Gut Microbiota Regulate Motor Deficits and Neuroinflammation in a Model of Parkinson's Disease

Abstract

The intestinal microbiota influence neurodevelopment, modulate behavior, and contribute to neurological disorders. However, a functional link between gut bacteria and neurodegenerative diseases remains unexplored. Synucleinopathies are characterized by aggregation of the protein α-synuclein (αSyn), often resulting in motor dysfunction as exemplified by Parkinson's disease (PD). Using mice that overexpress αSyn, we report herein that gut microbiota are required for motor deficits, microglia activation, and αSyn pathology. Antibiotic treatment ameliorates, while microbial re-colonization promotes, pathophysiology in adult animals, suggesting that postnatal signaling between the gut and the brain modulates disease. Indeed, oral administration of specific microbial metabolites to germ-free mice promotes neuroinflammation and motor symptoms. Remarkably, colonization of αSyn-overexpressing mice with microbiota from PD-affected patients enhances physical impairments compared to microbiota transplants from healthy human donors. These findings reveal that gut bacteria regulate movement disorders in mice and suggest that alterations in the human microbiome represent a risk factor for PD.

Keywords: Parkinson’s disease; gut-brain axis; microbiome; microglia; mouse model; short chain fatty acids; synuclein.

Copyright © 2016 Elsevier Inc. All rights reserved.

Figures

Figure 1. Gut microbes promote motor and gastrointestinal dysfunction

(A) Time to traverse beam apparatus (B) Time to descend pole (C) Time to remove adhesive from nasal bridge (D) Hind-limb clasping reflex score (E) Time course of fecal output in a novel environment over 15 minutes (F) Total fecal pellets produced in 15 minutes Animals were tested at 12-13 weeks of age. N=4-6, error bars represent the mean and standard error from 3 trials per animal. Data are representative of 2 experiments. *p ≤ 0.05; **p≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001. SPF=specific pathogen free, GF=germ-free, WT=wild-type, ASO=Thy1-α-synuclein genotype. See also Figure S1.

Figure 2. αSyn pathology is increased in mice harbouring a gut microbiota

(A) Representative images of the caudoputamen (CP) from SPF-ASO or GF-ASO animals stained with aggregation-specific αSyn antibody (red), Phospho-Ser129-αSyn antibody (green), and Neurotrace/Nissl (blue) (B) Representative images of the substantia nigra (SN) from SPF-ASO or GF-ASO animals, stained as above (C) Representative western blot of triton soluble and insoluble brain homogenates, immunostained with anti-αSyn antibody (D, E) Densitometry quantification of anti-αSyn western blots for (D) all αSyn and (E) ratio of insoluble to soluble αSyn staining (F) qRT-PCR analysis of human αSyn in the CP or inferior midbrain (Mid) (G) ELISA analysis of total αSyn present in homogenates from the the CP or inferior midbrain (Mid) Tissues collected from mice at 12-13 weeks of age. N=3-4, error bars represent the mean and standard error. *p ≤ 0.05; **p≤ 0.01; ***p≤ 0.001. SPF=specific pathogen free, GF=germ-free, WT=wild-type, ASO=Thy1-α-synuclein genotype. See also Figure S2.

Figure 3. αSyn-dependent microglia activation by the microbiota

(A) Representative 3D reconstructions of Iba1-stained microglia residing in the caudoputamen (CP) of SPF-WT, SPF-ASO, GF-WT, and GF-ASO animals (B) CP-resident microglia parameters diameter, number of branch points, and total branch length (C) Substantia nigra (SN)-resident microglia parameters diameter, number of branch points, and total branch length (D) ELISA analysis for TNF-α and IL-6 present in homogenates from the CP (E) ELISA analysis for TNF-α and IL-6 present in homogenates from the inferior midbrain (Mid) (F) qPCR analysis of CD11b+ cells derived from brain homogenate for tnfa and il6 (G) Diameter of microglia residing in the frontal cortex (FC) or cerebellum (CB) (H) ELISA analysis for TNF-α present in homogenates from the FC or CB Tissues collected from mice at 12-13 weeks of age. N=3-4, (with 20-60 cells per region per animal analyzed) error bars represent the mean and standard error. *p ≤ 0.05; **p≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001. SPF=specific pathogen free, GF=germ-free, WT=wild-type, ASO=Thy1-α-synuclein genotype. See also Figure S2.

