Inclusion and ethics statement
The research described here adhere with all relevant ethical regulations. All experiments involving mice were approved by the local authorities at the Regierung von Unterfranken, Würzburg, Germany under experiment numbers: 55.2.2-2532-2-631-101, 55.2.2-2532-1455-34, and 55.2-2-2532-2-992. Patient autopsies were obtained according to the guidelines of the Local Ethics Committee of the University Hospital of Marburg.
Mice were kept at the animal facility of the Center for Experimental Molecular Medicine, University of Würzburg, under barrier conditions and at a constant cycle of 12 h in the light and 12 h in the dark. Colonies were maintained at 20–24° Celsius and 40–60% humidity, with free access to food and water. Ten-to-12-week-old male C57Bl/6J or SNCAKO (JAX stock#016123)45 mice were obtained from Charles River Laboratories (Sulzfeld, Germany) and stereotactically injected unilaterally into the right SN with 2 μL of either empty AAV1/2 (EV), an AAV1/2 encoding the human mutated A53T-αSyn, or an AAV1/2 encoding GFP (GeneDetect) at a concentration of 5.1 × 1012 genomic particles/mL using the coordinates: −3.1 mm anteroposterior (AP), −1.4 mm mediolateral (ML), and 4.4 mm dorsoventral (DV)12. Both hαSyn and GFP were expressed under the CMV promoter. For the transgenic A30P/A53T PD mouse model, male and female heterozygous hm2a-Syn-39 were obtained from Jackson Laboratory (Jax stock# 008239)46 and 16–17-month-old animals used for the experiments. Homozygous male C57Bl/6 mito-Dendra2 mice were obtained from Jackson Laboratories (Jax stock# 018397)47. C57Bl/6J CD11c.DOG18 mice were kindly provided by G. Hämmerling, interbred with albino C57Bl/6J mice and maintained in the Center for Experimental Molecular Medicine (ZEMM) animal facility at Würzburg University. Both the Dendra2 and the CD11c.DOG mice were male 10–12 weeks old at the time of stereotactic injection. The local authorities at the Regierung von Unterfranken, Würzburg, Germany approved all animal experiments. The Dendra2 experiments were performed under experiment number 55.2.2-2532-2-631-101 and the CD11c.DOG experiments were performed under experiment number 55.2.2-2532-1455-34. All other experiments were performed under experiment number 55.2-2-2532-2-992.
Mice were sacrificed at either 1 week, 5 weeks, or 10 weeks following stereotactic surgery or at 16–17 months of age in the case of A30P/A53T transgenic animals. After transcardial perfusion with ice cold 0.1 M phosphate-buffered Saline (PBS)/heparin (Ratiopharm #X34331), mouse brains, spleens, cervical lymph nodes, thoracic section of the vagus nerve, distal ilea and distal colons were isolated and immediately embedded in TissueTek and snap frozen. Ten micrometer thick sections were obtained from the various organs and sections were stored at −20 °C. Following fixation with 4% Paraformaldehyde (Sigma #8.18715.1000) and a block with 2% Bovine Serum Albumin (BSA) (Sigma #A4503) and 10% Normal Goat Serum (NGS) (Dako #X0907), sections were incubated overnight with chicken anti-TH (1:500, Abcam #ab76442), rabbit anti-αSyn (1:10,000 brain and 1:5000 other organs, Sigma #S3062), rabbit anti-GFP (1:500, Invitrogen #A11122), rat anti-HA (1:100, Roche, #11867423001), rabbit anti-phospho αSyn S129 (1:100, Abcam, #ab59264), hamster anti-CD11c Alexa Fluor 488 (1:300, Invitrogen, #53-0114-82), rat anti-CD11b (1:100, Serotec, #MCA74G), goat anti-CHAT (1:300, Millipore, #Ab144P), rabbit anti-P2RY12 (1:200, Invitrogen, #PA5-77671), rabbit anti-LRP1 (1:100, Abcam, #Ab92544), rat ant-CD8 (1:500, Serotec, #MCA609G), rat anti-F4/80 (1:300, Serotec, #MCAP497), or rat anti-CD4 (1:1000, Serotec, MCA1767) in 2%NGS/1%BSA. Following three washes with 1× PBS, sections were then incubated for 1 h with the appropriate secondary antibodies diluted 1:300 in 2%NGS/1%BSA: goat anti-rabbit Cy3 (Abcam, #ab150175), goat anti-chicken AF647 (Abcam #ab150175), goat anti-rat AF647 (Abcam, #ab15016), donkey anti-goat (Dianova, #705-165-147). Sections were then washed and mounted with Fluoromount-G with DAPI (Invitrogen #00-495952). To measure proteinase K-resistant aggregations, sections were pre-treated with PK (20 μg/mL, Sigma #P2308) for 10 min at 37 °C prior to the blocking step. Complete digestion was verified by lack of TH signal.
