A Role for Neuronal Alpha-Synuclein in Gastrointestinal Immunity

Abstract

Background: Alpha-synuclein (αS) is a nerve cell protein associated with Parkinson disease (PD). Accumulation of αS within the enteric nervous system (ENS) and its traffic from the gut to the brain are implicated in the pathogenesis and progression of PD. αS has no known function in humans and the reason for its accumulation within the ENS is unknown. Several recent studies conducted in rodents have linked αS to immune cell activation in the central nervous system. We hypothesized that αS in the ENS might play a role in the innate immune defenses of the human gastrointestinal (GI) tract.

Methods: We immunostained endoscopic biopsies for αS from children with documented gastric and duodenal inflammation and intestinal allograft recipients who contracted norovirus. To determine whether αS exhibited immune-modulatory activity, we examined whether human αS induced leukocyte migration and dendritic cell maturation.

Findings: We showed that the expression of αS in the enteric neurites of the upper GI tract of pediatric patients positively correlated with the degree of acute and chronic inflammation in the intestinal wall. In intestinal allograft subjects who were closely monitored for infection, expression of αS was induced during norovirus infection. We also demonstrated that both monomeric and oligomeric αS have potent chemoattractant activity, causing the migration of neutrophils and monocytes dependent on the presence of the integrin subunit, CD11b, and that both forms of αS stimulate dendritic cell maturation.

Interpretation: These findings strongly suggest that αS is expressed within the human ENS to direct intestinal inflammation and implicates common GI infections in the pathogenesis of PD.

Keywords: Alpha-synuclein; Cell activation; Innate immunity; Parkinson disease.

Fig. 1

Representative biopsies of pediatric duodenum immune-stained for αS. The 3 top panels are serial sections (×20), stained with HE, immunostained for αS, or for CD68. The specimen exhibits severe acute and chronic inflammation. Lower panels: αS colocalizes with PGP 9.5 by double immunofluorescence demonstrating expression within neurons. A representative biopsy from the population in online suppl. Table 1 is shown. Primary antibodies: α-syn, ab27766 from Abcam; PGP 9.5, Z5116 from DAKO. Secondary antibodies: α-syn, Envision anti-mouse HRP polymer from Agilent; PGP 9.5, goat anti-rabbit biotin conjugated from Vector. Tertiary (fluorescent tag): α-syn, PerkinElmer Tyramide Signal Amplification with Cy5; PGP 9.5, Life Technologies streptavidin conjugated with AlexaFluor488. Procedures were as specified by the protocols provided with the reagents. Cy5 was imaged at 555 nm and GFP at 488 nm. Images were taken using Slidebook 6.0 and the overlay was an output from the Slidebook System.

Fig. 2

The degree of acute and chronic inflammation correlates with αS immune positivity. Scores for acute and chronic inflammation for all specimens are plotted relative to the associated αS scores (listed in online suppl. Table 1). Statistical significance between the inflammation scores associated with high αS scores (2 or 3) versus an αS score of 1 were determined by a 1-way ANOVA with a post hoc t test, with a Bonferroni correction. ns, not significant.

Fig. 3

Representative biopsies from intestinal transplant recipients with documented norovirus infection stained for αS (×20). a “Case 16, Pre” (native duodenum): a biopsy taken 1 month prior to the norovirus infection shows minimal expression of αS. b “Case 16, During” (native duodenum): a biopsy taken at the time of norovirus infection shows a robust expression of αS in neurites within the lamina propria. c “Case 3, During” (allograft jejunum): a biopsy taken at the time of norovirus infection shows areas with high expression of αS. d “Case 3, Post” (allograft jejunum): a biopsy taken 4 months after the initial norovirus infection shows continued high expression of αS. The patient still tested PCR positive for norovirus. Images 1 and 2 are enlargements of the outlined areas.

Fig. 4

αS is a chemoattractant dependent on CD11b. Assays were conducted as described in the Methods section. The average of 3 independent assays for each sample is presented. The concentration of the αS aggregate is in terms of the monomer. A positive control, FMLP, is noted in black. NAc 1–21, the N-acetylated peptide corresponding to the first 21 amino acids of αS; 1–25, a peptide corresponding to the first 25 amino acids. a Human neutrophils. b Human monocytes. c, d Representative photos of the cellular response. e Neutrophils from CD11b^−/− or wild-type mice (0.5 × 10⁶/mL), assayed as described in the Methods section. f Human neutrophils incubated in the presence of anti-CD11b antibody or the control (50 µg/mL each). CM, control media (negative control); HPF, high-powered field.

Fig. 5

αS stimulates phenotypic maturation of human dendritic cells. Human monocyte-derived dendritic cells were incubated at 5 × 10⁵/mL in the presence of recombinant monomeric or aggregated αS, and the NAc 1–21 amino terminal peptide of αS (each at a final concentration of 10 μM), for 48 h before they were immunostained and analyzed by flow cytometry for the expression of the indicated surface molecules. E. coli lipopolysaccharide (LPS; 100 ng/mL) was included as a positive control. The results of 1 experiment representative of 3 are shown. The ordinate of each flow cytometric analysis corresponds to the fluorescent intensity/cell, the abscissa to the cell number in a log scale.