Interaction of an α-synuclein epitope with HLA-DRB1∗15:01 triggers enteric features in mice reminiscent of prodromal Parkinson's disease

Abstract

Enteric symptoms are hallmarks of prodromal Parkinson's disease (PD) that appear decades before the onset of motor symptoms and diagnosis. PD patients possess circulating T cells that recognize specific α-synuclein (α-syn)-derived epitopes. One epitope, α-syn_32-46, binds with strong affinity to the HLA-DRB1^∗15:01 allele implicated in autoimmune diseases. We report that α-syn_32-46 immunization in a mouse expressing human HLA-DRB1^∗15:01 triggers intestinal inflammation, leading to loss of enteric neurons, damaged enteric dopaminergic neurons, constipation, and weight loss. α-Syn_32-46 immunization activates innate and adaptive immune gene signatures in the gut and induces changes in the CD4⁺ T_H1/T_H17 transcriptome that resemble tissue-resident memory (T_RM) cells found in mucosal barriers during inflammation. Depletion of CD4⁺, but not CD8⁺, T cells partially rescues enteric neurodegeneration. Therefore, interaction of α-syn_32-46 and HLA-DRB1^∗15:0 is critical for gut inflammation and CD4⁺ T cell-mediated loss of enteric neurons in humanized mice, suggesting mechanisms that may underlie prodromal enteric PD.

Keywords: CD4(+) T cells; HLA-DRB1(∗)15:01; Parkinson’s disease; antigen-experienced T(RM); constipation; enteric dopaminergic neurons; mucosal barriers; neo-antigen; prodromal disease; α-synuclein.

Figure 1.. α-Syn_32–46 immunizations induce weight loss in HLA DRB1*15:01, but not wild-type, mice.

(A) A schematic diagram of the experimental design. (B) Pie charts show the fraction of WT (top) - and HLA (bottom) - immunized mice that were healthy (light purple), became ill (dark purple) or died (gray). (C, D) Graphs of percentage weight change from the initial weight (0 DPI) for WT (C), and HLA (D) mice immunized with either PBS (grey triangles), CFA only (black circles), or CFA/α-syn_32–46 (green triangles). The red dashed lines indicate the time when mice received an immunization boost and PTx injections at 14 and 16 DPI, respectively. (E) Graph depicting the percentage of weight loss for CFA only and sick CFA/α-syn_32–46 HLA mice. The data in (E) were analyzed using a mixed-effect ANOVA for repeated measurements followed by Bonferroni post-hoc correction; * p<0.05. WT: CFA only (n= 23 mice), CFA/ α-syn_32–46 (n=25 mice). HLA: PBS (n=16 mice), CFA only (n=34 mice), CFA/ α-syn_32–46 (n=35 mice). See also Figure S1.

Figure 2.. Total GI transit time is delayed in CFA/α-syn_32–46 -immunized HLA mice.

(A, B) Diagrams of the experimental design for immunization and GI transit time. (C, D) Dotted bar graphs of total GI transit time at 21 DPI for either all (C), or sick (D), PBS (empty circles), CFA only (black) and CFA/α-syn_32–46 (green) HLA mice. (E, F) Dotted bar graphs of total GI transit time of CFA only (black) and CFA/α-syn_32–46 (green) HLA mice at 25 (E) and 35 (F) DPI. (G, H) Correlation graphs between peak weight loss and total GI transit time of CFA only (G), and CFA/α-syn_32–46 (H) HLA mice. Dark purple circles represent sick and light purple circles represent healthy mice. (I) Bar graphs of the total GI transit time of WT (dark bars) and HLA (light bars) immunized with CFA only (black) or CFA/α-syn_32–46 (green). Only sick CFA/α-syn_32–46 HLA mice are shown. Data were analyzed by Mann-Whitney test (C, D), Pearson correlation (G, H) and two-way ANOVA (I); ** p<0.01, *** p<0.001, **** p<0.0001. Dotted bar graphs represent the mean and SEM. Each symbol represents data from one animal. WT: CFA only n=13 mice, CFA/α-syn_32–46 n=14 mice. HLA: PBS n=7 mice, CFA only n=20 mice, CFA/α-syn_32–46 n=21 mice. WT: 2 independent experiments, HLA: 5 independent experiments. See also Figure S2.

Figure 3.. α-Syn_32–46 immunizations induce enteric neuron loss in HLA DRB1*15:01 mice.

