Human iPS cells
For control lines, we used two human iPS cell lines: 201B7 iPSCs were purchased from RIKEN BRC and kindly provided by Dr. Shinya Yamanaka  at Kyoto University and WD39 iPSCs were established  at Keio University. For PD (PARK2) lines, the patient A (PA9 and PA22) and patient B (PB2 and PB20) iPSCs were established by Dr. Imaizumi . All of the iPSCs were maintained on feeder cells in iPSC culture media, as described previously . All of the experimental procedures for iPS cell production were approved by the Ethics Committee of Keio University School of Medicine. All of the experimental procedures for cell differentiation and analysis were approved by the respective Ethics Committees of Keio University School of Medicine (Approval Number: 20–16-28) and Hoshi University School of Medicine (Approval Number: 28–008).
In vitro differentiation of human iPSCs (hiPSCs)
DA neuron differentiation from iPSCs was performed according to a previously reported protocol [16,17,18]. Neural induction was initiated through the inhibition of both BMP and TGFβ signaling using the small molecules Dorsomorphin (DM, Sigma-Aldrich, St. Louis, MO, USA) and SB431542 (SB, Tocris Bioscience, Bristol, UK). The small molecule CHIR99021 (CHIR, Stemgent, Lexington, MA, USA), a GSK3β inhibitor, was added to stimulate the canonical WNT signaling pathway. For neural induction from single hiPSCs, hiPSCs were incubated with TrypLE™ Select (Gibco, Life Technologies, CA, USA) for 5–10 min and dissociated into single cells by pipetting. Cells were plated into a T75 flask and cultured in KBM (KOHJINBIO, Saitama, Japan) supplemented with B27 (Gibco, Life Technologies), 20 ng/mL basic-FGF (bFGF, PeproTech. Inc., Rocky Hill, NJ, USA), 10 μM Y-27632 (Wako, Tokyo, Japan), 10 ng/mL hLIF (Millipore, Billerica, MA, USA), 1 μM Purmorphamine (Calbiochem, San Diego, CA, USA), 2 μM SB (Tocris Bioscience), 100 ng/ml CHIR, 100 ng/ml Sonic hedgehog (Shh, R&D Systems Inc., Minneapolis, MN, USA) and 100 ng/ml FGF8b (PeproTech) in 4% oxygen for 7 or 12 days. Neurospheres were repeatedly passaged by dissociation into single cells, and then cultured in the same manner. Neurospheres at passage 3 were typically used for analysis. For terminal differentiation, dissociated neurospheres were allowed to adhere to poly-L-ornithine (Sigma-Aldrich)- and fibronectin (Sigma-Aldrich)-coated coverslips and cultured in KBM (KOHJINBIO) containing B27 (Gibco, Life Technologies), 20 ng/mL brain-derived neurotrophic factor (BDNF, R&D Systems), 20 ng/mL glial cell-derived neurotrophic factor (GDNF, R&D Systems), 200 μM ascorbic acid (Sigma-Aldrich), and 500 μM dibutyryl-cAMP (Sigma-Aldrich) for 10 days.
Cells were fixed with 4% paraformaldehyde (PFA) and then washed three times with PBS. After cells were incubated with blocking buffer (PBS containing 5% normal fetal bovine serum and 0.3% Triton X-100) for 1 h at room temperature, they were incubated overnight at 4 °C with primary antibodies diluted with blocking buffer. The details of the primary antibodies and the dilution conditions are listed in Additional file 1: Table S1. The cells were again washed three times with PBS and incubated with secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 546 for 1 h at room temperature. After cells were washed three times with PBS, samples were mounted on slides with DAPI-Fluoromount-G™ (SouthernBiotech, Birmingham, AL, USA). Fluorescence of immunolabeling was detected using a light microscope BZ-X710 (KEYENCE, Osaka, Japan) or IX-73 (Olympus, Tokyo, Japan) and photographed with a digital camera using BZ-X Analyze software (KEYENCE) or cellSens software (Olympus).
Total RNA was isolated from cells using an RNeasy mini kit (QIAGEN, Hilden, Germany) with DNase I treatment, and cDNA was prepared by using a SuperScript® VILO™ cDNA Synthesis Kit (Invitrogen, Waltham, MA, USA). The qRT-PCR analysis was performed with Fast SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) on a StepOne Plus™ System (Applied Biosystems Inc., Foster City, CA, USA). Values were normalized to β actin (ACTB). The primer sets used in these experiments are listed in Additional file 2: Table S2.
