Distinct gut microbiome characteristics and dynamics in patients with Parkinson's disease based on the presence of premotor rapid-eye movement sleep behavior disorders

Abstract

Background: Alpha-synuclein aggregation, a hallmark of Parkinson's disease (PD), is hypothesized to often begin in the enteric or peripheral nervous system in "body-first" PD and progresses through the vagus nerve to the brain, therefore REM sleep behavior disorder (RBD) precedes the PD diagnosis. In contrast, "brain-first" PD begins in the central nervous system. Evidence that gut microbiome imbalances observed in PD and idiopathic RBD exhibit similar trends supports body-first and brain-first hypothesis and highlights the role of microbiota in PD pathogenesis. However, further investigation is needed to understand distinct microbiome changes in body-first versus brain-first PD over the disease progression.

Results: Our investigation involved 104 patients with PD and 85 of their spouses as healthy controls (HC), with 57 patients (54.8%) categorized as PD-RBD(+) and 47 patients (45.2%) as PD-RBD(-) based on RBD presence before the PD diagnosis. We evaluated the microbiome differences between these groups over the disease progression through taxonomic and functional differential abundance analyses and carbohydrate-active enzyme (CAZyme) profiles based on metagenome-assembled genomes. The PD-RBD(+) gut microbiome showed a relatively stable microbiome composition irrespective of disease stage. In contrast, PD-RBD(-) microbiome exhibited a relatively dynamic microbiome change as the disease progressed. In early-stage PD-RBD(+), Escherichia and Akkermansia, associated with pathogenic biofilm formation and host mucin degradation, respectively, were enriched, which was supported by functional analysis. We discovered that genes of the UDP-GlcNAc synthesis/recycling pathway negatively correlated with biofilm formation; this finding was further validated in a separate cohort. Furthermore, fiber intake-associated taxa were decreased in early-stage PD-RBD(+) and the biased mucin-degrading capacity of CAZyme compared to fiber degradation.

Conclusion: We determined that the gut microbiome dynamics in patients with PD according to the disease progression depend on the presence of premotor RBD. Notably, early-stage PD-RBD(+) demonstrated distinct gut microbial characteristics, potentially contributing to exacerbation of PD pathophysiology. This outcome may contribute to the development of new therapeutic strategies targeting the gut microbiome in PD. Video Abstract.

Keywords: Biofilm; Carbohydrate-active enzymes; Microbiome; Parkinson’s disease; Rapid eye movement sleep behavior disorders.

Fig. 1

Different gut microbiome alteration patterns in PD-RBD(+) and PD-RBD(−) depending on disease progression. A, B Principal coordinate analysis (PCoA) plots based on the unweighted UniFrac distance matrix using (A) shotgun metagenome and (B) 16S amplicon sequencing data from patients with early-stage PD and HC. Overall and pairwise permutational analysis of variance (PERMANOVA) were utilized to ascertain the significance of gut microbiome differences across groups. Bonferroni correction was implemented to adjust the p-value in pairwise PERMANOVA. Large circles represent the centroids for each group. The ellipses represent the 95% confidence interval. C, D The unweighted UniFrac distance-based PCoA illustrates gut microbiome variations across the disease stages: early (less than 2 years after PD diagnosis) and late (greater than or equal to two years after PD diagnosis). E, F Results of pairwise PERMANOVA analyses encompassing early- and late-stage of PD-RBD(+) and PD-RBD(−). Age was used as a covariate due to the age differences observed in late-stage samples, as shown in Supplementary table S6. G, H Unweighted UniFrac distances from PD-RBD(+) (blue) and PD-RBD(−) (red) to HC across the disease duration, employing (G) shotgun metagenome and (H) 16S rRNA gene amplicon data, respectively. I, J Unweighted UniFrac distances of PD-RBD(+) (blue) and PD-RBD(−) (red) from the late stages to their respective early stages, using (I) shotgun metagenome and (J) 16S rRNA gene amplicon data, respectively. ***p < 0.001 for PERMANOVA. *q < 0.05; **q < 0.01 for pairwise PERMANOVA. ns not significant, r Pearson correlation coefficient, PD Parkinson’s disease, RBD rapid eye movement sleep behavior disorder, HC healthy control

