Mice
All experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and following local ethical advice. Experimental procedures were approved by the UK Home Office and ethical approval was granted through consultation with veterinary staff at University College London (UCL). For all experiments, mice were obtained from the following sources and subsequently bred side by side in cages: C57BL/6J (WT) mice were obtained from Charles River UK, C57BL/6-Tg(Thy1-SNCA*E35K*E46K*E61K)3798Nuber/J (3KL) were obtained from The Jackson Laboratory. VHD (B6.tg(HD2)) mice were obtained from V. Cerovic (Institute of Molecular Medicine, RWTH Aachen). Sncatm1.1Kluk/J (SncaWT/GFP) were obtained from K. Luk (Perelman School of Medicine, University of Philadelphia). Cx3cr1CreERT2.Tgfb1LoxP animals were obtained from M. Greter (University of Zurich). Different strains were bred in the same location to harmonize environmental factors. All animals were housed under temperature-controlled (temperature, 23.1 °C; humidity, 30–60%) and pathogen-free conditions with 12 h light–12 h dark cycle with an ad libitum supply of food and water. Both male and female age-matched mice were used in this study: WT, VHD, SncaWT/GFP. Both male and female 3KL mice were used. Animals of 3–4 months were used for ME injection experiments.
Human tissue samples
All tissue samples were donated with the full, informed consent. Accompanying clinical and demographic data of all cases used in this study were stored electronically in compliance with the 1998 Data Protection Act. Ethical approval for the study was obtained from the NHS Research Ethics Committee and in accordance with the Human Tissue Authority’s code of practice and standards under licence number 12198, with an approved material transfer agreement. Consent has been obtained for sharing of individual-level data. Use of Translational Pathology Core Laboratory-derived human tissues was approved by the University of California Los Angeles Institutional Review Board, which waived the informed consent requirement for specimens acquired from the Translational Pathology and Core Laboratory (TPCL) (IRB 11-002504). Specimens were deidentified and age was provided as a 5-year age range.
Tamoxifen treatment
Cx3cr1CreERT2.Tgfb1LoxP and littermate control mice were orally gavaged 3 times (every other day) with 5 mg of tamoxifen (Sigma-Aldrich T5648) dissolved in corn oil at around 6 weeks of age.
Tissue isolation for histology
Mice were deeply anaesthetized using pentobarbital and were transcardially perfused with 15 ml of ice-cold filtered PBS (Gibco) followed by 15 ml of ice-cold 4% paraformaldehyde (PFA) (ThermoFisher). To obtain tissue from the ME, the small intestine was isolated, stored in ice-cold PBS and cut open longitudinally. Ice-cold PBS was used to clean the tissue from luminal debris. The tissue was stretched on a Sylgard plate, and the ME was carefully peeled off using surgical tools. Isolated ME was further fixed in ice-cold 4% PFA for 20 min and preserved at −20 °C in cryoprotectant solution (10% sucrose, ethylene glycol) until stained. From the lamina propria (cross-sections), 2-cm long pieces of small intestine were isolated and luminal contents were gently flushed with ice-cold PBS using a syringe. The tissue was further fixed in 4% PFA at 4 °C overnight and excess PFA was washed with 3 × 10 min in ice-cold PBS. The tissue was dehydrated using 30% sucrose solution in PBS and frozen vertically in embedding moulds (Epredia) with optimal cutting temperature medium (Sakura) before cryo-sectioning. Sections of 20–25 μm were mounted on microscopy slides (Epredia) until staining. The brain was isolated from the skull and was further fixed, frozen and sectioned as described for cross-sections above. Sections were preserved at −20 °C in cryoprotectant solution until stained. To obtain whole mounts of dura mater, the skull cap containing the dura mater was removed from the mouse skull and stored fixed in ice-cold 4% PFA at 4 °C for 2 h. After fixation, the dura mater was carefully removed.
Tissue isolation for FACS and cell sorting
Mice were anaesthetized using pentobarbital and were transcardially perfused with 15 ml of ice-cold filtered PBS. Collected tissues were stored in ice-cold Roswell Park Memorial Institute medium 1640 (RPMI-1640) (Gibco) completed with 5% FBS (Gibco) and 20 mM HEPES (Gibco). To obtain single-cell suspensions from the ME, peeled ME was cut in 1–2-mm pieces and digested with 2 mg ml−1 Collagenase type IV and 0.8 mg ml−1 dispase in RPMI (Gibco) supplemented with 2% HEPES (Gibco), 2% FBS (Gibco) and 50 μg ml−1 DNase for 1 h at 37 °C with continuous agitation. The single-cell suspensions were then homogenized with a potter grinder and strained through 70-μm cell strainers (BD Falcon). From the lamina propria, the remaining tissue following isolation of the ME, containing the lamina propria and submucosa layers, was washed with ice-cold Hank’s buffered saline solution (Gibco) supplemented with 1 mM dithiothreitol (Sigma-Aldrich), 1 mM EDTA (Invitrogen) and 20 mM HEPES for 8 min. Single-cell suspensions of lamina propria were prepared by digestion with 0.85 mg ml−1 Collagenase Type V (Sigma-Aldrich) in MEMα (Lonza) supplemented with 2% HEPES, β-mercaptoethanol (Gibco) and DNase for 30 min at 37 °C with continuous agitation. For the brain and dura, brains were quickly isolated from the skull, and brainstem was dissected from the rest of the brain tissue on ice using chilled instruments. The dorsal part of the skull was carefully removed, and meningeal dura mater was peeled off of the skull cap. The tissues were finely chopped using chilled razorblades and transferred to tubes containing ice-cold RPMI-1640 supplemented with 10% FBS and 20 mM HEPES medium. Single-cell suspensions from brainstem were prepared using the Dissociation kit (Miltenyi Biotech) according to the manufacturer’s instructions. Tissue chunks were pelleted by centrifugation at 300g for 2 min at 4 °C followed by medium removal and resuspension in a mix of buffer Z with enzymes A, P and Y. For digestion of meningeal dura mater, chopped tissue was resuspended in 0.5 mg ml−1 Collagenase P, 0.8 mg ml−1 Dispase II and 250 U ml−1 DNase1 and digested for 30 min. Single-cell suspensions from all tissues were then blocked with rat anti-mouse CD16/CD32 antibodies (BD Biosciences) used at the recommended dilution for 12 min before incubation with primary antibodies diluted at recommended dilutions (Supplementary Table 3) in FACS buffer (PBS, 2% FBS, 0.78 mM EDTA) containing Fc block for 20 min at 4 °C. Dead cells were excluded using Live/Dead near-infrared dye staining (Invitrogen) diluted in PBS or 4,6-diamidino-2-phenylindole (DAPI). Precision Count Beads (BioLegend) were used for absolute cell counting and analysed using the following formula: absolute cell count (cells per μm) = cell count/beads count × bead concentration. Flow cytometry data were analysed using FACSDiva software (v.4.0) and FlowJo software (Treestar).