(A, B) Diagrams of the experimental design and the MP and SP of the ileum used for flat mount analysis. (C-H) Representative images of the MP (C-E), and SP (F-H) stained for DAPI (blue), TH (green), and ANNA1 (red). Thin white arrows indicate cell bodies of dopaminergic neurons; white arrowheads indicate dopaminergic neuronal processes. Scale bars = 75 μm. (I-N) Dotted bar graphs of ANNA1⁺ (I) and TH⁺ (J) neurons per mm² in the MP, ANNA1⁺ (K), or TH⁺ (L) neurons per mm², area covered (M) and MFI (N) of the TH signal in the SP of PBS (clear), CFA only (black) and CFA/α-syn_32–46 (green) HLA mice. Dotted bar graphs show the mean and error bars the SEM. Data were analyzed by one-way ANOVA test. * p<0.05, ** p<0.01. PBS, n= 14; CFA only n=15 mice; CFA/α-syn_32–46 n=20 (myenteric) and 18 (submucosal) HLA mice; data collected from 6 independent experiments. See also Figure S3.

Figure 4.. Neuronal loss begins after 18 days post-immunization in CFA/α-syn_32–46 -immunized HLA DRB1*15:01 mice.

(A, B) Diagram of the experimental design (A) and the MP and SP of the ileum (B) used for analysis. (C-F) Representative images of the MP and SP at 18 DPI stained for DAPI (blue), TH (green), and ANNA1 (red). (G, H) Dotted bar graphs depicting the number of ANNA1⁺ (G) and TH⁺ (H) neurons in the MP of CFA only (black circles) and CFA/α-syn_32–46 (green circles) mice at 18 DPI. (I, J) Dotted bar graphs of ANNA1⁺ (I) and TH⁺ cells (J) in the SP of CFA only (black) and CFA/α-syn_32–46 (green) mice at 18 DPI. (K, L) Dotted bar graphs of the TH area (K) and MFI (L) of TH signal in the SP of CFA only (black) and CFA/α-syn_32–46 (green) mice at 18 DPI. (M-P) Representative images of the MP and SP at 42 DPI stained for DAPI (blue), TH (green), and ANNA1 (red). (Q, R) Dotted bar graphs of ANNA1⁺ (Q) and TH⁺ cells (R) in the MP of CFA only (black) and CFA/α-syn_32–46 (green) mice at 42 DPI. (S-V) Dotted bar graphs of ANNA1⁺ (S), and TH⁺ cells (T), TH area (U) and MFI (V) of TH signal in the SP of CFA only (black) and CFA/α-syn_32–46 (green) mice at 42 DPI. Dotted bar graphs show the mean and error bars the SEM. Each symbol represents data collected from a mouse. Data were analyzed by a two-tailed Student t-test. * p<0.05, ** p<0.01. 18 DPI: CFA only n=5 mice, CFA/α-syn_32–46 n=5 mice; 42 DPI: CFA only n=6–7 mice, CFA/α-syn_32–46 n=6 mice (data were collected from one experiment). (C-F; M-P) Scale bars = 50 μm. See also Figures S3 and S4.

Figure 5.. Gene signatures associated with the innate and adaptive immune responses and altered T_H1/T_H17 gene signatures are present in the small intestine of α-syn_32–46-immunized HLA mice.

(A) Diagram of the experimental design for bulk and scRNAseq (bulk RNA sequencing: CFA only n=4 mice, CFA/α-syn_32–46 n=4 mice; scRNAseq: PBS n=2 mice, CFA n=3 mice, CFA/α-syn_32–46 n=3 mice). (B) A volcano plot of DEG from bulk RNA seq of the ileums of CFA/α-syn_32–46 versus CFA only HLA mice at 21 DPI. Significantly down-regulated (blue) and up-regulated (red) genes are plotted by fold change on the x-axis and the adjusted p-value on the y-axis. Red lines indicate adjusted p values (p_adj) ≤ 0.05 and |log₂ fold change| ≥ 0. Genes of interest are highlighted in green and labeled. (C) GO enrichment bar graph depicting five functional categories of upregulated genes from the ileum of α-syn_32–46 HLA mice at 21 DPI. Red diamonds indicate the number of genes represented by the GO term, and gray bars indicate −log₂(FDR) values. (D) UMAP representation of scRNAseq data of CD45⁺ cells sorted from the small intestine of all three conditions (PBS, CFA, CFA/α-syn_32–46). (E) Bar graph of the proportion of sequenced CD45⁺ cells for each cluster of the UMAP. Each bar shows the relative proportions of CD45⁺ cells for each condition [PBS (purple), CFA only (teal), CFA/α-syn_32–46 (orange)] corrected for the number of cells sequenced from each condition. The underlined clusters are overrepresented (>45% of cluster) by cells isolated from CFA/α-syn_32–46 HLA mice. (F) UMAP projections of T_H1 and T_H17 clusters isolated from the dataset [PBS (purple), CFA only (teal), CFA/α-syn_32–46 (orange)]. (G) Heatmap of the average expression of 20 T_RM-associated genes in T_H1 and T_H17 cells, separated by condition. Bolded and underlined gene names are significantly upregulated in the T_H1 and T_H17 population (p_adj < 0.05) from CFA/α-syn_{32–46 -} compared to CFA only HLA mice. (H) UMAP projection of the T_H1 cell subset with a bar graph of the proportion of T_H1 subclusters as a percent of total T_H1 cells by condition. Each bar shows the relative proportions of cells from each condition [PBS (purple), CFA only (teal), CFA/α-syn_32–46 (orange)] corrected for the number of total T_H1 cells from each treatment. (I) Feature plots of T_RM genes from panel G, Tnfrsf4, and Fosl2 in the T_H1 cluster. (J) Violin plots of Bcl2, Il7r, Tnfrsf4, Ramp3, Crem, Ifngr1, Vps37b, and Fosl2 gene expression in the T_H1 cluster, separated by condition. Statistical analyses were done by Wilcoxon Rank Sum test, followed by Bonferroni correction for multiple comparisons. (K) UMAP projection of the T_H17 cell subset with a bar graph of the proportion of T_H17 subcluster as a percent of total T_H17 cells by condition. Each bar shows the relative proportions of cells from condition [PBS (purple), CFA only (teal), CFA/α-syn_32–46 (orange)] corrected for the number of cells from each treatment within the T_H17 cluster. (L) Feature plots of T_RM genes (from Fig. 5G), Ifng, and Il17a in the T_H17 cluster. (M) Violin plots of Bcl2, Il7r, Ramp3, Rgs2, Samsn1, Crem, Cd69, Ifng, and Il22 gene expression in the T_H17 cluster, separated by immunization treatment. For statistical significance: * p_adj < 0.05, ** p_adj < 0.01, *** p_adj < 0.001, **** p_adj < 0.0001. See also Figure S6.

