Cell culture - Human neuroblastoma SH-SY5Y (RRID: CVCL_0019) cell lines either non-transduced (nts) or stably overexpressing wild-type ATP13A2 (Addgene plasmid #171485, RRID: Addgene_171485 or Addgene plasmid #213697, RRID: Addgene_213697), a catalytically dead variant (D508N) (Addgene plasmid #171820, RRID: Addgene_171820), or the Pro652_Glu653del ATP13A2 variant (Addgene plasmid #213700, RRID: Addgene_213700) (the nomenclature is based on the sequence of ATP13A2 splice variant 2, which is 5 amino acids shorter at the N-terminal region and has been historically used for biochemical analysis [5]) were generated via lentiviral transduction as described previously [14]. A detailed protocol can be found at https://doi.org/10.17504/protocols.io.bw57pg9n. Cells were maintained at 37 °C in the presence of 5% CO2 and incubated in high-glucose Dulbecco’s modified Eagle medium supplemented with 1% penicillin/streptomycin (Sigma), 15% fetal calf serum (heat inactivated) (Sigma), 1% non-essential amino acids (Sigma), 1% sodium pyruvate (Gibco), and selection antibiotic (160 µg/mL hygromycin or 2 µg/mL puromycin (Invivogen)). The treatments were performed in the same medium, with the exclusion of selection antibiotic.

Immunofluorescence - Cells were seeded at 75 000 cells/well in a 12-well plate with coverslips and allowed to attach and grow for 48 h. Thereafter, cells were washed with ice-cold PBS and fixed with 4% paraformaldehyde (Thermo Fisher Scientific) (30 min, 37 °C). Cells were washed twice more with ice-cold PBS before permeabilization and blocking with a mixture of 5% BSA (Roth) and 0.5% saponin (Sigma) (referred to as blocking buffer) for 1 h. Next, cells were incubated with primary antibody (anti-ATP13A2, A3361, Sigma, RRID: AB_10597403; anti-CD63, 11-343-C100, ExBio, RRID: AB_10733918) (diluted 1/200 in blocking buffer) for 2 h and then subjected to secondary antibody (Alexa-488 goat anti-rabbit IgG, R37116, Invitrogen, RRID: AB_2556544; Alexa-594 goat anti-mouse IgG, A11005, Invitrogen, RRID: AB_2534073) (diluted 1/1000 in blocking buffer) for 30 min on a shaker. Subsequently, cells were immersed in 200 ng/ml DAPI (D9542, Sigma) for 15 min. Samples were thoroughly washed with PBS in between the different steps. Finally, the samples were mounted and images were acquired using a LSM880 microscope (Zeiss) with a 63x objective and Airyscan detector. A detailed protocol can be found at: https://doi.org/10.17504/protocols.io.bp2l6xy3klqe/v1.

SDS-page and immunoblotting – 70% confluent cells were harvested and subsequently lysed with radio-immunoprecipitation assay buffer (Thermo Fisher Scientific) supplemented with protease inhibitors (Sigma). 10 µg of protein (concentration determined with a bicinchoninic acid protein assay) was then loaded on a NuPage 4–12% Bis-Tris gel (Thermo Fisher Scientific, Bio-Rad) and subjected to an electrophoresis run at 130 V in MES running buffer (Thermo Fisher Scientific). The proteins were subsequently transferred to a PVDF membrane in NuPage transfer buffer (Thermo Fisher Scientific) supplemented with 10% v/v methanol (Roth). Immunoblots were blocked by incubation in 5% w/v milk powder (1 h, room temperature). Next, immunoblots were probed with primary antibodies (anti-GAPDH, G8795, Sigma, RRID: AB_1078991; anti-ATP13A2, A3361, Sigma, RRID: AB_10597403) (diluted 1/5000 and 1/1000 in 1% w/v BSA, respectively) and incubated O/N (4 °C). After thorough washing, immunoblots were subjected to secondary antibodies (HRP-linked anti-mouse IgG, 7076 S, Cell Signaling, RRID: AB_330924; HRP-linked anti-rabbit IgG, 7074 S, Cell Signaling, RRID: AB_2099233) (diluted 1/2000 in 1% w/v milk powder) (1 h, room temperature). All dilutions and wash steps were performed with TBS supplemented with 0.1% v/v Tween-20 (PanReac AppliChem). Detection was performed by means of chemiluminescence (Bio-Rad ChemiDoc) and protein levels were quantified with Image Lab software (RRID: SCR_014210, version 6.0.1, Bio-Rad, https://www.bio-rad.com/en-be/product/image-lab-software?ID=KRE6P5E8Z). A detailed protocol can be found at: https://doi.org/10.17504/protocols.io.e6nvwdqz2lmk/v1.

14C-labeled polyamine uptake – This protocol is based on a previous publication [15], with minor modifications. Briefly, 70% confluent SH-SY5Y cells in a 12-well plate were incubated (30 min, 37 °C) either with 5 µM 14C-labeled spermine (3139-50 µCi, ARC) or with a mixture of 5 µM 14C-spermine and 100 µM unlabeled spermine in cell culture medium. The medium was subsequently aspirated and cells were washed twice with ice-cold PBS (without Ca2+ and Mg2+). Next, the cells were lysed by incubation in 200 µl radio-immunoprecipitation assay buffer (10 min, room temperature) (Thermo Fisher Scientific) before scraping. The cell lysate was added to scintillation vials filled with 7 ml EcoLite Liquid Scintillation Cocktail (01882475-CF, MP Biomedicals). Thereafter, the wells were washed with 200 µl ice-cold PBS (without Ca2+ and Mg2+), which was added to the accompanying scintillation vial. Finally, 14C radioactivity in counts-per-minute (CPM) was measured with liquid scintillation counting (TRI-CARB 4910TR V Liquid Scintillation Counter, PerkinElmer). A detailed protocol can be found at: https://doi.org/10.17504/protocols.io.j8nlkorm5v5r/v1.

