Plasmids and vectors

To generate human LRRK2 and PINK1 stable transfected human dopaminergic SH-SY5Y cell lines, wild-type (WT) FL human LRRK2 and PINK1 cDNA was cloned into pcDNA3.1(-) mammalian expression vectors (Invitrogen) using In-Fusion cloning protocol (TaKaRa Bio Inc) and G2019S LRRK2 mutation, and A339T and E231G PINK1 mutations were created by polymerase chain reaction (PCR)-based technique using QuikChange® site-directed mutagenesis kit (Stratagen) as previously reported [26]. For transient transfection of PC12 cells, 2XMyc-LRRK2-WT (Addgene plasmid 25361), 2XMyc-LRRK2-G2019S (Addgene plasmid 25362), 2XMyc-LRRK2-3XKD (Addgene plasmid 25366), 2XMyc-LRRK2-kinase (Addgene plasmid 25071) and 2XMyc-LRRK2-RCK (Addgene plasmid 25065) plasmids developed by Dr. Mark Cookson were purchased from Addgene. 2XMyc-LRRK2-Kinase-G2019S, 2XMyc-LRRK2-Kinase-3XKD, 2XMyc-LRRK2-RCK-G2019S and 2XMyc-LRRK2-RCK-3XKD vectors were created using 2XMyc-LRRK2-kinase (Addgene plasmid 25071) and 2XMyc-LRRK2-RCK (Addgene plasmid 25065) as templates, respectively, based on QuikChange® site-directed mutagenesis protocol (Stratagen). Primers were designed and manipulated by Primer Premier 5.0, SimVector 4 and DNAMAN softwares. Primers used for plasmids cloning, site-directed mutagenesis and sequencing validation experiments are shown in Supplementary Table 1, Supplementary Table 2 and Supplementary Table 3. All positive clone sequencing were verified before performance of transfection protocols.

LRRK2 and PINK1 SH-SY5Y stable cell lines

The protocol to create human dopaminergic SH-SY5Y LRRK2 and PINK1 stable cell lines used in the current study had been reported previously [26]. Briefly, SH-SY5Y cells were transfected with pcDNA3.1 vector, pcDNA3.1-LRRK2-WT, pcDNA3.1-LRRK2-G2019S, pcDNA3.1-PINK1-WT, pcDNA3.1-PINK1-A339T or pcDNA3.1-PINK1-E231G vectors, respectively, using Lipofectamine reagents. After transfection, cells were treated with selection medium containing 0.6 g/L (w/v) G418 antibiotics (Promega) for 2–3 weeks before resistant cells were separated by serial dilution and allowed to grow from single cells. The stably transformed clones were verified by real time RT-PCR and Western blot analysis. Cells were maintained at 37 °C in a 5% CO2 incubator with 0.4 g/L (w/v) G418.

Drug administrations

To challenge cells with H2O2 or iron species, cells were treated with 100 µM H2O2, 100 µM FeSO4 or 100 µM FeCl3, respectively, and incubated for 24 h at 37 °C before analysis of cell viability or rate of cell death. To deplete DA in dopaminergic SH-SY5Y cells, cells were maintained in Dulbecco's modified Eagle medium (DMEM) in the presence of 1 mM α-MT for several passages before being used for experiments. To modulate DA content in Drosophila heads, Drosophila were cultured in culture mediums with or without 15 µM, 500 µM, 2 or 4 mM α-MT for different time periods before withdrawal of α-MT or checking of Drosophila PD symptoms and monitoring DA content in Drosophila heads. To study the reciprocal regulations of PINK1 and LRRK2 protein levels via the proteasome pathway, LRRK2 or PINK1 stable SH-SY5Y cells were cultured in the presence or absence of 3 µM MG132 for 6 h before analysis of LRRK2 or PINK1 protein levels by Western blot analysis. All chemicals were purchased from Sigma-Aldrich.

