For the experimental use of lipids, lipid stocks were prepared in 100% chloroform and a lipid film was formed by evaporation under nitrogen. Finally, lipids were re-suspended in either 95% ethanol (translocation assays) or reaction buffer supplemented with 10 mM of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS, ATPase assay).

Autophosphorylation assay

HeLa microsomes were prepared according to [49]. The assay was performed as previously described [48] with minor adaptations. Firstly, during microsomal preparation ATP10B was isolated in styrene maleic acid co-polymer (SMA) lipid particle-based microenvironment (SMALP), whereby the SMA co-polymer (PolySoMA, Uispac) was added to a final concentration of 2.5% (wt/vol) before the final ultracentrifugation step. Secondly, for the detection of ATP10B phospho-enzyme a reaction time of 5 min was used.

ATPase assay

HeLa microsomes were prepared [49]. ATPase activity was assessed using the commercially available ADP-Glo MAX assay (Promega). Briefly, 5 µg of microsomes were plated per well in a white 96-well plate, in 50 mM 3-(N-Morpholino)propanesulfonic acid (MOPS), 100 mM KCl, 11 mM MgCl 2 , 1 mM Dithiothreitol (DTT), 195 µM n-dodecyl-d-maltoside (DDM, pH 7, KOH) and allowed to equilibrate on ice for 1 h, placed at 37 °C for 5 min prior to the stimulation of ATPase activity by the addition of 5 mM ATP for 30 min (37 °C). Reactions were terminated by Glo MAX assay reagent addition and luminescence was monitored after 1 h using a Flexstation 3.0 microplate reader. To determine the effect of lipid substrate addition on ATP10B ATPase activity, 10 µg of microsomes were plated per well in a white 96-well plate, in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM KCl, 12.5 mM MgCl 2 , 1 mM DTT (pH 7, KOH) with 500 µM phosphatidylcholine, 500 µM glucosylceramide or the combination (250 µM of each lipid) and reactions were performed as described above.

Lipid translocation assay

HeLa microsomes were prepared according to [49]. ATP-dependent nitrobenzoxadiazole (NBD) phospholipid translocase activity was measured as described by [77], with some minor adaptations. In brief, microsomal membranes, harvested from stable HeLa cells overexpressing ATP10B WT or p.D433N, were assayed for ATP10B-dependent flippase activity using a back-extraction method. Equal volumes of microsomes, NBD-lipids (10 µM) and ATP regenerating system (without ATP) were mixed together and incubated for 1 h on ice to allow incorporation of the NBD-lipid into the cytosolic membrane leaflet. Subsequently, samples were incubated at 37 °C and after 2 h, ATP was added to stimulate ATP10B-dependent flippase activity. At hourly time points, 3 aliquots of 15 μl each were removed and added to one tube (A) containing 7.5 μl of buffer H (10 mM HEPES, pH 7.5, 150 mM NaCl) and two tubes (B, C) containing 7.5 μl of 3% fatty acid-free bovine serum albumin (BSA, Merck-Millipore) in buffer H. After 5 min on ice, 177.5 μl of ice-cold buffer H was added to each tube, and samples A and B were centrifuged at 150,000×g for 15 min (4 °C), while sample C was left on ice. The supernatants from samples A and B and the entire sample C were transferred to a 96-well plate and mixed with 200 μl 2% Triton X-100 in buffer H. The NBD fluorescence of each sample was measured using a fluorescence plate reader (FlexStation, Molecular Devices) with excitation at 467 nm and emission at 534 nm (cut-off 530 nm). The percentage of NBD-phospholipid in the cytosolic membrane leaflet was calculated as described in [77]. To investigate the effect of disease mutations or substrate specificity experiments were performed as 4 h endpoint measurements. For substrate specificity, competition experiments were performed with the aforementioned procedure; however the membrane fraction were incubated with NBD-PC or NBD-GluCer alone or in combination with a spectrum on non-NBD labeled lipids (10 µM), equilibrated on ice for 1 h followed by 37 °C for 2 h. Flippase activity was activated by the addition of ATP and terminated 2 h later. Samples were collected and assessed as described above.

Co-immunoprecipitation

HEK293T cells were lysed 48 h post-transfection in lysis buffer (25 mM Tris–HCl, pH 8.0, 150 mM NaCl, 2% Nonidet P-40, supplemented with complete protease (Roche) and Phospho-STOP (Sigma) inhibitor mixtures) for 30 min on ice and cleared by centrifugation (10 min, 20,000×g). Protein concentration of the supernatant was determined using a bicinchoninic acid (BCA) protein assay (Pierce™). 1 mg/ml of all cell lines was incubated overnight in the presence of protein G Dynabeads™ (Thermo Fisher Scientific) and HA antibody (Cell signaling, 3724) or normal rabbit IgG antibody (Santa Cruz, sc-2027) at 4 °C on a rotating device. Afterwards, the beads were collected and washed repeatedly with the lysis buffer. Finally, beads were re-suspended in lysis buffer supplemented with 4 × LDS sample buffer and 100 mM DTT, boiled for 10 min at 95 °C, and loaded on 4–12% Bis–Tris NuPAGE gels (Life Technologies).

