All animal experiments were conducted with approval from the Animal Care and Use Committee of the Graduate School of Frontier Biosciences, Osaka University.
Mice were kept under standard laboratory conditions with controlled temperature and 12 h light/dark cycle with ad libitum access to food and water intake. All mice used in this study, including ICR and C57BL/6J, were purchased from Japan SLC (Hamamatsu, Japan). The day the vaginal plug was observed was defined as embryonic day 0.5 (E0.5).
Vector Construction For Ddc-hko1 Knock-in
Knock-in of the hKO1 cassette into the Ddc locus was conducted using CRISPR/Cas9-mediated homology-directed repair genome editing. The construction design of the Ddc-hKO1 donor vector is shown in Fig. 1a. The 5′ homologous arm (HA), which is 732 bp upstream before the stop codon, and 3′ HA, which is 602 bp downstream after the stop codon, were amplified from mouse genomic DNA using the following primer sets: 5′ HA: forward, 5′-TCGAATTCGCGGATCCTTAGTCATTGGGAGTGGAG-3′; reverse, 5′-TAGTAGCTCCGGATCCTTCTTTCTCTGCCCTCAGC-3′; 3′ HA: forward, 5′-ACGAAGTTATCTTAAGAGGCATCAGGATTCCAGC-3′, reverse, 5′-CGGTGGCGGCCTTAAGAGCTGGCAATGTAGCTCAG-3′. The single guide RNA target sequence of the insertion site was 5′-CAGGTAAGCTAGCTGCACCA-3′.
Cell Culture And Transfection
A G4 mouse embryonic stem cell line was used in this study50. ESCs were maintained under serum-free conditions (0.5× N-2 [Thermo Fisher Scientific, MA, USA], 0.5× B-27 [Thermo Fisher Scientific, MA, USA], 100 U/mL mouse LIF [Merck, NJ, USA], 3 µM CHIR99021 [Funakoshi, Tokyo, Japan], 1 µM PD0325901 [Stemgent, MA, USA], 1 mM L-glutamine [Nacalai Tesque, Kyoto, Japan], and 1× penicillin/streptomycin [Invitrogen, MA, USA] in DMEM/F-12 with GlutaMAX [Thermo Fisher Scientific, MA, USA]) in the absence of feeder layers. The donor vector and pX330 Cas9/single guide RNA vector were transfected into ESCs using Lipofectamine 2000 (Thermo Fisher Scientific, MA, USA). Successful knock-in of the reporter cassette was confirmed by PCR amplification. Primer sets for 5′ and 3′ insertion sites were as follows: forward, 5′-TGAAGCCTGAAACCAGCCCC-3′, reverse, 5′-GCTCGAAGCAGTTGCCCCTCA-3′; and forward, 5′-ACGGCAGTTGGGATTCGTGA-3′, reverse, 5′-CATGATGACCAAGTGTCTGAAAGGG-3′, respectively.
Cardiac Lineage Differentiation Of Escs
Cardiac cell differentiation using Ddc-hKO1 ESCs was performed as previously described51. Briefly, ESCs were treated with 2.5 ng/mL of BMP-2 (Bio-Techne, Minnesota, USA) in propagation medium (100 mM non-essential amino acids [Thermo Fisher Scientific, MA, USA], 100 U/mL mouse LIF, 20 µM β-mercaptoethanol [Nacalai Tesque, Kyoto, Japan], 1× penicillin/streptomycin [Invitrogen, MA, USA], and 10% FBS [Nichirei, Tokyo, Japan] in DMEM/F-12 with GlutaMAX) for 24 h. ESCs were then passaged for embryoid body formation using the hanging drop method for 3 days. Embryoid bodies were collected in differentiation medium (100 mM non-essential amino acids, 20 µM β-mercaptoethanol, 1× penicillin/streptomycin (Invitrogen, MA, USA), and 20% FBS in DMEM/F-12 with GlutaMAX) and transferred to an ultra-low adhesion plate (Wako, Osaka, Japan) for 3 days to allow further differentiation under floating culture conditions. Embryoid bodies were plated on a cell culture dish coated with 0.1% gelatin (Sigma-Aldrich, Missouri, USA), and spontaneous beating cells were observed at approximately seven days after attachment.