αSyn, pαSyn, and PK resistant-containing villi were counted on two to three sections per animal, averaged and were quantified as per mm2 of ileal tissue. As αSyn was more diffuse in the SN, the median fluorescent intensity (MFI) of PK-resistant αSyn was quantified using ImageJ on a minimum of three sections per animal. Due to the lack of TH signal following PK digestion, the SNpc borders on the digested slices were defined with the selection tool in Fiji (ImageJ v. 2.3.0) by the TH signal from undigested slice of the same region. The mean gray value was measured within the selection and background was subtracted by measuring the mean gray value on an area without signal. Values are depicted as relative to control EV animals that were stained and imaged on the same day.
Immunohistochemistry on patient autopsies
Formalin-fixed and paraffin-embedded archived human brain tissue from the SN pars compacta were consecutively collected from neuropathologically characterized PD cases (n = 10) and controls (n = 4) matched for age, sex, and interval from death to tissue fixation were collected from the Department of Neuropathology Marburg with written informed consent from the respective responsible dependent and according to the guidelines of the Local Human Ethics Committee of the University Hospital of Marburg. The mean (±standard deviation) age was 74.3 ± 7.7 years in the control group and 73.0 ± 12.4 years in the PD group. The sex was obtained from the autopsy report and the male/female ratio in the control group was 3/1, in the PD group 7/3.
Double immunohistochemistry was performed on 3 µm thick sections with rabbit anti-CD11c (1:1000; Abcam EP1347Y; ab52632) combined with mouse alpha-synuclein (1:5000; Roboscreen; Mab 5G4) on the Leica Bond III platform. For the red staining of CD11c, the Bond Polymer Refine Red Detection Kit (DS9390) was used. For the green staining of alpha-synuclein, the Bond Polymer Refine detection Kit (DS9800) and Histogreen were used. CD11c+ cells with a visible nucleus within both SN of midbrains were counted using ×40 magnification and the amount of double labeled CD11c+αSyn+ cells of all CD11c+ cells per area was calculated.
Gut transit test
A 6% solution of Carmine Red (Sigma, #C1022) was freshly prepared in 0.5% methylcellulose (Sigma, #M0512). Two hundred microliters of this solution was then administered to the mice using a 21 gauge round tip feeding needle. Mice were immediately single housed and stools were continuously monitored for Carmine Red. Total gut transit time is recorded as the interval between administration of Carmine Red and the first observance of it in the stool.
Fecal pellet output
Mice were placed in an empty cage and fecal pellets were collected and weighed in a pre-weighed tube after 1 h. Pellets were dried at 65 °C overnight and tubes were re-weighed to assess the dry fecal pellet weight. The difference between the pre-dried weight and the post-dried weight was calculated to assess the water content percentage.
Depletion of CD11c+ cells
Ten-to-12-week-old wildtype and transgenic B6a.CD11c.DOG mice were stereotactically injected with an AAV1/2 encoding the human mutated A53T-αSyn as described above. Starting 1 week after injection, mice were injected with 20 ng/g Diphtheriatoxin (Sigma, #D0564) in 200 μL PBS every second day for 2 weeks. Following the 2 weeks of treatment mice were sacrificed and organs harvested for further analysis.
Following isolation of WBCs from 200 μL of whole blood (described below). Samples were resuspended in 50 μL of Ripa Buffer (25 mM Tris pH 8.0, 10 mM Hepes, 150 mM NaCl, 145 mM KCl, 5 mM MgCl2, 0.1% SDS, 1% NP-40, 10% Glycerol, 2 mM EDTA), sonicated and stored at −20 °C.
