MeCP2-deficient rats have altered microbiota across development in parallel with behavioral symptoms of RTT
We first sought to characterize changes in microbiota at different developmental time points selected to parallel previously identified behavioral symptoms present in the female rat model, which range in appearance from p21 (time of weaning) to 4-12 months of age. Alpha diversity measured by observed operational taxonomic units (OTUs) was not significantly different between the MeCP2-deficient rats at any of the selected timepoints (Fig. 1 a). Beta diversity measured by bray-curtis difference was also similar across groups at p21 (Fig. 1 b). However, Mecp2ZFN/+ rats begin to diverge from WT rats in beta diversity at p49, and continue to be significantly distinct through p196 (Fig. 1 c-i). These clustering changes in beta diversity despite lack of changes in alpha diversity indicate that although the Mecp2ZFN/+ and WT rats have similar numbers of bacterial taxonomies in their guts throughout development, the makeup of bacterial taxonomies is altered across development at multiple timepoints associated with the development of behavioral symptoms in Mecp2ZFN/+ rats.
MeCP2-deficient rats show developmental shifts in the microbiome
The human RTT microbiome has previously been characterized by two groups (15,16). In a large cohort of humans with RTT, Strati et al. found decreases in the relative abundance of Bacteroidetes in RTT compared to healthy controls, as well as increased Firmicutes/Bacteroidetes ratio in RTT. Strati et al. also characterized RTT through genus level changes in the relative abundance of Actinomyces, Bifidobacterium, Clostridium XIVa Eggerthella, Enterococcus, Erysipelotrichaceae incertae sedis, Escherichia/Shigella, and Megasphaera. Borghi et al. found that although a cohort of humans with RTT had similar phylum level microbial communities to controls, RTT participants differed in family Bacteroidaceae as well as Clostridium and Suterella species, and that differences in Bacteroidaceae correlate with disease severity.
In the current study, we also sought to characterize specific microbial shifts of RTT rats compared to WT rats across development as shown in Table 1. (For full taxonomies see Table S1). At p21, the gut microbiota of both Mecp2ZFN/+ and WT rats is characterized by dominance of Bacteroidetes and Firmicutes phyla, as is also apparent in the aforementioned human studies. As expected, there are no significant differences in relative abundance across any level in p21 RTT rats and WT rats, possibly due to cohousing of RTT and WT rats until weaning at p21. At p35, the gut microbiota of both RTT and WT rats is still characterized by dominance of Bacteroidetes and Firmicutes phyla. At this age, however, the RTT rat microbiome begins to diverge from WT rats with the inclusion of the family Barnesiellaceae, the abundance of which is increased in sedentary women and is predicted by increased body fat percentage (29). This is of interest given that previous studies show that RTT rats are significantly heavier than WT rats by p60-90, despite being housed under the same dietary conditions.
When the animals reach p49, divergence appears at the class Epsilonproteobacteria and order Campylobacterales in the Proteobacterium phylum, and class RF39 in the phylum Tenericutes. These broad category changes in relative abundance likely drive the separation in beta diversity that also begins at this time point. By p77, however, RTT rats and WT rats no longer show changes in relative abundance of Proteobacteria, but differences in the Phylum Tenericutes remain significant. With regard to previously reported differences in RTT body weight, it should be noted that changes in Mollicutes, a class of Tenericutes, has been associated with rodent obesity related to the western diet (30).
Of note, the gut microbiomes of RTT and WT rats significantly diverge at p105 across multiple diverse taxonomies. Changes in order RF39 in phylum Tenericutes still persist. Additionally, we note shifts in multiple orders in classes Bacilli and Clostridia in phylum Firmicutes, and class Bacteroidia in phylum Bacteroidetes. By p133, though relative abundances of order RF39 and class Bacilli still trend toward significant differences, the microbiomes of WT and RTT rats do not significantly diverge at any level of taxonomy. This indicates that the microbial changes in RTT exist in a specific developmental window independent of age or weight related microbial shifts previously documented in rats (31). At p196, changes in classes Bacilli and Clostridia in phylum Firmicutes re-appear, and changes in the order Bacteroidales in phylum Bacteroidetes appear. This may indicate that the microbiomes of RTT rats are broadly characterized by shifts in Firmicutes and Bacteroides bacteria. Another possibility is that the reemergence of microbiota changes are related to disease progression, as we have previously described the appearance of significant motor abnormalities in female RTT rats at ~6 months of age (26).
Specific microbial shifts flag p105 as an important developmental stage in MeCP2-deficient rats
As higher level analyses indicated large taxonomy shifts at p105 in RTT rats, we examined differences in abundance of specific bacterial species at this time point. At this age, abundance of B. acidifaciens, which has been shown to promote IgA production in the large intestine (33), is significantly increased in RTT rats compared to WT (Table 1). Serum IgA counts are associated with gastrointestinal inflammation in individuals with RTT (15) and gut IgA content is increased in children with autism relative to typically developing children (34), suggesting a potential role for IgA-mediated gut inflammation in these neurologic conditions. C. perfringens is also significantly increased in p105 RTT rats compared to WT (Table 1). This species is known to produce epsilon toxin (35), which has deleterious effects on neurons, among other cell types (36). Increases in Clostridium genera abundance have also been previously identified in children with autism (37). Additionally, A. muciniphila trend lower in abundance in Mecp2ZFN/+ rats compared to WT (FDR adj. p = 0.07). Notably, A. muciniphila has been described as protective against epilepsy in rodents (10), and its reduction in abundance has also been reported in children with autism (38).