Cell sorting was performed on a BD Aria III (BD Biosciences). For scRNA-seq (Fig. 4) and proteomics (Fig. 1), live CX3CR1hiCD11b+CD11c− ME-Macs and CD3+ cells (Extended Data Fig. 6a) were sorted in DMEM supplemented with 30% FBS and resuspended in PBS with 0.04% BSA.
Immunofluorescence
For free-floating sections, fixed ME or brain sections were permeabilized with 0.5% (ME) or 0.3% (brain) Triton X-100 at room temperature for 1 h followed by blocking with blocking buffer (5% donkey or goat serum (Abcam) and 0.5% or 0.3% Triton X-100 (ThermoFisher Scientific)) at room temperature for 1 h. Primary antibodies diluted at their recommended concentrations (Supplementary Table 3) in blocking buffer were added and the tissue was incubated at 4 °C overnight. Following 4 15-min PBS washes, the tissue was incubated with secondary antibodies diluted in blocking buffer for 2 h at room temperature and was washed again with PBS. Secondary antibody aliquots were centrifuged at 15,000g for 15 min before dilution in blocking buffer. Tissue was incubated for 5 min with DAPI (Roche) before being mounted on Prolong Gold Antifade Mounting Medium (ThermoFisher Scientific). For on-slide staining, for the gut cross-sections, slides were washed in PBS to remove excess optimal cutting temperature medium before permeabilizing with 0.3% Triton and blocking with 5% goat or donkey serum for 15 min. For dura mater whole-mount images, sections were washed 2 times with PBS and blocked for 1.5 h at room temperature with 10% normal donkey serum (Sigma-Aldrich) in PBS containing 0.3% Triton (Sigma-Aldrich) (PBS-T). Subsequently, the sections were stained with primary antibodies in 3% normal donkey serum/PBS-T (overnight, 4 °C). The following day, the samples were washed three times with PBS-T and once with PBS. The tissues were then stained with DAPI (Sigma-Aldrich)/PBS solution for 30 min at room temperature and washed twice with PBS. Finally, the sections were mounted using Mowiol 4-88 (Polysciences Inc.) mounting solution (prepared according to the manufacturer’s instructions) and stored at 4 °C. Dura mater whole mounts were imaged using a Phenoimager (Akoya Biosciences), or Nikon SoRa Spinning Disk Confocal using a ×10/×20 objective for large overview images and ×40/×60 objective for zoom-ins, all other tissues were analysed using a LSM800 (Zeiss), a LSM980 or Leica Stellaris STED microscope using a ×100 1.4 numerical aperture (NA) oil immersion Plan-Apochromat objective. All images were processed and analysed with Fiji (National Institutes of Health, NIH) and QuPath51. For T cell quantification (Figs. 3–5), we analysed 1–2 regions of interest (ROIs) (159.72 µm × 159.72 µm) per 4–5 adjacent ganglia in the duodenum, with data represented per ROI. Glial fibrillary acidic protein-positive myenteric glial cells were 3D-reconstructed using Imaris software (Bitplane), and their volume was calculated following background subtraction. For each mouse, four ROIs were analysed per ganglion. HuC/D+ neurons were quantified in two adjacent ganglia within the duodenum, with a ganglion defined as a cohesive aggregate of HuC/D-positive cells. Extraganglionic cells were excluded from the analysis.
Single molecule fluorescence in situ hybridization (smFISH) (RNAscope) and smFISH combined with IHC
To detect single RNA molecules, the RNA probe for Tgfb1 (407751) as well as the RNAscope Multiplex Fluorescent Detection Reagents v2 (323110) kit were purchased from ACD BioTechne. ME tissue was cryosectioned at 20 μm and was collected on Superfrost Plus Gold Slides (ThermoFisher K5800AMNZ72) and dried at room temperature overnight. The assay was performed according to the manufacturer’s instructions, and the standard IHC protocol as described above was used.