Ileal and SN sections were flash frozen in liquid nitrogen, weighed, and resuspended in RIPA buffer. Ileal samples were disrupted using the stainless-steel beads in the TissueLyser LT (Qiagen), then centrifuged at 15,781 G for 5 min. Supernatant was collected and stored at −20 °C.
For all samples, protein concentration was measured using the Lowry reagent and samples were mixed with the appropriate amount of 5× Loading buffer (250 mM Tris/HCl pH6.8, 25% Beta-mercaptoethanol, 20% SDS, 50% Glycerin, 0.20% Bromphenolblue). Ten micrograms of protein were loaded onto a 12% SDS-Gel and after running, protein was transferred onto a pre-wet Nitrocellulose membrane. Membranes were blocked with 5% milk and incubated overnight at 4 °C with rabbit anti-phospho αSyn S129 (1:1000, Abcam, #ab59264), rabbit anti-αSyn antibody (1:200, Abcam #212184), rabbit anti-P2RY12 (1:500, Invitrogen, #PA5-77671), rabbit anti-GFP (1:500, Invitrogen #A11122), or mouse anti-GAPDH (1:15,000, Calbiochem, #CB1001). After washing, membranes were incubated for 1 h at RT with the appropriate peroxidase-conjugated secondary antibodies diluted 1:5000: anti-rat IgG (Jackson Immuno research, #712-035-150), anti-rabbit IgG (Jackson Immuno Research, #711-035-152), or anti-mouse IgG (Jackson Immuno Research, 715-035-150). Blots were then developed using Western Lightning Plus (Perkin Elmer, NEL103E001EA).
Isolation of lamina propria leukocytes
Ten-centimeter-long sections of the ileum were isolated as described above and placed on a PBS soaked paper towel. Feces, fat, and peyers patches were removed and ilea were placed in ice cold RPMI (Sigma #R8758) supplemented with 10% Fetal Bovine Serum (Sigma #F7524) (complete RPMI). The epithelial layer was removed by incubating the tissues at 37 °C for 30 min in complete RPMI supplemented with 2 mM EDTA (Millipore #324506). Ilea were then washed three times with PBS and incubated for 15 min at 37 °C in Accutase (Sigma #A6964). Digested samples were then passed over a 70 μm cell strainer, centrifuged, and resuspended in 40% Percoll (GE Healthcare #17-0891-02). Samples were centrifuged at 800 G for 25 min at RT and the pellet containing the leukocytes was collected for further experiments.
After blocking with CD16/32 (1:200, Biolegend #101302) for 10 min, cells were stained with Fixable Violet Dead Cells Stain kit (1:1000, Invitrogen, L34955) together with rat anti-I-A/I-E Cy7(1:100, Biolegend, 107630), rat anti-CD86 PE (1:50, Invitrogen, 12-0862-82), hamster anti-CD103 AF488 (1:100, Biolegend, 121408), hamster anti-CD11b PerCP/Cy55.5 (1:50, Biolegend, #101228), hamster anti-CD11c APC (1:100, Biolegend, 117310), rat anti-CD45 APC (1:200, Biolegend #103111), rat anti-CD4 PE (1:100, BD Biosciences #553049), rat anti-CD8 PerCp/Cy5.5 (1:100, Biolegend #100733) for 30 min at 4 °C. After a wash, cells were either analyzed immediately or fixed and permeabilized using the FoxP3 staining kit. Cells were then incubated with a monoclonal rabbit anti-αSyn antibody (1:200, Abcam #212184) for 30 min followed by incubation with an goat anti-rabbit Cy5 antibody (1:300, Jackson Biozol #711-175-152). Flow cytometry analysis was performed using Flowjo v. 10.8.1 (BD Biosciences).
Isolation of immune cells
Brains were isolated as described above and placed in ice-cold PBS. After finely mincing the brains, Accutase was added and the brains were incubated at 37 °C for 30 min while shaking. Following digestion, brains were pressed through a 70 μm cell strainer and resuspended in 40% Percoll. Samples were centrifuged at 650 G for 25 min and the pellets were isolated and used for further experiments.