Figure 6.. CD4⁺ T cell depletion partially rescues neuronal loss in α-syn_32–46 immunized HLA mice.

(A) Diagram of the experimental design. Mice received α-CD4 or isotype control antibodies (white arrows) or α-CD8 antibodies (black arrows) intraperitoneally. (B) Graph showing the levels of CD4⁺ or CD8⁺ T cells as % of CD45⁺ cells at three distinct DPIs in CFA/α-syn_32–46 HLA mice for each treatment: α-CD4 (red), α-CD8 (teal), or isotype control (gray). (C) Kaplan-Meier curve depicting the probability of survival and (D) pie charts displaying the proportion of α-CD4 (red)-, α-CD8 (teal)-, or isotype control (gray)-treated CFA/α-syn_32–46 HLA mice that remained healthy (light purple), became ill (dark purple) or died (gray). (E) Graph depicting the percent of weight change from the initial weight (0 DPI) of α-CD4 (red)-, α-CD8 (teal)-, or isotype control (gray)-treated CFA/α-syn_32–46 HLA mice. (F) Dotted bar graph depicting the total GI transit time of α-CD4 (red)-, α-CD8 (teal)-, or isotype control (gray)-treated CFA/α-syn_32–46 HLA mice. (G-I) Representative fluorescent images of the SP of Isotype control (G), α-CD4 (H), α-CD8 (I) treated CFA/α-syn_32–46 HLA mice stained with DAPI (blue), TH (green) and ANNA1 (red). White arrowheads indicate TH⁺ cell bodies. Scale bar = 100 μm. (J-Q) Dotted bar graphs depicting the number of dopaminergic neurons (J, K), TH⁺ MFI (L), TH⁺ area (M), all neurons (N, O), and macrophages (P, Q) in the MP and SP of α-CD4 (red)-, α-CD8 (teal)-, or isotype control (gray)-treated CFA/α-syn_32–46 HLA mice. Data analyzed by repeated-measure ANOVA (B, C), and two-way ANOVA (G-N). Bar graphs depict the mean and error bars the SEM. Each symbol in the bar graphs represents the data collected from one mouse. *p<0.05, ***p<0.001, **** p<0.0001. Isotype control n = 8 mice, α-CD4 n = 10 mice, α-CD8 n = 8, data collected from 3 independent experiments. See also Figure S5.

Figure 7.. The role of α-syn-specific T cells in PD pathogenesis.

α-Syn-specific T cells have been identified in PD patient carriers of the HLA DRB1*15:01 allele. Humanized mice, lacking MHCII^−/− and expressing HLA DRB1*15:01, that are immunized with the α-syn_32–46 may produce α-syn-specific T cells (T_H1 and T_H17 lymphocytes; blue) in the peripheral lymphoid organs (spleen and lymph nodes). These T cell populations (blue) leave the circulation to home to the gut where they are converted to mucosal T_RM (yellow) found normally during infection or chronic inflammation based on our single cell RNA sequencing data. In addition, signatures of innate and adaptive immune responses are upregulated in the gut with CFA/α-syn_32–46 immunizations based on bulk RNA sequencing. The activation of both innate and adaptive immune responses in CFA/α-syn_32–46 HLA mice may trigger enteric neurodegeneration in the SP. There is neither inflammation nor T cell infiltration into the brains of α-syn_32–46 HLA mice, suggesting that additional factors are required to induce CNS phenotypes characteristic of PD.