Brain IHC
Free-floating 15-μm or 25-μm (for brainstem and dorsal motor nucleus of the vagus) or 30-μm (SNpc) serial coronal sections were cut with a Leica CM1860 cryostat. The tissue was then treated with 0.3% H2O2 for 30 min. For staining with mouse anti-s129p antibodies, the samples were then incubated in M.O.M. blocking solution from mouse-on-mouse Elite Peroxidase Kit (Vector laboratories, PK-2200) prepared according to the manufacturer’s instruction for 1.5 h. The tissue was then blocked for another 5 min in M.O.M. diluent from the kit. Then, the sections were incubated with primary antibodies against s129p αS (1:10 000; BioLegend, clone P-syn/81A, 825701) for 12 h at 4 °C. The following day, the sections were treated with M.O.M. Biotinylated Anti-Mouse IgG Reagent from M.O.M. Elite Peroxidase Kit (PK-2200) prepared according to the manufacturer’s instructions for 1 h at room temperature, and subsequently transferred into ABC solution (Vectastain Elite ABC Kit, Vector Laboratories) for 10 min at room temperature, and visualized with 3,3′-diaminobenzidine (DAB) (DAB Substrate Kit, Vector Laboratories). Sections were then transferred onto the glass slides, air dried, sequentially dehydrated in PBS, 70%, 100% ethanol and 100% xylene, and mounted. The images were taken on an Axioscope 5 microscope (Zeiss). For staining with rabbit anti-s129p antibodies, the free-floating sections were treated with 0.3% triton for 30 min, 0.3% H2O2 for 30 min and blocked in 20% normal goat serum, 1% BSA, 0.3% triton for 1 h at room temperature. The samples were then incubated with primary antibodies against s129p αS (1:20 000; Abcam, EP1536Y) for 12 h at 4 °C. After washing with PBS, the sections were incubated with the biotinylated secondary antibodies (1:200, Vector, Goat Anti-Rabbit IgG Antibody (H+L), Biotinylated, BA-1000-1.5) for 1 h at room temperature, subsequently transferred into ABC solution (Vectastain Elite ABC Kit, Vector Laboratories, PK-6100) for 45 min at room temperature and visualized with DAB (DAB Substrate Kit, Vector Laboratories).
s129p αS analysis
Fiji (NIH) was used to measure the total area of s129p αS signal in the total myenteric plexus and brainstem.
Myenteric plexus
Individual channels were first Z-projected and background-subtracted (10-pixel rolling ball radius). Next, threshold channels were empirically determined and then consistently applied to each channel for all subjects. For quantifying s129p, the Analyze Particles tool in ImageJ was used to quantify the total area of s129p (per myenteric ganglia or calculated per mm2). For the brainstem (for immunofluorescence), Z-stacks were taken of the entire tissue thickness within the dorsal motor nucleus of the vagus at 1-μm Z-step increments. Images were analysed in Fiji (NIH), in which individual channels were first Z-projected and thresholded empirically and a median filter of 0.5 pixels was applied to determine only punctate staining of total and s129p αS. This was consistently applied to each channel for all subjects. ROIs were drawn around choline acetyltransferase-positive cells with the freehand selections tool. The Analyze Particles tool in Fiji was used to quantify the total number of puncta and the percentage of punctate staining within each choline acetyltransferase-positive cell by applying ROIs to the corresponding total and s129p αS channels. For the brainstem (for DAB), first, colour deconvolution with H DAB vectors was used. Then, the background was subtracted with a rolling ball radius of 50 pixels from the images, followed by adjusting the threshold to measure the fibrillar inclusions of s129p αS. Total signal area was measured for each image, and the data were normalized to the average of the control group in each staining batch. Alternatively, the optical density of the DAB signal was measured in Fiji by first calibrating the software according to the ImageJ User guide. The optical density of individual cell bodies was then measured and the background optical density was subtracted. Mean values for each mouse were then calculated.
SNpc
Z-stacks were taken of the entire tissue thickness within the SNpc at 1-μm Z-step increments. Images were analysed in Fiji (NIH), where channels were split, the tyrosine hydroxylase (TH) channel was Z-projected and ROIs were drawn around TH+ cells as well as at least one ROI to measure background fluorescence. These ROIs were applied to the total and s129p αS channels and area, mean fluorescence and integrated density were measured for each cell at each plane of the Z-stack. Corrected total cell fluorescence (CTCF) for each TH+ cell was determined with the following equation: CTCF = integrated density − (area of selected cell × mean fluorescence of background readings). For SNpc s129p αS analysis, data were normalized to total αS and all data were then normalized to the mean of the NHC-αS group value.
TH and Nissl IHC
For TH IHC, brains were sagittally hemisected at the midline following removal of the hindbrain. The left hemisects were embedded in 10% sucrose, 0.5% glutaraldehyde in chicken egg yolk. The sample was allowed to polymerize for 5 min before being stored at −80 °C until sectioning. Embedded brains were sectioned into 30-μm slices using a Leica CM1860 cryostat and stored in cryoprotection solution at −20 °C until used for staining. All staining was performed in free-floating baskets (made in-house). Free-floating sections were washed once in PBS pH 7.5 for 5 min at room temperature on a horizontal rocker. Sections were then blocked in 5% BSA + 0.3% Triton X-100 in PBS for 1 h at room temperature. Following 3 × 5 min wash in PBS, sections were incubated in anti-TH antibody (Immunostar 22941) diluted 1:1,000 in 2% BSA + 0.1% Triton X-100 in PBS for roughly 16 h at 4 °C. Following 3 × 5-min washes in PBS, sections were incubated in biotinylated anti-mouse secondary antibody for 2 h at room temperature. Avidin-biotin complex elite kit (ABC, Vector labs PK-6100) (1:50 in PBS) was prepared 30 min before use according to the manufacturer’s instructions. Sections were then washed 3× for 5 min in PBS and incubated for 1 h at room temperature in ABC. Following a final 3 × 5-min wash in PBS, sections were incubated for 10 min in DAB solution (1 tablet Sigma D4418-50 set, 15 ml of PBS + 4.5 μl of H2O2). Sections were then washed once in PBS (5 min) and once in dH2O before being mounted onto slides and dried for roughly 30 min at 37 °C, and then covered with a glass coverslip with a polyvinyl alcohol-based mounting media (made in-house). All counting was performed blinded for treatment type and images were taken using a ×20 objective on a Leica Mica microscope acquiring z-stacks with a 0.7 μm z-step size. Stacked image tiles were loaded into StereoInvestigator desktop (MBF Bioscience). Stereological quantification was performed using the optical fractionator method from StereoInvestigator, analysing every sixth 30-μm coronal section serially for the SNpc and VTA ROIs. Using a counting frame of 50 × 50 μm snapped to 1-μm increments and a systematic random sampling grid size of 100 × 100 μm, TH+ cells were quantified unbiasedly52,53.
