Mice. Mice expressing EGFP in CCK cells [Tg(Cck-EGFP)BJ203Gsat/Mmmh], referred to as Cck-eGFP, were obtained from Mutant Mouse Resource and Research Center (RRID:MMRRC_000249-MU), and the colonies were maintained on a Swiss Webster background (Taconic Biosciences) (73). FVB;129S6-Snca1nbma Tg(SNCA*A53T)1Nbm Sncatm1NbmTg(SNCA*A53T)2Nbm/J mice (PAC-SNCAA53T) (32) and Snca–/– mice (74) were obtained from Robert L. Nussbaum, University of California, San Francisco, San Francisco, California, USA (RRID:IMSR_JAX:010799). The generation of SNCAA53T Cck-eGFP mice has been described previously (46) (referred to herein as SNCAA53T).

Human wild-type, A53T, or A30P forms of α-synuclein were expressed from a modified brainbow gene construct that also harbored 3 fluorescent proteins (blue, turquoise, and orange). In this manner only 1 fluorescent protein could be expressed from each copy of the construct. Cre recombinase is driven by the Villin promoter–targeted expression to mucosal cells of the gastrointestinal tract with different fluorescent proteins expressing in each stem cell. SNCAbow mice were generated using a technique that was recently described (52). Briefly, C-terminal tagged α-synuclein genes (SNCA tagged with V5, hSNCA-A30P mutant tagged with 3XHA, and SNCA-A53T mutant tagged with Myc) were amplified by PCR and pENTR plasmids were generated. These plasmids were sequenced in entirety at Massachusetts General Hospital Center for Computational & Integrative Biology DNA Core (https://dnacore.mgh.harvard.edu/new-cgi-bin/listing.action) and cloned by Infusion cloning into a ROSA26 mouse targeting vector adapted for Gateway cloning. Plasmid DNA harvested from bacterial colonies was mapped by restriction enzyme digestion analysis and positive colonies were identified. The entire plasmid was sequenced, linearized with XhoI, and transfected into G4 embryonic stem (ES) cells (129/B6N hybrid ES line) (MMRRC catalog MMRRC:011986-UCD, RRID:CVCL_E222). Putative positive ES cell clones were processed and validated across the homology arms by PCR. DNA from 2 selected bacterial colonies was amplified using LA Taq DNA polymerase (TaKaRa), and the region between the homology arms was sequenced. ES cells from the selected clone were microinjected into ICR/Hsd morulae to produce chimeric mice. The Tg mouse was mated with ROSA FLPe [Jax-129S4/SvJaeSor-Gt(ROSA)26Sortm1(FLP1)Dym/J (Jackson Laboratory, RRID:IMSR_JAX:003946)] to excise the neomycin cassette, then mated with Vil-Cre mouse [B6.Cg-Tg(Vil1-cre)997Gum/J (Jackson Laboratory, RRID:IMSR_JAX:004586) or Tg(Vil1-cre/ERT2)23Syr (gift of Sylvie Robine, Institut Curie-CNRS, Paris, France, RRID:IMSR_JAX:020282)] for expression of fluorescent proteins and associated α-synuclein transgenes in intestinal mucosal cells.

Vagotomy and tamoxifen treatment. Surgical subdiaphragmatic vagotomy was performed in 1-month-old male and female SNCAbow Vil-CreERT2 mice. Mice were anesthetized with ketamine (50–100 mg/kg), and an abdominal laparotomy was performed. Immediately below the diaphragm, the vagus nerve was identified and isolated from surrounding connective tissue and vessels. A 2 mm section of the vagus nerve was excised, and the surgical wound was closed with surgical clips. Mice were administered analgesics and observed daily for 5 days for any signs of distress. In sham-operated animals, abdominal laparotomy was performed, and the vagus nerve was exposed but not excised. Weight loss of ~15% was noted in mice undergoing vagotomy compared with sham surgery. One week following the surgery mice were treated with tamoxifen (5 mg/kg) or vehicle administered by intraperitoneal injection daily for 5 days.

