Gastrointestinal Dysfunction and Low-Grade Inflammation Associate With Enteric Neuronal Amyloid-β in a Model for Amyloid Pathology

Abstract

Background: Patients suffering from Alzheimer's disease, a progressive neurodegenerative disorder involving cognitive decline and memory impairment, often present with gastrointestinal comorbidities. Accumulating data also indicate that alterations in the gut can modulate Alzheimer's disease pathology, highlighting the need to better understand the link between gastrointestinal abnormalities and neurodegeneration in the brain.

Methods: To disentangle the pathophysiology of gastrointestinal dysfunction in Alzheimer's disease, we conducted a detailed pathological characterization of the gastrointestinal tract of 5xFAD mice by performing histological analyses, gene expression studies, immunofluorescence labeling and gut function assays.

Results: We found that 5xFAD mice have elevated levels of intestinal amyloid precursor protein and accumulate amyloid-β in enteric neurons. Histopathology revealed that this is associated with mild intestinal inflammation and fibrosis and accompanied by increased expression of proinflammatory cytokines. While overall enteric nervous system composition and organization appeared unaffected, 5xFAD mice have faster gastrointestinal transit.

Conclusion: Our findings indicate that amyloid-β accumulation in enteric neurons is associated with low-grade intestinal inflammation and altered motility and suggest that peripheral pathology may cause gastrointestinal dysfunction in Alzheimer's disease patients.

Keywords: enteric glia; enteric nervous system; gut function; intestinal motility; neuroinflammation.

FIGURE 1

APP expression in small intestinal (left) and colonic (right) myenteric plexus preparations isolated from WT and 5xFAD mice as determined by RT‐qPCR. Increased expression of APP was detected in both regions in 5xFAD as compared to WT mice (N = 5 per genotype, **p < 0.01).

FIGURE 2

Aβ presence in the myenteric plexus. (A) Representative confocal images (N = 4 per genotype) of small intestinal and colonic myenteric plexus preparations immunolabeled for S100ß and WO2, showing the enteric glia network (magenta) and Aβ (turquoise), respectively. Aβ accumulation can be found in colonic myenteric ganglia (arrows). (B) Representative (N = 4 per genotype) confocal images of small intestinal and colonic myenteric plexus preparations immunolabeled for HuCD and 4G8, showing enteric neurons (magenta) and Aβ (turquoise), respectively. Intracellular accumulation of Aβ was detected in myenteric neurons in the colon of 5xFAD mice (arrowheads). Scale bars: 20 μm.

FIGURE 3

Aβ‐positive myenteric neurons in the small intestine and colon of 5xFAD mice. Representative confocal images (N = 4 per genotype) of myenteric neurons in the small intestine and colon immunolabeled for WO2 (turquoise) and the enteric neuron subtype markers calbindin (yellow) and nNOS (magenta). Intracellular accumulation of Aβ was detected both in calbindin (arrowheads) and nNOS (arrows) expressing myenteric neurons. The asterisk depicts a myenteric neuron that is positive for WO2, calbindin, and nNOS. Scale bars: 20 μm.

FIGURE 4

Enlarged Peyer's patches and fibrosis in the gastrointestinal tract of 5xFAD mice. (A) Low‐power microscopic images of H&E‐stained sections (N = 6 per genotype) showing an increased number of Peyer's patches (arrows) in the small intestine of 5xFAD mice. Scale bars: 1 mm. (B) Zoomed‐in H&E‐stained sections of Swiss rolls (N = 6 per genotype) illustrate pathological changes in the intestinal wall of 5xFAD mice. Arrows in the top panels indicate scar tissue accumulation in the small intestine. Middle panels show enlarged and activated Peyer's patches with prominent germinal centers (indicated by arrowheads) distributed along the gut in 5xFAD mice. No abnormalities were observed in the colon. Scale bars: 50 μm. (C) Representative images of Sirius red staining of the small intestine. Arrows indicate regions of fibrotic tissue. Scale bars: 50 μm.

FIGURE 5

Expression of proinflammatory cytokines in 5xFAD mice. (A) Elevated levels of proinflammatory cytokine mRNAs were found in whole gut tissue samples from both the small intestine and colon by RT‐qPCR (N = 6 per genotype, **p < 0.01). (B) Fecal lipocalin‐2 levels were not significantly increased in 5xFAD mice (N = 6 per genotype).

FIGURE 6

Gastrointestinal dysfunction in 5xFAD mice. (A) Whole gut transit is faster in 5xFAD mice relative to their WT littermates (N = 10–13 per genotype). This is accompanied by (B) an increase in the average wet weight (N = 6–13 per genotype) and (C) water content of stools (N = 6–13 per genotype). No differences were observed between 5xFAD and WT littermates regarding (D) mouse weight (N = 6–13 per genotype), (E, F) gut length (N = 3–7 per genotype), and (G) defecation frequency (N = 6–13 per genotype). (H) intestinal permeability is not affected in 5xFAD mice (N = 9–12 per genotype). *p < 0.05, **p < 0.01.

FIGURE 7

Enteric glial cells are not affected in 5xFAD mice. (A) Analysis of enteric glial network organization revealed no differences between the two genotypes (N = 6 per genotype). (B) Representative immunofluorescence images of small intestinal and colonic myenteric plexus preparations labeled for S100B (yellow) and GFAP (blue) (N = 6 per genotype). Scale bars: 20 μm.

FIGURE 8

Enteric neuron numbers and specific subtype composition are not affected in 5xFAD mice. (A) Representative immunofluorescence images showing HuCD (magenta), calbindin (blue), and nNOS (green)‐positive myenteric neurons in the small intestine and colon. Scale bars: 100 μm. (B) Comparison of the number of myenteric neurons (HuCD, N = 11 per genotype) and (C, D) proportion of Calbindin (N = 3 per genotype) and nNOS (N = 3 per genotype) positive myenteric neurons in 5xFAD mice and WT littermates.