For Nissl staining, mounted sections containing SNpc and VTA used in the TH+ cell body quantifications were Nissl stained on top using a cresyl violet acetate-based solution to stain Nissl bodies (nucleoli and rough ER) purple. Slides were washed for 5 min in dH2O and then submerged in Nissl staining solution (4.5 g cresyl violet acetate, 45 ml of 100% ethanol, 450 ml of distilled water) for 8 min before destaining for 10 s in 2% acetic acid in ethanol, 30 s in 100% ethanol and 1 min in 100% ethanol. Sections were cleared in xylene for 10–15 min, before mounting with DPX mounting media (Cellpath SEA-1304-00A) and glass coverslips. TH+ neurons and Nissl+ neurons only (not glial cells) were quantified by unbiased stereology using the same parameters as the TH+ cell body quantifications53,54. The Nissl stain is most intense in nucleoli and in the rough endoplasmic reticulum of neurons and less in glia cells, enabling differentiation on the basis of staining patterns.
Nanostring DSP
Here, 5-μm-thick formalin-fixed paraffin-embedded sections of the myenteric plexus were deparaffinized and incubated with antibodies against bIII-tubulin to select myenteric ganglia as segmented ROI, according to the manufacturer’s instructions and ref. 55. Cell nuclei were stained with DAPI. Each ROI was manually drawn around myenteric ganglia in the myenteric plexus (geometric segments), encompassing both neurons and glial cells, resulting in a total of 2–3 analysed ROIs per slide. Each slide was incubated with an antibody panel conjugated to a unique UV-photocleavable oligonucleotide tag. Selected ROIs were individually illuminated with UV light using NanoString’s GeoMx Digital Spatial Profiler System (NanoString Technologies). Cleaved oligonucleotides were collected in 96-well plates and optical barcodes were analysed using the NanoString nCounter system. Obtained digital counts were normalized for signal-to-noise ratio using Rat IgG2b and Rabbit IgG. Internal housekeeping proteins included Histone H3, S6 and GAPDH.
TCR sequencing and analysis
RNA extracted was extracted from tissue samples and the TCR α and β genes were sequenced using an established quantitative sequencing pipeline, which uses unique molecular barcodes to correct for PCR bias, and sequencing errors56,57. The fastq files were processed and annotated using the in-house open-source computational pipeline Decombinator V5 (ref. 58). Plots were prepared using Python v.3.12.4, Plotly and Scipy. The NHC-αS- and PD-αS-injected groups were compared using a Bonferroni-adjusted unpaired t-test with Welch’s correction. The Jaccard index is defined as
$$J=\frac$$
(1)
where A and B are each a set of clonotypes in a tissue. To compute the expanded index, let c(σ) be the count of clonotype σ transcripts in the sample it is found in, and EA be the expanded subset of A such that
$$E_A=\c(\sigma ) > 1\,\rm\forall \sigma \in A\$$
(2)
Then the expanded index is computed by
$$\varepsilon =\frac$$
(3)
Mass spectrometry on sorted ME-Macs
Protein lysates from sorted duodenal ME-Macs were digested using the single-pot, solid-phase-enhanced sample-preparation method59. Samples were processed in accordance with the published methodology. Briefly, samples were reduced and alkylated using tris(2-carboxyethyl)phosphine and iodoacetamide, respectively. Alkylated proteins were then bound to the Sera-Mag SpeedBeads (ThermoFisher Scientific) by raising the organic solvent content of the buffer to above 70%. Proteins could then be washed using ethanol and acetonitrile before digestion within the same sample tube to reduce protein loss. Proteins were digested through the addition of 100 ng of Trypsin/Lys-C mix (Pierce) in 50 mM ammonium bicarbonate and leaving it at 37 °C at 300g overnight. Digested peptides were cleaned by binding them to the beads by means of increasing the organic solvent content of the buffer to above 95%. Samples were washed using 100% acetonitrile before being eluted from the beads using 2% DMSO. Peptide samples were then acidified to a final concentration of 0.1% Formic acid before injection onto the mass spectrometer. Acidified peptide samples were analysed on a Bruker TIMS-TOF Pro 2 using an EvoSep One. Samples were prepared onto an EvoTip in accordance with the manufacturer’s instructions. Samples were loaded onto the mass spectrometer using the ‘20 samples per day’ HPLC method provided by EvoSep using 100% water as a buffer A and 100% Acetonitrile as buffer B. The samples were acquired in data-independent acquisition mode with trapped ion mobility spectrometry on. Resultant data files were searched using DIA-NN v.1.8 (ref. 60). Search was performed against the UniProt SwissProt Mouse database in direct search mode. The double-pass neural network classifier was used and protein inference was using the protein IDs from the FASTA file. Precursor ion generation for the library used the default settings with a precursor false discovery rate at 1%. Mass accuracy and MS1 accuracy were set at 10 ppm each and the match between runs setting was enabled. Differential analysis data were analysed and visualized using CURTAIN v.2.0 (ref. 61).
Whole gastrointestinal transit function
An amount of 6% carmine red dye containing 1% methylcellulose was administered by oral gavage in 3-month-old 3KL mice or at 1 month and 3 months post-ME injection to determine the total gastrointestinal transit time. This parameter represents the time required for the mice to expel faeces containing the carmine red dye, starting from the moment of oral gavage.
