Leaky intestine and impaired microbiome in an amyotrophic lateral sclerosis mouse model

Abstract

Emerging evidence has demonstrated that intestinal homeostasis and the microbiome play essential roles in neurological diseases, such as Parkinson's disease. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons and muscle atrophy. Currently, there is no effective treatment. Most patients die within 3-5 years due to respiratory paralysis. Although the death of motor neurons is a hallmark of ALS, other organs may also contribute to the disease progression. We examined the gut of an ALS mouse model, G93A, which expresses mutant superoxide dismutase (SOD1(G93A)), and discovered a damaged tight junction structure and increased permeability with a significant reduction in the expression levels of tight junction protein ZO-1 and the adherens junction protein E-cadherin. Furthermore, our data demonstrated increased numbers of abnormal Paneth cells in the intestine of G93A mice. Paneth cells are specialized intestinal epithelial cells that can sense microbes and secrete antimicrobial peptides, thus playing key roles in host innate immune responses and shaping the gut microbiome. A decreased level of the antimicrobial peptides defensin 5 alpha was indeed found in the ALS intestine. These changes were associated with a shifted profile of the intestinal microbiome, including reduced levels of Butyrivibrio Fibrisolvens, Escherichia coli, and Fermicus, in G93A mice. The relative abundance of bacteria was shifted in G93A mice compared to wild-type mice. Principal coordinate analysis indicated a difference in fecal microbial communities between ALS and wild-type mice. Taken together, our study suggests a potential novel role of the intestinal epithelium and microbiome in the progression of ALS.

Keywords: Antimicrobial peptides; Paneth cells; ZO‐1; autophagy; cell biology; dysbiosis; intestinal permeability; microbiome; signal transduction; tight junction.

© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Figures

Figure 1

Disrupted intestinal junctions in the 2-month-old amyotrophic lateral sclerosis (ALS) mice. (A) Western blots of ZO-1 and E-cadherin in the colon. The relative band intensities of ZO-1 and E-cadherin are presented as the means ± SD (n = 3 per group, *P < 0.05). Western blot bands were quantified using Image Lab 4.01 (Bio-Rad). (B) Immunostaining of ZO-1 and E-cadherin in the colon. The tight junction protein ZO-1 shows discontinuity in the colon of ALS model mice. (C) H&E staining of the colon. Data are from a single experiment and are representative of n = 3 mice per group.

Figure 2

Enhanced IL-17 and intestinal permeability in amyotrophic lateral sclerosis (ALS) mice. (A, B) The inflammatory cytokine IL-17 in mouse serum and the small intestine (n = 3, *P < 0.05). (C) Intestinal permeability is increased in ALS mice (n = 3, *P < 0.05).

Figure 3

Defects in Paneth cells in the small intestine of amyotrophic lateral sclerosis (ALS) mice. (A) Lysozyme staining in the small intestine of ALS mice. (B) Number of Paneth cells/crypt in the small intestine. Ten slides from each mouse were counted. (C) Percentage of normal and abnormal Paneth cells in ALS mice (n = 3/group, *P < 0.05). (D) Defensin 5 alpha mRNA level in 3-month-old mice with symptoms (n = 3, *P < 0.05).

Figure 4

Shift of fecal microbial communities in amyotrophic lateral sclerosis (ALS) mice. (A) Butyrivibrio Fibrisolvens was decreased in G93A amyotrophic lateral sclerosis model mice. Real-time PCR of the bacterial universal 16S rRNA gene and 16S rRNA for Escherichia coli, Bacteroides Fragilis, and Butyrivibrio Fibrisolvens in fecal samples from both G93A and wild-type mice. Primers specific to universal 16S rRNA were used as an endogenous control to normalize loading between samples. The relative amount of 16S rRNA in each sample was estimated using the ΔΔCT method (n = 3, *P < 0.05). (B) Bacterial community of fecal samples from ALS and WT mice using 454 16S rRNA sequencing data (n = 3/group). (C) Principal coordinates analysis (PCoA) of unweighted UniFrac distances of 16S rRNA genes. An analysis was conducted for ALS (red) and WT (blue) mice. The results indicate that fecal microbial communities differ in ALS and WT mice.