Interactions of the enteric nervous system with the gut microbiome in the neuroligin-3 R451C mouse model of autism
Document TypePhD thesis
Access StatusOpen Access
© 2020 Madusani Herath Maduwelthanne Herath Mudiyanselage
Autism patients are four times more likely to be hospitalized due to gastrointestinal (GI) dysfunction compared to the general public. However, the exact cause of GI dysfunction in individuals with autism is currently unknown. Genetic predisposition to autism spectrum disorder (ASD) has been highlighted in various studies and mutations in genes that affect nervous system function can drive both behavioural abnormalities and GI dysfunction in autism. Neuroligin-3 (NLGN3) is a postsynaptic membrane protein and the R451C missense mutation in the NLGN3 gene is associated with ASD. Recent studies revealed that the NLGN3 R451C mutation induces GI dysfunction in autism patients as well as in mice but, the cellular localization and the effects of this mutation on NLGN3 production in the enteric nervous system (ENS) have not been reported to date. The intestinal mucosal barrier is the interface separating the external environment from the interior of the body. Mucosal barrier functions are directly regulated by the enteric nervous system. Therefore, ENS dysfunction can induce mucosal barrier impairments. An impaired intestinal barrier has been reported in autism patients, but neurally-mediated barrier dysfunctions have not been assessed in transgenic autism mouse models with an altered nervous system. The intestinal mucus layer is the outermost layer of the mucosa which separates the intestinal microbiota from the intestinal epithelium. The mucus layer also serves as an energy source for mucus-residing microbes in the intestine. Although the composition of mucus-residing microbiota is altered in a subset of autism patients, the underlying physiological interactions between the host and these microbes are unclear. Identifying the precise spatial location of microbial populations in the gut is essential in order to understand host-microbial interactions but this has not been investigated in Nlgn3R451C mice. In Chapter 3, I developed a method combining RNAScope in situ hybridization and immunohistochemistry technique to localize Nlgn3 mRNA in enteric neuronal subpopulations and glia. Further, a 3-dimensional quantitative image analysis method was developed to measure the Nlgn3 mRNA expression in the ENS. The same protocol was used to determine the effects of the Nlgn3 R451C mutation on Nlgn3 mRNA synthesis in the enteric nervous system. Findings from this study showed that, Nlgn3 mRNA is expressed in most submucosal and myenteric neurons in the ENS. Interestingly, this study revealed for the first time that Nlgn3 mRNA is expressed in enteric glia. In addition, analysis from this study demonstrated that the R451C mutation reduces Nlgn3 mRNA expression in most enteric neurons in mutant mice compared to WT. In Chapter 4, I investigated the effects of the Nlgn3 R451C mutation on intestinal mucosal barrier functions including the paracellular pathway and mucosal secretion in the small intestine. The Ussing chamber technique was used to measure the paracellular permeability and mucosal secretion ex vivo. Since the paracellular pathway is regulated by tight junctions, effects of this mutation on tight junction protein gene expression were measured using real-time (RT) PCR array and droplet digital (dd) PCR approaches. The impact of the Nlgn3 R451C mutation on the neurochemistry of the submucosal plexus was examined using immunocytochemistry. Results from these experiments indicated that the R451C mutation increases paracellular permeability and decreases transepithelial resistance (TER) in the distal ileum. However, ileal tight junction protein gene expression is unchanged in mutant mice compared to WT. Pharmacological stimulation of submucosal ganglia decreased the neurally-evoked mucosal secretion in mutant mice compared to WT. In addition, immunohistochemistry data revealed increased numbers of non-cholinergic and decreased cholinergic neuronal populations in the submucosal plexus in the distal ileum but not in the jejunum in Nlgn3R451C mutant mice. Given that, I identified altered barrier functions in the distal ileum of Nlgn3R451C mutant mice, in chapter 5, I also analysed the spatial distribution of the mucus-residing microbial populations in this region. To determine the spatial distribution of total bacteria, phylum Bacteroidetes, phylum Firmicutes, Akkermansia muciniphila (A. muciniphila) and Bacteroides thetaiotamicron (B. thetaiotamicron) were labelled using fluorescent in situ hybridization. Immunofluorescence for the mucin-2 protein was incorporated to co-stain the mucus and determine the thickness of the mucus layer. Both the spatial pattern of microbial populations and mucus layer thickness were analysed using MATLAB-based BacSpace software. Immunofluorescence experiments revealed that the R451C mutation increases mucus density adjacent to the epithelium. Along with increased mucus density, the total bacterial density was higher in the mucosa in mutant mice. Further, a decreased ratio of Bacteroidetes/Firmicutes, a decreased A. muciniphila density and increased density of B. thetaiotamicron were observed in mutant mice. Overall, findings from this thesis revealed that NLGN3 is expressed in the enteric nervous system and that the R451C mutation reduces Nlgn3 mRNA expression levels in enteric neurons. Furthermore, the Nlgn3 R451C mutation impairs intestinal mucosal barrier integrity. Findings from this study also revealed that this mutation alters mucus density as well as the spatial distribution and composition of the microbial community in the distal ileum in mice. Therefore, these findings highlight that an autism-associated gene mutation that affects nervous system function impairs the mucosal barrier and may contribute to the pathophysiology of GI dysfunction in ASD.
KeywordsEnteric nervous system; Autism spectrum disorder; Neuroligin-3; Gastrointestinal dysfunction; Intestinal mucosal barrier; Gut microbiome
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