Spleens and LN were pressed through a 70 μm filter and incubated with red blood cell (RBC) lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA) for 3 min. Cells were centrifuged down and used for further experiments.
Peripheral blood was incubated in RBC lysis buffer for 5 min prior to centrifugation.
Single-cell sequencing and analysis
Five animals per group were sacrificed and single cell suspensions from the brain, ileum, and spleen cells were made. An equal number of cells from similar organs were pooled and 20,000 CD4+, CD8+, and CD11c+ cells/organ/condition were FACS-sorted using a FACSAria III (BD). Single cell sequencing libraries were generated from the pooled biological replicates with the Chromium Single Cell 3’ v.3 assay (10× Genomics) according to the company’s protocol. Libraries were sequenced with the NovaSeq 6000 platform (S1flow cell, 100 cycles; Illumina) in paired-end mode to reach an average depth of 125,000 reads and 2300 genes per single cell. Data were demultiplexed using the CellRanger software v. 6.1.2. The reads were aligned to a custom mm10 reference genome which included the complete hαSyn gene using the STAR aligner.
Data analysis was performed using the Seurat v. 4.1.1 R package48. Low quality cells with more than 20% mitochondrial genes and <200 or >7500 genes were removed. EV and hαSyn data for each organ was merged together and the data was then log-normalized. The FindVariableFeatures function was used to select variable genes. The ScaleData function was then used to scale the data prior to Principal Component Analysis, UMAP dimensional reduction, and clustering using 15 principal components and a resolution of 0.5. The cluster markers were found using the FindAllMarkers function and T cells were removed based on cluster markers. Brain, Spleen, and Ileum CD11c+ cells were then integrated together using the IntegrateData function. CD11c+ clusters were then formed using the FindClusters function and a resolution of 0.6. FindAllMarkers was again performed and the clusters identified using the Mygeneset tool in the Immunological Genome Project19. Gene Ontology analysis and subsequent plots were created using cluster profiler v. 4.8.3 on the respective marker gene sets49.
Total protein was isolated from SN and Ileal samples as described above. Protein concentrations were measured and samples were diluted with RIPA Buffer to 5 μg/μL for the SN and 8 μg/μL for the ileum. In total 11 μL of each sample was loaded onto a CodePlex Chip accordingly to manufacturer’s instructions (PhenomeX, #CODEPLEX-2L04-2), followed by loading the CodePlex Chip into the Isolight reader (IsoLight software version 1.10.0, PhenomeX). Cytokine concentrations were then measured using the corresponding CodePlex Secretome Adaptive Immune – Mouse Kit (PhenomeX). Raw data was analyzed by the automated IsoSpeak software.
Photoconversion of cells in the SN
Ten-to-12-week-old Dendra2 mice were stereotactically injected with AAV as described above. Immediately after injection, a mono fiber-optic cannula was implanted directly above the SN (AP:3.1, ML:1.4, DV: 4.1) and secured. Starting one week following the surgery, the SN of the mice were exposed weekly to 40 mW of light at a wavelength of 473 nm for 30 s via the fiber-optic cannula. Mice were sacrificed one day following the last photoconversion session, and the blood, cervical lymph nodes, spleen, ileum, and brain were harvested.
To determine the normality of the data, a Q-Q plot was analyzed in GraphPad v. 9.4.0. For normally distributed data, parametric methods were utilized. For non-normal distributed data, we utilized non-parametric methods. For the TH+ and Nissl+ stereological investigations, one-way ANOVA with Tukey’s multiple comparison post hoc test was utilized. Two-way ANOVA with Bonferroni’s post hoc test was utilized for αSyn+ and pαSyn+ villi assessment, Fecal Pellet Output, Fecal Water Content, CD86 expression, and CD11c+ photoconverted cells. Unpaired one-tailed Student’s t test was utilized to analyze αSyn MFI, αSyn+CD11c+ cell numbers, cytokine expression, and percent photoconverted cells in the ileum. p Values of <0.05 were considered significant. GO Term Enrichment was determined using a one-sided hypergeometric test and the Benjamini–Hochberg correction for multiple comparisons.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.