Split luciferase assay

A protein complementation assay based on firefly luciferase was used to show interaction between ATP10B and CDC50A, with eGFP as a control. Plasmids for the N- and C-terminal parts of luciferase were generated that fused to ATP10B WT, CDC50A and eGFP was used as a control. 20,000 HEK293T cells were plated 20,000 cells/well in a 96-well plate and transfected with different combinations of the aforementioned plasmids. Luciferase activity was measured 48 h after transfection using the ONE-Glo Luciferase Assay System (Promega) and is used as a measure for protein interaction between the N- and C-terminal parts of luciferase.

Immunoblotting

HeLa microsomes were prepared according to [49] and immunoblotting was performed as previously described [113]. For total cell lysate investigation, cells were lysed using RIPA buffer (ThermoFisher Scientific, 89900) and DNA excluded by centrifugation (15,000×g for 15 min). Briefly, western blots of typically 20–40 µg of protein were ran on 4–12% Bis/Tris gel (NuPage, Thermo Scientific, NP0323BOX) and transferred to a 0.45 µm PVDF membrane (Immobilon-P, Thermo Scientific, 88518) and probed for ATP10B (Sigma-Aldrich, HPA034574), CDC50A (anti-FLAG antibody; Sigma-Aldrich, F3165). Cell death was assessed using a cleaved caspase 3 antibody (Cell signaling, 9661). All blots were probed for either GAPDH (Sigma-Aldrich, G8795) or β-actin (Sigma-Aldrich, C6198) as a loading control. Detection was performed using HRP-conjugated secondary antibodies (BIOKE, 7074S and 7076S). Detections were performed on a Bio-Rad Chemidoc Imager with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, 32106). For co-immunoprecipitation experiments, PVDF membranes (Hybond P; Amersham Biosciences) were blocked in 5% skimmed milk in PBS and probed with primary antibodies against HA-tag (Covance, MMS-101P) and FLAG-tag (Novus Biologicals, NBP1-06712).

Immunofluorescence

After fixation with 4% PFA, cells were washed twice with PBS containing 0.5% tween 20 (PBS/T; Sigma-Aldrich, P1379) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T9284) and 0.02% SDS (Acros Organics, 230425000) containing PBS/T for 20 min. To minimize non-specific binding, 0.1 M Glycine in PBS/T was added to the cells after washing followed by blocking in PBS/T containing 1% BSA and 2% FBS. After washing, cells were incubated overnight at 4 °C with various primary antibodies: Rab11 (Thermo Fisher Scientific, 71-5300), Rab5 (Santa Cruz Biotechnology, Sc-46692), EEA-1 (Becton Dickinson, 610457), CD63 (BIO-CONNECT Diagnostics, 11-343-C100), LAMP-1 (Abcam, ab24170), Rab7 (Abcam, ab137029 and ab25245), LAMP2 (Abcam, ab25339 and ab25631), ATP10B (Sigma-Aldrich, HPA034574) or cleaved caspase 3 (Cell Signaling, 9661) diluted according to manufacturer’s recommendations in PBS/T containing 0.1% BSA and 0.2% FBS. Finally, after the samples were washed with PBS-T, cells were incubated with Alexa Fluor dyes (Thermo Fisher Scientific, 1/2000) for 30 min. Nuclei were stained with DAPI (Sigma-Aldrich, D9542-10 mg). Cells were visualized using an LSM780 confocal microscope.

Cell viability

Cells were seeded at 5000 cells per well in a 96-well plate. After treatment, cells were washed with PBS and incubated with 0.01 mg/ml MUH (4-methylumbelliferyl heptanoate; Sigma-Aldrich, M2514; dissolved in PBS) for 30 min at 37 °C. Fluorescence was measured with a Flexstation 3.0 plate reader (Molecular Devices, Wokingham, UK) at an excitation of 355 nm, emission of 460 nm, and cut off value of 455 nm.