Fig. 2

Differential abundance analyses reveal gut microbiome differences in PD-RBD(+) compared to PD-RBD(−) and HC. A Overall results of pairwise DA analyses utilizing shotgun metagenome data. Rows were hierarchically clustered using the complete linkage method. Taxa referenced in the main text are emphasized in bold. B, C Taxa (B) enriched or (C) depleted in PD-RBD(+) compared to PD-RBD(−) and/or HC as identified by the shotgun metagenome data at the genus level. D Taxa enriched in PD-RBD(−) than PD-RBD(+) or HC. E Scatter plot depicts the results of DA analyses comparing PD-RBD(+) with PD-RBD(−) (x-axis) and with HC (y-axis). Positive values on both axes represent a positive correlation with PD-RBD(+), while negative values imply a reverse association. The overall results illustrate a substantial positive correlation, suggesting a similar association of taxa with PD-RBD(+) compared to PD-RBD(−) and HC. Red and green represent the taxa depicted in (B) and (C), respectively. q < 0.1; *q < 0.05; **q < 0.01; ***q < 0.001. ns not significant, ρ Spearman’s rank correlation coefficient, PD Parkinson’s disease, RBD rapid eye movement sleep behavior disorder, HC healthy control, DA differential abundance, RA relative abundance

Fig. 3

Distinct functional characteristics of PD-RBD(+) compared to PD-RBD(−) and HC. A Bar plot displaying the counts of differentially abundant KEGG orthology (KO) across the group comparisons. The orientation and color of the bars signify which group the KOs are enriched in. B Venn diagrams illustrate the counts of differentially abundant KOs identified in the DA analyses comparing PD-RBD(+) with both PD-RBD(−) with HC. C Scatter plot illustrates the results of DA analyses of functional profiles comparing PD-RBD(+) with PD-RBD(−) (x-axis) and HC (y-axis). Positive values on both axes denote a positive association with PD-RBD(+), while negative values suggest an inverse one. Red represents genes engaged in Biofilm formation–Escherichia coli pathway, while green indicates those involved in the UDP-GlcNAc synthesis/recycling pathway. D, E Results of KEGG enrichment analyses using differentially abundant KOs in PD-RBD(+) compared to (D) HC and (E) PD-RBD(−). Only results with adjusted p < 0.1 are presented. The pathways discussed in the main text are emphasized in bold. PD Parkinson’s disease, RBD rapid eye movement sleep behavior disorder, HC healthy control, KEGG Kyoto Encyclopedia of Genes and Genomes, ρ Spearman’s rank correlation coefficient

Fig. 4

Differential gene abundances and diverse curli sequence characteristics in the gut microbiome of patients with PD and HC. A The abundance of the genes involved in the biofilm formation–E. coli pathway (KEGG pathway map02025) was found to be significantly elevated in PD-RBD(+) compared to those in HC and/or PD-RBD(−). The csgD gene (red box) serves as a master regulator for the biofilm formation pathway, and the ydaM gene is an upregulator of csgD. CsgD facilitates curli production by activating the csgBAC (encoding curli subunits and a curli chaperone) and csgEFG (encoding a curli secretion system) operons (orange box). Furthermore, CsgD enhances the expression of adrA, which in turn triggers the cellulose synthase operon bcsABZC through the signaling molecule c-di-GMP (blue box). Both curli and cellulose constitute bacterial biofilms. The curli fibrils produced during this process promote pathologic aggregation of alpha-synuclein. B The sequence logo of curli major subunit CsgA sequences retrieved from the MAGs or from the reference DB (UniProt P28307 for E. coli K12 and A0A9Q7ZLG0 for Citrobacter youngae). The multiple sequence alignment used to build the sequence logo is visualized in Fig. S6B (Additional file 1). All CsgA sequences exhibited conserved repeat regions (R1–R5 indicated by black arrows; only R2-R4 are shown in this figure). The gatekeeper residues are highlighted and indicated by purple arrows. q < 0.1; *q < 0.05. PD Parkinson’s disease, RBD rapid eye movement sleep behavior disorder, HC healthy control, KEGG Kyoto Encyclopedia of Genes and Genomes, RA relative abundance, MAG metagenome-assembled genome

