Neomycin

C57BL6/J mice were housed under a 12-h light–dark cycle and given regular chow (MF, Oriental Yeast Co). All experimental procedures involving mice were performed according to protocols approved by the Committee on the Ethics of Animal Experiments of the Tokyo University of Agriculture and Technology. (Permit Number: 28–87). For HFD studies, 4-week-old male mice were placed on a D12492 diet (60% kcal fat, Research Diets) for 12 weeks. The generation of Gpr43^-/- was described previously[18 (link)]; the mice were maintained on a C57BL6/J genetic background. aP2-Gpr43TG mice were generated as previously[18 (link)]. For antibiotic treatment, 4-6-week-old mice were treated with ampicillin (Nacalai Tesque; 0.4 mg/ml), neomycin (Nacalai Tesque; 0.4 mg/ml), metronidazole (Wako; 0.4 mg/ml), gentamicin (Sigma; 0.4 mg/ml) and vancomycin (Sigma; 0.2 mg/ml) in drinking water for 3 weeks. For collection of blood and tissue samples, mice were sacrificed by anesthesia with somnopentyl. All efforts were made to minimize suffering.

PGCs were isolated from blood of BPR embryos at stage 14–16 (Hamburger-Hamilton, HH) and incubated in forced-air incubators (P-008B Biotype; Showa Furanki, Saitama, Japan) at 38.5 °C and 60–80% relative humidity. Cells were cultured on Mitomycin C–treated buffalo rat liver (BRL) feeder cells in BRL-conditioned medium with supplements as described36 (link). PGC transfection for genome editing was carried out using Lipofectamine 2000 according to the manufacturer’s protocol with these minor modifications: (i) 1.6 μg of pX330-sgRNA sequence–carrying plasmid, and (ii) Lipofectamine 2000 reagents were diluted in 100 μl of OPTI-MEM (Thermo Fisher Scientific, Waltham, MA) and then were incubated with 0.5–1 × 10⁵ PGCs for 5 min, followed by a 2-h incubation at 37 °C with 400 μl of antibiotic-free BRL-conditioned medium. Then, PGCs were transferred to 6-well plates with BRL feeder cells and were cultured at 37 °C, 5% CO₂. PGCs were treated with or without 500 μg/ml neomycin (Nacalai, Kyoto, Japan), 1 μg/ml puromycin (InvivoGen, San Diego, CA), or 50 μg/ml zeocin (InvivoGen) from day 2 to day 4 after transfection to enrich for pX330-transfected cells. The remaining PGCs were then transferred to antibiotic-free medium and allowed to proliferate for transplantation and analysis.

FMT was performed as previously described [20 (link)]. For the bacterial transfer experiments, donor mice received PEG or saline orally for two weeks, and 4–5 fecal pellets (~100 mg) were collected fresh for daily FMT. The collected feces were suspended in 1 mL of phosphate-buffered saline, followed by centrifugation at 2000× g for 10 s. To minimize the changes in microbial composition of the supernatant, the 100 μL aliquots of the supernatant containing fecal bacteria were orally administered to recipient mice immediately after centrifugation. Recipient mice were fed a HFD for four weeks, treated with a mixture of four antibiotics for four days and subsequently rested for three days prior to transfer. For antibiotic treatment, an antibiotics cocktail containing ampicillin, neomycin, metronidazole, and vancomycin (Nacalai Tesque) was diluted in sterilized water to final concentrations of 5, 5, 5, and 2.5 mg/mL, respectively. The antibiotics cocktail (200 mL) was orally administered for four days. Oral antibiotic administration via ad libitum administration via drinking water was avoided to minimize individual differences in body weight change and antibiotic doses [26 (link)]. After three days of rest following antibiotic administration, FMT from donor mice was performed five times weekly for six weeks.

As previously described [36 (link)], mice were treated with antibiotics, which were administered daily starting from seven days before the injection of α-synuclein PFF until the end of the experiments. The antibiotics were administered as a cocktail provided in sterile drinking water and included ampicillin (1 g/L; Nacalai Tesque, Kyoto, Japan), vancomycin (0.5 g/L; Shionogi Pharma), neomycin (0.5 g/L; Nacalai Tesque), gentamycin sulfate (100 mg/L; Nacalai Tesque) and erythromycin (10 mg/L; Sigma-Aldrich Japan, Tokyo, Japan). We prepared stock solutions of ampicillin, vancomycin and neomycin by dissolving them in sterilized water at a concentration of 50 mg/mL. erythromycin was dissolved in 99.5% ethanol (FUJIFILM Wako Pure Chemical) at a concentration of 50 mg/mL, while gentamycin was dissolved in sterilized water at concentration of 25 mg/mL. The stock solutions were aliquoted and stored at −30 °C for two months. For preparation of an antibiotic cocktail, the stock solutions were mixed with 300 mL of sterilized water. The average amount of water intake was 5 to 7 mL/mouse/day.

All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Metropolitan University and all experiments were conducted in accordance with the National Research Council Guide for Care and Use of Laboratory Animals (A2-2, A3-22, A4-19).
As shown in Figure 1, mice were randomly divided into three groups, the control group (Con group, n = 5), the antibiotics control group (AB group, n = 5), and the FMT group (n = 5). The FMT group received FMT from S. murinus three times after treatment with antibiotics in the third week. The AB group was treated with a combination of antibiotics without receiving transplantation. The Con group did not receive antibiotic treatment or transplantation. To remove indigenous gut microorganisms, we referred to previous reports on antibiotic administration (Chen et al., 2020 (link); Gudi et al., 2020 (link); Su et al., 2020 (link)). The antibiotics included a combination of ampicillin (1.0 g/L; Nacalai Tesque, Kyoto, Japan), vancomycin (0.5 g/L; Shionogi, Osaka, Japan), neomycin (1.0 g/L; Nacalai Tesque, Kyoto, Japan), and metronidazole (1.0 g/L; Nacalai Tesque, Kyoto, Japan), which was added to drinking water for 10 consecutive days to remove indigenous gut microorganisms. After a 3-day recovery period, FMT was performed on the next 3 consecutive days.

CERT1 KO HCT116 cells were generated as described previously (7 ). In KI cell lines, CRISPR guide RNA sequences, which were designed for human CERT1 genes, were cloned into the pX458 vector. The target sequence was 5′-GGCACCAGCCTCAGTGTTAA-3′. A donor sequence with or without the patient-derived mutation flanked by approximately 350-bp homology arms of CERT1 was subcloned into the donor vector. This vector contains the simian virus 40 (SV40) PGK promoter, a human growth hormone (hGH) polyA signal, and a neomycin-resistant gene (Fig. S1). Each donor sequence was engineered to contain sufficient silent mutations to avoid recognition and cleavage by Cas9. HCT116 cells were cotransfected with the pX458 vector carrying the abovementioned guide RNA and the donor vector with a transfection reagent (ViaFect; Promega). After 48 h, GFP-positive cells were isolated using a cell sorter (BD FACSMelody; BD Biosciences). Gene-edited cells were selected by 10 μg/ml neomycin (Nacalai Tesque) and single clones were obtained. Heterozygous and homozygous clones with the desired gene insertion in CERT1 alleles were identified by genomic PCR and DNA sequencing. Genomic PCR primers were 5′-GCTAATTGGTCCAATGTGAGG-3′ and 5′-GGTTGCAGTGAGCCGTGATGGTGTCATTG-3′.