Cell death

HeLa cells treated with rotenone or MnCl 2 were assessed for cell death induction as previously described [48]. Briefly, treated cells were collected at 48 h by trypsinization, PBS-washed and exposed to 1 µg/ml propidium iodide (PI; Sigma-Aldrich, P4170-25MG) for 5 min. PI positivity was captured using an Attune flow cytometer (Life Technologies). Neuronal cell apoptosis was assessed by cleaved caspase 3 staining in accordance with the manufacturer’s guidelines (Cell Signaling, 9661). Cortical neurons were plated with or without cover slips. Cells plated without coverslips were ultimately assessed by flow cytometry using an Attune Flow Cytometer. Samples seeded on coverslips were imaged using a LSM780 confocal microscope (Leica). DAPI staining was used to visualize the nucleus.

TUNEL staining

Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) was assessed according to the manufacturer’s protocol for the Click-iT Plus TUNEL assay (ThermoFisher, C10617). Images were captured using a Leica LSM780 confocal microscope.

Fluorescein isothiocyanate-dextran based lysosomal pH

WM115 cell lines were plated in 12 well plates (100,000 cells per well) and allowed to attach overnight 37 °C. Cells were then exposed to 50 µg/ml fluorescein isothiocyanate (FITC)-dextran for 72 h. Samples were then washed, placed in fresh media, and treated with the appropriate stressors (1 µM Rotenone and 1 µM BAF A1) for 4 h. Samples were then collected by centrifugation (450×g, 5 min) and washed in PBS containing 1% BSA. Cells were finally re-suspended in 50 µl of PBS/BSA and FITC dual emission assessed by flow cytometry (excitation 488 nm, emission 530 (BL1) and 600 (BL2) using an Attune NXT flow cytometer (invitrogen). The emission (BL1/BL2) of all samples were compared to the signals obtained for control cells re-suspended in monensin (100 µM) containing Britton Robinson buffer with increasing pHs (3.0–8.0).

Lysosomal degradative capacity (DQ-BSA)

Cells were seeded in 12-well plates (1.0 × 105 cells per well) and the next day, cells were pre-treated with rotenone for 1 h at 37 °C. 50 nM BAF A1 was used as an internal control. Subsequently, 5 µg/ml DQ Green BSA was added to the cells for a further 3 h (37 °C). Finally, cells were collected and the mean fluorescent intensities of 10,000 events were assessed using an Attune Nxt (Thermo Scientific) flow cytometer. For the assessment of lysosomal degradation capacity in isolated cortical neurons, cells were seeded in 12-well plates containing cover slips (3.0 × 105 cells per well) and the next day ATP10B was knocked down via lentiviral transduction. mirFluc was used as an internal transduction control. 48 h post lenti-viral exposure. ATP10B WT, p.D433N or Fluc were over-expressed via lentiviral transduction and allowed to incubate for a further 24 h at 37 °C. Following rescue, cells were treated with 10 µg/ml DQ-BSA for 1 h prior to treatment with rotenone (50 nM), and incubated further for 23 h at 37 °C. Cells were subsequently fixed, DAPI stained and images captured via confocal microscopy (Zeiss LSM780).

Lysosomal membrane integrity

To assess lysosomal membrane integrity, WM-115 cells were seeded in 12-well plates (1.0 × 105 cells per well) and the next day, cells were incubated with 5 µg/ml acridine orange (dissolved in media) for 15 min at 37 °C. Thereafter, medium was discarded, cells were washed and fresh medium was added. Cells were then treated with rotenone or the positive control BAF A1 (1 µM) for 4 h at 37 °C. Finally, cells were collected and resuspended in PBS containing 1% BSA. The mean fluorescence of 10,000 events was captured using an Attune Nxt (Thermo Scientific) flow cytometer.

Statistics

To investigate association between recessive ATP10B mutations and PD, mutation frequencies were statistically compared between the early-onset patient group and control individuals using Fisher’s exact statistics. ATP10B gene expression levels in PD patient brains versus brains of neurologically healthy individuals were statistically compared using a Mann–Whitney U test. Statistical significance of lipid translocation, cell death, and viability were performed by t test and one/two way analysis of variance within GraphPad Prism 6.01. Pearson’s coefficients were generated using Image J. Unless otherwise specified, data are represented as the average ± standard error of the mean (SEM) of a minimum of three independent experiments.

Ethical assurances

The genetic studies were approved by the ethic committee of the Antwerp University Hospital and the University of Antwerp. Clinical protocols were approved by the ethics committees of the main participating hospitals i.e. the Hospital Network Antwerp and the University Hospital Antwerp, as well as by the ethical review boards of the participating general hospitals which are participating via the BELNEU consortium. All human biological samples were collected in accordance with the written informed consents signed by the participants. All mouse primary neuron experiments were carried out in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC) and approved by the Bioethical Committee of the KU Leuven (Belgium) (ECD project P185-2014).