Fig. 5

The UDP-GlcNAc synthesis/recycling pathway is depleted in PD-RBD(+) and negatively correlated with bacterial biofilm formation. A Schematic representation of the UDP-GlcNAc synthesis and recycling pathway, which is related to the amino sugar and nucleotide sugar metabolism (KEGG pathway map00520) and peptidoglycan biosynthesis (map00550) pathways. Solid red arrows denote reactions catalyzed by enzymes encoded by genes exhibiting significant differences in the DA analyses, whereas solid black arrows signify reactions facilitated by those without significant differences. B Relative abundances of significantly different genes marked by solid red arrows in (A). Pie charts represent taxonomic contributions to each gene across groups. C Scatter plot of centered log-ratio (clr) transformed abundances for csgD and glmU genes, which are essential for biofilm formation and UDP-GlcNAc synthesis, respectively. These genes show a negative correlation (ρ = − 0.32, p = 4.0E-4). D Distribution of the four subject categories based on the clr-transformed abundances of csgD and glmU genes across groups. Fisher’s exact test was used for statistical analysis. q < 0.1; *q < 0.05; ***q < 0.001 for DA analysis. **p < 0.01 for Fisher’s exact test. ns not significant, ρ Spearman’s rank correlation coefficient, GlcN-6P glucosamine 6-phosphate, GlcN-1P glucosamine 1-phosphate, GlcNAc N-acetylglucosamine, GlcNAc-6P GlcNAc 6-phosphate, GlcNAc-1P GlcNAc 1-phosphate, UDP-GlcNAc uridine diphosphate-GlcNAc, DA differential abundance, PD Parkinson’s disease, RBD rapid eye movement sleep behavior disorder

Fig. 6

Differentially abundant CAZymes between PD-RBD(+) and HC, and the associated substrates and taxa. A A waterfall plot of DA analysis comparing the CAZyme profiles between PD-RBD(+) and HC. Only significant results are shown. CAZymes targeting dietary fibers and host mucins are indicated by green and red colors, respectively. Error bars represent the standard error from the model. B, C (B) Target substrate and (C) taxonomic contribution profiles of differentially abundant CAZymes. The order of CAZymes along the x-axis is consistent with that in (A). GH glycoside hydrolase, GT glycosyl transferase, PL polysaccharide lyase, CE carbohydrate esterase, AA auxiliary activity, CMB carbohydrate-binding module, PD Parkinson’s disease, RBD rapid eye movement sleep behavior disorder, HC healthy control, DA differential abundance

Fig. 7

Graphical summary of the current study. Left panel: In both HC and early-stage PD-RBD(−) compared to early-stage PD-RBD(+), the gut microbiome tends to favor degradation of dietary fibers over host mucin. In line with this, the abundance of fiber-associated bacteria (i.e., Prevotella, Faecalibacterium, and Agathobacter) is increased, whereas that of mucin degradation-associated bacteria (i.e., Akkermansia, Barnesiella, and Desulfovibrio) is decreased. In HC and PD-RBD(−), fiber-associated bacteria are associated with enhanced capability to discharge N-acetylglucosamine (GlcNAc) into the environment, which in turn suppresses biofilm formation by biofilm-producing bacteria (i.e., Escherichia). Right panel: Conversely, in early-stage PD-RBD(+) compared to both in HC and early-stage PD-RBD(−), the gut microbiome shows a preference for degrading host mucin over dietary fibers. Correspondingly, abundance of fiber-associated bacteria is reduced, and that of mucin degradation-associated bacteria is increased. The reduction in fiber-associated bacteria may lead to a scarcity of GlcNAc, thereby facilitating easier biofilm formation by bacteria. Consequently, these characteristics of PD-RBD(+) may be associated with increased susceptibility to infection and inflammation, potentially contributing to the exacerbation of PD pathophysiology, such as alpha-synuclein aggregation. PD Parkinson’s disease, RBD rapid eye movement sleep behavior disorder, HC healthy control. Created with BioRender.com