Microsome collection - SH-SY5Y cells overexpressing wild-type or mutant ATP13A2 were seeded in 500 cm2 plates. Once they reached 70–80% confluency, cells were collected. Next, cells were lysed by resuspending the cell pellet in 3 ml hypotonic LIS buffer (10 mM Tris.HCl pH 7.5, 0.5 mM MgCl2.6H2O, 1 mM DTT, 1x SigmaFast protease inhibitors) (S8830, Merck), which was incubated on ice for 15 min. The suspension was transferred to a Dounce homogenizer and 60 up-and-down strokes were applied, followed by addition of 3 ml 1 M solution (0.5 M sucrose, 10 mM Tris.HCl pH 7.3, 40 µM CaCl2, 1 mM DTT, 1x SigmaFast protease inhibitors) and another 30 up-and-down strokes. The nuclear (1000 x g, 10 min, 4 °C), mitochondrial-lysosomal (12 000 x g, 20 min, 4 °C), and microsomal fractions (140,000 x g, 35 min, 4 °C) were then collected. Fractions were suspended in 0.25 M sucrose with 1x SigmaFast protease inhibitors. A detailed protocol can be found at: https://doi.org/10.17504/protocols.io.5qpvo3w7dv4o/v1.

ADP-Glo assay - ATPase activity was assessed using a commercially available luminescence assay (ADP-Glo Max assay, V7002, Promega) according to manufacturer’s instructions. A reaction mixture (final volume 25 µl) was made in a 96-well plate and contained 50 mM MOPS-KOH (pH 7), 100 mM KCl, 11 mM MgCl2, 1 mM DTT, 0.1 mg/ml DDM, 5 µg microsomes (1:2 ratio DDM: microsomes) and various concentrations of polyamines. The microsomes were collected from SH-SY5Y cells overexpressing ATP13A2 (wild-type or mutants). Next, the reaction was incubated (20 min, 37 °C followed by 10 min, room temperature) following the addition of 5 mM ATP and terminated by adding 25 µl of ADP-Glo Reagent. The 96-well plate was subsequently incubated (40 min, room temperature), followed by the addition of 50 µl of ADP-Glo Detection Reagent. After 60 min, luminescence was detected using the Bio Tek plate reader. A detailed protocol can be found at: https://doi.org/10.17504/protocols.io.3byl4q1x8vo5/v1.

RNA collection and qPCR - RNA was isolated using the NucleoSpin RNA plus kit (Macherey-Nagel) following manufacturer’s instructions. RNA concentration and purity were determined using a Nanodrop spectrometer (Thermo Fisher Scientific). 5 µg RNA was then converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). A 96-well plate was prepared with a ten-fold dilution of each cDNA sample in duplicates (with a volume of 5 µl cDNA per well), to which the following reaction mixture was added (volumes are given per well): 10 µl SYBR Green master mix (Roche), 1 µl of 5 µM forward primer, 1 µl of 5 µM reverse primer, and 3 µl distilled water. The cDNA was replaced by an equivalent volume of distilled water for the negative controls. Primers targeting the ATP13A2 gene were 5’-CATGGCTCTGTACAGCCTGA-3’ (forward) and 5’ CTCATGAGCACTGCCACTGT-3’ (reverse). Primers targeting the β-actin gene were 5’-CACTGAGCGAGGCTACAGCTT-3’ (forward) and 5’-TTGATGTCGCGCACGATTT-3’ (reverse). The qPCR read-out was performed, in which the reaction was initiated at 95 °C for 10 min, followed by 50 cycles at 95 °C for 10 s and 55 °C for 30 s, and ended at 95 °C for 1 min. A melting curve was determined from 55 to 95 °C. A detailed protocol can be found at: https://doi.org/10.17504/protocols.io.14egn3ymzl5d/v1.

PubMed and Embase searches were completed in April 2023 (and updated in December 2023) using the terms “kufor-rakeb”, “kufor rakeb”, “KRPPD”, ”parkinson disease-9”, “parkinson disease 9”, “PARK9”, “pallido-pyramidal degeneration with supranuclear upgaze paresis and dementia”, “ATP13A2”, or “1p36.13”, in combination with “schizophrenia”, “psychosis”, “psychotic”, “hallucinations”, “delusions”, “paranoia”, “antipsychotic”, or “neuroleptic”. The Pubmed search yielded nine results and the Embase search yielded 26. After duplicates were removed, 33 results remained. All results were manually reviewed, including their reference lists, for additional relevant articles. OMIM was also reviewed. Only articles that described the use of antipsychotic medications in individuals with Kufor-Rakeb syndrome were included in the review. The search process is outlined in Fig. 2.

Literature Search flow diagram for antipsychotic use in Kufor-Rakeb syndrome