Figure 4. Postnatal microbial signals promote motor and gastrointestinal dysfunction

(A) Time course schema for animal treatment and testing (B) Time to traverse beam apparatus (C) Time to descend pole (D) Time to remove nasal adhesive (E) Hindlimb clasping reflex score (F) Time course of fecal output in a novel environment over 15 minutes (G) Total fecal pellets produced in 15 minutes (H) Representative 3D reconstructions of Iba1-stained microglia residing in the caudoputamen (CP) of Abx-ASO or Ex-GF-ASO animals (I) Diameter of microglia residing in the CP or substantia nigra (SN) Animals were tested at 12-13 weeks of age. N=6-12, error bars represent the mean and standard error from 3 trials per animal, and compiled from 2 independent cohorts or 20-60 microglia per region analyzed. #0.05<p<0.1; *p ≤ 0.05; **p≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001. SPF=specific pathogen free, GF=germ-free, Abx=antibiotic-treated, Ex-GF=recolonized germ-free animals, WT=wild-type, ASO=Thy1-α-synuclein genotype. See also Figure S3.

Figure 5. SCFAs promote αSyn-stimulated microglia activation and motor dysfunction

(A) Representative 3D reconstructions of Iba1-stained microglia residing in the caudoputamen (CP) of wild-type or ASO SCFA-treated animals (B) Diameter of microglia residing in the CP or substantia nigra (SN) (C) Time to traverse beam apparatus (D) Time to descend pole (E) Time to remove nasal adhesive (F) Hindlimb clasping reflex score (G) Time course of fecal output in a novel environment over 15 minutes (H) Total fecal pellets produced in 15 minutes Animals were tested at 12-13 weeks of age. N=6-12, error bars represent the mean and standard error from 3 trials per animal, and compiled from 2 independent cohorts or 20-60 microglia per region analyzed. Data are plotted with controls from Figure 4 for clarity. *p ≤ 0.05; **p≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001. SPF=specific-pathogen free, GF=germ-free, SCFA=short-chain fatty acid-treated, WT=wild-type, ASO=Thy1-α-synuclein genotype. See also Figures S3-5.

Figure 6. Microbiome dysbiosis of PD patient samples after transplant into germ-free mice

(A) Unweighted UniFrac Principle Coordinate Analysis of microbial communities of human donors (large circles) and recipient mice (small circles). Each donor and recipient sample are matched by color. (B) Unweighted and weighted UniFrac analysis of microbial communities in recipient animals based on donor identity (C) Unweighted and weighted UniFrac analysis of microbial communities in recipient animals based on mouse genotype (D) Comparison of unweighted and weighted UniFrac analysis of microbial communities in recipient animals (E) Taxa-level analysis of individual genera altered between PD and healthy donors as a function of recipient mouse genotype. Left column indicates percentage with significant differences observed; right column indicates fold change between PD and healthy donors. Light colors indicate non-statistically significant differences N=3-6, over 3 time points post-colonization. ***p≤ 0.001, 999 permutations. HC=germ-free mice colonized with fecal microbes from healthy controls, PD=germ-free mice colonized with fecal microbes from Parkinson's disease patients, WT=wild-type, ASO=Thy1-α-synuclein genotype. See also Figure S6.

Figure 7. Microbiota from PD patients induce increased αSyn-mediated motor deficits

(A-F) Time to cross a beam, time to descend the pole, time to remove nasal adhesive, and hindlimb clasping reflex scores of mice humanized with microbiota from either PD patients or matched healthy controls (G) Compilation of all independent cohorts in each motor task: beam traversal, pole descent, adhesive removal, and hindlimb clasping reflex score, grouped by health status of fecal donor Animals were tested at 12-13 weeks of age. N=3-6, error bars represent the mean and standard error from 3 trials per animal. #0.05<p<0.1; *p ≤ 0.05; **p≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001. HC=germ-free mice colonized with fecal microbes from healthy controls, PD=germ-free mice colonized with fecal microbes from Parkinson's disease patients, WT=wild-type, ASO=Thy1-α-synuclein genotype. See also Figure S7 and Table S1.