Reprogramming of induced pluripotent stem cells (iPSC)

The collection of human peripheral blood samples followed the inclusion criteria as per the SingHealth CIRB Ref NO: 2018/2920. Briefly, 2 ml of peripheral blood of patients with or without G2019S LRRK2 mutation was collected and lysed in 2 ml of 1 × red blood cell lysis buffer (eBioscience, San Diego, CA) for 10 min. After lysis, peripheral blood mononuclear cells were spun down and harvested. About 30,000 cells with or without G2019S LRRK2 mutation were suspended in 500 µl of StemSpan expansion medium (StemCell Technologies, Vancouver) in 1 well of a 24-well tissue culture plate and infected with OCT4, SOX2, KLF4 and cMYC Sendai virus (CytoTune-iPS Reprogramming Kit, Thermo Fisher Scientific) with a multiplicity of infection of ten. 24 h later, the infected cells were cultured in 0.5 ml fresh cell expansion medium and plated onto Matrigel (BD Biosciences)-coated dish 2 days later. The induced pluripotent stem cell colonies with an embryonic stem cell (ESC)-like appearance were identified and isolated manually between day 18 to day 25 post-infection. The iPSCs were maintained on Matrigel-coated plates in mTESR-1 medium (Stem Cell Technologies). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2.

Neural progenitor cell (NPC) induction and dopaminergic neuron differentiation

For NPC induction, iPSCs with or without G2019S LRRK2 mutation were harvested using a cell scraper and cultured in suspension as embryoid bodies (EBs) for 8 days in StemPro defined medium (Thermo fisher scientific) minus fibroblast growth factor 2 (FGF2). EBs will then be cultured for an additional 2–3 days in suspension in neural induction medium containing DMEM/F12 with Glutamax, NEAA, N2 and 20 ng/ml FGF2 before attachment on Matrigel-coated culture plates. Neural rosettes were isolated manually with glass Pasteur pipette 3 days later and dissociated into single cells under Accutase digestion and replated onto culture dishes to obtain a homogeneous population of NPC. The NPCs were expanded in Neurobasal media containing nonessential amino acids (NEAA), 2 mM glutamine, B27 and 20 ng/ml FGF2. To induce dopaminergic differentiation, NPCs were plated onto poly-D-lysine and mouse laminin-coated dishes and cultured in DA medium I: Neurobasal medium supplemented with 2 mM NEAA, l-glutamine, 2% B27, 200 ng/ml sonic hedgehog (SHH) and 100 ng/ml FGF8 for 7 days. After 7 days, the DA progenitor cells were then fed with DA medium II: neurobasal medium supplemented with 2 mM NEAA, L-glutamine, 2% B27 and brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) (20 ng/ml of each), 1 ng/ml transforming growth factor beta-3, 200 µM ascorbic acid and 1 mM cAMP for 6 weeks before Western blot analysis or high-performance liquid chromatography (HPLC) analysis of DA content in DA neurons.

Generation of human middle brain-like organoids (hMLOs)

hMLOs were generated from human pluripotent stem cells using a previously reported protocol with minor modifications [47]. Briefly, hPSCs were dissociated from intact colonies to single cells using TrypLETM Express (Gibco), and 10,000 cells were plated in each well of a low-cell-adhesion 96-well culture plate with V-bottomed conical wells (Sumitomo Bakelite) to form uniform embryoid bodies (EBs) in a medium containing DMEM/F12:Neurobasal (1: 1), 1:100 N2 (Invitrogen), 1:50 B27 without vitamin A (Invitrogen), 1% GlutaMAX (Invitrogen), 1% minimum essential media–nonessential amino acid (Invitrogen) and 0.1% β-mercaptoethanol (Invitrogen) supplemented with 1 μg/ml heparin (Sigma-Aldrich), 10 μM SB431542 (Stemgent), 200 ng/ml Noggin (R&D Systems), 0.8 μM CHIR99021 (STEMCELL Technologies) and 10 μM ROCK inhibitor thiazovivin (Tocris). On day 4, midbrain patterning media that contain additional components (100 ng/ml SHH-C25II (R&D Systems), 100 ng/ml fibroblast growth factor 8 (FGF8) (R&D Systems) and 0.5 μM purmorphamine (Stemgent)) were added. After 3 days, the resulting neurospheroids were embedded in 30 μl of reduced growth factor Matrigel and placed in a 37 °C incubator for 30 min to solidify Matrigel, and the tissue growth induction media containing Neurobasal medium, 1:100 N2 supplement, 1: 50 B27 without vitamin A, 1% GlutaMAX, 1% minimum essential media–nonessential amino acid, and 0.1% β-mercaptoethanol supplemented with 2.5 μg/ml insulin (Sigma-Aldrich), 200 ng/ml laminin (Sigma-Aldrich), 100 ng/ml SHH-C25II, 100 ng/ml FGF8 and 0.5 μM purmorphamine were added. After 24 h, the hMLOs were transferred into ultralow-attachment six-well-plates (Costar) containing the final differentiation media, which consisted of Neurobasal medium, 1:100 N2 supplement, 1: 50 B27 without vitamin A, 1% GlutaMAX, 1% minimum essential media–nonessential amino acid, 100 U/ml penicillin G and 100 μg/ml streptomycin, and 0.1% β-mercaptoethanol with supplements [10 ng/ml BDNF (Peprotech), 10 ng/ml GDNF (Peprotech), 100 μM ascorbic acid (Sigma-Aldrich) and 125 μM db-cAMP (Sigma-Aldrich)], and cultured on an orbital shaker. From day 30, the amount of N2 and B27 without vitamin A was reduced into half, and the hMLOs were maintained without supplements from day 60 onward. The medium was replaced every 3 days.