Establishment Of Reporter Mice
Ddc-hKO1 chimeric mice were generated using the aggregation method. Briefly, 8-16-cell stage embryos were collected from the oviduct ampulla and uterus by flushing with M2 medium (Sigma-Aldrich, Missouri, USA) at E2.5. Zona pellucida was digested using 0.5% pronase (Sigma-Aldrich, Missouri, USA), and each morula was aggregated with Ddc-hKO1 ESCs. Following overnight incubation, chimeric blastocysts were transferred into the uterine of pseudo-pregnant ICR female mice. Chimeric mice were crossed with C57BL/6 female mice for several generations to maintain their C57BL/6J background. The primer sets for genotyping were as follows: forward, 5′-GTTTGTGCTACGCTTTGCTG-3′; reverse, 5′-CCTCAGGGTCATCTCCTGGT-3′.
Vibratome Sectioning And Imaging
Brains were collected from Ddc-hKO1 homozygous mice and fixed in 4% paraformaldehyde in phosphate buffered saline (4% PFA/PBS) overnight at 4°C. Fixed brains were embedded in freshly prepared 3% low-melting temperature agar before sectioning. Embedded brains were sectioned using a vibratome (Leica VT1000s; Leica Microsystems, Wetzlar, Germany) with a thickness of 200 µm and placed in chilled PBS. Sections were treated with 0.5× CUBIC-1, an animal tissue clearing reagent, for 24h, followed by 1× CUBIC-1 for another 24h52. Sections were mounted in 1× CUBIC-1 and imaged using a fluorescence microscope (BZ-X700; Keyence, Osaka, Japan).
Brain Dissociation And Cell Sorting Using A Facs
The brain dissociation protocol was performed with modification as previously described53,54. Homozygous knock-in Ddc-hKO1 mouse brains were harvested and placed in chilled 95% oxygenated hibernate A solution (Thermo Fisher Scientific, MA, USA). The VTA and SNc of the midbrain were micro-dissected in chilled dissection medium (20 mM HEPES [Nacalai Tesque, Kyoto, Japan], 10% [w/v] D-(+)-trehalose dihydrate [Nacalai Tesque, Kyoto, Japan] in HBSS (+) with phenol red [Nacalai Tesque, Kyoto, Japan]) saturated with 95% oxygen. The recovered VTA and SNc were minced into smaller blocks covered with dissection medium and immediately transferred into pre-warmed dissociation medium (10 U papain [Worthington, Ohio, USA], 2 mg DNase I [Sigma-Aldrich, Missouri, USA], and 4 U Dispase II [Sigma-Aldrich, Missouri, USA] in HBSS (+) with phenol red). After a 30-min incubation at 37 ºC, tissues were gently dissociated using a fire-polished glass pipette (approximately 40 µm of internal diameter). Brain suspension was resuspended with solution A (20 mM HEPES, 40 mg/mL BSA [Nacalai Tesque, Kyoto, Japan], 10% [w/v] D-(+)-trehalose dihydrate, 4 µL of RNase inhibitor [Nacalai Tesque, Kyoto, Japan] in HBSS without phenol red), followed by filtration using a cell strainer (pore size 40 µm). The filtrate was subjected to centrifugation, and the collected pellet was resuspended in solution B (0.9 M sucrose [Nacalai Tesque, Kyoto, Japan], 10% (w/v) D-(+)-trehalose dihydrate, 4 µL of RNase inhibitor in HBSS without phenol red, pH 7.5). Neuronal cells were concentrated by centrifugation in solution B. Finally, the cell pellet was washed again with solution A and resuspended in sorting medium (2% FBS [Thermo Fisher Scientific, MA, USA], 10% [w/v] D-(+)-trehalose dihydrate, 40 U RNase inhibitor [Thermo Fisher Scientific, MA, USA] in DMEM/F-12 without phenol red [Thermo Fisher Scientific, MA, USA]). All procedures were conducted on ice except for the digestion process. The hKO1-positive and -negative cells were sorted using a FACS (BD FACSAria III, BD Bioscience, USA) with 100 µm flow cells at a flow rate of 1.