Previous studies have additionally utilized LEfSe scores to identify statistically significant differences in microbiota classifications (39) (Fig. 2). We performed LEfSe analysis on p105 rats to supplement our abundance data. The epsilon toxin-producing family Clostridiaceae has a significantly large linear discriminant analysis (LDA) score in Mecp2ZFN/+ rats compared to WT, with biological relevance of the sub classification genera Clostridium noted as above. A variety of clostridium-related species, which have been broadly described as associated with RTT (15,16) also demonstrate large LDA scores between genotypes. The SCFA-producing genus Lachnobacterium and family Lachnospiracaea in turn are similarly reduced in Mecp2ZFN/+ rats, again paralleling findings in Strati et al. and Borghi et al. Other taxonomies of note with large LDA score differences indicating relative reduction in Mecp2ZFN/+ vs WT rats include A. muciniphila, noted above, and Ruminococcus gnavus, which is involved in tryptophan metabolism in the gut (40). The broader Ruminococcus genus was also noted to be depleted in RTT participants in Borghi et al. and elevated in IBD patients (41). As the changes in abundance of specific bacterial species at p105 in RTT rats most closely mimics reported changes noted in humans with RTT, this suggests that p105 is a key translational time point in the presented RTT model.
Gut microbiota changes in p105 MeCP2-deficient rats are reflected by changes in predicted microbiome function
It is not unexpected that an animal model of a complex condition such as RTT would demonstrate differences in significant disease-associated bacterial species relative to human studies. Despite these differences, the general functions that significantly altered bacteria perform in the body may be similar between rats and humans and thus identify common biologically relevant pathways in RTT. We utilized PICRUSt (42) to predict functional differences between RTT and WT rat microbiota at p105. KEGG level 1 pathway analysis predicted that RTT rats have significant decreases in pathways related to cellular processes and environmental information processing, and inversely showed increases in pathways related to metabolism and genetic information processing (Fig. 3a).
Strati et al. previously showed enrichment in KEGG pathways related to SCFA metabolism, including carbohydrate metabolism, in humans with RTT. Similarly, RTT rats demonstrate enriched carbohydrate metabolism as well as differences in KEGG pathways that are related to SCFA production. Other altered pathways of note include purine metabolism and fatty acid elongation in mitochondria (Fig. 3 b-d).
Humans with RTT and Mecp2ZFN/+ rats share common alterations in microbiota and resulting predicted functional pathways
To further assess the relationship between the Mecp2ZFN/+ model of RTT and human RTT, we recruited 6 individuals with RTT and their mothers to examine gut microbiome changes. All RTT participants were females with various MECP2 mutations. Most experienced constipation as a gastrointestinal comorbidity, as well as neurological comorbidities including seizures and sleep problems. All RTT participants were taking a variety of medications for various comorbidities, and one patient was taking probiotics in an attempt to ease constipation (Table 2).
To determine changes in microbiome function in our patient samples relative to control samples, we examined differences in LDA score. Generally, anaerobic bacteria had higher LDA scores in mothers compared to RTT participants. Of note, the SCFA producing taxonomies Lachnobacterium and Faecalibacterium are decreased in RTT participants compared to their mothers. In contrast, the family Clostridiaceae, which can produce epsilon toxin, is increased in those with RTT (Fig. 4).
Given these significant LEfSe findings, we next utilized QIIME (43) to map taxonomies in each group to KEGG Orthologs (KOs), and mapped KOs to KEGG pathways with PICRUSt. Consistent with a decrease in SCFA producing bacteria, RTT participants show a decrease compared to their mothers in KEGG pathways related to microbial SCFA production including the pentose phosphate pathway and purine metabolism. (Fig. 5). The data reveal clear functional similarities between RTT patient microbiomes and the microbiomes of MeCP2-deficient rats, inviting the potential for increased therapeutic relevance from RTT rat microbiome studies.
Impact of MecP2 mutation on fecal SCFA levels at p105
As previous studies in humans with RTT showed alterations in fecal SCFA content (15,16) and our sequencing results indicate alterations at p105 in SCFA-related microbial pathways, we next examined the content of 12 SCFAs in fecal samples from p105 RTT and WT rats. Unbiased hierarchical clustering of total SCFA profiles in RTT rats and WT shows clear clustering of RTT samples separately from WT (Fig. 6A). There were no measurable levels of 3-methyl valeric acid or octanoic acid in our samples. Additionally, there were no differences between RTT and WT feces in levels of propionic acid, butyric acid, iso-burtyic acid, 2-methyl butyric acid, iso-caproic acid, valeric acid, or iso-valeric acid (Fig. 6A-G). However, RTT samples do contain significantly lower levels of acetic acid (p = 0.0062), caproic acid (p= 0.0044 ), and heptanoic acid (p=0.0186) (Fig 6 H-J). Our findings suggest that RTT rats have a distinct fecal SCFA profile.