Transgenic (TG) LRRK2 mice lines

LRRK2 G2019S TG mice were generated using a bacterial artificial chromosome containing the entire mouse mutant G2019S LRRK2 and were purchased from the Jackson Laboratory (#012467) [48]. LRRK2 R1441G TG mice generated from BAC containing the entire human mutant R1441G LRRK2 were also provided by the Jackson Laboratory (#009604) [49]. Mice genotypes were verified by PCR using genomic DNA from mice tails. Mice were maintained in a pathogen-free facility and exposed to a 12 h light/dark cycle with food and water. All procedures were performed in accordance with institutional guidelines, and all protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Neuroscience Institute (NNI) of Tan Tock Seng Hospital (TTSH).

Drosophila stocks, preparation and behavioral assays

Promoter lines containing elav-GAL4, ddc-GAL4 and 24B-GAL4 were obtained from Bloomington Stock Center (Bloomington, IN, USA). Transgenic human LRRK2 Drosophila were generated by Drosophila embryo microinjection protocols as previously described [50]. Briefly, WT or mutant G2019S LRRK2 human cDNA containing a myc-tag at the C terminus was cloned into pUAST plasmid and microinjected into Drosophila embryos (BestGene. USA). Positive lines were selected and sequence confirmed before culture for experiments. The UAS-HA-PINK1/cyo Drosophila line to express Drosophila PINK1 was a gift from Prof Alexander J Whitworth (MRC Centre for Developmental and Biomedical Genetics, Sheffield S10 2TN, UK). The UAS-hPINK1 309D7C1 II Drosophila line to overexpress mutant G309D PINK1 was a gift from Prof Zhuo hua Zhang (Centre for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan 410008, China). The UAS-hPINK1 and dPINK1 RNAi (II, III) Drosophila lines were gifts from Prof Lu Bing Wei (Stanford University. USA). To create the LRRK2 plus PINK1 double transgenic Drosophila lines, stable LRRK2 and PINK1 1 Drosophila were first achieved, respectively, via the following protocol. The promoter ddc-GAL4 line was crossed with the UAS controlled LRRK2 and PINK1 lines, respectively. The red eye virgin offspring Drosophila were selected and crossed with TM3/TM6B balancer males. Then progeny males with the reddest eye color were single picked and crossed with balancer lyshid/TM6B virgins in individual vials. On day 6, the individual vials were heat shocked at 37 ºC to achieve pure stable gene stocks. Stable lines were verified using single Drosophila PCR and Western blot experiments. The stable LRRK2 and stable PINK1 lines were then crossed to obtain the double LRRK2/PINK1 Drosophila lines. Drosophila were routinely raised at 25 °C on cornmeal media that were replaced every 3 days. Drosophila heads were harvested at the respective time points for HPLC analysis of DA content, confocal microscopy detection of TH-positive DA neurons and Western blot analysis of TH, PINK1 or LRRK2 proteins in Drosophila heads. For longevity experiments, 1 day-old Drosophila were transferred to fresh media every 3 days. The life span was assessed by monitoring the survival of 50 Drosophila in five cohorts for each genotype. The triplicate cohorts of 50 Drosophila per genotype were monitored for survival daily. For the climbing assay, motor ability was assessed at 15 min intervals using the negative geotaxis assay. Briefly, three cohorts of 20 female and age-matched control Drosophila were anesthetized and placed in a vertical plastic column (length, 25 cm; diameter, 1.5 cm). After a 2 h recovery period, Drosophila were tapped to the bottom and the percentage of Drosophila that climbed to or above the top column line in 1 min was calculated. Triplicate trials were performed in each experiment at 15 min intervals.