Generation of PARK2 gene knock-in/knock-out (PARK2-KIKO line) by CRISPR-Cas9
We previously generated CRISPR/Cas9-dependent PARK2-KIKO line using 201B7 as control iPSCs, to evaluate parkin loss of function on DA neurons-derived from iPSCs (Kuzumaki et al., in submission). In brief, a targeting donor DNA plasmid (pUC- 5’3’PARK2- PurTK) was used to disrupt exon 2 of PARK2 gene by homologous recombination. The CSIV-U6-PARK2 (Ex2)-sgRNA-L&R-EF-Csy4-2A-Cas9 was used as a house-made all-in-one vector. The 201B7 was suspended in Opti-MEM (Thermo Fisher) containing Y-27632, house-made all-in-one vector and targeting donor DNA vector plasmid. Electroporation of plasmid DNA was performed using a NEPA21 electroporator (Nepa Gene Co., Ichikawa, Japan). As shown in Additional file 3: Figure S1, PARK2-KIKO clone was identified by PCR method with the primers listed in Additional file 4: Table S3.
The present study was conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, Hoshi University, as adopted by the Committee on Animal Research of Hoshi University, which is accredited by the Animal Research Committee of Hoshi University. Male C57BL/6 J mice (Jackson Laboratory) were used in this study. All mice were housed at up to 6 mice per cage and kept in a temperature-controlled room (24 ± 1 °C) maintained on a 12 h light-dark cycle (light on at 8 a.m.). Food and water were available ad libitum.
[D-Lys-3]-GHRP-6 (Tocris, Bristol, United Kingdom) and morphine hydrochloride (Daiichi-Sankyo Co., Ltd., Tokyo, Japan) were used in this study.
Intracerebroventricular (i.c.v.) administration was performed according to the method described previously . A 2 mm double needle (Natsume Seisakusho) attached to a 25 μl Hamilton microsyringe was inserted into the unilateral injection site using a V-shaped holder to hold the head of the mouse. On the day of the assay, [D-Lys-3]-GHRP-6 (0.3 to 10 nmol/ mouse) was injected into the hole. The injection volume was 4 μl for each mouse.
Cannula implantation into the SNc
Stereotaxic injections were performed under isoflurane (3%) anesthesia and using small-animal stereotaxic instruments (RWD Life Science, Shenzhen, China). Mice were placed in a stereotaxic apparatus and the skull was exposed. A small hole was then made in the skull using a dental drill. A guide cannula (EIM-54; Eicom, San Diego, CA, USA) was implanted into the SNc (from bregma: AP -3.0 mm, ML ±1.2 mm, DV -4.3 mm). [D-Lys-3]-GHRP-6 (1 to 5 nmol/side) was microinjected at a rate of 0.25 μl min− 1 for 4 min. At the end of injection, the injection cannula was kept in the SNc for an additional 2 min before removal and then replaced by a stylet.
Rotarod assay test
Motor coordination was assessed using the rotarod test. Mice were individually placed on a slowly rotating rod (4 rpm/min), and subjected to continuous acceleration at 20 rpm/min; the time at which the mouse fell off the rod was recorded. The test was performed 10 min after i.c.v. injection of either saline vehicle or [D-Lys-3]-GHRP-6 (0.3 to 10 nmol/mouse), or 15 min after microinjection of either saline vehicle or [D-Lys-3]-GHRP-6 (1 to 5 nmol/side).
Balance beam test
The apparatus consisted of a 1 m–long bar (28 or 11 mm in diameter) with a black escape box on one end (O’HARA & Co., LTD., Tokyo, Japan). Mice were acclimated to enter the escape box on the 28 mm-diameter bar for 2 days before testing. The latency to reach the box on the 11 mm-diameter bar was measured (cut off time = 60s). The test was performed 10 min after microinjection of either saline vehicle or [D-Lys-3]-GHRP-6 (1 to 5 nmol/side).
After 30 min of habituation to the apparatus, the locomotor activity of mice was measured by a Three-point Meter (O’HARA & Co., LTD). With this device, the position of the mouse is detected when the infrared beams positioned along the X and Y axes around the cage are interrupted. This device detects the movement of the whole body of the target animal, without being misled by the movement of the tail or any other part of the mouse. Counts of hyperlocomotor activity were obtained at 1-min intervals for 120 min after the injection of morphine hydrochloride (Daiichi-Sankyo Co., Ltd., Tokyo, Japan).
The data are presented as the mean ± S.E.M. The statistical significance of differences between groups was assessed by an unpaired t-test or one-way analysis of variance (ANOVA) test followed by the Bonferroni’s multiple comparison test. All statistical analyses were performed with GraphPad Prism (GraphPad Software, La Jolla, CA, USA). A p value of < 0.05 was considered to reflect significance.