Preparation and culture of mouse organoids. Mouse small intestine was dissected, gently flushed with ice-cold phosphate-buffered saline (PBS) (pH 7.4)/Primocin (1:1,000) (InvivoGen, catalog ant-pm-1), cut into ~0.5 cm pieces that were placed in 7.5 mL cold PBS/EDTA (3 mM)/Primocin/Y27632 (1:1,000) (ApexBio, catalog A3008-200) containing penicillin-streptomycin (Gibco, catalog 15140-122), and gently shaken for 15 minutes at 4°C. The intestinal tissue was transferred to fresh EDTA/PBS/Primocin/Y27632, shaken for 25 minutes at 4°C, and transferred to PBS. Tissue was then transferred to PBS/Y27632, shaken for 2 minutes, and filtered through a 70 μm mesh, examined under a microscope, and aliquoted at a density of 50 crypts in 15 μL growth factor–reduced Matrigel (Corning, catalog 354230). The suspension was centrifuged at 475g for 5 minutes at 4°C, and the pellet was resuspended in cold growth factor–reduced Matrigel and aliquoted (15 μL/well) in a 48-well plate (Eppendorf, catalog 0030723113). Matrigel was left to polymerize for 30 minutes at 37°C. To each well, we added 200 μL of prewarmed (37°C) Intesticult Media (StemCell Technologies, catalog 06005) containing Primocin. Media were changed every 2 days and organoids were split weekly.

Isolation and coculture of nodose ganglion neurons. Nodose ganglia were dissected from Snca–/– mice and placed in 300 μL ice-cold mouse Intesticult Media containing nerve growth factor-2 (NGF) (25 ng/mL, MilliporeSigma, catalog N6009) and liberase (0.156 mg/mL). After incubating at 37°C for 30 minutes, the supernatant was replaced with 500 μL Intesticult Media containing NGF. Tissue was dissociated by pipetting, filtered through a 70 μm mesh, and centrifuged at 211g for 2 minutes at room temperature. The pellet was resuspended in fresh media, mixed with growth factor–reduced Matrigel (Corning, catalog 354230), and added to intestinal organoid cultures (at least 4 weeks old). The nodose ganglia/organoid mixture was incubated in an 8-well Chamber slide (Thermo Fisher Scientific) at 37°C for 30 minutes to allow polymerization. Subsequently, prewarmed Intesticult Media containing NGF was added, and the cell mixture were grown for an additional 5–8 days prior to imaging.

Immunostaining of organoids. Whole-mount organoid staining was performed as described previously (75) with slight modifications. After removal of media, organoids were fixed in 4% paraformaldehyde in PBS (prewarmed at 37°C to prevent Matrigel depolymerization) for 20 minutes at room temperature. Organoids were permeabilized with prewarmed 0.5% Triton X-100 in PBS, followed by 3 washes with 100 mM glycine (Invitrogen, catalog 10977-023). After blocking with 5% bovine serum albumin/5% donkey serum/PBS for 2 hours at room temperature, primary antibody was added, and incubation continued in a humidified chamber for 16 hours at 4°C. Primary antibodies used for immunostaining included rabbit CCK (73), rabbit α-synuclein (Abcam catalog ab138501, RRID:AB_2537217, at 1:1,000), guinea pig PGP9.5 (Abcam catalog ab10410, RRID:AB_297150, at 1:100), chick β-tubulin III (Tuj1; Neuromics catalog CH23005, RRID:AB_2210684, at 1:100), and chick GFP (Abcam catalog ab13970, RRID:AB_300798, at 1:1,000). Slides were washed 3 times for 20 minutes each in IF buffer (PBS containing 0.1% BSA, 0.2% Triton X-100, 0.05% Tween 20). Secondary antibodies (in IF) were applied for 1 hour at room temperature in the dark. Secondary antibodies included donkey anti-chicken Alexa Fluor 488 (Jackson ImmunoResearch Labs catalog 703-545-155, RRID:AB_234037, at 1:500), donkey anti-mouse Alexa Fluor 568 (Jackson ImmunoResearch Labs catalog 715-006-150, RRID:AB_234037, at 1:500), and donkey anti–guinea pig Alexa Fluor 647 (Jackson ImmunoResearch Labs catalog 706-605-148, RRID:AB_2340476, at 1:250). Slides were washed 3 times, 20 minutes each wash, with IF buffer. DAPI was applied for 5 minutes at room temperature, washed in PBS, and mounted with ProLong Gold (Thermo Fisher Scientific, catalog P36930).

IF of duodenal and nodose tissue sections. To characterize expression of transgenes and fluorescent proteins, mice were anesthetized with a mixture of xylazine and ketamine and perfused with ice-cold 3.5% freshly depolymerized paraformaldehyde. Intestinal tissue was harvested, postfixed, and cryopreserved in graded sucrose solutions. The tissue was embedded in OCT, and cryosections (10–20 μm thickness) were collected on Fisherbrand Superfrost Plus Microscope Slides (Thermo Fisher Scientific). Immunostaining was performed as described previously (46). The following primary antibodies were used: chick GFP (Abcam catalog ab13970, RRID:AB_300798, at 1:1,000), rabbit α-synuclein (Abcam catalog ab138501, RRID:AB_2537217, at 1:1,000), sheep α-synuclein (Abcam catalog ab6162, RRID:AB_2192805, at 1:1,000), and rabbit PGP9.5 (MilliporeSigma catalog AB1761-I, RRID:AB_2868444, at 1:50).

