Disruption of the microbiota-gut-brain axis is a defining characteristic of the α-Gal A (-/0) mouse model of Fabry disease

Abstract

Fabry disease (FD) is an X-linked metabolic disease caused by a deficiency in α-galactosidase A (α-Gal A) activity. This causes accumulation of glycosphingolipids, especially globotriaosylceramide (Gb3), in different cells and organs. Neuropathic pain and gastrointestinal (GI) symptoms, such as abdominal pain, nausea, diarrhea, constipation, and early satiety, are the most frequent symptoms reported by FD patients and severely affect their quality of life. It is generally accepted that Gb3 and lyso-Gb3 are involved in the symptoms; nevertheless, the origin of these symptoms is complex and multifactorial, and the exact mechanisms of pathogenesis are still poorly understood. Here, we used a murine model of FD, the male α-Gal A (-/0) mouse, to characterize functionality, behavior, and microbiota in an attempt to elucidate the microbiota-gut-brain axis at three different ages. We provided evidence of a diarrhea-like phenotype and visceral hypersensitivity in our FD model together with reduced locomotor activity and anxiety-like behavior. We also showed for the first time that symptomology was associated with early compositional and functional dysbiosis of the gut microbiota, paralleled by alterations in fecal short-chain fatty acid levels, which partly persisted with advancing age. Interestingly, most of the dysbiotic features suggested a disruption of gut homeostasis, possibly contributing to accelerated intestinal transit, visceral hypersensitivity, and impaired communication along the gut-brain axis.

Keywords: Fabry disease; gastrointestinal disorders; gut microbiota; gut-brain axis; short-chain fatty acids; visceral pain; α-Gal A null mice.

Figure 1.

Diarrhea-like phenotype in α-Gal A (-/0) mice. stool analysis was carried out on α-Gal A (-/0) mice (gray bars) and α-Gal A (+/0) controls (white bars) at the age of 8–10-week-old (T1), 16–20-week-old (T2), and 12-month-old (T3). The fecal output was measured as number of pellets (a) and mg produced in 24 h (b). The water content (c) was calculated after one hour according to the equation: water content (%) = 100 (wet weight – dry weight)/wet weight. Data are expressed as mean ± SEM of 10 animals per group (n = 10). Two-way ANOVA test with Bonferroni post-correction, *p < 0.05, **p < .01 and ***p < .001 VS α-Gal A (+/0) mice. (d) whole gut transit time was measured by the carmine red method in control and FD mice. Data are presented as mean ± SEM and the number of animals per group is 3. Data were compared using two-way ANOVA followed by Tukey’s post hoc test. Differences were considered significant at the p < .05 level.

Figure 2.

Visceral hypersensitivity in α-Gal A (-/0) mice. visceral sensitivity was assessed in 8–10-week-old (T1), 16–20-week-old (T2), and 12-month-old (T3) α-Gal A (-/0) mice (gray) and α-Gal A (+/0) controls (white), by measuring the electromyography (EMG) amplitude of abdominal contraction (VMR, visceral-motor response) under light anesthesia (left panel, A-B-C) and scoring behavioral responses (AWR, abdominal withdrawal reflex) in awake animals (right panel, D-E-F) to colorectal distension with increasing volumes (50–300 µl balloon inflation). Each value represents the mean ± SEM of 10 animals per group (n = 10). *p < .05, **p < .001 and ***p < .001VS α-Gal A (+/0) animals.

Figure 3.

Anxiety-like behavior and locomotor activity in α-Gal A (-/0) mice. experiments were carried out on 8–10-week-old (T1), 16–20-week-old (T2), and 12-month-old (T3) α-Gal A (-/0) mice (gray) and α-Gal A (+/0) controls (white). Data are representative of at least three independent experiments performed on 8–11 animals (n = 8–11) per group per genotype. Anxiety-like behavior was measured as (a) frequency in the periphery (%); (b) time in periphery and (c) total distance moved (cm). Values represent means ± SEM. Two-way ANOVA followed by Tukey’s post hoc test was applied. *p < .05; **p < .01; ***p < .001 VS α-Gal A +/0; ##p < .01; ###p < .001 VS same genotype.

Figure 4.

Alpha and beta diversity of the gut microbiota in α-Gal A (-/0) mice. a, Boxplots showing the distribution of alpha diversity, according to Shannon entropy and Simpson index, in the gut microbiota of α-Gal A (-/0) mice (orange hues) and α-Gal A (+/0) controls (blue hues) at 8–10-week-old (T1), 16–20-week-old (T2), and 12-month-old (T3) (n = 10 each group). Tukey test, ***p < .001. b, Principal component analysis (PCA) of beta diversity, based on Aitchison distance, of all fecal samples. A significant separation was found between groups of mice at each age and within each mouse group over time (PERMANOVA, p ≤ .005).

Figure 5.

Potential compositional signatures of gut microbiota dysbiosis in α-Gal A (-/0) mice. boxplots showing the relative abundance distribution of differentially represented phyla (a), families (b) and genera (c) between α-Gal A (-/0) mice (orange hues) and α-Gal A (+/0) controls (blue hues) at each age (8–10-week-old (T1), 16–20-week-old (T2), and 12-month-old (T3) (n = 10 each group)), and within each mouse group over time (Wilcoxon test, *p < .05, **, p < .01). Only taxa with relative abundance > 0.5% in at least 1 sample are shown.

Figure 6.

Alterations in predicted gut microbiota functions related to the gut-brain axis in α-Gal A (-/0) mice. Heatmap showing the differential abundance of significantly altered neuroactive gut-brain modules between α-Gal A (-/0) mice and α-Gal A (+/0) controls at each age (at 8–10-week-old (T1), 16–20-week-olg (T2), and 12-month-old (T3) (n = 10 each group)). Stars indicate Benjamini-Hochberg-adjusted p-values (*p_adj < 0.1, **p_adj < .01, ***p_adj < .001). Colors of the cells indicate the effect size (β); red hues indicate higher levels in α-Gal A (-/0) mice, whereas blue hues indicate higher levels in α-Gal A (+/0) controls.

Figure 7.

Alterations in fecal short-chain fatty acid levels in α-Gal A (-/0) mice. experiments were carried out on 8–10-week-old (T1), 16–20-week-old (T2), and 12-month-old (T3) α-Gal A (-/0) mice (gray) and α-Gal A (+/0) (white). Short-chain fatty acid (SCFA) levels were measured in µmol/g. (a) total SCFAs; (b) butyric acid; (c) acetic acid; (d) propionic acid; (e) iso-butyric acid; (f) valeric acid; (g) isovaleric acid. Data are shown as means ± SEM. Two-way ANOVA followed by Tukey’s post hoc test was applied (n = 10, each group). *p < .05; **p < .002; ***p < .001 VS α-Gal A +/0; #p < .05 ##p < .002; ###p < .001 VS the same genotype.