Immunochemistry staining of TH-positive DA neurons in Drosophila heads

Drosophila brain fixation and antibody staining were carried out as described previously [51]. Briefly, adult brains were dissected in phosphate-buffered saline (PBS), then fixed with 4% formaldehyde and washed a few times with PBT (PBS + 0.1% Triton X-100). After blocking with 3% BSA (in PBT), Drosophila brain samples were incubated in primary antibody (rabbit anti-TH; Sigma-Aldrich; T8700, 1: 1000 dilution) overnight at 4 °C with rotation, washed with PBT and incubated in secondary antibody (Cy3-conjugated goat anti-rabbit; Jackson Immunoresearch; 1:500). Samples were washed a few times with PBT, incubated with Vectashield and TH-positive DA neurons were analyzed by Carl Zeiss Upright Confocal Microscope (Zeiss).

Analysis of insoluble ROS-modified and DAQ-conjugated proteins in Drosophila heads by Western blot analysis

Drosophila heads were homogenized and lysed in buffer (25 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% (w/v) sodium dodecyl sulfate (SDS)) containing protease inhibitors (Roche Applied Science) and phenylmethylsulfonyl fluoride and incubated on ice for 30 min. The lysate was then sonicated before centrifugation at 13,200 rpm (16,000 g) for 30 min to obtain the soluble (supernatant) and insoluble (pellet) fractions. The pellet was solubilized in 30 µl of 4 × SDS loading buffer (161–0737, BIO-RAD) with continuous shaking at 4 °C overnight, then boiled and loaded for gel electrophoresis, SDS-PAGE and Western blot analysis with anti-DA antibody (Abcam, ab8888, 1: 1000–2000) to detect DA-conjugated proteins or with anti-dinitrophenylhydrazone (DNP) antibody (Cell biolabs, 230801, 1:1000) to detect ROS-modified carbonylated proteins, respectively.

Western blot analysis

Cells, Drosophila heads, brain organoids or mice brain tissues were collected in lysis buffer (100 mM HEPES pH 7.5, 5 mMmagnesium chloride, 150 mMsodium chloride, 1 mMEDTA, 1% (v/v) Triton X-100 and 1% (v/v) protease inhibitor cocktail (Calbiochem)) and centrifuged at 12,000 g at 4 °C for 10 min. From the supernatant, 30 μg proteins was resolved by 15% SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane using an electric blotting apparatus (Biorad), blocked with washing buffer A (150 mM sodium chloride plus 13 mMTris–hydrochloric acid, pH 7.5, and 0.1% (v/v) Tween 20) containing 5% (w/v) skim milk for 30 min (for TH, GFP and PINK1) or 1 h (for caspase-3) at room temperature before incubation with primary antibody in washing buffer A with 2% (w/v) skim milk (rabbit-anti-PINK1 antibody, Novas Biologicals. 1:1000; or anti-TH (for Drosophila samples), Sigma-Aldrich, T8700, 1:1000; anti-TH (for brain organoids, mouse brain and DA cell samples), Santa Cruz Biotechnology, sc-25269, 1:1000; anti-LRRK2 (for mice brain samples), Abcam, ab133474, 1:10,000, anti-LRRK2 (for cells and Drosophila samples), Sigma-Aldrich, HPA014293, 1:1000) overnight at 4 °C. The membrane was washed 5 × 5 min each with washing buffer A and subsequently incubated with the secondary antibody (anti-mouse antibody, Santa Cruz Biotechnology; sc-2005, 1:5000 or anti-rabbit antibody, Sigma-Aldrich, A4914, 1: 10,000) for 2 h at room temperature. Following subsequent washes, the blots were developed with the enhanced chemiluminescent kit (Pierce) on Kodak CL-Xposure™ films.

Quantitative densitometric analysis of Western blot data

Quantitative analysis of the densities of protein bands in Western blot gels was done by densitometric analysis using the Image software Bandscan 4.30. The relative densities of the respective protein bands of LRRK2, PINK1 and TH in control lanes in Western blot gels were set as 50%. The relative densities of protein bands of LRRK2, PINK1 and TH of other lanes in Western blot gels were expressed as the ratio of the densities for LRRK2, PINK1 and TH, after automatic comparison with the ratio of densities of control lanes by the software.