α-Synuclein protein expression and purification. Plasmid construct (pRK172-human-α-synuclein) encoding wild-type human α-synuclein was expressed in BL21-CodonPlus (DE3-RIL) cells (Agilent, catalog 230245-41). Protein expression was induced with 0.1 mM isopropyl β-d-1-thiogalactopyranoside at cell density OD (600 nm) 0.8 and overnight incubation at 18°C with continuous shaking. Cell pellets were lysed in 0.75 M NaCl, 10 mM Tris HCl pH 7.6, 1 mM EDTA, and 1 mM PMSF and sonicated at 30% power (Thermo Fisher Scientific 500 Dismembrator) for 1 minute followed by boiling the cell suspension 15 minutes. Centrifuged and filtered samples were dialyzed against 10 mM Tris HCl, pH 7.6, with 50 mM NaCl, 1 mM EDTA, and 1 mM PMSF. The suspension was passed through a HiPrep Q HP 16/10 column, 1 × 20 mL, on an ÄKTA pure protein purification system (both Cytiva, formerly GE Healthcare) with running buffer composed of 10 mM Tris pH 7.6 and 25 mM NaCl, then eluted with a linear gradient application of high-salt buffer (10 mM Tris HCl, pH 7.6, 1 M NaCl). Fractions containing a single band of α-synuclein were identified by Coomassie staining of SDS-PAGE gels and were dialyzed and concentrated. Purified monomer protein was subjected to 2 to 3 rounds of endotoxin removal (Endotoxin removal kit, GenScript) until a level of < 0.1 EU/mg was achieved. Endotoxin levels were determined using an LAL chromogenic endotoxin quantification kit (GenScript).

Preparation of mouse tissue homogenates for RT-QuIC assay. Mouse tissues (10% weight/volume) were prepared by homogenizing the tissue in PBS supplemented with 1% Triton X-100 followed by probe tip sonication for 1 minute (5 seconds on, 15 seconds off) at 10% amplitude (Thermo Fisher Scientific 500 Dismembrator). Sonicated samples were centrifuged for 20 minutes at 20,000g at 4°C, and supernatants were aliquoted for further applications. For RT-QuIC analysis, 1% of homogenates were added into the reaction.

RT-QuIC. RT-QuIC analysis was performed in clear-bottom, ultra-low-binding, 384-well plates (Corning) loaded with 1 zirconia silica bead (2.3 mm, OPS Diagnostics) per well. Reaction conditions included 10 μM ThT in sterile PBS, 20 μM of human α-synuclein monomer, and 10% of tissue homogenates (1% weight/volume). Human α-synuclein monomer was passed through an ultra-0.5 centrifugal filtration system (100 kDa MWCO, Amicon) to remove high–molecular-weight aggregates. Reactions were performed in triplicate, and each technical and biological experiment was repeated 3 times. Standard curves with serial dilutions of recombinant α-synuclein fibrils in 1% of matrix homogenate from Snca–/– mice were included for each reaction in order to confirm the efficiency, sensitivity, and specificity of the reaction (Supplemental Figure 1). Serial dilutions of recombinant fibrils were prepared according to approximate molecular weight calculated via Dynamic Light Scattering measurements and estimated structural arrangements as described (47). Negative control samples including the lysates from Snca–/– tissues were used to exclude any background signals likely occurring due to tissue origins. The RT-QuIC assay was conducted using an OMEGA BMG plate reader for 15–90 hours with 60 seconds of shaking at 700 rpm and 60 seconds of rest. The ThT signal was monitored every 30 minutes at 448-10 nm excitation and 482-10 nm emission.

Chemiluminescence-enhanced ELISA measurements for human α-synuclein. Determination of human α-synuclein concentration in nodose ganglia was performed according to manufacturer’s instructions (BioLegend, catalog 844101). The 1:10 diluted samples were run in triplicates, and reference standard curve ranged 6.1–1,500 pg/mL. Luminescent signal was recorded on a ClarioStar plate reader (BMG).

Statistics. Statistical analyses were performed via the GraphPad Prism 9 software. Unpaired 2-tailed t test was applied for comparison between 2 groups, and 1-way ANOVA with a Dunnett’s post hoc analysis was performed for multiple groups as indicated in figure legends. Statistical significance was considered with P values less than 0.05.

Study approval. All experiments were performed with approval by the Duke University Institutional Animal Care and Use Committee.

Data availability. Values for all data points found in graphs are in the Supporting Data Values file, which has also be deposited at https://doi.org/10.7924/r4542x03c