RNA Purification And RNA-seq Analysis
hKO1-positive and -negative cells were collected by sorting into TRIzol LS reagent (Thermo Fisher Scientific, MA, USA). Cells from the two brains were pooled as one biological replicate. RNA purification was performed using a Direct-zol RNA microprep kit (Zymo Research, CA, USA) according to the manufacturer’s protocol. Library preparation was performed using the SMARTer Ultra-Low RNA Kit (Clontech, CA, USA), and cDNA was amplified according to the manufacturer’s protocol. Sequencing was conducted using a next-generation sequencer, an Illumina NovaSeq 6000 platform in 101-base single-end mode. Sequence reads were mapped to mouse reference genome sequences (mm10) using TopHat software (v 2.0.13) combined with Bowtie2 (v 2.2.3) and SAM tools (v 0.1.19). The FPKM values were calculated using Cufflinks software (v 2.2.1). Gene ontology enrichment analysis was performed using DAVID functional annotation bioinformatics microarray analysis (https://david.ncifcrf.gov) and ranked GSEA (v 4.1.0) (http://www.gsea-msigdb.org/gsea/index.jsp).
Stereotaxic injection of G51D α-synuclein into the SNc of mice
Generation and intranigral injection of pre-formed G51D α-synuclein fibrils were performed as described previously19. Adult Ddc-hKO1 male mice at 7–8 weeks old were used for the injection. Mice were anesthetized, and saline or G51D α-synuclein (20 µg at 5 µg/µL) were injected into the SNc using a Hamilton microsyringe (Hamilton Co, NV, USA) under stereotaxic surgery (1.3 mm lateral, -2.8 mm posterior from the bregma, 4.3 mm below the dural surface). Unilateral injections were performed for immunohistochemistry, and bilateral injections were performed for RNA-seq analysis.
At 7 or 12 weeks post-injection, α-synuclein-inoculated mice were deeply anesthetized and perfused transcardially with 4% PFA/PBS. Dissected brains were post-fixed overnight in 4% PFA/PBS and then immersed in PBS containing 30% sucrose solution (30% sucrose/PBS) until sinking as reported previously55. Immunohistochemistry was performed on 20 µm serial section cut with a cryostat (Leica Microsystems, Wetzlar, Germany). The primary antibodies used were as follows: mouse anti-phosphorylated-α-synuclein (Ser-129, 1:10,000; Wako, Osaka, Japan), rabbit anti-TH (1:1,000; Calbiochem, CA, USA), and mouse-anti-fatty acid-binding protein 1 (FABP1) (1:200, Abcam, UK). For double immunofluorescence staining, appropriate fluorescent secondary antibodies conjugated to Cy3 and FITC (1:500; Jackson ImmunoResearch) were used. Incubation was performed in PBS for 1 h at room temperature. Sections were washed with PBS three times, counterstained with 4′,6-diamidino-2-phenylindole mounting medium (Vectashield, Vector Laboratories), and observed using BZ-9000 (Keyence, Osaka, Japan). For histological analysis, coronal sections were incubated with a biotinylated secondary antibody (1:500; Vector Laboratories), and the reaction products were visualized with avidin-biotin-peroxidase complex (Vector Laboratories) using 3′-diaminobenzidine (Sigma-Aldrich, Missouri, USA) as a chromogen. For the stereological assessment of the total number of TH-positive neurons, serial sections were prepared as reported previously55. Every fourth section was stained through the entire extent of the SNc. Cells were counted based on the method described by Furuya et al.55. For the assessment of FABP1, three sections from each mouse were analyzed, and the average percentage of FABP1- and Ddc-hKO1-double positive cells was calculated.