Directed depletion of ME-Macs
Depletion of ME-Macs was carried out using a published protocol adapted to our purpose9. Mice were injected with 15 µg g−1 αCSF1R (clone AFS98, Bio XCell) versus IgG2a control (clone 2A3, Bio XCell) into the ME, 24 h before injection with NHC-αS versus PD-αS. Mice were orally gavaged with 15 µg αCCR2 (MC21, kindly provided by M. Mack, Universität Regensburg) daily for 7 days after AFS98 treatment to restrain ME-Mac replenishment.
Fingolimod treatment
T cell egression was targeted by oral administration of fingolimod (FTY720, Cambridge Biosciences) at a dose of 0.5 mg kg−1 through gavage on days 1, 3, 5 and 7 following PD-αS treatment. Fingolimod was dissolved in ethanol (2 mg ml−1) and saline. Saline containing 2 mg ml−1 ethanol was used as vehicle control.
Quantification of s129p αS engulfment
Engulfment analysis was performed as previously described but adapted to ME62. Mouse ME was immunostained with MHCII, LAMP1 and s129p αS, and human ME was immunostained with CD209 (ref. 63), LAMP1 and 2F12. Two regions and 3–6 ROIs were acquired for each mouse and human sample, respectively, on a ×63 1.4 NA objective Zeiss 800 microscope. For mouse, 60–80 z-stack planes were taken with 0.27-μm spacing and raw images were processed in Imaris (Bitplane) for analysis, after background subtractions. A mask was applied in MHCII+ LAMP1+ reconstructed lysosomes for s129p αS, and the percentage engulfment of s129p αS within lysosomes was calculated using the following formula: volume of engulfed material (s129p or 2F12 within LAMP1)/total ME-Mac volume × 100.
Sequential extraction of insoluble fraction from human brain
Sequential extraction from human brain was performed according to ref. 24. Briefly, 0.5 mg of brain tissue was homogenized in high salt (HI) buffer (50 mM Tris-HCl, 750 mM NaCl, 5 mM EDTA, 10 mM NaF, pH 7.40) containing protease inhibitors. After ultracentrifugation at 100,000g for 30 min at 4 °C, supernatant was removed and fresh HI buffer added. The same steps were subsequently repeated with HI buffer containing 1% Triton, HI buffer with 1% Triton and 30% sucrose, HI buffer with 1% sarkosyl and finally in PBS to resuspend the sarkosyl-insoluble fraction of the brain homogenate enriched in aggregated αS. In the absence of pathology, the sample is expected to contain minimal random contaminants, with samples showing comparable total αS levels between disease and NHC groups64. Postmortem brain samples used in this study are summarized in Supplementary Table 1b.
αS SAA
Flash-frozen PD brain tissue (500 µg, frontal cortex, Braak VI, absent copathology) was homogenized and subjected to serial extraction as described earlier. For the SAA reaction to amplify and monitor αS aggregates, 10 μl of brain-derived seed was incubated with recombinant monomeric αS at 42 °C in a BMG FLUOstar Omega plate reader to amplify amyloid αS by incorporating monomeric αS into the growing aggregate22. Before each SAA experiment, lyophilized monomeric protein was dissolved in 40 mM phosphate buffer (pH 8), filtered using a 0.22-mm filter and the concentration of recombinant protein was measured by use of absorbance at 280 nm using a Nanodrop One spectrophotometer. Brain-derived insoluble protein was tip-sonicated for 30 s (1 s off, 1 s on) at 30% of amplitude and added to a 96-well plate with 230 mM NaCl, 0.4 mg ml−1 αS and a 3-mm glass bead (Millipore Sigma, no. 1040150500). Repeated shaking (1 min incubation, 1 min double-orbital shaking at 400 rpm) disrupts the aggregates to produce an expanded population of converting units. The amyloid dye thioflavin T was used in adjacent wells to monitor the increase in fibrillar content through fluorescence readings at 480 nm every 30 min until the signal plateaued towards the end of the amplification interval of 4 days. The lag time of each sample was defined as the time at which the average, background corrected thioflavin T fluorescence of the sample reached 5% of its own maximum fluorescence. For SAA on isolated ME-Macs and neurons, single-cell suspensions were isolated from the ME using the magnetic-activated cell sorting method (Miltenyi Biotec, 130-097-142). ME tissues were first homogenized as described earlier and centrifuged at 400g for 7 min. The resulting cell suspension was incubated with a CD11b antibody (Miltenyi Biotec) for 30 min at 4 °C to label CD11b+ cells. The suspension was then passed through a magnetic-activated cell sorting magnetic column, and the flow-through containing CD11b− cells was collected for subsequent neuronal isolation. For the isolation of enteric neurons, CD11b− cells were processed using the Adult Neuron Isolation Kit (Miltenyi Biotec, catalogue no. 130-126-602). Briefly, cells were incubated with the Adult Non-Neuronal Cell Biotin-Antibody for 30 min, followed by centrifugation at 400g for 7 min. The pellet was incubated with anti-biotin microbeads for 10 min and subsequently applied to a magnetic-activated cell sorting magnetic column. The flow-through from this step represented the enriched enteric neuronal fraction. Isolated ME-Macs and enteric neurons were then lysed to obtain protein extracts. Tip sonication was performed for 30 s (1 s off, 1 s on) at 30% amplitude to ensure efficient lysis. Lysed cells were subjected to SAA as described above.
Recombinant αS expression and purification
Briefly, plasmid pET21a-SNCA was expressed in BL21(DE3) Escherichia coli. After cell lysis, αS was purified by use of ion exchange chromatography (5 ml HiTrap Q HP columns, GE Life Sciences, 17516301) and size exclusion chromatography (13 ml of HiPrep 26/60 Sephacryl S-200 HR, GE Life Sciences) using the ÄKTAprime plus fast protein liquid chromatography system and subsequently lyophilized in protein low binding tubes (Eppendorf)65.