Quantitative real-time reverse transcription PCR (RT-PCR)

Expression levels of LRRK2 and TH in SH-SY5Y and PC12 cells were analyzed using real-time quantitative RT-PCR. Total RNA was isolated using NucleoSpin RNA II (Macherey–Nagel). A two-step quantitative RT-PCR was carried out. Reverse transcription was performed with Maxima First-Strand cDNA synthesis kit for RT-PCR (Fermentas); 1 µg RNA sample was used for each reverse transcription. RT-PCR primers used in this study were acquired from PrimerBank (http://pga.mgh.harvard.edu/primerbank/) as shown in Supplementary Table 4. All real-time PCR reactions were performed using the CFX96 Real-Time PCR detection system (Bio-Rad Laboratories) and the amplifications were done using Maxima SYBR Green/Fluorescein qPCR master mix (2X) (Fermentas). The thermal cycling conditions were composed of 50 ºC for 2 min, followed by an initial denaturation step at 95 ºC for 10 min, 40 cycles at 95 ºC for 15 s and 60 ºC for 45 s. The experiments were carried out in triplicate for each data point. Expression data were normalized to the geometric mean of housekeeping gene β-actin to control the variability in expression levels. The relative quantification in gene expression was determined using the 2−ΔΔCT method [52]. The expression levels of TH or LRRK2 of cells transfected with expression or shRNA vectors of PINK1 were expressed as % of control cells, set as 100%.

HPLC analysis of DA contents

The HPLC analysis was performed using SHIMADZU prominence LC-20 HPLC system (SHIMADZU) equipped with an Esa Coulochem 5300 electrochemical detector and a reversed-phase column (70–0636 MD-150 3.2 × 150 mm 3 µm, Dionex) and analyzed under the control of LC Solution program. The setting of ECD was as follows: guard cell: Model 5020, analytical cell: Model 5014B, potentials: E1 = − 300 mV, E2 =  + 200 mV, EGC =  + 350 mV, and analyzing range was set as 5 uA. The SHIMADZU HPLC system was formed by LC-20AD Liquid Chromatograph, SIL-20AC Prominence Autosampler, CTO-20AC Prominence Column Oven, CBM-20A Prominence Communication Bus Module and DGU-20A5 Prominence Degasser. During the HPLC procedure, isocratic elution with elution buffer (100 mM sodium phosphate, 10 mM sodium hepatosulfate, 0.1 mM EDTA, adjusted by phosphoric acid to pH 2.95, 8% (v/v) methanol) was performed. All solutions for HPLC analysis were filtered through GNWP04700 NYLON 20UM WH PL 47MM membrane (Millipore) before use. Cells, Drosophila heads and mice brain or human brain organoid samples were harvested, homogenized in 100 µl ice-cold 0.5 N perchloric acid (Roche), sonicated (Vibra-Cell VCX130, Sonics & Materials) for 1 min in ice and centrifuged; 40 μl of filtered lysate was loaded and analyzed under a flow rate of 0.5 ml per min for 15 min for each sample. The DA peak appeared in the HPLC chromatograph at about 13 min of elution time. The DA content in the solutions was acquired based on the DA peak areas in HPLC chromatography. DA content in cells transfected with empty vectors or pcDNA3-EGFP only acted as control groups. The DA peak areas of control groups were set as 100%, while the DA peak areas of other groups were calculated and expressed as % of control groups.

MTT assay

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) was prepared as 2 mg/ml stock solution in PBS and stored at 4ºC. The MTT/DMEM solution (15% (v/v) of 2 mg/ml MTT stock solution mixed with 85% (v/v) DMEM) was prepared freshly. Cells in 24-well dishes were washed with PBS and incubated in 500 µl of MTT/DMEM solution in the dark at 37 ºC for 3 h. The solution was then aspirated without disrupting the cells and 500 µl of solubilizing solution (0.04 M hydrochloric acid/isopropanol plus 3% (w/v) SDS) was added and mixed well. The plates were incubated at room temperature for 1 h in the dark. The solution optical density was measured at 595 nm using a spectrophotometer (Elisa Reader Spectra Max 340, U.S.A.) in a 96-well plate (Iwaki). The cell viability of control groups was set as 100%, while the cell viability of other groups was set as a percentage of that of control cells.

Trypan blue exclusion analysis

Cells were harvested by trypsinization. The cell suspension was mixed with the same volume of 2% w/v trypan blue solution and cells were counted under a light microscope (Axiovert 25 Zeiss). The numbers of total cells and dead cells were counted, respectively, and the percentage of dead cells was determined. At least, 200 cells were counted for each cell suspension sample. At least six replicates were used per sample.

Statistical analysis

Statistical analyses were conducted using one-way or two-way ANOVA, followed by post hoc Dunnett’s test using software Minitab 14. Graphs were constructed with SigmaPlot 2001.