αS ELISA
Samples were processed and analysed as described before using 2F12 (MABN1817, Merck) as a capture, SOY1 (Merck, MABN1818) as a sulfo-tagged detection antibody on an MSD enzyme-linked immunosorbent assay (ELISA) platform65. For sulfotag labelling of detection antibodies, 200 μl of SOY1 antibody (1.37 mg ml−1 in PBS) was incubated at room temperature for 2 h with 16 μl of 3 nmol μl−1 MSD NHS-Sulfotag reagent (150 nmol freshly suspended in 50 μl of PBS). Next, 250 μl of PBS was added to antibody solutions, concentrated using Amicon ultra filter tubes (10,000 molecular weight cut off), and brought up to 500 μl of PBS again. This was repeated five times to dilute out the tag reagent. Protein concentration was subsequently measured using BCA assay. For plate preparation, MSD Standard plates were coated with 30 µl of 200 ng filtered 2F12 (1 mg ml−1) from recently filtered batches diluted in PBS and stored overnight at 4 °C. Plates were then tapped out, blocked with 150 µl per well in 5% MSD blocker A in 0.01% PBS-T, sealed and placed on an orbital shaker for 1 h at room temperature. Plates were subsequently washed 5 times with 150 µl of PBS-T per well, samples were added in 2% SDS in PBS-T with 1% MSD blocker A, as well as recombinant αS at different concentration gradients in PBS-T with 1% MSD blocker A (0.5% NP-40) (Meso Scale Diagnostics LLC) and incubated for 2 h at room temperature with orbital shaking. Plates were washed 5 times with 150 µl of 0.1% PBS-T per well before the addition of detection antibody solution: that is, 30 µl per well of 200 ng sulfo-tagged SOY1 antibody in PBS-T with 1% MSD blocker A. Plates were incubated for 1 h at room temperature with orbital shaking and protected from light. After 5 washes with PBS-T, 150 µl of 2× MSD reader buffer diluted in MilliQ water was added and the plate was read using a Meso Sector S 600.
Proteinase K digest
Sarkosyl-insoluble and SAA-amplified samples were treated with 1 µg ml−1 of proteinase K (Roche, 3115836001) at 37 °C for 1 h with gentle shaking. The digestion was stopped by adding NuPAGE LDS sample buffer (Invitrogen, NP0007) and boiling the sample at 95 °C for 7 min. Samples were then loaded onto a Novex 16% Tricine gels (Invitrogen) for protein separation. After electrophoresis, gels were incubated in 20% ethanol for 5 min at room temperature and blotted onto iBlot 2 Nitrocellulose Regular Stacks (Invitrogen) using the iBlot 2 Dry Blotting system (Invitrogen). The membrane was rinsed in ultrapure water and incubated in 4% PFA/PBS for 30 min at room temperature. The membranes were blocked in Odyssey blocking buffer (PBS)/PBS buffer 1:1 (LI-COR) or casein buffer 0.5% (BioRad) for 1 h at room temperature. After blocking, membranes were incubated overnight at 4 °C with anti-αS clone 42 (BD Biosciences). After three washes in PBS-T 0.1%, the membrane was incubated for 1 h at room temperature with the secondary antibody (goat anti-mouse IgG F(Ab)2 conjugated with horseradish peroxidase (HRP), Abcam) in blocking solution. Membranes were washed in PBS-T 0.1% and then the signal was detected using the Invitrogen iBright imaging system and the Luminata Crescendo Western HRP substrate (Millipore).
Endotoxin analysis of insoluble fractions
Lipopolysaccharide levels were measured with Pierce Chromogenic Endotoxin Quantification Kit (ThermoFisher, no. A39552). All experiments were conducted within a Laminar Flow cabinet using pyrogen (endotoxin) free pipette tips. The pH of each sample was measured using pH Strips and adjusted to within 6–8 using endotoxin-free sodium hydroxide (Merck) or hydrogen chloride (Merck). Before starting the assay all samples and kit components were left to warm to room temperature. The E. coli endotoxin standard vial was reconstituted with room temperature endotoxin-free water by adding 1:10 ml of the EU amount indicated on the vial to make a 10 EU ml−1 solution, then vortexed at 1,200 rpm for 15 min. Standards were prepared according to table 2 in the manufacturer’s instructions. Samples were diluted to between 1:2 and 1:30 (10–50 µl of sample) with room temperature endotoxin-free water. Lyophilized Amoebocyte was reconstituted immediately before loading the plate with 1.7 ml of endotoxin-free water. Next, 50 µl of each standard, and samples were loaded in triplicate onto prewarmed plate (plate kept at 37 °C on a Thermoblock for the duration of the assay). Following sample and standard loading, 50 µl of reconstituted amoebocyte was added to each well. The plate was covered and left to incubate based on the manufacturer’s instructions. Before the amoebocyte incubation window closing, chemogenic substrate was reconstituted with 3.4 ml of endotoxin-free water and incubated at 37 °C for 5–10 min. When amoebocyte incubation was finished, 100 µl of warm chemogenic substrate was added to each well and plate was left to incubate at 37 °C for 6 min. Finally, 25% acetic acid was added to each well (diluted in endotoxin-free water). The plate was read at an optical density of 405 nm before analysis.
Transmission electron microscopy
Transmission electron microscopy specimens were prepared on carbon-coated formvar grids (Electron Microscopy Sciences). Samples were adsorbed on the grids for 5 min and then negatively stained with 1% (w/v) aqueous uranyl acetate. Images were taken on a JEOL 1400 transmission electron microscope (JEOL USA Inc.) at an accelerating voltage of 100 kV.
Microinjections of human αS extracts into ME
Mice were anaesthetized with isoflurane (2–4%) and kept at constant body temperature with a conventional heat pad. Abdominal hair was removed, and an incision was made along the midline. A 10-μl 36 G Hamilton syringe (World Precision Instruments) was used to inject a total of 7 μl of human brain insoluble fraction (containing roughly 87 pg fibrillar αS by ELISA, Extended Data Fig. 3a) into the wall of the duodenum at 4 sites. The injection sites were located 0.5–1 cm away from pyloric stomach and at least 0.3 cm from each other. Injection sites were marked with 0.2% Fast Green FCF (Sigma-Aldrich). Following the injections, animals were sutured and single housed for 5 days to allow recovery. Animals received carprofen (33.33 mg ml−1) in their drinking water for 4 days postsurgery. Mice were euthanized at 1 month or 3 months postinjection.
scRNA-seq library preparation, expression and NicheNet analysis
For scRNA-seq, four biologically independent samples (duodenal ME) were used per genotype, and one sample represents pooled ME from four mice. Single-cell libraries were prepared using 10X Genomics Chromium Next GEM Single Cell 5′ kit v.3.1, according to the manufacturer’s instructions. Libraries were sequenced using an S2 flowcell on an Illumina NovaSeq S6000 instrument, with sequencing parameters recommended by 10X Genomics, aiming for 40,000 reads per cell. Raw Fastq read files were processed using cellranger count (v.6.0) for each dataset to generate count matrices that were used for subsequent analysis (filtered_feature_bc_matrix). Integrated analysis of multimodal single-cell data in R (v.4.0) were used to first examine each dataset individually using Seurat (v.4.3) and SeuratObject (v.4.1.3)66. Cells expressing fewer than 200 or more than 2,500 features were excluded from further analysis, as well as cells expressing more than 30% mitochondrially encoded features (‘MT’ chromosome in GRCm39). Each dataset was normalized individually using SCTtransform (v.0.4.1) and datasets were integrated together (IntegrateData) using 3,000 genes. Principle component analysis was carried using the ‘RunPCA()’ command on 100 principal components and the appropriate number of principal components evaluated using the and ElbowPlot function. Clusters of transcriptionally related cells were identified using the Louvain method and the ‘FindClusters’ command at 0.2 resolution and were visualized using uniform manifold approximation and projection for dimension reduction. Cell clusters corresponding to macrophages were identified through the expression of marker genes Csf1r, Aif1 (Iba1), Fcgr3 (CD16) and H2-Eb1 (MHCII). T cells were identified through expression of Cd3e (CD3), Cd8a (CD8) and Cd4. Cell clusters that did not express any of these markers above were not considered for further analyses. Differentially expressed genes were computed for each cluster using the ‘FindMarkers’ command. ClusterProfiler (v.4.12.6) was used for Gene Ontology analysis considering differentially expressed genes with log2 fold change greater than 0.75 and Bonferroni-adjusted P value less than 0.05. Ligand–receptor interaction networks were inferred using nichenetr (v.2.2.0) considering differentially expressed genes in the receiver population (T cells) and the top ten ligands based on the area under the precision–recall curve expressed by 3KL macrophages.
Mouse multiplex cytokine array
Mice were anaesthetized and perfused with ice-cold PBS. The gut was dissected, and 75 mg of duodenal tissue was collected in 300 μl (4 times wet weight) ice-cold PBS with cocktail protease inhibitors (ThermoFisher Scientific, 11814111). The tissue was homogenized at 50 Hz for 5 min using a TissueLyser LT (Qiagen) and centrifuged at 10,000g for 10 min at 4 °C. The supernatant was collected and mixed well in an equal volume of RIPA lysis buffer (Thermo Scientific, 10017003), and left for 20 min on ice. The samples were then centrifuged at 10,000g for 10 min at 4 °C and the supernatant was collected and sonicated (70% amplitude, 30 s on and 30 s off for 10 min) (Q800R3 Sonicator, QSONICA). A standard BCA protein assay was performed on the supernatant fraction to obtain the amount of protein (ThermoFisher Scientific, 10678484). Cytokine levels were assessed using the proteome profiler array (Mouse Cytokine Array Panel A, BioTechne, ARY006), which detects the levels of 40 mouse cytokines. The analysis was performed using the manufacturer’s instructions and 200 ng ml−1 of the samples were used. The dot blots were visualized by chemiluminescence detection using an Amersham Imager 680 (Bioke) system.
ME photoconversion
For photoconversion of ME T cells, VHD mice were first injected with NHC-αS versus PD-αS into ME. Five days later, mice were re-anaesthetized and small intestine was exposed to the site of injection, marked with 0.2% Fast Green FCF (Sigma-Aldrich). The rest of the body was carefully covered with aluminium foil to avoid undesired exposure. Next, the intestine was illuminated with UV light (405 nm) 1 cm distally from injection site for total of 2 min (2 intervals of 60 s each) using a BlueWave QX4 high-intensity spot-curing system (DyMax) equipped with a 3 mm diameter focusing lens. During the illuminations, the intestine was kept wet with PBS. Abdominal incisions were carefully closed with separate sutures on both peritoneal layer and skin. Animals received carprofen (33.33 mg ml−1) in their drinking water for 4 days postsurgery. Animals were euthanized at 1 month postsurgery.
Human gut tissue IHC
Postmortem formalin-fixed paraffin-embedded jejunal sections (ten from patients with PD, ten from NHC participants) were obtained from the Banner Sun Health Research Institute Brain and Body Donation Program of Sun City, Arizona, USA67 (Supplementary Table 1a). The sections were incubated in a 60 °C oven overnight before staining after which they were deparaffinized in xylene and rehydrated in decreasing grades of alcohol. The sections were then stained using Leica Bond RX under standard IHC protocols within UCLA’s TPCL. Briefly, automated detection was performed based on protocol F using the Bond Polymer Refine Detection kit (Leica Biosystems). Heat-induced antigen retrieval was performed using the BOND Epitope Retrieval Solution 2 (Leica Biosystems, catalogue no. AR9640) buffer for 30 min. For chromogenic IHCs, the primary CD4 (Cell Marque, 104R-14, 1:50) was incubated for 60 min. Sections were incubated with Dakocytomation Envision System Labelled Polymer HRP anti-rabbit (Agilent Technologies, catalogue no. K4003) for 10 min followed by BOND Polymer Refine Detection DAB chromogen (Leica, catalogue no. DS9800) for 10 min. Images were acquired with a Lionheart LX Automated Microscope (Agilent Technologies). In some cases, QuPath was used to view slides scanned with an Aperio ScanScope AT (Leica) in the UCLA TPCL51. For fluorescent IHC, the sections were blocked in 10% donkey serum, 1% BSA and 0.1% Tween in tris-buffered saline (TBS) for 30 min at room temperature, and then incubated with the primary antibodies overnight (Supplementary Table 3). The sections were washed in 0.1% Tween TBS and incubated with the secondary antibodies for 1 h at room temperature. The sections were then washed in 0.1% Tween TBS, treated with an autofluorescence quenching kit (Vector, SP-8400-15) following the manufacturer’s instructions, incubated in 1:10,000 DAPI in PBS for 10 min, washed again and mounted onto slides with ProLong Glass mounting medium. Information regarding sex is included in Supplementary Table 1a; however, sex was not taken into consideration when including patient samples. Findings did not apply to only one sex.
Rotarod coordination test
All behavioural testing was performed under a handling hood with lights off. On the first day, the mice were trained on a rotarod (Ugo Basile) with a constant speed of 8 rpm for a total of 5 min. Then, on the next three consecutive days, the animals were placed on an accelerating rotarod. The speed of rotation constantly accelerated from 4 rpm to 40 rpm in 3 min, and a trial lasted for a maximum of 5 min. The time was recorded when the animal fell off the apparatus or spun on it twice without attempts to resume running (latency to fall). On each day, the animals did 2 trials with 15–25 min break, and the results are presented as the mean of latency to fall (6 trials).
Statistics
All statistical analyses were performed in Prism (GraphPad Software, v.9.3.1) or R (v.4.3.2) and a complete overview is provided in Supplemental Table 4. Outliers were identified in Fig. 1j and Extended Data Fig. 3a and removed from the dataset using GraphPad Prism (ROUT, Q = 1%). For some datasets, bimodality was observed, probably reflecting variability introduced by different researchers performing experiments on separate days (for example, Figs. 1p, 3d and 5k and Extended Data Fig. 2i). Normal distribution and equality of variance of the residuals were tested using the Shapiro–Wilk normality test and the F-test, Spearman’s test, Bartlett’s test, Brown-Forsythe test or Levene’s test, using significance level α = 0.05. To stabilize variances on heteroscedastic residuals, we performed log10 or square root transformations on the data for Figs. 1c, 2d,g, 3p, 4f and 5c,d,g,j,n and Extended Data Figs. 2d, 4i, 6h,l,n and 7h,j (linear data were used for plotting to facilitate interpretation of the outcome). For Fig. 1p, to test the association between two binary variables (SAA negative versus positive), the Fisher’s exact test was used. A one-sample t-test was performed to assess whether the mean αS levels in enteric neurons (Extended Data Fig. 2l) significantly differed from zero, analysed independently for each group. Two groups were compared using two-sided unpaired Student’s t-test, two-sided Welch’s t-test or two-sided Mann–Whitney test, depending on the data structure. For clonotype overlap (Jaccard and Expanded Index) and effective number of species between ME and dura mater, Bonferroni-adjusted unpaired t-test with Welch’s correction was used (Fig. 3r,t and Extended Data Fig. 5a). To compare more than two groups (IgG, anti-CSF1R, anti-CCR2 or anti-CSF1R/anti-CCR2), one-way analysis of variance (ANOVA) with Bonferroni’s multiple-comparison posthoc test, Kruskal–Wallis test with Dunn’s multiple comparisons posthoc test was used, depending on the data structure. Two-way ANOVA with Bonferroni’s multiple-comparison test was used to assess the interaction of PD-αS versus NHC-αS injection with (1) anti-CSF1R/anti-CCR2 versus IgG treatment, (2) UV versus non-UV illumination, (3) Cx3cr1CreERT2.Tgfb1LoxP versus Cx3cr1+/+.Tgfb1Lox or at (4) 1 month or 3 months post-treatment. Two-way repeated measures ANOVA was used when values were matched (for example, different ME-Mac clusters or intestinal regions from the same mouse in Fig. 4f and Extended Data Figs. 2b and 6j,l; within variable, mouse). Multiple Mann–Whitney tests, with the Benjamini–Krieger–Yekutiel correction to control for the false discovery rate, were used to compare αS levels between cytosolic and insoluble fractions, as well as to compare cytokine levels in ME at 1 month postinjection. For brain region (SNpc versus VTA) versus treatment (NHC versus PD-extract injection) analysis in Fig. 2k and Extended Data Fig. 3f, we used a linear mixed-effect model with multivariate t-distribution posthoc (Fig. 2k), using mouse as a nested variable to incorporate the intermouse differences across region. All data are presented as mean ± s.e.m., statistical significance was set at α = 0.05. Statistical methods were not used to predetermine study sizes but were based on similar experiments previously published. Experiments were blinded to the genotype of the animal as well as the treatment of the animal. Experiments involving human sections were blinded to the demographics of the patients. Independent experiments were performed to confirm the reproducibility of the data.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
